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https://openalex.org/W2751146544
https://europepmc.org/articles/pmc5574549?pdf=render
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Visual affective classification by combining visual and text features
PloS one
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RESEARCH ARTICLE Ningning Liu1, Kai Wang2*, Xin Jin3, Boyang Gao4, Emmanuel Dellandre´a5, Liming Chen5 Ningning Liu1, Kai Wang2*, Xin Jin3, Boyang Gao4, Emmanuel Dellandre´a5, Liming Chen5 1 School of Information technology and Management, University of International Business and Economics, BJ, P.R.China, 2 School of Manufacturing Science and Engineering, Sichuan University, Chengdu, P.R. China, 3 Department of Computer Science and Technology, Beijing Electronic Science and Technology Institute, BJ, P.R.China, 4 Department of Advanced Robotics, Istituto Italiano di Tecnologia (IIT), via Morego, Genova, Italy, 5 LIRIS, Ecole Centrale de Lyon, Universite´ de Lyon, CNRS, UMR5205, France * kai.wang@scu.edu.cn * kai.wang@scu.edu.cn a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Liu N, Wang K, Jin X, Gao B, Dellandre´a E, Chen L (2017) Visual affective classification by combining visual and text features. PLoS ONE 12(8): e0183018. https://doi.org/10.1371/journal. pone.0183018 Editor: Christos Papadelis, Boston Children’s Hospital / Harvard Medical School, UNITED STATES Published: August 29, 2017 Copyright: © 2017 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Affective analysis of images in social networks has drawn much attention, and the texts sur- rounding images are proven to provide valuable semantic meanings about image content, which can hardly be represented by low-level visual features. In this paper, we propose a novel approach for visual affective classification (VAC) task. This approach combines visual representations along with novel text features through a fusion scheme based on Dempster- Shafer (D-S) Evidence Theory. Specifically, we not only investigate different types of visual features and fusion methods for VAC, but also propose textual features to effectively cap- ture emotional semantics from the short text associated to images based on word similarity. Experiments are conducted on three public available databases: the International Affective Picture System (IAPS), the Artistic Photos and the MirFlickr Affect set. The results demon- strate that the proposed approach combining visual and textual features provides promising results for VAC task. * kai.wang@scu.edu.cn Visual affective classification by combining visual and text features Funds for the Central University in UIBE (14QD21) and the Sichuan Province Science and Technology Support Program Project under Grant 2015JY0172. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. In existing literatures, most works on VAC so far focus on investigating visual representa- tions, e.g. color factors [10, 11], texture attributes [12], shape elements [9] as well as aesthetic features [13, 14]. Specifically, Colombo et al. [11] developed expressive and emotional level fea- tures based on Ittens theory [15] and semiotic principles. Machajdik et al. [5] investigated four groups of visual features for VAC including color, texture, composition and content. Liu et al. [16] proposed an emotion descriptors by using a novel affective probabilistic latent semantic analysis (affective-pLSA) model. Above works investigate visual representations for emotional concepts mainly by using traditional visual features [5], machine learning strategies [16] or human perceptual rules [11]. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. With the popular use of social networks in recent years, the increasing literatures have exposed rich resources of semantic information conveyed by online user generated content: the images and the associated texts(captions or tags) [9, 17–20]. Sivic et al. [18] investigated a text retrieval approach that can be successfully applied to VOC. Wang et al. [17] built a text- based feature by using the tags of an auxiliary dataset from internet, and demonstrated that it consistently improves performance on VOC problems. Liu et al. [21] proposed a multimodal approach effectively fusing visual and text modalities to predict various concepts (including 9 emotional ones) in images. All above methods improve the performance of different visual- based concept classification by making use of the texts from user generated content (UGC), as the texts surrounding an image (tags, discussions, group names) provide valuable information that can hardly be represented by the visual features [9]. In fact, the conventional visual fea- tures are hard to handle the unpredictability of objective concepts’ positions, sizes, appear- ances, lightings, and unusual camera angles, not to mention the emotional concepts. In a word, how to leverage the text and visual information to help perceiving the visual emotional semantics is one of the promising directions for VAC. Introduction Visual object classification (VOC) targets on classification of objects in images at the cognitive level. By contrast, visual affective classification (VAC) aims at identifying the emotions that are expected to arise in image reviewers at the affective level, which proves to be extremely chal- lenging due to the semantic gap between the low level visual features and the high level emo- tion-related concepts [1, 2]. VAC topic involves multiple research fields, including computer vision, pattern recognition, artificial intelligence, psychology, and cognitive science. Studies on psychology and affective computing [3–5] indicate that the prediction of emotions in image viewer is of subjectivity, which suggests that the emotions rely on individual feelings. The rea- son is that people from different backgrounds or cultures might perceive the same visual con- tent quite differently. However, recent works on affective computing [3, 6–9] argue that certain features in images, as a universal validity to classify images in terms of affective con- cept, are believed to evoke some human feelings more easily, and have certain stability and generality across different people and different cultures. Data Availability Statement: All relevant data is available through the following contacts: 1) The IAPS, Ph.D M.M. Bradley, Coordinator CSEA Media Core, media@cseamedia.org 2) The Artistic Photo Set, Ph.D Jana Machajdik, Faculty of Informatics, jana@caa.tuwien.ac.at 3) The Mirflick Affect Set, Ph.D Emmanuel Dellandra, e´cole Centrale de Lyon, emmanuel.dellandrea@ec-lyon.fr. Funding: This work was supported by the National Natural Science Foundation of China under Grant (51435011, 51505309), the Fundamental Research 1 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 In this paper, we target on proposing an effective approach combining visual and text infor- mation for VAC. Fig 1 shows the flowchart of our framework. For each of image, visual Fig 1. The framework of proposed approach. Note that if there is no tags associated to the image, only the visual classifiers are combined to predict the results. of proposed approach. Note that if there is no tags associated to the image, only the visual classifiers are combined Fig 1. The framework of proposed approach. Note that if there is no tags associated to the image, only the visual classifiers are combined to predict the results. https://doi.org/10.1371/journal.pone.0183018.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 2 / 21 Visual affective classification by combining visual and text features descriptors produce visual features for visual classifiers. Meanwhile, if available, the associated text is preprocessed to build the text features for text classifiers. All classifiers are then com- bined to predict the semantic emotion category of the input test image. In order to evaluate the effectiveness of proposed methods, we not only testify combining visual feature within two emotion models on the IAPS [22] and the Artistic Photos database [5], but also test fusing of visual and text features on the MirFlickr Affect set [8]. As a summary, the contributions of our paper can be summarized as following three aspects: • We propose a late fusing scheme for VAC based on D-S Evidence Theory, whose interesting properties allow fusing different ambiguous sources of information. This scheme is proved to be efficient to fuse different features for VAC. • We propose a late fusing scheme for VAC based on D-S Evidence Theory, whose interesting properties allow fusing different ambiguous sources of information. This scheme is proved to be efficient to fuse different features for VAC. • We build a textual feature, namely the emotional Histogram of Textual Concepts (eHTC), to effectively capture emotional semantics for the short text, and we also try to measure the emotional metrics of text based on the Affective Norms for English Words(ANEW) data set. • We investigate various visual features for VAC, including mid-level features related to aes- thetic quality, harmony, dynamism etc., and evaluate their efficiency within two emotion models. The rest of this paper is organized as follows. First, we describe the related works. Next, we present the proposed text features and other four groups of visual features for emotional semantics respectively. Then, we carry out the experiments and show the corresponding results. Finally, we give the discussion and draw the conclusion. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 Visual affective classification by combining visual and text features Fig 2. The two types of emotion models. While (a) is a discrete emotion model from Kate Hevners Adjective Circle [23], (b) is a dimensional model from Wundt’s [26]. https://doi org/10 1371/journal pone 0183018 g002 Fig 2. The two types of emotion models. While (a) is a discrete emotion model from Kate Hevners Adjective Circle [23], (b) is a dimensional model from Wundt’s [26]. Fig 2. The two types of emotion models. While (a) is a discrete emotion model from Kate Hevners Adjective Circle [23], (b) is a dimensional model from Wundt’s [26]. https://doi.org/10.1371/journal.pone.0183018.g002 from Colombo et al. [28], who proposed expressive and emotional level features based on Ittens theory [15] and semiotic principles. Ke et al. [14] proposed high level features for image aesthetic classification based on a group of principles, including simplicity, realism and basic photographic technique. On the other hand, the machine-learning based methods try to learn a mapping function between visual features and high level emotional semantics. Wang wei- ning et al. [29] firstly developed an orthogonal three-dimension emotional model using 12 pairs of emotional words, and then predict the emotional factor using SVM regression based on three fuzzy histograms. Machajdik et al. [5] employed two feature selection algorithms and investigated various features including color, texture, composition, and content features for affective image classification. In recent years, many works [30–36] based on deep learning [37] have been shown to achieve remarkable improvements on the performances of various VOC tasks [36, 38, 39], as these models can be trained to capture powerful features for visual objects. However, when it comes to emotions, the existing literatures based on deep learning mainly focus on face image based [40–42] or multiple physiological signals based emotion recognition [43]. The application of deep learning in VAC task is limited as it usually requires large amounts of data and time to train a robust model. Thus, the applicability of deep learning in domains are different but related to that of the training set [44]. In short, above approaches for VAC have largely demonstrated their effectiveness within different dataset, but the major shortcoming is still that their visual features are lack of descriptive power as regard to the high level emotional concepts. from Colombo et al. [28], who proposed expressive and emotional level features based on Ittens theory [15] and semiotic principles. Ke et al. Related works As far as emotion recognition is concerned, researchers mainly focus on emotion recognition in audio (speech or music) and facial expressions (visual or 3D based). Limited contributions are concerned with the recognition of affective semantics carried by images, and a lot of issues need to be addressed particularly concerning following three fundamental problems: How to build emotion models to describe human moods in compute? How to extract features to repre- sent high-level semantics and how to establish classification schemes to handle the distinctive characteristics of emotions [3]? Accordingly, the related works can be summarized as follows: The emotion models. In literatures, several emotion models have been proposed and can generally be categorized into two types: the discrete one and the dimensional one. The discrete emotion models take adjectives or nouns to specify the emotions, such as happy, sadness, fear, anger, disgust and surprise. A common example is Kate Hevner’s Adjective Circle [23], as depicted in Fig 2(a). The dimensional models regard emotions as a coincidence of values on a number of different strategic dimensions [22], such as valence, arousal or dominance. A very early approach has been proposed by Wundt [23, 24], as shown in Fig 2(b). In practice, most works in VAC employ the discrete models, as it can be easily applied to image tagging or label- ing [25], but they usually require a heavy dictionary, and they cannot represent a wide range of emotions compared with the dimensional ones, which allow percentage based ratings to assign specific emotion dimensions [3]. In this paper, both emotion models are employed to testify our approaches for VAC. The affective features. The state-of-arts for VAC have so far proposed a large set of visual features, which generally can be divided into two types: the hierarchy approaches and the machine-learning approaches [27]. The approaches belonging to the first category build a hier- archical inference model based on the domain knowledge or rules. One of the initial works is PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 3 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 [14] proposed high level features for image aesthetic classification based on a group of principles, including simplicity, realism and basic photographic technique. On the other hand, the machine-learning based methods try to learn a mapping function between visual features and high level emotional semantics. Wang wei- ning et al. [29] firstly developed an orthogonal three-dimension emotional model using 12 pairs of emotional words, and then predict the emotional factor using SVM regression based on three fuzzy histograms. Machajdik et al. [5] employed two feature selection algorithms and investigated various features including color, texture, composition, and content features for affective image classification. In recent years, many works [30–36] based on deep learning [37] have been shown to achieve remarkable improvements on the performances of various VOC tasks [36, 38, 39], as these models can be trained to capture powerful features for visual objects. However, when it comes to emotions, the existing literatures based on deep learning mainly focus on face image based [40–42] or multiple physiological signals based emotion recognition [43]. The application of deep learning in VAC task is limited as it usually requires large amounts of data and time to train a robust model. Thus, the applicability of deep learning in domains are different but related to that of the training set [44]. In short, above approaches for VAC have largely demonstrated their effectiveness within different dataset, but the major shortcoming is still that their visual features are lack of descriptive power as regard to the high level emotional concepts. As current visual representations for high-level visual concepts (e.g. objects, events, emo- tions) appear to be reaching the ceiling of performance, there exists an increasing works inter- ested in web data mining [14, 17] or multimodal approaches [21, 45–48], which manage to utilize both the visual and associated text data from Internet. While the main approaches for PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 4 / 21 Visual affective classification by combining visual and text features representing the textual content are the word frequency statistic models (TF) with different variants (TF/IDF), these models have developed several extensions, including latent semantic analysis (LSA) [49], probabilistic LSA [50], and Latent Dirichlet allocation (LDA) [51]. As described in detail previously [21], the major drawback of these word frequency statistic-based approaches is lack of semantic sensitivity for three reasons: First, a text document is simply interpreted as an unordered collection of words, thereby disregarding grammar and even word order. Second, a text document is further summarized as a vector of term frequencies, thereby failing to capture the relatedness between words. Third, the tags for a given image from Internet is highly sparse (8.7 tags on average per image in MIR FLICKR), thereby hardly represented by the frequency statistic models. Recently, the word2vec [52] have provided state-of-the-art performances on many natural language processing (NLP) task [53, 54]. How- ever, the different model parameters and different corpora sizes can greatly affect the quality of a word2vec model, which makes it hard to capture word sentiment from the small image tags data set [55]. As a consequence, popular text mining techniques (word2vec, LSTM) developed for text classification or retrieval are not applicable for the short text data directly. To tackle these problems, we propose novel textual representations, which can effectively handle the image tags and can improve the performance of VAC by effectively fusing with visual features. g g p p y y g The classification scheme. In the review of classification schemes for VAC, a number of works [16, 56, 57] build their classifying methods by employing the traditional classifiers. Yanulevskaya et al. [56] employed support vector machines (SVM) to build an emotion classi- fication approach for art works. Guo et al. [57] proposed an emotion recognition system based on neuro-Hidden Markov Model (HMM) to classify the emotion contained in images. Liu et al. [16] built an emotion categorization system via a multilabel k-nearest-neighbor (KNN) classifier based on the visual descriptors. Among them, some works have tried to investigate fusion methods to improve the performance of VAC. One of works is from Machajdik [5], who simply concatenated low-level features to one vector and fed it to naive Bayers classifiers for VAC task. Ke [14] combined the quality metrics by linear fusion method for photo aes- thetic classification. In recent years, many works [40, 42, 45] have proposed multimodal approaches to fuse visual and text data to analysis various concepts in images. Ba¨nziger et al. [40] established the multimodal emotion recognition test (MERT) to measure the emotional competence in multimodal approaches that combining the visual and auditory sense modali- ties (audio/video, audio only, video only, still picture). M. Malinowski [45] proposed a multi- modal approach for automatic question answering by combining semantic segmentations of real-world scenes with symbolic reasoning about questions in a Bayesian framework within Visual Turing Challenge [58, 59]. Ngiam et al. [42] proposed a novel application of deep net- works to learn features by multiple modalities from multiple sources. Above works indicated that the performance of VAC or VOC task can be further improved by simple early fusing methods [5, 14] or by multimodal approaches [40, 42, 58, 59]. As emotions are high-level semantic concepts and by nature highly subjective and ambiguous, it is need to build a classifi- cation scheme to handle the information that may be uncertain, incomplete and leading to conflicts. In this paper, we manage to solve this issue by introducing a fusion method for VAC task based on the Evidence Theory, which allows to handle ambiguity and uncertainty in the emotion characteristics especially dealing with the small data set. Text features for emotional semantics In this section, we first present a text feature for emotional semantics, namely emotional histo- gram of textual concepts(eHTC) which extends the histogram of textual concepts(HTC) [21] to capture the emotional tendency by employing an affective dictionary ANEW and a semantic PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 5 / 21 Visual affective classification by combining visual and text features similarity measurement. Moreover, we also propose a new text feature, namely emotional Met- rics of Textual Concepts(eMTC) which measures the projection of tags in the three dimen- sional affective space [60] based on the affective ratings of the ANEW concepts. eHTC: emotional Histogram of Textual Concepts In recent years, there is an strong increasement on sharing websites particularly related to pho- tos and videos, and most of them (e.g. Flickr, Facebook, Weibo) allow users to share images and to contribute descriptions in the form of tags or captions. These texts provide valuable resources of information describing the visual data. Based on these data, Wang et al. [17] built a text-based feature (TF) by using an auxiliary dataset of images annotated with tags, and it improves the performance of VOC particularly when the training dataset is small. Mensink et al. [47] also employed the TF feature combing with the visual features to improve the perfor- mance of visual concept classification. In contrast to these conventional Bag-of-Words approaches, we have proposed HTC to capture the relatedness of semantic concepts through a three-step process as depicted in previously [21], showing in Fig 3. In this paper, we propose the eHTC for VAC, which is to calculate a histogram of textual concepts towards an emotional dictionary, and each bin is the contribution of each word toward the underlying concept according to a predefined semantic similarity measurement. The calculation of eHTC needs a definition of dictionary and a proper words similarity. In practice, we use DANEW as the dictionary, which is being developed to provide a set of norma- tive emotional ratings for a large number of words [61], and we employ the Resnik’s measure- ment as the words similarity, which uses the term probability based on the information content of a term distance. The algorithm of eHTC is detailed as following Algorithm 1: Fig 3. The three steps process of our HTC algorithm. As demonstrated in [21], the HTC describes the meaning of a word by its atoms, components, attributes, behavior, related ideas, etc., and is in clear contrast to the BoWs approaches where the relatedness of textual concepts are simply ignored as word terms are statistically counted. For instance, the concept of “peacock” can be described by “bird”, “beautiful”, “pretty”, “feathers”, “monsoon”, “animal”, “plumage”, etc. Fig 3. The three steps process of our HTC algorithm. As demonstrated in [21], the HTC describes the meaning of a word by its atoms, components, attributes, behavior, related ideas, etc., and is in clear contrast to the BoWs approaches where the relatedness of textual concepts are simply ignored as word terms are statistically counted. eHTC: emotional Histogram of Textual Concepts fi ¼ fi= Pd j¼1 fj. 1 When an image has no tag, we set each bin value of eHTC as 0.5, which is at the middle between 0 (no connection to di in the dictionary) and 1 (sameness as di in the dictionary). 1 When an image has no tag, we set each bin value of eHTC as 0.5, which is at the middle between 0 (no connection to di in the dictionary) and 1 (sameness as di in the dictionary). There are two main differences between HTC and eHTC. First, in contrast to HTC’s fre- quency-based words from the training data [21], the eHTC employs an emotional dictionary ANEW, which contains a relative large set of emotional words. Compared to other sentimental dictionaries e.g. POMS [62], SentiStrength [63], SentiWordNet [64], the ANEW is the most appropriate choice. Moreover, the eHTC uses the Resnik’s words similarity measurement [65], which performs well in a wide range of applications such as word sense disambiguation. In fact, we also evaluated other popular words similarity measurements [20] by using Natural lan- guage toolkit [66], such as Path [67], Wup [68] and Lin [69] distance measurements, but in our case the Resnik one proved to be the best choice. Compared to the conventional term frequency-based features, the advantages of eHTC are multiple as the HTC’s [21]. First, for the sparse text such as image tags, eHTC offers a smooth description of the semantic measurements of user tags over a set of textual concepts defined by the dictionary. Second, for the case of polysemy or synonyms, eHTC helps disambiguate tex- tual concepts according to the context. For example, the concept of “bank” can refer to a finan- cial institution but also to the sloping land of a river. However, when a tag “bank” comes with a photo showing a financial institution with tags such as “finance”, “building”, “money”, etc., thereby clearly distinguishing the concept “bank” in finance from that of a river where corre- lated tags can be “water”, “boat”, “river”, etc. This is also the reason that we improve the perfor- mance of visual features and rank the first out of 80 runs within the ImageCLEF 2012 photo annotation challenge. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 eHTC: emotional Histogram of Textual Concepts For instance, the concept of “peacock” can be described by “bird”, “beautiful”, “pretty”, “feathers”, “monsoon”, “animal”, “plumage”, etc. Fig 3. The three steps process of our HTC algorithm. As demonstrated in [21], the HTC describes the meaning of a word by its atoms, components, attributes, behavior, related ideas, etc., and is in clear contrast to the BoWs approaches where the relatedness of textual concepts are simply ignored as word terms are statistically counted. For instance, the concept of “peacock” can be described by “bird”, “beautiful”, “pretty”, “feathers”, “monsoon”, “animal”, “plumage”, etc. https://doi.org/10.1371/journal.pone.0183018.g003 https://doi.org/10.1371/journal.pone.0183018.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 6 / 21 Visual affective classification by combining visual and text features Algorithm 1: emotional Histogram of Textual Concepts (eHTC) Algorithm 1: emotional Histogram of Textual Concepts (eHTC) Input: The tags W = {wt} and the dictionaryDANEW = {di} as t 2 [1, T] and i 2 [1, d]. Output: Histogramf composedof values fi with 0  fi  1 and i 2 [1, d]. Input: The tags W = {wt} and the dictionaryDANEW = {di} as t 2 [1, T] and i 2 [1, d]. Output: Histogramf composedof values fi with 0  fi  1 and i 2 [1, d]. • Preprocessthe tags by filtering with a stop-wordslist. • Preprocessthe tags by filtering with a stop-wordslist. • If the image has no tags, return f as fi = 0.5, 8i 2 [1, d].1 • Preprocessthe tags by filtering with a stop-wordslist. • If the image has no tags, return f as fi = 0.5, 8i 2 [1, d].1 • If the image has no tags, return f as fi = 0.5, 8i 2 [1, d].1 • For each word wt in W: • For each word wt in W: 1. Compute the dist(wt, di) between concepts wt and di, where dist is the Resnik measure. 1. Compute the dist(wt, di) between concepts wt and di, where dist is the Resnik measure. 2. Obtain the matrix M as: M(t, i) = dist(wt, di). • Compute the feature f as: fi ¼ PT t¼1 Mðt; iÞ, and normalize to [0 1] as: d • Compute the feature f as: fi ¼ PT t¼1 Mðt; iÞ, and normalize to [0 1] as: d • Compute the feature f as: fi ¼ PT t¼1 Mðt; iÞ, and normalize to [0 1] as fi ¼ fi= Pd j 1 fj. Algorithm 2: emotional Metrics of Textual Concepts (eMTC) Algorithm 2: emotional Metrics of Textual Concepts (eMTC) Input: Tag data W = {wt} with t 2 [1, T], dictionaryDANEW = {di} with i 2 [1, d], the ratings of words in DANEW including valence Vi, arousal Ai and control Ci. Output: Metrics in valence, arousal and dominance dimeansions(mv, ma, mc). • Preprocessthe text by using a stop-wordsfilter. • If the input image has no tags (W = ;), return m with 8i mi = 0.5. • If the input image has no tags (W = ;), return m with 8i mi = 0.5. • If the input image has no tags (W = ;), return m with 8i mi = 0.5 • Do for each word wt 2 W: • If the input image has no tags (W ;), return m with 8i mi 0.5. • Do for each word wt 2 W: • Do for each word wt 2 W: 1. Calculate dist(wt, di), where dist is the Resnik measure between concepts wt and di. 1. Calculate dist(wt, di), where dist is the Resnik measure between concepts wt and di. 2. Obtain the semantic matrix S as: S(t, i) = dist(wt, di). • Calculate the eHTC feature f as: fi ¼ PT Sðt iÞ and normalize 2. Obtain the semantic matrix S as: S(t, i) = dist(wt, di). • Calculate the eHTC feature f as: fi ¼ PT t¼1 Sðt; iÞ, and normalize it to [0 1] as: fi ¼ fi= Pd j¼1 fj. • Calculate the eHTC feature f as: fi ¼ PT t¼1 Sðt; iÞ, and normalize it to [0 1] as: fi ¼ fi= Pd j¼1 fj. • Calculate the eMTC feature m as: mv = (1/d)∑i(fi.Vi), ma = (1/d)∑i(fi.Ai) and mc = (1/d)∑i(fi.Ci). • Calculate the eMTC feature m as: mv = (1/d)∑i(fi.Vi), ma = (1/d)∑i(fi.Ai) and mc = (1/d)∑i(fi.Ci). Visual features for emotional semantics According to the study [5, 70], the VAC approaches are fundamentally different from the dominant VOC approaches, in which the SIFT-related features are the standard descriptors. The features based on global image statistics (global histograms) perform better than local image descriptors (bag-of-words models) for emotional categories [70]. In this paper, we com- pute a set of global features to represent the layout and the atmosphere of an image. All these features can be categorized into four groups: the color, texture, shape and mid-level, showing in Table 1: Color. According to [5], colors can be effectively used by artists to induce emotional effects. Studies [15] shows that HSV (Hue, Saturation, and Value) color space is more related to human color perceptions, and different color is associated with different emotions, such as red is connected to happiness, dynamism and power whereas its opposite color is green. In this Table 1. Summary of 4 group visual features. Category Features(Short name) # Short Description Color Color moments(color_Mom) 144 It is the three central moments(Mean, Standard deviation and Skewness) on HSV channels. Color histogram(color_Hist) 192 This histogram is concatenated from 64 bins on each HSV channel. Color correlograms(color_Corr) 256 It is a three-dimensional table expresses how the spatial correlation of color changes in a image. Texture Tamura(texture_Tamu) 3 Tamura [12] proposed visual features describing the coarseness, contrast, directionality. Grey level Co-occurrence matrix (texture_GCM) 16 It is defined as the distribution of co-occurring values at a given offset over an image. Local binary pattern(texture_LBP) 256 A compact multi-scale texture descriptor analysis of textures with multiple scales by combining neighborhoods with different sizes. Shape Histogram of line orientations (shape_Hist) 12 It is the number of lines in different orientations by using Hough transform [73]. Mid-level Face information(mid_Face) 2 Number of faces in images and their emotion categories, based on Viola and Jones face detector [75] and Adaboost face expression recognition [76]. Harmony(mid_Harm) 1 It is to describe the color harmony atmosphere of a image based on Itten’s color theory. Dynamism(mid_Dyna) 1 It is calculated as the ratio of oblique lines against horizontal and vertical ones. Y. Ke(mid_Ke) 5 We implement the aesthetic criterion from Ke [14] including: spatial distribution of edges, hue count, blur, contrast and brightness. R. Datta(mid_Datta) 44 We implement most of Datta’s aesthetic features [74] (44 of 56) except those related to the integrated region matching(IRM) calculation. Table 1. eMTC: emotional Metrics of Textual Concepts The eMTC is designed to measure the emotional metrics on valence, arousal, and dominance dimensions based on the ANEW set, in which each word is valuated with scores from 1 to 9 in terms of three affective dimensions valence(ranging from pleasant to unpleasant), arousal (ranging from calm to excited) and dominance(ranging from controlled to arbitrary). For instance, the “adorable” has a mean valence of 8.12, a mean arousal of 4.96 and a mean domi- nance of 6.00. By using the affective ratings of the ANEW set, we compute the projection of an document on the three dimensional affective space, in terms of valence, arousal and dominance metrics by a linear combination between the ANEW concept’s ratings and the corresponding eHTC values. More precisely, based on the eHTC f extracted from a text, the emotional metrics of a text document in valence mv, arousal ma and dominance md dimensions eMTC can be com- puted as follows in Algorithm 2: PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 7 / 21 Visual affective classification by combining visual and text features Algorithm 2: emotional Metrics of Textual Concepts (eMTC) Visual affective classification by combining visual and text features paper, different features based on HSV color space are employed to describe color information in image including: color histograms, moments of color, and correlograms. Texture. Textures in images also contain emotional expressions, e.g. Tamura features have been proven to correlate with human visual perception [5, 9]. In this paper, Tamura feature [12], co-occurrence [71], together with local binary pattern (LBP) [72] are employed to repre- sent visual texture semantics. Shape. Studies on artistic paintings have brought to the fore semantic meanings of shape and lines, and it is believed that shapes influence the degree of aesthetic beauty perception [11]. In this paper, the Hough transform is employed to build a histogram of line orientations in 12 different orientations [73]. Mid-level. We have proposed features to describe the harmony and dynamism characteris- tics in an image [8, 9]. The harmonic feature is computed according to Itten’s color theory [15], where colors are organized into a chromatic sphere and harmonious color positions on the sphere are connected thanks to regular polygons [21]. Meanwhile, a ratio has been pro- posed to characterize the dynamism in an image, defined as the numbers of oblique lines divided by the total number of lines in an image. Moreover, we implemented the works for image aesthetic classification [14, 74], which are expected to help measure the factor of pleas- ant in images, as a good aesthetic images usually can induce people pleasant feelings. Experiments and results In this section, we conduct experiments on three datasets: the IAPS set (only images and the dataset is available at: http://csea.phhp.ufl.edu/media/requestform.html), the Artistic photo set (Only images and the dataset is available at: http://www.imageemotion.org/) and the MirFlickr Affect photos (Images and texts, and the dataset is available at: http://liris.cnrs.fr/membres? idn=edelland). After describing the experimental setup, we carry out several experiments with an in-depth analysis on: (1) the performance of visual features on IAPS and Artistic photo set; (2) the performance of visual and text features on MirFlickr Affect photos, and (3) the perfor- mance of combination approach based on D-S evidence theory. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 Visual features for emotional semantics Summary of 4 group visual features. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 8 / 21 Visual affective classification by combining visual and text features Table 2. Number of images per dataset and emotion category in IAPS and artistic photo. Amusement Anger Awe Contentment Disgust Excitement Fear Sad sum IAPS* 37 8 54 63 74 55 42 61 394 Artistic P. 101 77 103 70 70 105 115 166 807 Please note that IAPS* is a subset of 394 images from the IAPS dataset. Table 2. Number of images per dataset and emotion category in IAPS and artistic photo. Please note that IAPS* is a subset of 394 images from the IAPS dataset. https://doi.org/10.1371/journal.pone.0183018.t002 https://doi.org/10.1371/journal.pone.0183018.t002 MirFlickr Affect: This affective dataset [79] was collected of about 2000 photographs selected from MIRFLICKR25000 Collection [80]. Compared to the IAPS and Abstract photo set, this dataset is much realistic, as the photos and tags are from the Flickr users uploaded and tagged. This collection supplies all original image tag data, which has an average number of 8.94 words per image. The emotion model of dataset relies on a dimensional view in two primary dimensions: valence one (ranging from pleasant to unpleasant) and arousal one (ranging from calm to excited), which improve the applicability for navigation and visuali- zation [3]. In order to obtain the ground truth of affective space ratings in terms of valence and arousal, the selected 2000 images were rated from 1 to 9 by using a web-survey, where each one was assigned in average 20 times by 20 people. Meanwhile, we preprocess the rat- ings by abandoning instability samples, leaving with 1172 images [8]. Affective image database The available datasets for VAC are rather limited, and the available public database are described as below: IAPS: According to [5, 22], the International Affective Picture System has being developed to provide a set of normative emotional stimuli for experimental investigations of emotion and attention. It is characterized along the dimensions of valence, arousal, and dominance. The image set contains various pictures depicting mutilations, snakes, insects, attack scenes, accidents, contamination, illness, loss, pollution, puppies, babies, and landscape scenes, among others. This data has been widely used in studies of emotion and VAC tasks [5, 8, 16, 27, 77]. Artistic Photos: According to [5], the artistic photos set was downloaded from an art sharing site [78], and was built to investigate whether the conscious use of colors and textures dis- played by the artists can improve VAC. This dataset was obtained by using the emotion cat- egories as search terms in the art sharing site, so the emotion category was determined by the artist who uploaded the photo. These photos were taken by people who attempt to evoke a certain emotion in the viewer of the photograph through the conscious manipula- tion of the image composition, lighting, colors, etc. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 9 / 21 Experimental setup The above databases are built for the studies focusing on emotional concepts, and are much professional compared with other benchmarks, e.g. PASCAL, ImageNET, ImageCLEF dataset. However, the main drawback is that these datasets are relatively small and highly unbalanced. Therefore, we need to carefully setup the experiments to get a convincing results. • To evaluate the performance of visual features on IAPS and Artistic Photo set, we followed the work from Machajdik [5], who employed a discrete emotion model with 8 emotions: anger, awe, disgust, fear, sadness, excitement, contentment, and amusement [81]. Table 2 indicates that both datasets are relatively small (less than 400 images for each class) and with a quit unbalanced distributions. In order to leverage these problems, the experimental setup is done as follows: for the classifiers, we employ the Support Vector Machine framework (SVM) with Radial Basis Functions (RBFs) using one against all scheme, and choose the average true positive rate (ATPR) per class over the positive and negative classes as the evalu- ation measurement defined by [5]. We carry out the experiments in 5-fold cross validation, and evaluate the validity of visual features on IAPS and Artistic Photo sets. • To evaluate the performance of visual and text features on MirFlickr Affect dataset, we firstly build six classes by equally dividing each dimension into three levels: low, neutral and high, showing in Table 3. The experiments are then set as follows: we build six SVM classifiers for each classes using one against all scheme. More specifically, LIBSVM tool [82] are employed, • To evaluate the performance of visual and text features on MirFlickr Affect dataset, we firstly build six classes by equally dividing each dimension into three levels: low, neutral and high, showing in Table 3. The experiments are then set as follows: we build six SVM classifiers for each classes using one against all scheme. More specifically, LIBSVM tool [82] are employed, Table 3. The description of the MirFlickr affect dataset. Database Size Text LV NV HV LA NA HA MirFlickr Affect dataset 1172 8.93/image 257 413 502 261 693 218 Please note that LA is the acronym of low arousal, NA is neutral arousal, HA is high arousal, LV is low valence, NV is neutral valence and HV is high valence. Table 3. The description of the MirFlickr affect dataset. https://doi.org/10.1371/journal.pone.0183018.t003 Experimental setup Database Size Text LV NV HV LA NA HA MirFlickr Affect dataset 1172 8.93/image 257 413 502 261 693 218 Please note that LA is the acronym of low arousal, NA is neutral arousal, HA is high arousal, LV is low valence, NV is neutral valence and HV is high valence. Table 3. The description of the MirFlickr affect dataset. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 10 / 21 Visual affective classification by combining visual and text features and the input features are normalized to train the RBF kernel based SVM classifiers, which produce measurement vector as the degree of input belongs to different classes. To obtain reliable results, we measure the ATPR performance of visual and text features within 5-fold cross validation. • To combine the features, we employ a powerful classifier combination technique based on the D-S evidence theory, whose interesting properties allow to fuse different ambiguous sources of information. The experimental setup is carried out as follows: First, we extract fea- tures on MirFlickr Affect set and train the SVM classifiers on a training set (50% data set), which produces measurement vector as the degree of input belongs to different classes. Then, we define the belief function dn(θk) according to the following formulas: mnðykÞ ¼ dnðykÞ=ð X k dnðykÞ þ 0:5Þ ð1Þ ð1Þ ð1Þ where dnðykÞ ¼ expðk wn k yn k2Þ, wn k is a reference vector and will be initialized ran- domly. Finally, as inspired by the Al-Ani’s work [83], the classifiers are combined by adjust- ing wn k so that minimizing the mean square error (MSE) of the combined classification results zk as Err ¼ jjz tjj 2 ð2Þ zk ¼ mnðykÞ ð3Þ ð2Þ zk ¼ mnðykÞ ð3Þ ð3Þ where t is the target output. Meanwhile, we make a comparison with different types of com- bination approaches, including: meanscore : zk ¼ 1 N X N n¼1 yn k ð4Þ ð4Þ majorityvoting : zk ¼ argmaxðy1 k; y2 k; :::; yN k Þ ð5Þ ð5Þ where yn k represent the kth measurement of classifier cn. In order to testify the fusion methods for VAC, we combine the visual features for IAPS and Artistic Photo set, and fuse the visual and text features for MirFlickr Affect dataset. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 Performance of visual features on IAPS and artistic photos Fig 4 shows the performance of each visual feature on the IAPS (a) and Artistic Photos sets (b) respectively. Fig 4(a) shows the results of the IAPS, and we can see that the texture and color features with average 55% ATPR perform better than the shape and mid-level ones for most categories. The reason is that the shape and mid-level descriptors are low dimensions and with low discriminative power to classify the high-level concepts. Also, we can see that the mid-level feature face information (mid_Face) with 56% ATPR is among one of the most powerful features for the “Amusement” category. The texture-related features (texture_GCM, texture_LBP) perform better than the others especially for “Disgust” and “Fear” categories in the IAPS set. The reason is that the images in the IAPS set are highly content related, for exam- ple, the “Amusement” images usually include happy people with smiling faces which can easily be identified by face detector, whereas “Fear” and “Disgust” images often show insects, snakes or injuries with certain textures which can be distinguished by texture descriptors. However, the face information(mid_Face) does not make a contribution to other classes in which few images have human face. Fig 4(b) shows the performance of each visual features on the Artistic PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 11 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 Visual affective classification by combining visual and text features Fig 5. Performance of each visual features on MirFlickr affect sets. https://doi.org/10.1371/journal.pone.0183018.g005 https://doi.org/10.1371/journal.pone.0183018.g005 https://doi.org/10.1371/journal.pone.0183018.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 Visual affective classification by combining visual and text features Photos. We can see that the color features(color_Moment, color_HSVhistogram) are the most effective ones among the visual features, following by aesthetic-related features(mid_Ke, Fig 4. Performance of each visual feature on IAPS (a) & artistic photo (b). https://doi.org/10.1371/journal.pone.0183018.g004 Photos. We can see that the color features(color_Moment, color_HSVhistogram) are the most effective ones among the visual features, following by aesthetic-related features(mid_Ke, mid_Datta) and texture features(texture_LBP). This indicates that the color information plays important role for classification of the artistic photos. Meanwhile, the face information (mid_Face) fails to detect “Amusement” and other categories in the Artistic Photos set, as there is no strong correlation between faces and classes in the set. Fig 4. Performance of each visual feature on IAPS (a) & artistic photo (b). https://doi.org/10.1371/journal.pone.0183018.g004 Fig 4. Performance of each visual feature on IAPS (a) & artistic photo (b). Fig 4. Performance of each visual feature on IAPS (a) & artistic photo (b). https://doi.org/10.1371/journal.pone.0183018.g004 Fig 4. Performance of each visual feature on IAPS (a) & artistic photo (b). https://doi.org/10.1371/journal.pone.0183018.g004 https://doi.org/10.1371/journal.pone.0183018.g004 Photos. We can see that the color features(color_Moment, color_HSVhistogram) are the most effective ones among the visual features, following by aesthetic-related features(mid_Ke, mid_Datta) and texture features(texture_LBP). This indicates that the color information plays important role for classification of the artistic photos. Meanwhile, the face information (mid_Face) fails to detect “Amusement” and other categories in the Artistic Photos set, as there is no strong correlation between faces and classes in the set. Photos. We can see that the color features(color_Moment, color_HSVhistogram) are the most effective ones among the visual features, following by aesthetic-related features(mid_Ke, mid_Datta) and texture features(texture_LBP). This indicates that the color information plays important role for classification of the artistic photos. Meanwhile, the face information (mid_Face) fails to detect “Amusement” and other categories in the Artistic Photos set, as there is no strong correlation between faces and classes in the set. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 12 / 21 Visual affective classification by combining visual and text features Fig 6. Performance of text features on MirFlickr affect set. Fig 6. Performance of text features on MirFlickr affect set. https://doi.org/10.1371/journal.pone.0183018.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 Performance of visual and text features on MirFlickr affect set Fig 5 shows the performance of visual features on the MirFlickr Affect set. We can see that the mid-level aesthetic features (mid_Datta, mid_Ke) with average 50% APTR perform better on valence dimension, while the color features (color_HSVhistogram, color_Correlograms) with average 56% APTR perform better on arousal dimension. It can be interpreted as the aesthetics more likely influence human pleasant feelings that related to the valence, and the colors more probably induce human active emotions that related to arousal. This is also confirmed by Machajdik’s work [5]. Fig 6 shows the performance of text features on MirFlickr Affect set. We can see that the eHTC achieves the best performance with average 57% ATPR, which is better than HTC with average 54% ATPR. The reason is that firstly the dictionary of eHTC is much more interrelated to the affective concepts compared to the HTC’s word frequency based dictionary, and secondly the Resnik’s words similarity measurement also contributes to strengthen the discriminative power on VAC concepts. It also shows that the performance of eMTC is among the lowest one in terms of ATPR and is unfortunately closing to random. It can be explained as the empiric ratings of the ANEW words are highly subjective, and many terms have high standard deviations, which imply less confidence associated to the rating values. Meanwhile, we make a comparison with other popular text features, such as the TF and the Latent Dirichlet allocation (LDA) topic model [51]. However, the results on Fig 6 indicate that the LDA, learned with 64 topics, do not work well with the image tags and receives the worst performance with average 46% ATPR. The main reason lies in the fact that image tags or captions are generally short with less than 10 tags per image (e.g 7.8 words for MirFlickr tags). This makes TF feature very sparse with many zeros, and causes inadequate training on LDA topic model. In a word, the results confirm that HTC [21] and its variant eHTC are proven to be effective in particular when handling short texts in social networks. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 13 / 21 Performance of combining features based on D-S evidence theory In this section, we firstly show the performance of combining visual features on IAPS and Artistic Photo set, then present the performance of fusing visual and text features on MirFlickr Affect dataset. Meanwhile, we make a comparison with the state-of-arts. Fig 7 shows the results of different fusion methods on IAPS set and the comparison with the state-of-arts [5, 29, 77]. We can see that the performance show an improvement by employing fusion methods compared to the case of best feature used, especially highlighted for “amusement”, “awe”, “disgust” and “sad” concepts with average 5% ATPR improvement. More specifically, the D-S evidence theory method obtains the best result with average 61% ATPR compared with other conventional methods, e.g. mean-score and majority voting. Also, it outperforms the state-of-arts by Yanulevskaya [77] and Machajdik [5] for five of eight categories (except “anger”, “contentment” and “sad”). All these indicate that the D-S evidence theory fusion approach is suitable for VAC task, as it is based on adjusting the evidence of different classifiers by minimizing the MSE of training data. The accurate estimation of evidence of each classifier will lead to minimizing the MSE of the combined results, and hence resolve the conflicts between classifiers. However, one should be noted that for “anger” category, the result is only a bit better than random chance(52% for one-versus-all), which can be expected in this challenging task with such a small set of train- ing images. Fig 8 shows the combining results on Artistic Photo set. It is clear that the performances of D-S evidence theory fusion method with average 63% ATPR outperforms the best individual feature and the other conventional methods. It also performs better than the state-of-art [5] in PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 14 / 21 Visual affective classification by combining visual and text features Table 4. The performance with different settings on MirFlickr affect set by combining textual and visual features based on the D-S evidence theory. The best performance in each panel is indicated in bold. LV(%) NV(%) HV(%) LA(%) NA(%) HA(%) txtf_eMTC 43.3 49.8 54.6 45.2 53.4 48.8 txtf_eHTC 51.4 53.1 56.1 48.7 56.4 52.8 Color 51.2 48.6 50.5 58.6 59.3 56.4 Color+txtf_eMTC 53.5 48.8 51.5 58.3 59.5 56.3 Color+txtf_eHTC 52.4 49.5 52.4 59.7 60.5 57.5 Texture 50.5 49.3 50.2 53.2 52.1 52.5 Texture+txtf_eMTC 51.2 50.7 50.4 55.5 53.4 53.7 Texture+txtf_eHTC 54.3 53.6 52.1 53.1 54.5 56.7 Shape 49.2 47.8 48.1 51.2 48.5 48.0 Shape+txtf_eMTC 50.1 47.5 51.6 52.4 48.8 52.1 Shape+txtf_eHTC 52.3 50.3 53.8 52.2 52.5 53.8 Mid-level 55.6 54.2 55.8 54.5 55.3 53.4 Mid-level+txtf_eMTC 55.3 56.2 56.7 55.2 56.8 54.5 Mid-level+txtf_eHTC 57.3 57.6 58.3 57.5 58.4 56.2 All visual 57.8 55.3 56.4 60.7 60.2 59.8 All visual+txtf_eMTC 58.2 59.3 58.6 63.5 63.3 61.4 All visual+txtf_eHTC 56.6 58.9 46.5 59.1 62.4 59.1 All visual+ All text 59.9 65.4 55.6 64.5 64.5 63.2 https://doi.org/10.1371/journal.pone.0183018.t004 t settings on MirFlickr affect set by combining textual and visual features based on the D-S evidence theory. cated in bold Table 4. The performance with different settings on MirFlickr affect set by combining textual and visual feat The best performance in each panel is indicated in bold. Table 4 shows the combing results on MirFlickr Affect dataset based on D-S evidence the- ory. It shows that the visual features perform better than textual features with average 4% ATPR, and the eHTC outperforms the eMTC except at the neutral valence class. Specifically, the fusion of mid-level group and text features perform better on the valence dimension, while the combination of color features and text features work well on the arousal dimension. When combined with textual features, the performance of the shape feature group improves obvi- ously with average 4% ATPR improvement. Moreover, the combination of all visual and tex- tual features achieves the best classification accuracy for all classes. These results indicate that the proposed textual features can help to improve the performance of conventional visual based affective classification by employing the D-S evidence theory fusion approach, which exploits the complementary information provided by the different classifiers. Visual affective classification by combining visual and text features Fig 7. Performance of the various combination methods on the IAPS dataset. https://doi.org/10.1371/journal.pone.0183018.g007 Fig 7. Performance of the various combination methods on the IAPS dataset. https://doi.org/10.1371/journal.pone.0183018.g007 https://doi.org/10.1371/journal.pone.0183018.g007 the cases of “Amusement”, “Anger”, “Disgust”, “Excitement”and “Sad”. This further demon- strate that the D-S evidence theory fusion approach has the ability to fuse different ambiguous sources of information for affective concepts, and can effectively improves the performance of VAC task. Fig 8. Performance of the various combination methods on the artistic photo Set. https://doi.org/10.1371/journal.pone.0183018.g008 Fig 8. Performance of the various combination methods on the artistic photo Set. https://doi.org/10.1371/journal.pone.0183018.g008 https://doi.org/10.1371/journal.pone.0183018.g008 https://doi.org/10.1371/journal.pone.0183018.g008 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 15 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 https://doi.org/10.1371/journal.pone.0183018.t004 Discussion With the rapid development in social networks, there is a constantly focus on utilizing multi- media resources to accomplish machine-learning tasks. Indeed, not only the images can help to text-based analysis, e.g. sentiment analysis [84, 85], but also the texts can improve image- based classification, such as VOC task [86–88] These works show that the multimodal approaches can combine the preponderance and complementary information of each sources, and achieve better classification results than single modality, which is also confirmed by our results on VAC task. The limitations of our approach involve following issues. First, the small size of training data sets makes the modalities trained insufficiency. To overcome this, we have employed 5-fold cross validation, but leaving with unstable standard deviations in the small training size classes. Second, the proposed fusion approach based on D-S evidence theory performs well for VAC task, but having complex parameters to be tuned and causing a time-consuming train PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 16 / 21 Visual affective classification by combining visual and text features process. Third, the texts from social networks usually include informal expressions, e.g Emoji, but our text features ignore them during the preprocessing stage. Acknowledgments This work was supported by the National Natural Science Foundation of China under Grant (51435011, 51505309), the Fundamental Research Funds for the Central University in UIBE (14QD21) and the Sichuan Province Science and Technology Support Program Project under Grant 2015JY0172. Conclusion and future work In this paper, we present a multimodal approach for the VAC task. Firstly, we proposed two text-based features to capture emotional semantics from image tags. We also evaluated various visual features, aiming at characterizing visual content related with emotional concepts. Finally, we employed a fusion method based on the D-S theory of evidence, which exploits the complementary information to resolve the conflicts between classifiers. The experiments were conducted on three databases: the IAPS, the Artistic Photo set and MirFlickr Affect set, and have shown promising results on visual affective classification. From the results, we can conclude as follows: (i) the fused method based on D-S evidence theory is proved to be useful for the VAC task in efficiently fusing different features; (ii) the proposed textual eHTC can effectively capture emotional semantics from image tags, and help to improve the performance of visual classifiers for VAC task; (iii) the classification of visual emotional concepts is still extremely challenging, and the average performance of this approach is 57%, which can be expected as the small training set. In the future, we plan to make more efforts on the following aspects: building a large data- base, exploring regression model for VAC in a dimensional emotion model, and testifying Word2Vector or other similarities to improve eHTC. At last, how to efficiently apply the pro- posed methods to web-images on a large scale will also be investigated. Project administration: Ningning Liu, Kai Wang. Project administration: Ningning Liu, Kai Wang. Resources: Xin Jin, Emmanuel Dellandre´a. Resources: Xin Jin, Emmanuel Dellandre´a. Resources: Xin Jin, Emmanuel Dellandre´a. Software: Ningning Liu. Supervision: Liming Chen. Visualization: Ningning Liu. Visualization: Ningning Liu. Writing – original draft: Kai Wang. Writing – original draft: Kai Wang. Conceptualization: Ningning Liu. Conceptualization: Ningning Liu. Formal analysis: Boyang Gao. Project administration: Ningning Liu, Kai Wang. PLOS ONE | https://doi.org/10.1371/journal.pone.0183018 August 29, 2017 References 1. Picard RW. 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An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction
Kardiologiâ
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ОРИГИНАЛЬНЫЕ СТАТЬИ § Раннее эхокардиографическое прогнозирование функционального восста-новления миокарда после инфаркта миокарда с подъемом сегмента ST. Кардиология. 2021;61(1):66–71]. Corresponding author Alper Karakus. E-mail: alpkarakus06@gmail.com Alper Karakuş1, Berat Uğuz2 1 Department of Cardiology, Besni State Hospital, Adıyaman, Turkey 2 Department of Cardiology, Bursa City Hospital, Bursa, Turkey Alper Karakuş1, Berat Uğuz2 1 Department of Cardiology, Besni State Hospital, Adıyaman, Turkey 2 Department of Cardiology, Bursa City Hospital, Bursa, Turkey An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction Discrimination of the time course of functional recovery in early period following ST elevation myocar- dial infarction (STEMI) has prognostic importance. This study aims to establish the ability of the com- bined systolic-diastolic index (E / (Ea x Sa), for differentiating recoverable myocardium or persistent non-viability in subjects with STEMI. 77 patients with first acute STEMI were enrolled to the study. Serial transthoracic echocardiography (TTE) examinations were performed at the time of the admission (immediately after reperfusion), hospital discharge (5±1 days) and control examination (30±2 days). To assess the association between E / (Ea×Sa) index and myocardial recovery, two categories (Transient stunning and persistent non-via- bility) were defined on basis of improvement of left ventricle ejection fraction (LV EF) (Improvement was defined as an increase more than 10 % in LV EF at day 30 compared to baseline). 55 patients (71.4 %) had improvement of LV EF and 24 patients (28.6 %) had no recovery of systolic func- tion (30 days LV EF 48.78±6.1 vs. 39.31±8.1 %, p=0.01). The E / (Ea×Sa) index were significantly higher in the “non-viability” than in stunning group on predischarge and 1 month (1.61±0.64 vs 1.25±0.43, p=0.01 and 1.33±0.25 vs 1.14±0.21, p=0.01, respectively). The change in the E / (Ea×Sa) index in early period (within 5±1 days) was higher in the stunning group (26 %) compared to non-viability group (15 %) (p=0.033). E / (Ea×Sa) index was found to be a significant predictor of myocardial recovery in multivariable logistic regression (OR 0.91, 95 % CI 0.83–0.98, p=0.001). Roc curve showed that the cutoff value of E / (Ea×Sa) index is 1.29 with 73 % of sensitivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54–0.89). Our findings suggest that E / (Ea×Sa) index is a strong predictor of functional recovery; the odds of recovery decreases as E / (Ea×Sa) index value increases. Diastolic dysfunction; echocardiography; functional recovery; myocardial stunning; non-viability; ST elevation myocardial infarction Alper Karakuş, Berat Uğuz. An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction. Kardiologiia. 2021;61(1):66–71. [Russian: Айлпей Каракуш, Берат Угеузе. Раннее эхокардиографическое прогнозирование функционального восста-новления миокарда после инфаркта миокарда с подъемом сегмента ST. Кардиология. 2021;61(1):66–71]. age can be useful to detect this group of patients at the early period of STEMI following PPCI. ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477 ОРИГИНАЛЬНЫЕ СТАТЬИ § ОРИГИНАЛЬНЫЕ СТАТЬИ § Alper Karakuş1, Berat Uğuz2 1 Department of Cardiology, Besni State Hospital, Adıyaman, Turkey 2 Department of Cardiology, Bursa City Hospital, Bursa, Turkey An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction Goal Discrimination of the time course of functional recovery in early period following ST elevation myocar- dial infarction (STEMI) has prognostic importance. This study aims to establish the ability of the com- bined systolic-diastolic index (E / (Ea x Sa), for differentiating recoverable myocardium or persistent non-viability in subjects with STEMI. Material and methods 77 patients with first acute STEMI were enrolled to the study. Serial transthoracic echocardiography (TTE) examinations were performed at the time of the admission (immediately after reperfusion), hospital discharge (5±1 days) and control examination (30±2 days). To assess the association between E / (Ea×Sa) index and myocardial recovery, two categories (Transient stunning and persistent non-via- bility) were defined on basis of improvement of left ventricle ejection fraction (LV EF) (Improvement was defined as an increase more than 10 % in LV EF at day 30 compared to baseline). Results 55 patients (71.4 %) had improvement of LV EF and 24 patients (28.6 %) had no recovery of systolic func- tion (30 days LV EF 48.78±6.1 vs. 39.31±8.1 %, p=0.01). The E / (Ea×Sa) index were significantly higher in the “non-viability” than in stunning group on predischarge and 1 month (1.61±0.64 vs 1.25±0.43, p=0.01 and 1.33±0.25 vs 1.14±0.21, p=0.01, respectively). The change in the E / (Ea×Sa) index in early period (within 5±1 days) was higher in the stunning group (26 %) compared to non-viability group (15 %) (p=0.033). E / (Ea×Sa) index was found to be a significant predictor of myocardial recovery in multivariable logistic regression (OR 0.91, 95 % CI 0.83–0.98, p=0.001). Roc curve showed that the cutoff value of E / (Ea×Sa) index is 1.29 with 73 % of sensitivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54–0.89). Conclusions Our findings suggest that E / (Ea×Sa) index is a strong predictor of functional recovery; the odds of recovery decreases as E / (Ea×Sa) index value increases. Keywords Diastolic dysfunction; echocardiography; functional recovery; myocardial stunning; non-viability; ST elevation myocardial infarction For сitation Alper Karakuş, Berat Uğuz. An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction. Kardiologiia. 2021;61(1):66–71. [Russian: Айлпей Каракуш, Берат Угеузе. ОРИГИНАЛЬНЫЕ СТАТЬИ § Alper Karakuş1, Berat Uğuz2 1 Department of Cardiology, Besni State Hospital, Adıyaman, Turkey 2 Department of Cardiology, Bursa City Hospital, Bursa, Turkey An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction Goal Discrimination of the time course of functional recovery in early period following ST elevation myocar- dial infarction (STEMI) has prognostic importance. This study aims to establish the ability of the com- bined systolic-diastolic index (E / (Ea x Sa), for differentiating recoverable myocardium or persistent non-viability in subjects with STEMI. Material and methods 77 patients with first acute STEMI were enrolled to the study. Serial transthoracic echocardiography (TTE) examinations were performed at the time of the admission (immediately after reperfusion), hospital discharge (5±1 days) and control examination (30±2 days). To assess the association between E / (Ea×Sa) index and myocardial recovery, two categories (Transient stunning and persistent non-via- bility) were defined on basis of improvement of left ventricle ejection fraction (LV EF) (Improvement was defined as an increase more than 10 % in LV EF at day 30 compared to baseline). Results 55 patients (71.4 %) had improvement of LV EF and 24 patients (28.6 %) had no recovery of systolic func- tion (30 days LV EF 48.78±6.1 vs. 39.31±8.1 %, p=0.01). The E / (Ea×Sa) index were significantly higher in the “non-viability” than in stunning group on predischarge and 1 month (1.61±0.64 vs 1.25±0.43, p=0.01 and 1.33±0.25 vs 1.14±0.21, p=0.01, respectively). The change in the E / (Ea×Sa) index in early period (within 5±1 days) was higher in the stunning group (26 %) compared to non-viability group (15 %) (p=0.033). E / (Ea×Sa) index was found to be a significant predictor of myocardial recovery in multivariable logistic regression (OR 0.91, 95 % CI 0.83–0.98, p=0.001). Roc curve showed that the cutoff value of E / (Ea×Sa) index is 1.29 with 73 % of sensitivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54–0.89). Conclusions Our findings suggest that E / (Ea×Sa) index is a strong predictor of functional recovery; the odds of recovery decreases as E / (Ea×Sa) index value increases. Keywords Diastolic dysfunction; echocardiography; functional recovery; myocardial stunning; non-viability; ST elevation myocardial infarction For сitation Alper Karakuş, Berat Uğuz. An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction. Kardiologiia. 2021;61(1):66–71. [Russian: Айлпей Каракуш, Берат Угеузе. Раннее эхокардиографическое прогнозирование функционального восста-новления миокарда после инфаркта миокарда с подъемом сегмента ST. Кардиология. 2021;61(1):66–71]. Corresponding author Alper Karakus. E-mail: alpkarakus06@gmail.com ОРИГИНАЛЬНЫЕ СТАТЬИ § Alper Karakuş1, Berat Uğuz2 1 Department of Cardiology, Besni State Hospital, Adıyaman, Turkey 2 Department of Cardiology, Bursa City Hospital, Bursa, Turkey An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction Goal Discrimination of the time course of functional recovery in early period following ST elevation myocar- dial infarction (STEMI) has prognostic importance. This study aims to establish the ability of the com- bined systolic-diastolic index (E / (Ea x Sa), for differentiating recoverable myocardium or persistent non-viability in subjects with STEMI. Material and methods 77 patients with first acute STEMI were enrolled to the study. Serial transthoracic echocardiography (TTE) examinations were performed at the time of the admission (immediately after reperfusion), hospital discharge (5±1 days) and control examination (30±2 days). To assess the association between E / (Ea×Sa) index and myocardial recovery, two categories (Transient stunning and persistent non-via- bility) were defined on basis of improvement of left ventricle ejection fraction (LV EF) (Improvement was defined as an increase more than 10 % in LV EF at day 30 compared to baseline). Results 55 patients (71.4 %) had improvement of LV EF and 24 patients (28.6 %) had no recovery of systolic func- tion (30 days LV EF 48.78±6.1 vs. 39.31±8.1 %, p=0.01). The E / (Ea×Sa) index were significantly higher in the “non-viability” than in stunning group on predischarge and 1 month (1.61±0.64 vs 1.25±0.43, p=0.01 and 1.33±0.25 vs 1.14±0.21, p=0.01, respectively). The change in the E / (Ea×Sa) index in early period (within 5±1 days) was higher in the stunning group (26 %) compared to non-viability group (15 %) (p=0.033). E / (Ea×Sa) index was found to be a significant predictor of myocardial recovery in multivariable logistic regression (OR 0.91, 95 % CI 0.83–0.98, p=0.001). Roc curve showed that the cutoff value of E / (Ea×Sa) index is 1.29 with 73 % of sensitivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54–0.89). Conclusions Our findings suggest that E / (Ea×Sa) index is a strong predictor of functional recovery; the odds of recovery decreases as E / (Ea×Sa) index value increases. Keywords Diastolic dysfunction; echocardiography; functional recovery; myocardial stunning; non-viability; ST elevation myocardial infarction For сitation Alper Karakuş, Berat Uğuz. An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction. Kardiologiia. 2021;61(1):66–71. [Russian: Айлпей Каракуш, Берат Угеузе. ОРИГИНАЛЬНЫЕ СТАТЬИ § Alper Karakuş1, Berat Uğuz2 1 Department of Cardiology, Besni State Hospital, Adıyaman, Turkey 2 Department of Cardiology, Bursa City Hospital, Bursa, Turkey An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction Goal Discrimination of the time course of functional recovery in early period following ST elevation myocar- dial infarction (STEMI) has prognostic importance. This study aims to establish the ability of the com- bined systolic-diastolic index (E / (Ea x Sa), for differentiating recoverable myocardium or persistent non-viability in subjects with STEMI. Material and methods 77 patients with first acute STEMI were enrolled to the study. Serial transthoracic echocardiography (TTE) examinations were performed at the time of the admission (immediately after reperfusion), hospital discharge (5±1 days) and control examination (30±2 days). To assess the association between E / (Ea×Sa) index and myocardial recovery, two categories (Transient stunning and persistent non-via- bility) were defined on basis of improvement of left ventricle ejection fraction (LV EF) (Improvement was defined as an increase more than 10 % in LV EF at day 30 compared to baseline). Results 55 patients (71.4 %) had improvement of LV EF and 24 patients (28.6 %) had no recovery of systolic func- tion (30 days LV EF 48.78±6.1 vs. 39.31±8.1 %, p=0.01). The E / (Ea×Sa) index were significantly higher in the “non-viability” than in stunning group on predischarge and 1 month (1.61±0.64 vs 1.25±0.43, p=0.01 and 1.33±0.25 vs 1.14±0.21, p=0.01, respectively). The change in the E / (Ea×Sa) index in early period (within 5±1 days) was higher in the stunning group (26 %) compared to non-viability group (15 %) (p=0.033). E / (Ea×Sa) index was found to be a significant predictor of myocardial recovery in multivariable logistic regression (OR 0.91, 95 % CI 0.83–0.98, p=0.001). Roc curve showed that the cutoff value of E / (Ea×Sa) index is 1.29 with 73 % of sensitivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54–0.89). Conclusions Our findings suggest that E / (Ea×Sa) index is a strong predictor of functional recovery; the odds of recovery decreases as E / (Ea×Sa) index value increases. Keywords Diastolic dysfunction; echocardiography; functional recovery; myocardial stunning; non-viability; ST elevation myocardial infarction For сitation Alper Karakuş, Berat Uğuz. An early echocardiographic prediction for functional myocardial recovery after ST elevation myocardial infarction. Kardiologiia. 2021;61(1):66–71. [Russian: Айлпей Каракуш, Берат Угеузе. Раннее эхокардиографическое прогнозирование функционального восста-новления миокарда после инфаркта миокарда с подъемом сегмента ST. Кардиология. 2021;61(1):66–71]. Corresponding author Alper Karakus. E-mail: alpkarakus06@gmail.com Раннее эхокардиографическое прогнозирование функционального восста-новления миокарда после инфаркта миокарда с подъемом сегмента ST. Кардиология. 2021;61(1):66–71]. Corresponding author Alper Karakus. E-mail: alpkarakus06@gmail.com ОРИГИНАЛЬНЫЕ СТАТЬИ § Alper Karakuş1, Berat Uğuz2 1 Department of Cardiology, Besni State Hospital, Adıyaman, 2 Department of Cardiology, Bursa City Hospital, Bursa, Turk An early echocardiographic p myocardial recovery after ST Goal Discrimination of the time course dial infarction (STEMI) has prog bined systolic-diastolic index (E non-viability in subjects with ST Material and methods 77 patients with first acute STEM (TTE) examinations were perfo hospital discharge (5±1 days) an E / (Ea×Sa) index and myocardia bility) were defined on basis of im was defined as an increase more t Results 55 patients (71.4 %) had improvem tion (30 days LV EF 48.78±6.1 vs in the “non-viability” than in stu p=0.01 and 1.33±0.25 vs 1.14±0. period (within 5±1 days) was hi (15 %) (p=0.033). E / (Ea×Sa) in in multivariable logistic regressio the cutoff value of E / (Ea×Sa) in p=0.01, CI: 0.54–0.89). Conclusions Our findings suggest that E / (E of recovery decreases as E / (Ea×S Keywords Diastolic dysfunction; echocardi elevation myocardial infarction For сitation Alper Karakuş, Berat Uğuz. An e after ST elevation myocardial inf Берат Угеузе. Раннее эхокардио миокарда после инфаркта миока Corresponding author Alper Karakus. E-mail: alpkaraku The study exclusion criteria h • Patients with unsuccessful reperfusion (Thrombolysis in myocardial infarction (TIMI) score < III). • Any complication during PPCI or requiring re-PCI dur- ing follow-up. • Presence of pathological q / Q wave in admission electro- cardiogram. • History of previous coronary intervention. • Use of loop diuretics during follow-up. • Presence of previous MI, heart failure, cardiomyopathy, dysrhythmia (included atrial fibrillation) or any valvulop- athy, which is more than mild. The study inclusion criteria • First acute STEMI based on the 2018 European Society of Cardiology (ESC) guideline on Fourth Universal Defi- nition of Myocardial Infarction Guidelines [12]. Material and methods • The Third TTE was done at the time of the control exami- nation (30±2 days). Statistical Analysish • The onset of symptoms <12 hours. • The onset of symptoms <12 hours. The normality was analyzed by The Kolmogorov–Smir­nov test. According to the normality pattern, continuous variables were presented as mean±standard deviation, or median and in- terquartile range. Student’s T test was used when the assump- tion of normal distribution was met, and Mann–Whitney U test was used when normal distribution was not obtained. Categor- ical variables were compared by using the Chi-square test and the  results were presented as percentages. One-way repeated ANOVA was used in terms of echocardiographic parameters for each group. Additionally, post-hoc test was applied to determine the groups that are significant. To assess the association between E / (Ea×Sa) index and myocardial recovery, two categories were defined according to whether there was a 10 % improvement in EF. The effects of NT pro-BNP, creatinine, E / (Ea×Sa) index, high sensitive C reactive protein (Hs-CRP), door to balloon time, and age on myocardial recovery were determined with logistic regression analysis. The significant P-value was <0.05. SPSS 26.0 Statistical Package Software was used to perform all data analyses. Introduction ST segment elevation myocardial infarction (STEMI) is the foremost cause of cardiovascular mortality and mor- bidity [1]. Primary percutaneous coronary intervention (PPCI) is the standard mechanical re-vascularization tech- nique for patients with STEMI and aims to limit myocar- dial necrosis. Even if after a successful PPCI, re-perfused myocardium by the infarct-related coronary artery may re- main akinetic due to transient myocardial stunning or per- sistent non-viability [2]. The striking point, the initial studies limited the notion of myocardial stunning to systolic dysfunction. But the cur- rent studies support the presence of diastolic dysfunction (evidenced both by regional diastolic index [impaired left ventricular (LV) untwisting rate) and global diastolic index (increased E / e’ ratio)] [5, 6]. A wide range of diagnostic techniques (Including dobu- tamine stress echocardiography, positron emulsion tomog- raphy scanning, radionuclide myocardial perfusion imaging, and cardiovascular magnetic resonance imaging) can deter- mine the presence or absence of metabolic activity in that dysfunctional myocardial segment. However, these modali- ties are relatively costly and limited in availability. Stunning is a delayed response in myocardial recovery [3]. But the persistent non-viability is associated with adverse clinical outcomes and poor contractile function [4]. Over and above, patients who will not have a functional recovery will also be patients who are more likely to develop heart fail- ure. So, predicting myocardium with potential for improve- ment in contractility and myocardium with irreversible dam- The combined systolic-diastolic index, which is cal­cu­lated as E / (Ea×Sa), is a reliable estimate parameter of increased LV 66 Angiographic Procedureh The femoral or radial approach was used for coronary an- giography and PPCI. The patients who needed thrombus as- piration or glycoprotein IIb-IIIa inhibitors infusion exclu­ded from study. All patients received drug-eluting stents. TIMI III flow was achieved in all patients. All patients received medical treatment, including acetylsalicylic acid, P2Y12 in- hibitor, beta-blockers, statins, aldosterone antagonists as ap- propriate, and angiotensin converting enzyme inhibitors or angiotensin receptors blockers accor­ding to 2017 ESC guide- line on Acute Myocardial In­farction in Patients Presenting with ST-Segment Elevation (Management) Guidelines [13]. Echocardiographic Evaluation Echocardiographic Evaluation filling pressure and decreased systolic functions, particularly in patients with regional wall motion abnormalities (E is the early diastolic mitral velocity, Ea is the early diastolic mitral annular velocity, and Sa is the systolic mitral annular velocity) [7–11]. Echocardiographic Evaluation Serial transthoracic echocardiography (TTE) exa­mi­na­ tions were performed by using GE Vivid S60 system with 2.5 MHz transducer (GE Vingmed Ultrasound AS, Hor­ten, Norway). We believe that myocardial recovery may be reflected to improvement of diastolic filling and systolic functions at ear- ly days after PPCI. So, in the present study we tested the abili- ty of this combined systolic-diastolic index for differentiating recoverable myocardium or persistent non-viability. • The admission TTE was done immediately after reperfu- sion (with seen more than 50 % resolution in ST segment elevation on ECG in 1 hour after PPCI). t • The second TTE was done at the time of hospital dis- charge (5±1 days). Material and methods Study Protocol and Populationh The LV ejection fraction (EF) was calculated by modified Simpson (biplane) method. LV myocardial recovery was de- fined as an increase more than 10 % in LV EF between after PPCI and day 30. The study was conducted in Bursa City Hospital (Turkey) between July 2020 and August 2020 and collaborated with Adıyaman Besni State Hospital (Turkey) for study design and data analysis. Eighty-nine patients with acute STEMI were enrolled to this cross-sectional study. Ethics Committee ap- proved the study protocol (Registration number: 2020–5 / 4) and subjects who met study criteria consented informed. Peak early (E) and late (A) mitral entry velocities were re- corded from the apical four-chamber view by pulsed-wave Doppler with the 5‑mm sample volume. Tissue Doppler im- aging recordings, Sa, Ea, and Aa values were recorded from the medial and lateral annulus in an apical four-chamber view and taken average of at least 3 cardiac cycles. E / (Ea×Sa) in- dex value was calculated and presented as the arithmetic mean of the lateral and medial Ea and Sa velocities. The study inclusion criteria Results Eighty-nine patients were evaluated for the study. Twelve patients with STEMI were excluded from initial study pop- ulation (One patient dead, 5 patient had history of  previ- ous MI, 3 patients had history of previous PCI, 1  patient had missing data, 2 patients were not reached for follow up). The data of the remaining 77 patients were analyzed. 67 ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477 ОРИГИНАЛЬНЫЕ СТАТЬИ § Table 1. Baseline characteristics and laboratory findings of the study patients Variables Patients with myocardial stunning (Group 1, n=55, 71.4 %) Patients with non viability (Group 2, n=22, 28.6 %) p value Age (years) 56.8±9.5 60±13.1 0.24 Sex (n, %) females 10 (18.1) 5 (22.7) 0.53 Hypertension, n (%) 21 (38.1) 7 (31.8) 0.66 Dyslipidemia, n (%) 5 (9) 3 (13.6) 0.57 DM, n (%) 20 (36.3) 5 (22.7) 0.22 Smoker, n (%) 27 (49) 13 (59.1) 0.47 Body mass index (kg / m²) 23.9±2.4 24.7±3.5 0.88 Glucose (mg / dl) 174±43 153±44 0.10 Creatinine (mg / dl) Admission Control 0.83±0.2 0.93±0.2 0.98±0.3 1.08±0.3 0.034 0.018 Triglyceride (mg / dl) 171±92 143±87 0.23 LDL (mg / dl) 118±36 126±33 0.36 HDL (mg / dl) 37.9±18 37.2±8 0.87 TSH (µIU / mL) 1.73±1.3 1.89±1.3 0.64 Na (mmol / L) 137±3.6 138±3 0.37 K (mmol / L) 4.1±0.4 4.0±0.5 0.49 AST (U / L) 27±11 31±6 0.62 ALT (U / L) 24±7 26±9 0.42 Hs-CRP (nmol / L) Admission Control 7.7±1.9 3.45±0.9 7.1±1.6 5.2±0.6 0.75 0.15 NTproBNP (µmol / L) Admission Control 677±95 610±87 678±67 2235±564 0.99 0.026 Leukocyte (109 / L) 10.9±2.5 11.6±3.8 0.37 Hemoglobin (g / dl) 13.6±1.7 12.9±1.6 0.51 Platelet (109 / L) 276±77 259±72 0.38 DM: Diabetes mellitus, LDL: Light density lipoprotein, HDL: High density lipoprotein, TSH: Thyroid-stimulating hormone, Na: Sodium, K: Potassium, AST: aspartate aminotransferase, ALT: alanine aminotransferase, HsCRP: High sensitive C reactive protein, Admission: 1 hour after primary percutaneous intervention, Control: 30±2 days after admission. Table 2. The angiographic characteristics of study groups Variables Patients with myocardial stunning (Group 1, N=55, 71.4 %) Patients with Non viability (Group 2, N=22, 28.6 %) p value Stent length (mm) 28.5±11.8 27.1±9.9 0.63 Stent diameter (mm) 2.93±0.3 2.82±0.2 0.13 MI territory A i MI (%) 23 (41 8) 9 (41) 0 12 Study patients were categorized into two groups accord- ing to LV EF improvement compared to EF, which detected at first or second TTE examination at 1‑month follow up. Results Pa- tients with improvement in EF >10 % during follow-up com- pared to the basal echocardiographic study who considered to have stunning (Group I or transient stunning group) were 55 (71.4 %) while the other patients without improvement of EF were 22 (28.6 %) (Group II or permanent non-viabil- ity group). The Combined Systolic-Diastolic Indexh h The E / (Ea×Sa) index value in both groups is presented in table 3. The E / (Ea×Sa) index (Predischarge and 1‑month values) were significantly higher in the non-viability than in stunning group. On the other hand, in the stunning group, the index before discharge from the hospital decreased by 26 % compared to the early period index after PPCI (Admis- sion), while this decrease was observed by 15 % in the non- viability group. The percentage of change of E / (Ea×Sa) in- dex at hos­pitalization period (5±1  days) was compared between both groups and found statistically significant (p=0.033). Table 2. The angiographic characteristics of study groups Table 2. The angiographic characteristics of study groups Variables Patients with myocardial stunning (Group 1, N=55, 71.4 %) Patients with Non viability (Group 2, N=22, 28.6 %) p value Stent length (mm) 28.5±11.8 27.1±9.9 0.63 Stent diameter (mm) 2.93±0.3 2.82±0.2 0.13 MI territory Anterior MI, n (%) Non-anterior MI, n (%) 23 (41.8) 32 (58.2) 9 (41) 13 (59) 0.12 0.94 Door-to-balloon time (min) 38.5±17 41.5±16 0.86 IRA LAD, n (%) Non-LAD, n (%) 23 (41.8) 32 (58.2) 9 (41) 13 (59) 0.12 0.94 MI: Myocardial Infarction, IRA: Infarct related artery, LAD: Left anterior descending, Min: Minute. Angiographic Characteristicsif No significant difference was observed between the groups regarding stent length and diameter, myocardial infarction territory and door to balloon time. The angio­grap­ hic features were summarized in table 2. Baseline and Follow-Up Characteristics Baseline demographic and clinical characteristics of study populations are presented in table 1. In brief, there was no found significant difference between both groups re- garding the risk factors of coronary artery disease (includ- ing diabetes mellitus, hypertension, hyperlipidemia and smo­king). Creatinine level was significantly higher in non- viability group than stunning group at baseline (0.98±0.3 vs 0.83±0.2 mg / dl, p=0.034) and at 1 month (1.08±0.3  vs 0.93±0.2 mg / dl, p=0.018). NT pro-BNP was significantly higher in non-viability group compared to stunning group at follow up. Echocardiographic Characteristics Regarding the baseline echocardiographic characteris- tics, peak E (Admission), E / A ratio (Admission), and E / Ea ratio (Admission and 1 month) were significantly higher in the non-viability group. The results are shown in table 3. DM: Diabetes mellitus, LDL: Light density lipoprotein, HDL: High density lipoprotein TSH: Thyroid-stimulating hormone DM: Diabetes mellitus, LDL: Light density lipoprotein, h ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477 A LAD: Left anterior descending, Min: Minute. Identification of the Predictors for Myocardial Recovery Multivariable logistic regression analysis to assess factors predictive of myocardial recovery included NT-proBNP, cre- atinine, baseline E / (Ea×Sa) index, Hs-CRP, door to balloon ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477 68 ОРИГИНАЛЬНЫЕ СТАТЬИ § time, and age showed that E / (Ea × Sa) index was the only predictive factor for myocardial recovery (OR: 0.91, 95 % CI (0.83–0.98), P=0.001) (tab. 4). Roc curve showed that the cutoff value of E / (Ea x Sa) index is 1.29 with 73 % of sensi- tivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54– 0.89) (fig. 1). 0,8 1,0 0,2 0,4 0,6 0 0 0,2 0,4 0,6 0,8 1,0 AUC: 0.71, p=0.01, CI: 0.54-0.89 Sensitivity E / (Ea x Sa) Index 1 – Specifcity Figure 1. Roc curve showed that the cutoff value of E/(Ea×Sa) index is 1.29 with 73% of sensitivity and 61% of specificity Figure 1. Roc curve showed that the cutoff value of E/(Ea×Sa) index is 1.29 with 73% of sensitivity and 61% of specificity Figure 1. Identification of the Predictors for Myocardial Recovery Roc curve showed that the cutoff value of E/(Ea×Sa) index is 1.29 with 73% of sensitivity and 61% of specificity p g p parameters of the study patients Variables Patients with myocardial stunning (Group 1, n=55, 71.4 %) Patients with non viability (Group 2, n=22, 28.6 %) p* value LV EF (%) Admission 44.50±6.6 43.1±0.4 0.45 Predischarge 44.58±6.8 42.95±9.3 0.40 Control 48.78±6.1 39.31±8.1 0.01 p** value 0.02a,b 0.03a - Mitral E velocity# Admission 72.39±21.32 85.86±13.76 0.01 Predischarge 87.58±17.84 90.14±18.55 0.33 Control 76.30±16.38 80.04±11.83 0.27 p** value 0.01b,c 0.02b - Mitral A velocity# Admission 82.80±15.62 84.54±21.66 0.69 Predischarge 83.53±16.50 80.86±20.70 0.55 Control 82.60±16.41 82.36±20.33 0.95 p** value 0.75 0.30 - Mitral E / A Admission 0.90±0.35 1.07±0.27 0.04 Predischarge 1.03±0.37 1.08±0.35 0.56 Control 0.97±0.34 1.04±0.41 0.43 p** value 0.04c 0.28 - Mitral Ea velocity# Admission 9.20±2.1 9.07±1.5 0.79 Predischarge 9.76±2.3 9.76±2.3 0.31 Control 10.7±1.9 10.7±1.9 0.64 p** value 0.01a,b 0.02a,b - Mitral Aa velocity# Admission 11.1±1.7 11.5±2.1 0.34 Predischarge 11.5±2.1 10.6±2.1 0.11 Control 12.0±1.7 11.5±2.1 0.24 p** value 0.13 0.29 - Mitral Sa velocity# Admission 10.2±1.8 10.5±2.1 0.48 Predischarge 10.4±2.0 10.2±2.2 0.72 Control 10.9±1.7 11.0±1.9 0.98 p** value 0.04a 0.03b - LV E / Ea Admission 7.9±1.8 9.8±2.6 0.01 Predischarge 8.8±1.7 9.2±2.3 0.46 Control 8.1±1.8 9.1±2.5 0.04 p** value 0.03b,c 0.10 - E / (Ea×Sa) index Admission 1.83±0.63 2.02±0.92 0.30 Predischarge 1.25±0.43 1.61±0.64 0.01 Control 1.14±0.21 1.33±0.25 0.01 p** value 0.01a,c 0.01a,c - LV; Left ventricle, EF; ejection fraction, Admission: 1 hour after primary percutaneous intervention, Predischarge: 5±1 days after admission. Control: 30±2 days after admission. #; (cm / s), *; p values; Independent Samples-T Test, ***; p values; repeated measures ANOVA. a: p<0.05: Admission vs. Control; b: p<0.05: Predischarge vs. Control; c: p<0.05: Admission vs. Predischarge. 0,8 1,0 0,2 0,4 0,6 0 0 0,2 0,4 0,6 0,8 1,0 AUC: 0.71, p=0.01, CI: 0.54-0.89 Sensitivity E / (Ea x Sa) Index 1 – Specifcity Sensitivity AUC: 0.71, p=0.01, CI: 0.54-0.89 time, and age showed that E / (Ea × Sa) index was the only predictive factor for myocardial recovery (OR: 0.91, 95 % CI (0.83–0.98), P=0.001) (tab. 4). Roc curve showed that the cutoff value of E / (Ea x Sa) index is 1.29 with 73 % of sensi- tivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54– 0.89) (fig. 1). ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477 ОРИГИНАЛЬНЫЕ СТАТЬИ § Nevertheless, creatinine levels were not found to be associated with E / (Ea×Sa) in- dex and improvement of LV EF. So, we believe that these data did not reflect any selection bias against functional re- covery. Patients who did not have myocardial recovery had signif- icantly higher E / (Ea×Sa) index value pattern during entire follow up compared to patients who had improvement of LV EF. The one of the most striking findings of our study is that there was, on average, decrease of 26 % in the E / (E×Sa) in- dex value compared to the initial TTE measurement in tran- sient stunning group (within 5 days). This dramatic change was seen, on average, of 15 % in non-viability group. Like- wise, we did observe that E / (Ea×Sa) index was higher lev- el in non-viability group (1.33±0.25 vs. 1.14±0.21, p=0.01), similar to the predischarge measures. The literature suggests that most systolic myocardial recovery occurs within the first 2  weeks after MI, but the available data do not allow fur- ther discrimination of the time course of recovery [19–21]. So, our findings may have important implications about the prognostic value of clinical assessment of LV function early in the course of STEMI. This study has some strengths and limitations that are worth noting. One of strengths is that contrary to other imaging modalities, no contrast agent or inotropic agent was used. Another one is that predicting functional re- covery occurs relatively early. And also, we believe that our results can be used for the risk stratification of the pa- tient following STEMI and PPCI. First limitation, the all differences between stunning and non-viability groups were assessed by echocardiography and were limited by 30 days. Thus, no other imaging modality was used to re- veal the ultimate extent of functional recovery. Second, PPCI based successful reperfusion therapy was mandat- ed by the study protocol. Therefore, the data can only be interpreted for this patient group. Third, the  power of study is under limitation of relatively small sample size. On the other hand, our results could be pioneer for larg- er studies. At 1 month, we found that patients in the stunning group had a significantly better LV diastolic function, re- flecting in  a  lower ratio of E / Ea. ОРИГИНАЛЬНЫЕ СТАТЬИ § CI: 0.57–0.73)). Similar results were demonstrated by Lam  W. which evaluating LV suction functions in the dobutamine stress echocardiography -based study [5]. In contrast, Faustino M. et al. reported that E / Ea ratio was an independent predictor of myocardial recovery in patients with MI [6]. Consequently, our findings sug- gest that the severity of the E / (Ea × Sa) index and its ra- tio of change in early period was superior to other echo- cardiographic parameters in predicting recovery of  LV function at 1 month after STEMI and PPCI. The patient with lower decrease of initial E / (Ea×Sa) index (within 5  days) and the patients with predischarge E / (Ea×Sa) index >1.29 presented with poor functional recovery af- ter 1 month following STEMI. Besides, Mornos C. et al. demonstrated that E / (Ea×Sa) index is useful and reliable parameter to assess the  LV filling pressure, particularly in patients with regional wall motion abnormalities [7]. And also, it is more log- ical to  evaluate diastolic (relaxation) and systolic (con- traction) functions together as part of the ongoing cycle. So, in this present study, we assessed the early predictive value of E / (Ea×Sa) index for improvements in LV func- tions after STEMI. The substantial recovery of systolic function that oc- curred within 30 days after MI was the endpoint of our study; 71.4 % of patients had improvement of LV EF and 28.6 % had no recovery of systolic function (48.78±6.1 vs. 39.31±8.1 %, p=0.01). And, there was no statistically sig- nificant difference in term of LV EF in both groups before discharge from the  hospital (44.58±6.8 vs. 42.95±9.3 %, p=0.40). This extent of systolic recovery in our study pop- ulation appears to be consistent with that reported in most previous studies [17, 18]. Additionally, we did not find differences in the initial ex- amination of any inflammatory response (increased white blood cell counts and high level of Hs-CRP and plasma glu- cose) related to functional recovery in both groups. It is not surprising that control NT-proBNP level compared to ad- mission was higher in non-viability group. The increased NT-proBNP may play a role in why improvement of EF was lesser in this group. Likewise, creatinine level was higher in non-viability group than transient stunning group. But it may be arguable that creatinine level at admission was also significantly higher in this group. Discussioni Our findings suggest that persistent LV dysfunction and transient myocardial stunning can be distinguished in the early period after STEMI. Furthermore, E / (Ea×Sa) index value to be a strong predictor of functional recovery; the odds of full recovery decreases as E / (Ea×Sa) index val- ue increases. Myocardial stunning is typically defined as transient myo­cardial dysfunction in areas of restored coronary per- fusion after STEMI [3]. Acute occlusion of a coronary ar- tery rapidly impairs contractile function and may result in myocardial necrosis depending on  the  occlusion du- ration [14–16]. Previous research demonstrated the di- astolic dysfunction of LV during myocardial stunning. Table 4. Identification of the predictors for myocardial recovery with multiple re Table 4. Identification of the predictors for myocardial recovery with multiple regression analysis Variables OR (CI, 95 %) p value Age 1.03 (0.99–1.08) 0.08 E / (Ea x Sa) index 0.91 (0.83–0.98) 0.001 Creatinine 1.14 (1.06–1.23) 0.32 NT-proBNP 1.01 (0.94–1.07) 0.70 Hs-CRP 1.11 (0.97–1.19) 0.09 Door to balloon time 1.05 (0.97–1.11) 0.65 Hs-CRP: High sensitive C reactive protein, NT-proBNP: N-terminal pro-b-type natriuretic peptide. Table 4. Identification of the predictors Table 4. Identification of the predictors for myocardial recovery with multiple regression analysis ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477 69 ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477 REFERENCES pertension. European Heart Journal. 2018;39(33):3021–104. DOI: 10.1093/eurheartj/ehy339 1. Vogel B, Claessen BE, Arnold SV, Chan D, Cohen DJ, Giannit- sis E et al. ST-segment elevation myocardial infarction. Nature Re- views Disease Primers. 2019;5(1):39. DOI: 10.1038/s41572-019- 0090-3 13. Ibanez B, James S, Agewall S, Antunes MJ, Bucciarelli-Ducci C, Bue- no H et al. 2017 ESC Guidelines for the management of acute myo- cardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Soci- ety of Cardiology (ESC). European Heart Journal. 2018;39(2):119– 77. DOI: 10.1093/eurheartj/ehx393 2. Guaricci AI, Bulzis G, Pontone G, Scicchitano P, Carbonara R, Rab- bat M et al. Current interpretation of myocardial stunning. Trends in Cardiovascular Medicine. 2018;28(4):263–71. DOI: 10.1016/j. tcm.2017.11.005 3. Braunwald E, Kloner RA. The stunned myocardium: prolonged, post- ischemic ventricular dysfunction. Circulation. 1982;66(6):1146–9. DOI: 10.1161/01.CIR.66.6.1146 14. Tatli E, Aktoz M, Altun A. A Patient With Unstable Angina and With- out Abnormal Electrocardiographic Findings and Total Occlusion of the Left Anterior Descending Coronary Artery. Trakya Universite- si Tip Fakultesi Dergisi. 2010;27(1):83–5. [Av. at: https://www.re- searchgate.net/publication/288813603_A_Patient_With_Unstable_ Angina_and_Without_Abnormal_Electrocardiographic_Findings_ and_Total_Occlusion_of_the_Left_Anterior_Descending_Coro- nary_Artery] 4. Yap J, Lim FY, Gao F, Wang SZ, Low SCS, Le TT et al. Effect of Myocar­dial Viability Assessed by Cardiac Magnetic Resonance on Survival in Patients With Severe Left Ventricular Dysfunction. Circulation Reports. 2020;2(6):306–13. DOI: 10.1253/circrep.CR- 19-0126 5. Lam W, Leano R, Haluska B, Marwick TH. Effects of myocardi- al ischaemia on left ventricular untwist and filling pressure. Heart. 2011;97(9):757–61. DOI: 10.1136/hrt.2010.201699 15. Cokkinos DV. Myocardial Stunning. [P. 171-184. DOI: 10.1007/978- 3-319-98186-4_9]. In: Myocardial Preservation. [ISBN: 978-3-319- 98185-7] Cokkinos DV, editor -Cham: Springer International Publish- ing;2019. 6. Faustino M, Baptista SB, Freitas A, Monteiro C, Leal P, Nédio M et al. The Index of Microcirculatory Resistance as a Predictor of Echocardiographic Left Ventricular Performance Recovery in Patients With ST-Elevation Acute Myocardial Infarction Un- dergoing Successful Primary Angioplasty. Journal of Intervention- al Cardiology. 2016;29(2):137–45. DOI: 10.1111/joic.12278 16. Neri M, Riezzo I, Pascale N, Pomara C, Turillazzi E. Ischemia/Reper- fusion Injury following Acute Myocardial Infarction: A Critical Issue for Clinicians and Forensic Pathologists. Mediators of Inflammation. 2017;2017:7018393. DOI: 10.1155/2017/7018393 7. Mornos C, Cozma D, Rusinaru D, Ionac A, Maximov D, Petrescu L et al. A novel index combining diastolic and systolic Tissue Doppler parameters for the non-invasive assessment of left ventricular end-dia- stolic pressure. International Journal of Cardiology. 2009;136(2):120– 9. No conflict of interest is reported. No conflict of interest is reported. Acknowledgement ОРИГИНАЛЬНЫЕ СТАТЬИ § We found that other conventional echocardiographic measurements includ- ing mitral E velocity, mitral E / A ratio had shown differ- ences between groups. But they were less linked to im- provement in ventricular function. Our analysis demon- strated that the diagnostic accuracy of  the E / (Ea×Sa) index in predicting functional myocardial recovery af- ter PPCI is more sensitive and specific than that of tra- ditional diastolic parameters including E / Ea ratio and Sa wave (the E / (Ea×Sa) index = 73 % of sensitivity and 61 % of specificity (AUC: 0.71, p=0.01, CI: 0.54–0.89), the E / Ea ratio = 64 % of sensitivity and 41 % of specific- ity (AUC=0.67, p=0.03, CI: 0.61–0.74), Sa wave = 53 % of sensitivity and 56 % of specificity (AUC=0.65, p=0.03, In conclusion, our findings suggest that the combined sys- tolic-diastolic ([E / (Ea×Sa)]) index, is related to reco­very of ventricular function after STEMI and could be a more im- portant predictor of functional recovery than other early echocardiographic measures. And, using this index in early period for prognostic assessment may be the ideal. 70 ОРИГИНАЛЬНЫЕ СТАТЬИ § gy Depart­ment, Ankara, Turkey) for supervision, Assoc. Prof. Dr. Emrah CİNKARA (Gaziantep University, Turkey) for language control and Dr. Nurbanu BURSA (Hacettepe University, Turkey) for support. gy Depart­ment, Ankara, Turkey) for supervision, Assoc. Prof. Dr. Emrah CİNKARA (Gaziantep University, Turkey) for language control and Dr. Nurbanu BURSA (Hacettepe University, Turkey) for support. Informed consenth Acknowledgement We would like to thank Prof. Dr. Mustafa KILIÇKAP (Ankara University, School of Medicine, Cardiolo- Informed consent The study protocol was approved with registration number of 2020–5 / 4 by the Bursa City Hospital ethics committee. No conflict of interest is reported. No conflict of interest is reported. REFERENCES DOI: 10.1016/j.ijcard.2008.04.029h 17. Solomon SD, Glynn RJ, Greaves S, Ajani U, Rouleau J-L, Mena- pace F et al. Recovery of Ventricular Function after Myocardial Infarc- tion in the Reperfusion Era: The Healing and Early Afterload Reduc- ing Therapy Study. Annals of Internal Medicine. 2001;134(6):451–8. DOI: 10.7326/0003-4819-134-6-200103200-00009 18. Rifqi S, Sungkar S, Sobirin MA, Uddin I, Furuse Y, Nugroho MA et al. Early recovery of left ventricular function after revascularization of coronary artery disease detected by myocardial strain. Biomedical Re- search. 2017;28(4):1487–92. [Av. at: https://www.semanticscholar. org/paper/Early-recovery-of-left-ventricular-function-after-Rifqi-Sun gkar/77556515261b781086c9c8240a89d2746e946ff5] 8. Mornoş C, Petrescu L, Ionac A, Cozma D. The prognostic val- ue of a new tissue Doppler parameter in patients with heart failure. The International Journal of Cardiovascular Imaging. 2014;30(1):47– 55. DOI: 10.1007/s10554-013-0294-7 9. Ari H, Ari S, Sarigül OY, Coşar S, Doğanay K, Emlek N et al. A Nov- el Index Combining Diastolic and Systolic Tissue Doppler Parame- ters for Prediction of Atrial Fibrillation Recurrence. Echocardiography. 2016;33(7):1009–15. DOI: 10.1111/echo.13212h gf 19. Bhatt AS, Ambrosy AP, Velazquez EJ. Adverse Remodeling and Re- verse Remodeling After Myocardial Infarction. Current Cardiology Reports. 2017;19(8):71. DOI: 10.1007/s11886-017-0876-4 10. Camci S, Ari S, Karakus A, Ari H, Taner T. The predictive value of the combined systolic‐diastolic index for atrial fibrillation after cor- onary artery bypass surgery. Echocardiography. 2020;37(8):1177–83. DOI: 10.1111/echo.14806 20. Lesizza P, Prosdocimo G, Martinelli V, Sinagra G, Zacchigna S, Gi- acca M. Single-Dose Intracardiac Injection of Pro-Regenerative Mi- croRNAs Improves Cardiac Function After Myocardial Infarction. Circulation Research. 2017;120(8):1298–304. DOI: 10.1161/CIR- CRESAHA.116.309589 11. Ari S, Ari H, Tütüncü A, Çamci S, Vatansever Ağca F, Melek M. A nov- el index combining diastolic and systolic tissue Doppler parameters for predicting cardiac resynchronization therapy response. Echocar- diography. 2020;37(8):1184–91. DOI: 10.1111/echo.14797 21. Daubert MA, White JA, Al-Khalidi HR, Velazquez EJ, Rao SV, Crow- ley AL et al. Cardiac remodeling after large ST-elevation myocardi- al infarction in the current therapeutic era. American Heart Journal. 2020;223:87–97. DOI: 10.1016/j.ahj.2020.02.017 g 12. Williams B, Mancia G, Spiering W, Agabiti Rosei E, Azizi M, Burnier M et al. 2018 ESC/ESH Guidelines for the management of arterial hy- 71 ISSN 0022-9040. Кардиология. 2021;61(1). DOI: 10.18087/cardio.2021.1.n1477
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Blood glucose estimation using third person continuous glucose monitoring data via complete ensemble empirical mode decomposition with adaptive noise and long-short-term memory networks
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Blood glucose estimation using third person continuous glucose monitoring data via complete ensemble empirical mode decomposition with adaptive noise and long-short-term memory networks Bingo Wing-Kuen  (  Lingyongquanling@gdut.ed ) Guangdong University of Technology Junjian Liang  Guangdong University of Technology Weiren Zhao  Guangdong University of Technology Rui Li  Guangdong University of Technology Jiaqi Liu  Guangdong University of Technology Research Article Keywords: Micro-invasive glucose estimation, continuous glucose monitoring data of the third person, complete ensemble empirical mode decomposition with the adaptive noise, long-short-term memory network Posted Date: July 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3140386/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Open Access RESEARCH ARTICLE Blood glucose estimation using third person continuous glucose monitoring data via complete ensemble empirical mode decomposition with adaptive noise and long-short-term memory networks Junjian Liang1,2, Weiren Zhao2,3, *Bingo Wing-Kuen Ling1, Jiaqi Liu1, and Rui Li1 Junjian Liang1,2, Weiren Zhao2,3, *Bingo Wing-Kuen Ling1, Jiaqi Liu1, and Rui Li1 *Correspondence: Bingo Wing-Kuen Lingyongquanling@gdut.edu.cn 1Faculty of Information Engineering, Guangdong University of Technology, Guangzhou 510006, China 2School of Physics and Optoeletronic Engineering, Guangdong University of Technology, Guangzhou, 510006, China 3Guangdong Provincial Key Laboratory of Photonics Information Technology, Guangdong University of Technology, Guangzhou, 510006, China Introduction Introduction The total number of diabetes patients has been increasing worldwide. According to one survey [1], there were 451 million diabetes patients in 2017. This number is expected to reach 693 million by 2045. Diabetes is a chronic disease that severely affects human health. If it is not properly treated, it can cause complications such as kidney disease, heart disease, and neuropathy. In severe cases, diabetes can be life- threatening, and this condition is, unfortunately, incurable. Patients’ blood glucose levels can only be stabilized using drugs or insulin injections. These stabilizing treatments require regular estimation of patients’ blood glucose levels in order to determine the doses of drugs to be taken or the amount of insulin to be injected. Accurate estimation of blood glucose levels is therefore critical for the management of diabetes. In the study of diabetes, many methods have been proposed for estimating blood glucose levels. Traditionally, the most common approach has been an invasive one, with patients required to prick their fingers several times per day and thus draw small amounts of blood to be measured. However, this approach causes long-term pain in patients with diabetes. To address this issue, a micro-invasive approach has also been proposed. In this method, a 1- mm pin is pushed into the arm of the user to estimate blood glucose [2]. This method is still quite costly, however, and thus places a significant financial burden on patients. More recently, fully non-invasive approaches have also been proposed [3]. These approaches can be mainly categorized into two types. The first is based on physiological models [4]; however, many other physiological parameters must first be estimated in order to construct such models. Moreover, different diabetes patients can have very different parameters in these models, due to their inherent physiological differences. This makes both modeling and performing blood glucose estimation fairly difficult [5]. The second type is based on previously measured blood glucose values at past time- points. Models are easier to construct under this paradigm, and results are generally more reliable [6]. As a result, this type of estimation method has drawn considerable attention from diabetes researchers in recent years. In particular, many estimation methods based on artificial neural networks have been proposed in recent years [7]. It is worth noting that glucose data vary over time. Th f l h (LSTM) k The total number of diabetes patients has been increasing worldwide. Introduction The first is based on physiological models [4]; however, many other physiological parameters must first be estimated in order to construct such models. Moreover, different diabetes patients can have very different parameters in these models, due to their inherent physiological differences. This makes both modeling and performing blood glucose estimation fairly difficult [5]. The second type is based on previously measured blood glucose values at past time- points. Models are easier to construct under this paradigm, and results are generally more reliable [6]. As a result, this type of estimation method has drawn considerable attention from diabetes researchers in recent years. In particular, many estimation methods based on artificial neural networks have been proposed in recent years [7]. (PURE) within 30-days after discharge for patients that had a clinical admission at the department of Urology. Our second aim was to evaluate the difference perfor- mance of the PURE probability calculator developed using ML algorithms, comparing regression versus clas- sification algorithms. We hypothesized it is feasible to develop a strong performing PURE probability calcu- lator, and there is no difference in performance when developed with ML algorithms using classification ver- sus regression algorithms. Introduction According to one survey [1], there were 451 million diabetes patients in 2017. This number is expected to reach 693 million by 2045. Diabetes is a chronic disease that severely affects human health. If it is not properly treated, it can cause complications such as kidney disease, heart disease, and neuropathy. In severe cases, diabetes can be life- threatening, and this condition is, unfortunately, incurable. Patients’ blood glucose levels can only be stabilized using drugs or insulin injections. These stabilizing treatments require regular estimation of patients’ blood glucose levels in order to determine the doses of drugs to be taken or the amount of insulin to be injected. Accurate estimation of blood glucose levels is therefore critical for the management of diabetes. Using this approach, we proposed a method to map continuous glucose monitoring data from a third person to new continuous glucose monitoring data from a particular user, using only a few invasively derived initial measurements from that user [9]. We believe that the CEEMDAN-based de-noising method is tailor-made for this problem of blood glucose estimation problem, as we found that discarding the first six IMFs obtained, by applying the CEEMDAN approach to the mapped signal, achieved excellent performance with regard to de-noising. The outline of this report is as follows: Section 2 reviews the background on time-frequency analysis techniques such as the EMD and CEEMDAN, as well the background on LSTM networks. Section 3 presents our proposed method. Computer numerical simulation results regarding the proposed method are then presented in Section 4, and conclusions are drawn in Section 5. g In the study of diabetes, many methods have been proposed for estimating blood glucose levels. Traditionally, the most common approach has been an invasive one, with patients required to prick their fingers several times per day and thus draw small amounts of blood to be measured. However, this approach causes long-term pain in patients with diabetes. To address this issue, a micro-invasive approach has also been proposed. In this method, a 1- mm pin is pushed into the arm of the user to estimate blood glucose [2]. This method is still quite costly, however, and thus places a significant financial burden on patients. More recently, fully non-invasive approaches have also been proposed [3]. These approaches can be mainly categorized into two types. Abstract Background Blood glucose estimation is critical for monitoring the health of patients with diabetes. Traditionally, it has been performed invasively, via pricking a fingertip to draw a small amount of blood for analysis. However, this type of approach causes pain to patients in the long-term. More recently, micro-invasive approaches have been proposed as alternatives; however, these new methods can be quite costly. Methods To address this issue, we proposed a minimally invasive approach for obtaining blood glucose measurements. In our proposed approach, the continuous glucose monitoring data of a third person are mapped to more sparse data from a particular user, acquired through traditional invasive means. The complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) method is then applied to the mapped glucose data. The first six intrinsic mode functions are discarded, and a long short-term memory (LSTM) network is used to perform non-invasive blood glucose estimation. To demonstrate the effectiveness of our proposed method, a loss function was used as a performance metric. Our approach was compared to the LSTM network, both with and without an empirical mode decomposition (EMD) approach. Results Numerical simulation results showed that our approach achieved a higher accuracy than eith wo popular methods. Conclusions Our method can be used to improve the accuracy of blood glucose prediction. Keywords Micro-invasive glucose estimation, continuous glucose monitoring data of the third person, complete ensemble empirical mode decomposition with the adaptive noise, long-short-term memory network Page 2 of 8 from a third person to perform blood glucose estimation. Instead of using empirical mode decomposition (EMD) or ensemble empirical mode decomposition (EEMD) to perform signal decomposition, we used a complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) approach because of its robust nature. In CEEMDAN, instead of only adding white Gaussian noise directly to the original signal, as is done in the conventional EEMD approach, the white Gaussian noise is multiplied by a weight factor. Moreover, instead of taking the average of each IMF obtained from the various realizations in the conventional EEMD approach, CEEMDAN takes the average of only the first components obtained in the various realizations. Empirical mode decomposition Since the local characteristics of a signal in the time domain can be characterized by its time scales and its time scales can also be characterized by its extrema, the objective of EMD is to exploit the local characteristics of the signal through its extrema [10]. EMD assumes that the signal contains at least one local maximum and minimum. If there are no extrema in the signal, but there is a point of reflection, then the extrema can be obtained by differentiating the signal one or more times. Decomposition can then be performed on the differentiated signal, and the obtained components can be subsequently It is worth noting that glucose data vary over time. Therefore, long short-term memory (LSTM) networks using signal decomposition approaches have been used for blood glucose level estimations [8]. However, existing methods assume that both the training and estimated glucose data come from the same patient. Although relatively good estimation performance can be achieved, under this model, each patient has to wear the micro-invasive glucose measuring device at least once. As a result, the cost paid by the user is still high. To address this issue, we proposed using glucose data Page 3 of 8 𝑥𝑖(𝑡) = ∑ 𝑐𝑖,𝑙(𝑡) + 𝑟̃𝑖(𝑡) ∀𝑙 (3) integrated. Once the extrema of the signal are located, then cubic spline interpolation is performed on the local minima, to obtain the lower envelope of the signal. It is also similarly applied to the local maxima, to obtain the upper envelope. By subtracting the signal from the average of its upper and lower envelopes, a new signal is obtained. By repeating this process until convergence is achieved [11], the first IMF is formed. The rest of the IMFs are similarly obtained by subtracting the signal from the sum of the obtained IMFs and repeating the process. This process is known as sifting. Over the course of sifting, the total number of extrema in the IMFs decreases. Hence, the IMFs have different time scales. 8 (3) Further, if we let 𝑐̅1(𝑡) be the average of the first IMF obtained in K realizations, we can derive, 𝑐̅1(𝑡) = 1 𝐾∑ 𝑐𝑖,1(𝑡) 𝐾 𝑖=1 (4) (4) After 𝑐̅1(𝑡) is computed, let 𝑟1(𝑡) be the first residue defined as the difference between the original signal and the first averaged IMF. Empirical mode decomposition This yields, 𝑟1(𝑡) = 𝑥(𝑡) −𝑐̅1(𝑡) (5) (5) To illustrate the process mathematically, let 𝑖𝑡ℎ represent an acquired signal, M be the total number of IMFs, 𝑐𝑖(𝑡) be the 𝑐𝑖(𝑡) IMF, and r(t) be the residue. The signal can then be represented as the sum of the obtained IMFs and the residue, given by, After 𝑟1(𝑡) is computed, j is incremented by 1. The next averaged IMF and the corresponding residue can be obtained as follows: let 𝐵𝑗(𝑧(𝑡)) be the operator that the 𝑗𝑡ℎ IMF was extracted by applying the EMD to z(t) . This gives us, After 𝑟1(𝑡) is computed, j is incremented by 1. The next averaged IMF and the corresponding residue can be obtained as follows: let 𝐵𝑗(𝑧(𝑡)) be the operator that the 𝑗𝑡ℎ IMF was extracted by applying the EMD to z(t) . This gives us, 𝑥(𝑡) = ∑ 𝑐𝑖(𝑡) + 𝑟(𝑡) 𝑀 𝑖=1 (1) (1) 𝑐̅𝑗+1(𝑡) = 1 𝐾∑ 𝐵1(𝑟𝑗(𝑡) + 𝜀𝑗𝐵𝑗(𝑢𝑖(𝑡))) 𝐾 𝑖=1 (6) 𝑟𝑗+1(𝑡) = 𝑟𝑗(𝑡) −𝑐̅𝑗+1(𝑡) (7) (6) Here, the signal can be perfectly reconstructed using the obtained IMFs and the residue. Compared to the other time frequency representation methods, the EMD is nonlinear and adaptive. Because of these favorable properties, the EMD is widely used in many signal analysis applications. (7) For j ≥1.If we let 𝑀̅ be the total number of the IMFs obtained, we can see that Complete ensemble empirical mode decomposition with adaptive noise x(t) = ∑ c̅i(t) + rM̅ (t) M̅ i=1 (8) (8) Although EMD has some nice features, its use may invite the mode mixing phenomenon. EEMD was proposed to avoid this shortcoming. In EEMD, Gaussian-distributed white noise is added to the original signal. This avoids the occurrence of the mode mixing phenomenon; however, as the white noise has not been canceled, the reconstructed signal is corrupted by its presence. This shows us that the Gaussian white noise is added to the original signal in conventional EEMD, while the various Gaussian white noises are multiplied by varying weights before being added to the signal in CEEMDAN. Long short-time memory networks Page 4 of 8 mapped data to obtain a finite number of IMFs and the residue. The first six IMFs are discarded, to obtain the de- noised signal. The retained IMFs in the training set are used to make a prediction. Finally, the predicted continuous glucose monitoring data are obtained and used to estimate the user’s blood glucose level. j = 1,2. . . ,11 and b⃗⃗ j for j = 1,2. . . ,4 be the weight matrices and the weight vectors of the LSTM network, respectively. Figure 1. Structure of an LSTM network L t Figure 2. Flowchart of our proposed method. Figure 1. Structure of an LSTM network a⃗ cu[n] = W1x⃗ [n] + W2s⃗ [n −1] + W3v⃗ [n −1] + b⃗⃗ 1, (9) a⃗ cs[n] = W4x⃗ [n] + W5s⃗ [n −1] + W6v⃗ [n −1] + b⃗⃗ 2, (10) a⃗ cr[n] = W7x⃗ [n] + W8s⃗ [n −1] + W9v⃗ [n −1] + b⃗⃗ 3, (11) a⃗ du[n] = W10x⃗ [n] + +W11v⃗ [n −1] + b⃗⃗ 4, (12) (12) be the gate inputs of the LSTM network. Let 𝐺𝑑(∙) and 𝐺𝑐(∙) be the gate functions of the LSTM network [14]. Finally, let be the gate inputs of the LSTM network. Let 𝐺𝑑(∙) and 𝐺𝑐(∙) be the gate functions of the LSTM network [14]. Finally, let Figure 2. Flowchart of our proposed method. u⃗⃗ [n] = Gd(a⃗ du[n]), (13) g⃗ cu[n] = Gc(a⃗ cu[n]), (14) g⃗ cs[n] = Gc(a⃗ cs[n]), (15) g⃗ cr[n] = Gc(a⃗ cr[n]), (16) u⃗⃗ [n] = Gd(a⃗ du[n]), (13) g⃗ cu[n] = Gc(a⃗ cu[n]), (14) g⃗ cs[n] = Gc(a⃗ cs[n]), (15) g⃗ cr[n] = Gc(a⃗ cr[n]), (16) Long short-time memory networks The LSTM network is a type of recurrent neural network (RNN). Unlike other neural networks, RNNs represent a dynamic system with memory; that is, the current output of an RNN depends on the previous time instants of its input. Therefore, RNNs can be used to make predictions. However, these networks also have long-term dependencies. Hence, training RNNs can be difficult. The CEEMDAN approach was therefore proposed, in turn, to mitigate this shortcoming of EEMD [12]. In CEEMDAN, multiple noises are multiplied by varying weights and added to the original signal, to generate a set of noisy signals. To describe the process formulaically, let K be the total number of realizations, ui(t) be the Gaussian-distributed white noise at the ith realization, and j + 1 be the IMF index. Here, j is initially set to 0. Let εj be the gain multiplied by the synthesized noise when computing the (j + 1)tℎ IMF and xi(t ) be the noise-corrupted signal at the itℎ realization. This yields, LSTM networks were proposed to mitigate this difficulty. These networks enjoy the advantages of RNNs in terms of their capability to learn from previous time instants of the input signal. Moreover, as they flexibly add memory units to each neuron in the hidden layer, training becomes more feasible. This makes LSTMs even more suitable for making predictions. Because of this favorable feature, they are widely used for predicting financial data. xi(t) = x(t) + ε0ui(t) for i = 1, . . . , K (2) (2) The basic structure of an LSTM network is shown in Figure 1.Based on this structure, the training process for LSTM networks is based on the Vanilla rule, which is summarized as follows [13]: let x⃗ [n] be the input and s [n −1] and v⃗ [n −1]be the state vectors, and let Wj for EMD is then applied to 𝑥𝑖(𝑡). If we let 𝑐𝑖,𝑙(𝑡) be the 𝑙𝑡ℎIMF and 𝑟̃𝑖(𝑡) be the residue of the noise-corrupted signal at the 𝑖𝑡ℎ realization, we then obtain, Page 4 of 8 mapped data to obtain a finite number of IMFs and the residue. The first six IMFs are discarded, to obtain the de- noised signal. The retained IMFs in the training set are used to make a prediction. Finally, the predicted continuous glucose monitoring data are obtained and used to estimate the user’s blood glucose level. Dataset The data used in this study included both continuous glucose monitoring data from a third person, acquired using a micro-invasive device, as well as data from the particular user, acquired using a minimally invasive conventional glucose meter. The continuous glucose monitoring data were acquired over 12 days, with sampling performed every 15 min. Figure 3 shows a plot of the continuous glucose monitoring data. be the gate outputs of the LSTM network. The dynamic behavior of the network is then governed by s⃗ [n] = g⃗ cs[n]⨀s⃗ [n −1] + g⃗ cu[n]⨀u⃗⃗ [n], (17) v⃗ [n] = g⃗ cr[n]⨀r [n], (18) where denotes the vector cross product and be the gate outputs of the LSTM network. The dynamic behavior of the network is then governed by s⃗ [n] = g⃗ cs[n]⨀s⃗ [n −1] + g⃗ cu[n]⨀u⃗⃗ [n], (17) v⃗ [n] = g⃗ cr[n]⨀r [n], (18) where denotes the vector cross product and r [n] = Gd(s⃗ [n]), (19) O d th d Figure 3. A plot of the continuous glucose monitoring data used in this study. be the gate outputs of the LSTM network. The dynamic behavior of the network is then governed by where denotes the vector cross product and (19) Figure 3. A plot of the continuous glucose monitoring data used in this study. Mapping of the data In particular, 7-9 samples from the breakfast interval, 11-14 samples from the lunch interval, and 17-20 samples from the dinner interval are taken out every day, and the rest of the samples are grouped together. Once these neighborhoods are defined, then the local maxima can be found. Let Xbreakfast_b and Xbreakfast_a be the glucose values taken 15 min before and after the time instant corresponding to Xmax _breakfast on each day, respectively. Similarly Let Xluncℎ_b and Xluncℎ_a be the glucose values taken at 15 min before and after the time instant corresponding to Xmax _luncℎ in each day, respectively, and let Xdinner_b and Xdinner_a be the glucose values taken 15 min before and after Xmax _dinner in each day, respectively.Let Xbreakfast be the vector of the glucose values where the corresponding time intervals of these values are in the region between the corresponding time interval of Xmax _breakfast and that of Xnorm on both sides of in the breakfast interval. Let Xbreakfast_i be the itℎ element in Xbreakfast . Let Xluncℎ be the vector of the glucose values where the corresponding time intervals of these values are in the region between the corresponding time interval between Xmax _luncℎ and Xnorm , on both sides of the lunch interval. Let Xluncℎ_i be the itℎ element in Xluncℎ. Let Xluncℎbe the vector of the glucose values where the corresponding time intervals of these values are in the region between the corresponding time interval of Xmax _luncℎ and Xnorm, on both sides of the dinner interval. Let Xdinner_i be the itℎ element in Xdinner. Let Xsteady be the vector of the glucose values in the steady intervals on third person. To map the continuous glucose monitoring data of the third person to new glucose data from a particular user using only a few invasive measurements, the characteristic points of the third-party data must be identified. For example, glucose values typically rise after meals. After that, they drop and stay steady until the next meal is taken. Hence, the characteristic points are defined as the maximum glucose values in the breakfast interval, the lunch interval, and the dinner interval, as well as the value in the intervening steady states. Let Xmax _breakfast, Xmax _luncℎ and Xmax _dinner be the maximum glucose values in the breakfast interval, the lunch interval and the dinner interval, respectively, and let Xnorm be the steady- state value between meals. Mapping of the data Let X̃luncℎ_i itℎelement in X̃luncℎ.Let X̃dinner be the vector mapped glucose values based on Xdinner. Let X̃dinn the itℎ element in X̃dinner. Let X̃steady be the vector mapped glucose values based on Xsteady. Let X̃stea the itℎ element in X̃steady. This gives us, X̃breakfast_i = Xbreakfast_i(Ybreakfast_b+Ybreakf (Xbreakfast_b+Xbreakfast_a) (20) X̃luncℎ_i = Xluncℎ_i(Yluncℎ_b+Yluncℎ_a) (Xluncℎ_b+Xluncℎ_a) (21) X̃dinner_i = Xdinner_i(Ydinner_b+Ydinner_a) (Xdinner_b+Xdinner_a) , (22) and X̃steady_i = Xsteady_iYnorm Xnorm (23) To generate the new continuous glucose monitoring d time instants corresponding to the centers of X̃breakfast and X̃dinner are set to the time instants where the pa user takes the measurements at the breakfast, lun dinner intervals, respectively[16]. However, as the when the particular user takes their breakfast, lun dinner measurements are not exactly equal to the corresponding to Xmax _breakfast, Xmax _luncℎand Xmax for the third-party, the duration between the breakfa lunch intervals in the continuous third-party data is no to the corresponding duration between the meals particular user. Likewise, the duration between the lun dinner intervals is not necessarily equal between the u the third-party. Therefore, X̃steady is required to be resc order to fit the new steady interval between two cons new meal intervals in the user’s data. Let X̂steady re this rescaled vector. To address this issue, the discrete transform-based rescaling method is used. Let LCG LNEW be the total number of data-points in X̃stea X̂steady , respectively. If LCGM = LNEW , then set X̂s X̃steady. If LCGM < LNEW, then X̂steady = idct([dct(X̃steady) 0LNEW−LCGM X̃breakfast_i = Xbreakfast_i(Ybreakfast_b+Ybreakfast_a) (Xbreakfast_b+Xbreakfast_a) , (20) X̃luncℎ_i = Xluncℎ_i(Yluncℎ_b+Yluncℎ_a) (Xluncℎ_b+Xluncℎ_a) , (21) X̃dinner_i = Xdinner_i(Ydinner_b+Ydinner_a) (Xdinner_b+Xdinner_a) , (22) and X̃steady_i = Xsteady_iYnorm Xnorm , (23) X̃luncℎ_i = Xluncℎ_i(Yluncℎ_b+Yluncℎ_a) (Xluncℎ_b+Xluncℎ_a) (21) To generate the new continuous glucose monitoring data, the time instants corresponding to the centers of X̃breakfast, X̃luncℎ and X̃dinner are set to the time instants where the particular user takes the measurements at the breakfast, lunch and dinner intervals, respectively[16]. However, as the times when the particular user takes their breakfast, lunch and dinner measurements are not exactly equal to the times corresponding to Xmax _breakfast, Xmax _luncℎand Xmax _dinner for the third-party, the duration between the breakfast and lunch intervals in the continuous third-party data is not equal to the corresponding duration between the meals for the particular user. Likewise, the duration between the lunch and dinner intervals is not necessarily equal between the user and the third-party. Mapping of the data Figure 2 shows a flowchart of our proposed method. First, continuous glucose monitoring data from a third person, acquired using a micro-invasive device, are mapped to new continuous glucose monitoring data from a particular user, by taking a few initial, invasive measurements from that user. The CEEMDAN method is then applied to the The procedure for mapping the third-party continuous glucose monitoring data to new data from a particular user using only a few invasive measurements from that user were as follows [15]: Step 1: Obtain continuous glucose monitoring data from the Page 5 of 8 user. Let X̃breakfast be the vector of the mapped glucose values based on Xbreakfast . Let X̃breakfast_i be the itℎ element in X̃breakfast . Let X̃luncℎ be the vector of the mapped glucose values based on Xluncℎ. Let X̃luncℎ_i be the itℎ element in Xluncℎ. Let X̃luncℎ be the vector of the mapped glucose values based on Xluncℎ. Let X̃luncℎ_i be the itℎelement in X̃luncℎ.Let X̃dinner be the vector of the mapped glucose values based on Xdinner. Let X̃dinner_i be the itℎ element in X̃dinner. Let X̃steady be the vector of the mapped glucose values based on Xsteady. Let X̃steady_i be the itℎ element in X̃steady. This gives us, third person. To map the continuous glucose monitoring data of the third person to new glucose data from a particular user using only a few invasive measurements, the characteristic points of the third-party data must be identified. For example, glucose values typically rise after meals. After that, they drop and stay steady until the next meal is taken. Hence, the characteristic points are defined as the maximum glucose values in the breakfast interval, the lunch interval, and the dinner interval, as well as the value in the intervening steady states. Let Xmax _breakfast, Xmax _luncℎ and Xmax _dinner be the maximum glucose values in the breakfast interval, the lunch interval and the dinner interval, respectively, and let Xnorm be the steady- state value between meals. It is worth noting that the maximum glucose values in the meal intervals are the local maxima in the continuous glucose monitoring data of the third party. To find these local maxima, the neighborhoods around these local maxima must be defined. Therefore, four segments are taken from the third-party data for each day. Mapping of the data It is worth noting that the maximum glucose values in the meal intervals are the local maxima in the continuous glucose monitoring data of the third party. To find these local maxima, the neighborhoods around these local maxima must be defined. Therefore, four segments are taken from the third-party data for each day. In particular, 7-9 samples from the breakfast interval, 11-14 samples from the lunch interval, and 17-20 samples from the dinner interval are taken out every day, and the rest of the samples are grouped together. Once these neighborhoods are defined, then the local maxima can be found. Let Xbreakfast_b and Xbreakfast_a be the glucose values taken 15 min before and after the time instant corresponding to Xmax _breakfast on each day, respectively. Similarly Let Xluncℎ_b and Xluncℎ_a be the glucose values taken at 15 min before and after the time instant corresponding to Xmax _luncℎ in each day, respectively, and let Xdinner_b and Xdinner_a be the glucose values taken 15 min before and after Xmax _dinner in each day, respectively.Let Xbreakfast be the vector of the glucose values where the corresponding time intervals of these values are in the region between the corresponding time interval of Xmax _breakfast and that of Xnorm on both sides of in the breakfast interval. Let Xbreakfast_i be the itℎ element in Xbreakfast . Let Xluncℎ be the vector of the glucose values where the corresponding time intervals of these values are in the region between the corresponding time interval between Xmax _luncℎ and Xnorm , on both sides of the lunch interval. Let Xluncℎ_i be the itℎ element in Xluncℎ. Let Xluncℎbe the vector of the glucose values where the corresponding time intervals of these values are in the region between the corresponding time interval of Xmax _luncℎ and Xnorm, on both sides of the dinner interval. Let Xdinner_i be the itℎ element in Xdinner. Let Xsteady be the vector of the glucose values in the steady intervals on each day. Let Xsteady_i be the itℎ element in Xsteady . Step 2: Obtain blood glucose data from the particular user. Blood glucose values are acquired before and after breakfast, before and after the lunch, before and after the dinner, and before sleep on each day; let Ybreakfast b , pp g luncℎ luncℎ_i itℎ element in Xluncℎ. Let X̃luncℎ be the vector mapped glucose values based on Xluncℎ. Mapping of the data We therefore discarded the first 6 IMFs for both CEEMDAN and EMD when performing de-noising. Page 6 of 8 Here, dct(z) and idct(z) denote the discrete cosine transform and the inverse discrete cosine transform of z, respectively. If LCGM > LNEW, then set . X̂steadyidct ([ILNEW 0(LCGM−LNEW)×LNEW]dct(X̃steady)) ,(25) Here, Ia and 0b×c denote the a × a identity matrix and the b × c zero matrix, respectively. Step 4: Smooth the discontinuity. There is an inevitable discontinuity between the new meal interval and the new steady interval. To address this, the discontinuity point in the new continuous glucose monitoring data is taken as the f h l i b f d f hi the continuous glucose monitoring data of the user are reconstructed using only the residue. Figures 7 and 8 show the continuous glucose monitoring data of the user reconstructed using different retained IMFs, decomposed by EMD and CEEMDAN, respectively. It is worth noting that different datasets have different numbers of IMFs. Also, the number of IMFs obtained when data are decomposed by CEEMDAN is different from that obtained when data are decomposed by EMD, even for the same dataset. However, there were always ≥ 6 IMFs for these two approaches, for all of the glucose data in our database. We therefore discarded the first 6 IMFs for both CEEMDAN and EMD when performing de-noising. ,(25) Step 4: Smooth the discontinuity. There is an inevitable discontinuity between the new meal interval and the new steady interval. To address this, the discontinuity point in the new continuous glucose monitoring data is taken as the mean of the values one point before and after this discontinuity. Figure 5. IMFs of the continuous glucose monitoring data for the user, decomposed by EMD. Figure 4 shows a sample of continuous third-party glucose monitoring data and mapped glucose monitoring data of the user (the yellow line) over 12 days. It can be seen that these two datasets are quite different. This is because the new continuous glucose monitoring data contain the invasive measurements of that particular user. measurements of that particular user. Figure 4. Plots of two sets of continuous glucose data. Selection of the IMFs for performing de-noising Figure 5. IMFs of the continuous glucose monitoring data for the user, decomposed by EMD. Figure 4. Plots of two sets of continuous glucose data. Figure 5. Mapping of the data IMFs of the continuous glucose monitoring data for the user, decomposed by EMD. Figure 4. Plots of two sets of continuous glucose data. Figure 4. Plots of two sets of continuous glucose data. Mapping of the data Therefore, X̃steady is required to be rescaled in order to fit the new steady interval between two consecutive new meal intervals in the user’s data. Let X̂steady represent this rescaled vector. To address this issue, the discrete cosine transform-based rescaling method is used. Let LCGM and LNEW be the total number of data-points in X̃steady and X̂steady , respectively. If LCGM = LNEW , then set X̂steady = X̃steady. If LCGM < LNEW, then ̂ ̃ Step 2: Obtain blood glucose data from the particular user. Blood glucose values are acquired before and after breakfast, before and after the lunch, before and after the dinner, and before sleep on each day; let Ybreakfast_b , Ybreakfast_a , Yluncℎ_b , Yluncℎ_a , Ydinner_b , Ydinner_a and Ynorm denote these blood glucose values, respectively. Here, these invasive measurements need only be taken only on the first day. X̂steady = idct([dct(X̃steady) 0LNEW−LCGM]) , X̂steady = idct([dct(X̃steady) 0LNEW−LCGM]) , (24) Step 3: Map the continuous glucose monitoring data from the third person to the one-day data from the particular (24) Page 6 of 8 and idct(z) denote the discrete cosine d the inverse discrete cosine transform of z, f LCGM > LNEW, then set . [ILNEW 0(LCGM−LNEW)×LNEW]dct(X̃steady)) 0b×c denote the a × a identity matrix and the trix, respectively. oth the discontinuity. There is an inevitable between the new meal interval and the new l. To address this, the discontinuity point in the us glucose monitoring data is taken as the values one point before and after this ws a sample of continuous third-party glucose ta and mapped glucose monitoring data of the ow line) over 12 days. It can be seen that these are quite different. This is because the new ucose monitoring data contain the invasive of that particular user. the continuous glucose monitoring data of the user ar reconstructed using only the residue. Figures 7 and 8 show the continuous glucose monitoring data of the use reconstructed using different retained IMFs, decomposed by EMD and CEEMDAN, respectively. It is worth noting tha different datasets have different numbers of IMFs. Also, th number of IMFs obtained when data are decomposed by CEEMDAN is different from that obtained when data ar decomposed by EMD, even for the same dataset. However there were always ≥ 6 IMFs for these two approaches, for al of the glucose data in our database. Training the long short-term network The new minimally invasive data from the user, for the first 9 days, were used to train the LSTM. The trained model was then tasked with predicting the user’s data for the next 3 days. Selection of the IMFs for performing de-noising In this study, estimation accuracy in term the mean absolute percentage error (MAPE) was use evaluate the similarity between the estimated and ac blood glucose values; that is, Page 7 of 8 Figure 8. Continuous glucose monitoring data of the user, reconstructed using different retained IMFs decomposed by CEEMDAN. Figure 6. IMFs of the continuous glucose monitoring data for the user, decomposed by CEEMDAN. Figure 6. IMFs of the continuous glucose monitoring data for the user, decomposed by CEEMDAN. Figure 6. IMFs of the continuous glucose monitoring data for the user, decomposed by CEEMDAN. Figure 8. Continuous glucose monitoring data of the user, reconstructed using different retained IMFs decomposed by CEEMDAN. decomposed by CEEMDAN. Figure 7. Continuous glucose monitoring data of the user, reconstructed using different retained IMFs decomposed by EMD. Selection of the IMFs for performing de-noising Once the new glucose data from the particular user are available, CEEMDAN is applied to this mapped data to obtain a finite number of IMFs, as well as the residue. To perform a comparison, EMD is also applied to these data to obtain a new set of IMFs. Figures 5 and 6 show the IMFs of the continuous glucose monitoring data from the user decomposed by EMD and CEEMDAN, respectively. A progressive approach is used to determine which IMFs should be discarded. The first component is discarded and the continuous glucose monitoring data of the user are reconstructed by adding together the rest of the IMFs and the residue. Then, the first two IMFs are discarded and the user data are again reconstructed in the same way. This procedure is repeated until all of the IMFs are discarded and Page 7 of Figure 6. IMFs of the continuous glucose monitoring data for the user, decomposed by CEEMDAN. Figure 7. Continuous glucose monitoring data of the user, reconstructed using different retained IMFs decomposed by EMD. Figure 8. Continuous glucose monitoring data of the user, reconst using different retained IMFs decomposed by CEEMDAN. Training the long short-term network The new minimally invasive data from the user, for the 9 days, were used to train the LSTM. The trained model then tasked with predicting the user’s data for the next 3 Computer numerical simulation results The results from our proposed method were compare those of an LSTM with the EMD approach [17] and to t of an LSTM without the EMD approach [18], because are the two most used methods in the literature that are most comparable to our proposed method. It is notewo that IMFs and residues are estimated in both our method the LSTM-with-EMD approach [17]. Continuous glu monitoring data are estimated directly in the LSTM-with EMD approach [18]. Seven data-points of invasive blood glucose measurem were acquired each day, and the data from the last three were used to evaluate the performances of the var methods. Let be the total number of the samples in the set, which means that K = 21. Let 𝑦𝑖 and 𝑦̂𝑖 be the 𝑖𝑡ℎ blood glucose value and the 𝑖𝑡ℎ corresponding predi glucose value over the final 3 days in the dat respectively. Computer numerical simulation results The results from our proposed method were compared to those of an LSTM with the EMD approach [17] and to those of an LSTM without the EMD approach [18], because they are the two most used methods in the literature that are the most comparable to our proposed method. It is noteworthy that IMFs and residues are estimated in both our method and the LSTM-with-EMD approach [17]. Continuous glucose monitoring data are estimated directly in the LSTM-without- EMD approach [18]. Seven data-points of invasive blood glucose measurements were acquired each day, and the data from the last three days were used to evaluate the performances of the various methods. Let be the total number of the samples in the test set, which means that K = 21. Let 𝑦𝑖 and 𝑦̂𝑖 be the 𝑖𝑡ℎ true blood glucose value and the 𝑖𝑡ℎ corresponding predicted glucose value over the final 3 days in the dataset, respectively. In this study, estimation accuracy in terms of the mean absolute percentage error (MAPE) was used to evaluate the similarity between the estimated and actual blood glucose values; that is, Figure 7. Continuous glucose monitoring data of the user, reconstructed using different retained IMFs decomposed by EMD. MAPE = 1 K ∑ | yi−ŷi yi | K i=1 , (26) (26) Table 1 shows the MAPEs yielded by the various methods. The MAPE from the LSTM-without-EMD approach without performing data mapping was notably higher than the one from the LSTM-without-EMD but with data mapping. Page 8 of 8 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Methods MAPE The LSTM without performing the EMD and without performing the mapping 0.2955 The LSTM without performing the EMD but with performing the mapping 0.2231 The LSTM with discarding the first IMF in the EMD and with performing the mapping 0.1982 The LSTM with discarding the first two IMFs in the EMD and with performing the mapping 0.1501 The LSTM with discarding the first three IMFs in the EMD and with performing the mapping 0.1402 The LSTM with discarding the first four IMFs in the EMD and with performing the mapping 0.1527 The LSTM with discarding the first six IMFs in the CEEMDAN and with performing the mapping 0.1265 Page 8 of 8 Consent for publication Not applicable. 5. Chen. YH, Lu. SY, Zhang. SS, Li,.Y, Qu. Z, Chen. Y, Lu. BW, Wang.XY, Feng. X, Skin-like biosensor system via electrochemical channels for noninvasive blood glucose monitoring, SCIENCE ADVANCES, vol 3, No.12, Dec 2017, https://www.science.org/ doi/full/10.1126/sciadv.1701629 5. Chen. YH, Lu. SY, Zhang. SS, Li,.Y, Qu. Z, Chen. Y, Lu. BW, Wang.XY, Feng. X, Skin-like biosensor system via electrochemical channels for noninvasive blood glucose monitoring, SCIENCE ADVANCES, vol 3, No.12, Dec 2017, https://www.science.org/ doi/full/10.1126/sciadv.1701629 Table 1. The MAPEs yielded by the various methods. Publisher’s Note This implied that performing data mapping improved the estimation performance. Moreover, it can be seen that the MAPE from the LSTM-without-EMD with mapping approach was higher than that obtained when LSTM was used along with discarding some IMFs in the EMD and performing data mapping. This suggested that discarding some IMFs also improved the estimation performance. Furthermore, it was observed that the MAPE based on LSTM with the CEEMDAN approach and data mappingwas the lowest among all of the methods. This implied that performing CEEMDAN further improved the estimation performance. Not applicable. Not applicable. Conclusion In this study, continuous glucose monitoring data from a particular user were obtained via mapping the continuous glucose monitoring data from a third person to a small subset of data acquired from the user through minimally invasive means. The CEEMDAN method was then used to perform de-noising. The first six IMFs were discarded. An LSTM was then used to perform blood glucose estimation. Computer numerical simulation results show that our proposed method could yield a higher estimation accuracy compared to an LSTM that did not perform mapping or EMD, an LSTM that performed mapping but not EMD, and an LSTM that performed both mapping and EMD. Received: 25 July 2022 Accepted: 18 May 2023 Authors’ contributions p p 9. Gonzales. WV, Mobashsher. AT , Abbosh. A, The Progress of Glucose Monitoring A Review of Invasive to Minimally and Non-Invasive Techniques, Devices and Sensors, SENSORS, vol 19,2019, https://www.mdpi.com/1424- 8220/19/4/800 Junjian Liang had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Weiren Zhao and Bingo Wing-Kuen Ling contributed equally as co- authors. Jiaqi Liu, and Rui Li help process date. 10. Wang. YH, Yeh. CH, Young. HWV, Hu. K , Lo. MT , On the computational complexity of the empirical mode decomposition algorithm, PHYSICA A- STATISTICAL MECHANICS AND ITS APPLICATIONS, vol. 400, Apr 2014, https://www.sciencedirect.com/science/article/ abs/pii/S0378437114000247 Concept and design: Junjian Liang and Bingo Wing-Kuen Ling. References 1. Tuomi. T , Santoro. N, Caprio. S , Cai. MY , Weng. JP, Groop. L , The many faces of diabetes: a disease with increasing heterogeneity, LANCET, vol 383, March 2014, https://www.sciencedirect.com/science/article/abs/pii/S0140673613622199 In the future, a joint decision tree and the LSTM-based method will be used to perform blood glucose estimation. 2. Bruen. D, Delaney. C, Florea. L, Diamond. D, Glucose Sensing for Diabetes Monitoring: Recent Developments, SENSORS, vol 1866, AUG 2017, https://www.mdpi.com/1424-8220/17/8/1866 2. Bruen. D, Delaney. C, Florea. L, Diamond. D, Glucose Sensing for Diabetes Monitoring: Recent Developments, SENSORS, vol 1866, AUG 2017, https://www.mdpi.com/1424-8220/17/8/1866 Availability of data and materials The data that support the findings of this study are available fromgithub which were used under license for the current study, and so are not publicly available. Data are how- ever available from the authors upon reasonable request and with permission . The code is accessible via .https://github.com/hallen632 /blood-glucose-processing 6. Kim. J, Campbell. AS, Wang. J, Wearable non-invasive epidermal glucose sensors: A review, TALANTA, vol 177, Aug 2017, https://www.sciencedirect.com/science/article/abs/ pii/S0039914017309098 7 H ang DW Lee S Seo M Ch ng TD Recent ad ances in 6. Kim. J, Campbell. AS, Wang. J, Wearable non-invasive epidermal glucose sensors: A review, TALANTA, vol 177, Aug 2017, https://www.sciencedirect.com/science/article/abs/ pii/S0039914017309098 H DW L S S M Ch TD R d i 7. Hwang. DW, Lee. S ,Seo. M, Chung. TD, Recent advances in electrochemical non-enzymatic glucose sensors - A review, ANALYTICA CHIMICA ACTA, vol 1033, May 2018, https://www.sciencedirect.com/science/article/abs/pii/S0003267018306718 7. Hwang. DW, Lee. S ,Seo. M, Chung. TD, Recent advances in electrochemical non-enzymatic glucose sensors - A review, ANALYTICA CHIMICA ACTA, vol 1033, May 2018, https://www.sciencedirect.com/science/article/abs/pii/S0003267018306718 Competing interest The authors declare p g The authors declare that they have no competing interests. 8. Lin. PH, Sheu. SC, Chen. CW, Huang. SC, Li. BR, Wearable hydrogel patch with noninvasive, electrochemical glucose sensor for natural sweat detection, TALANTA, vol 241, 2021, https://www.sciencedirect.com/science/article/pii/S0039914021011097 8. Lin. PH, Sheu. SC, Chen. CW, Huang. SC, Li. BR, Wearable hydrogel patch with noninvasive, electrochemical glucose sensor for natural sweat detection, TALANTA, vol 241, 2021, https://www.sciencedirect.com/science/article/pii/S0039914021011097 Ethics approval and consent to participate 3. Lee. H ,Hong. YJ, Baik. S, Hyeon. T ,Kim. DH, Enzyme-Based Glucose Sensor: From Invasive to Wearable Device, ADVANCED HEALTHCARE MATERIALS, vol 7, 2017, https://onlinelibrary.wiley.com/doi/full/10.1002/adhm.201701150 3. Lee. H ,Hong. YJ, Baik. S, Hyeon. T ,Kim. DH, Enzyme-Based Glucose Sensor: From Invasive to Wearable Device, ADVANCED HEALTHCARE MATERIALS, vol 7, 2017, https://onlinelibrary.wiley.com/doi/full/10.1002/adhm.201701150 The data is aquire from study from GDUT and we have their consence of using their data. All methods were carried out in accordance with relevant guidelines and regulations.All experimental protocols were approved by ethics committee of Guangdong University of technology. 4. Yao. Y, Chen. JY, Guo. YH, Lv. T, Chen. ZL, Li. N, Cao. SK, Chen. BD, Chen. T, Integration of interstitial fluid extraction and glucose detection in one device for wearable non-invasive blood glucose sensors, BIOSENSORS & BIOELECTRONICS,vol 179, May 2021, https://www.sciencedirect.com/science/article/abs/pii/S0956566321001159 4. Yao. Y, Chen. JY, Guo. YH, Lv. T, Chen. ZL, Li. N, Cao. SK, Chen. BD, Chen. T, Integration of interstitial fluid extraction and glucose detection in one device for wearable non-invasive blood glucose sensors, BIOSENSORS & BIOELECTRONICS,vol 179, May 2021, https://www.sciencedirect.com/science/article/abs/pii/S0956566321001159 g g y gy Confirming that informed consent was obtained from all subjects . Acknowledgments We thank Guangdong University of technology for funding this work. And LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. 11. Jin. XB, Yang. NX,Wang. XY, Bai. YT, Su. TL, Kong. JL, Deep Hybrid Model Based on EMD with Classification by Frequency Characteristics for Long- Term Air Quality Prediction, MATHEMATICS, vol 8, 2020, Page 9 of 8 Page 9 of 8 https://www.mdpi.com/2227-7390/8/2/214 https://www.mdpi.com/2227-7390/8/2/214 12. Zhou. YR, Li.TY, Shi. JY, Qian. ZJ, A CEEMDAN and XGBOOST-Based Approach to Forecast Crude Oil Prices, IEEE Trans. Cybern, vol 4392785, 2019, https://www.hindawi.com/ journals/ complexity/2019/4392785/ 13. Sherstinsky, A, Fundamental of RNN and LSTM network, PHYSICA D- NONLINEAR PHENOMENA, vol 404, MAR 2020, https://www.sciencedirect.com/science/ article/abs/pii/S0167278919305974 14. Song. XY, Liu. YT , Xue. L, Wang. J, Zhang. JZ, Wang. JQ, Jiang. L ,Cheng. ZY, Time-series well performance prediction based on Long Short-Term Memory (LSTM) neural network model, JOURNAL OF PETROLEUM SCIENCE AND ENGINEERING, vol 186, March 2020, https://www.sciencedirect.com/science/article/pii/S0920410519311039 15. Wang. JQ, Tang. JL, Guo. K , Green Bond Index Prediction Based on CEEMDAN-LSTM, FRONTIERS IN ENERGY RESEARCH , vol 793413, 2022, https://www.frontiersin.org/ articles/ 10.3389/fenrg.2021.793413/full 16. Cao. J, Li. Z, Li. J, Financial time series forecasting model based on CEEMDAN and LSTM, PHYSICA A-STATISTICAL MECHANICS AND ITS APPLICATIONS, vol.519, 2019, https://www.sciencedirect.com/science/article/abs/pii/S0378437118314985 17. Karijadi. I, Chou. SY, A hybrid RF-LSTM based on CEEMDAN for improving the accuracy of building energy consumption prediction, ENERGY AND BUILDINGS, vol 259, March 2022, https://www.sciencedirect.com/science/article/abs/pii/S0378778822000792 18. Wang. MH, Wang. YQ, Prediction of Blood Glucose Concentration Based on EMD denoising and LSTM networks, 2019 CHINESE AUTOMATION CONGRESS (CAC2019), 2019, https://ieeexplore.ieee.org/abstract/document/8996472
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https://zenodo.org/record/2469857/files/article.pdf
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The District Nurse in Coöperative Work
˜The œAmerican journal of nursing/American journal of nursing
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public-domain
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The District Nurse in Cooperative Work.—Jammi 876 vanilla flavoring, and one tablespoonful of sugar. The nurture is then poured into a pint mason-jar with loose cover and set in a saucepan of cold water over a gas-burner for thirty to forty minutes or until the custard begins to thicken. With a mania for adapting and inventing there is a possibility that the private nurse will grow dissatisfied with more modern, up-to-date appliances, but the danger is not great, since there is room and need for both. THE DISTRICT NURSE IN COOPERATIVE WORK* By MARIK R. JAMMlS Graduate of the Johns Hopkins School for Nurses To discuss the district nurse in cooperative work is to approach a subject offering so many possibilities that it is difficult to determine just where to set one’s limitations. Her position in the field of charitable endeavor is peculiarly her own. For the nature of the work has in it a double relationship—that which is purely professional in its relation to acute disease, and that which is social through its constructive and preventive work. Just how this position can be used for mutual helpfulness is to-day a vital problem. In the past few years organized district nursing has developed rapidly and along several lines. There are in different parts of the country independent organizations, district nurses working in connection with City Health Departments in the public schools under the Board of Education, and as special departments of Charity Organi¬ zation Societies. The latter method is the one followed in Minneapolis, therefore I can speak with more assurance of that than of any other. The work there is a separate department of the Associated Charities and is under the direction of the Committee on District Nursing. The com¬ mittee, composed entirely of women, is responsible for raising the neces¬ sary funds and for the general direction of the work. There are three nurses in the field, one of whom devotes all her time to tuberculous patients under the general direction of the Anti-Tuberculosis Committee of the Associated Charities. This plan of work has been found better adapted to the needs of Minneapolis than an independent organization. Naturally there has existed from the first the closest cooperation between the Associated Charities and the district nurses, and as the work grows • Bead at the Conference of Corrections and Charities, Portland, July, 1805. 8^6 The American Journal of Nursing and becomes better known the cooperation with other organizations is most encouraging. Generally speaking, it would seem that the particular form of organization is a matter of minor importance. The most essential thing is to have it adaptable to the particular needs of the community, and the more vital question is, what shall be the nurses’ relation of helpful¬ ness to fellow-workers who are looking at the same problem with just as much interest, if from an entirely different standpoint of view. The gauge of a nurse’s work in the eyes of the medical profession is usually in the technical application of her calling. From the stand¬ point of organized charity, however, we have come to believe that the influence of the work on the social side, plus professional skill, is the important factor. A district nurse enters the home with somewhat of an advantage over the ordinary social worker. She frequently comes at a time when sickness has had more or less of a subduing influence. The kind of service she offers has a tendency to establish immediate confidence. It also creates a sense of dependence which gives an unusual opportunity to get at the hidden springs of family life. In addition to this, her train¬ ing should render her alive to conditions which might escape the social worker—conditions on the physical side of the problem which would have strong bearing upon the social. This is undoubtedly so, as we come more and more to realize that medical treatment is often the foundation-stone in social uplifting. In' many homes there is no actual disease, only a pitiful degree of moral and physical apathy, due largely to unsanitary housing, insufficient food, and a discouraging struggle for livelihood. In these homes the nurse will probably encounter more than one worker. The charity agent may be there, regarding the family from the standpoint of adjustment and relief; the settlement worker, as harboring future citizens; the rental agent, as unprofitable tenants, or the Probation Officer, as subjects for watchfulness. To them the family is largely a social problem. With the visit of the nurse it may still remain a social problem, but becomes possessed of a large medical element. Some slight physical ailment may be at the root of the father’s apathy; the mother may be shiftless because she is too weak to be otherwise, or it may be that the boy or girl is a constant truant because of some visional disturbance that makes school-life un¬ bearable. The uplifting of that family then becomes a question of physi¬ cal cure and prevention, without which the work of the charity agent and others would fail in its ultimate purpose. It is this preventive element in district nursing which offers a The District Nurse in Cooperative Work.—Jamine 877 chance for general helpfulness. The acutely sick will always require the immediate attention of the nurse upon the work. To extend the benefits of their care to an entire family with a view to building up healthy bodies, to know just when and where to apply the ounce of pre¬ vention which will save the future pound of cure, is to give to district nursing the dignity of broad aims and to greatly increase its utility. With this extended point of view will naturally come greater responsibilities, and the question arises, does the nurse entering the work require special qualifications, and is her general training a sufficient guarantee of success? In the June number of The American Journal of Nursing we have a very interesting letter from Mr. John Glenn, of Baltimore, in which he lays before us a condition in social service generally which has a strong bearing upon this question. It is, as he states it, “ The failure on the part of workers to see the whole social point of view, to consider the relationship of a family to the community, and the effect of example on other families in the neighborhood; that persons who have had special training in special lines do not understand what a thorough investigation of conditions means; that in the case of district nurses, they come to the work when they have not been long out of the trainingschool; they have had a fine training, and are full of splendid enthu¬ siasm, but have had little experience in the world. They have not studied the aims and the methods of others, so that when they step out of their own professional sphere their efforts to relieve often give a setback to the efforts of others who have had longer experience.” Fol¬ lowing up this thought, he adds, “ Nurses should be taught to understand what thorough investigation means, its scope and its value, that they might give as much support and sympathy as they can to trained workers.” There is probably among district nurses an intelligent appreciation of this condition as Mr. Glenn reviews it. Just how to provide a remedy that can be made uniform throughout is a matter for liberal discussion. If we accept the broad and liberal interpretation of district nursing, it would Seem that the successful nurse should possess a high standard of qualification. She would need to be a woman broad in education and experience, with a power of observation elastic enough to cover the question as a whole, and possessing a spirit of liberal compromise. The nature of the work will often carry her into the field of other workers, where she will require clear judgment and discernment to keep the line well defined between her work and theirs. She cannot afford to overstep it, for experience proves to us again and again that when a district nurse carries material relief or institutes regular investigations, her influence 878 The American Journal of Nursing in that particular family is greatly hampered. It is difficult to define just where the loss is. It is a something almost intangible, but it becomes keenly apparent to the thoughtful nurse. When the family fully understands that as a nurse she has nothing to give except her profes¬ sional skill and womanly sympathy, they quickly learn to accept her at her own valuation without question of further gain. To stand in this near relation to all branches of social work without intruding upon or compromising her own high standard must necessitate, as Mr. Glenn points out, some knowledge of the work as a whole. Up to this time there has been no definite plan by which nurses could obtain a broad insight into philanthropic aims and methods. What knowledge we possess has been acquired through hard experience and in the few odd hours snatched from a crowded round of duties, and it has proven very inadequate. If district nursing is to assume the dignity of a specialty, could not a certain amount of preliminary social training be required? The suggestion is not a new one; it has been made by our leaders in nursing at some of the recent noted meetings, but made in connection with the plan for a centralized training-school, a plan that will take a long time to mature. In the meantime our own individual need is pressing. It behooves us to meet this need that we may keep abreast with our fellow-workers, who are putting forth every effort to meet it on their side. It does not seem possible to add any more to the curriculum of a general training-school, and, moreover, a nurse usually has no definite idea while in training as to the particular line of work she wishes to follow. There are, however, a number of sources of instruction open to us which could be used for preliminary work. Schools of philanthropy, correspondence courses, volunteer service in charity organization socie¬ ties and in settlements, all offer methods of obtaining training. A preliminary training of this sort would have the advantage of starting a nurse in with at least a substantial theoretical knowledge of what social service involves, and would tend to inspire a still higher appreciation of district nursing and to encourage nurses to stay in the work for longer periods. With this broader knowledge an established principle district nurses will be more than ever in a position to ask of coworkers an equal degree of understanding, not of her professional methods as such, but a reali¬ zation that her work has both a medical and social aspect, that she has definite methods of work, and that her usefulness covers a wide field. Cooperation is, after all, a matter of understanding. There is an abundance of good-will and earnestness. If we can add to these essen- Our Duty in Small Things.—Lucas 878 tial qualities a comprehensive knowledge of one anothers’ methods, there will grow up among us that unity of action which is the strength of all work. OUR DUTY IN SMALL THINGS* By ALICE LUCAS Saiby Gamp, with her ignorance, volubility, and bibulous proclivi¬ ties, is a thing of the past. She was but a type of the old-time nurse who adopted the calling as a makeshift, having tried her hand at every¬ thing else and failed. Following closely upon her heels, in the dayB of our grandmothers, we see the care of the sick relegated to some old woman long since past her usefulness in other spheres. Later, if there happened to be a maiden aunt or indigent relative in the family, they were called upon to perform the offices of nurse, although oftentimes they knew no more of nursing than of Sanscrit. If none of these individuals could be procured, the poor unfortunate was confided to the tender mercies (?) of a hired attendant, generally a woman of the lower class, with no education and less common-sense, who generally looked out for her own comfort rather than that of her patient. But the day was fast drawing near when better times were at hand; and although it took the mighty struggle and agony of the Crimean War to bring before the public the large-hearted sympathy and heroic endeavors of Florence Nightingale, yet it establishes forever among the nations of the civilized world that important adjunct to modem existence—the trained nurse. Glancing back over a period of some forty years, we recall to mind the illiterate, immoral, and intemperate—in fact, thoroughly incom¬ petent in every way—woman who stood in place of our trained nurses of to-day. In contrast we have women of large intelligence, thoroughly trained faculties, and purity of life. Women were found who would subject themselves to strict discipline, severe physical and mental labor, the oftentimes uncongenial duties, that they might gain through ex¬ perience that knowledge that would enable them to become the faithful and competent assistants of the physicians, with whom they must work shoulder to shoulder in their labor of love to uplift and relieve suffering humanity. As the years have passed, the standard of the profession has been constantly advancing. While it is charged against us that many enter it from a love of romanticism, the hope of financial reward, or the * Read at the Graduate Nurses’ Association, Springfield, Mass., December, 1904.
https://openalex.org/W4379057783
https://www.frontiersin.org/articles/10.3389/fpsyt.2023.1179803/pdf
English
null
Behind the scenes of the healthcare COVID-19 pandemic crisis: potential affecting factors of healthcare work sustainability in Romania during 2020–2022
Frontiers in psychiatry
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cc-by
14,194
TYPE  Original Research PUBLISHED  01 June 2023 DOI  10.3389/fpsyt.2023.1179803 TYPE  Original Research PUBLISHED  01 June 2023 DOI  10.3389/fpsyt.2023.1179803 TYPE  Original Research PUBLISHED  01 June 2023 DOI  10.3389/fpsyt.2023.1179803 OPEN ACCESS EDITED BY Samer El Hayek, Erada Center for Treatment and Rehab, United Arab Emirates EDITED BY Samer El Hayek, Erada Center for Treatment and Rehab, United Arab Emirates REVIEWED BY Nicola Mucci, University of Florence, Italy Jerome Visperas Cleofas, De La Salle University, Philippines Agata Ewa Chudzicka-Czupała, SWPS University of Social Sciences and Humanities, Poland *CORRESPONDENCE Cristina Savu cristina.savu@s.unibuc.ro †These authors have contributed equally to this work RECEIVED 04 March 2023 ACCEPTED 02 May 2023 PUBLISHED 01 June 2023 CITATION Savu C, Armaș I, Burcea M and Dobre D (2023) Behind the scenes of the healthcare COVID-19 pandemic crisis: potential affecting factors of healthcare work sustainability in Romania during 2020–2022. Front. Psychiatry 14:1179803. doi: 10.3389/fpsyt.2023.1179803 Erada Center for Treatment and Rehab, United Arab Emirates REVIEWED BY Nicola Mucci, University of Florence, Italy Jerome Visperas Cleofas, De La Salle University, Philippines Agata Ewa Chudzicka-Czupała, SWPS University of Social Sciences and Humanities, Poland *CORRESPONDENCE Cristina Savu cristina.savu@s.unibuc.ro †These authors have contributed equally to this work Cristina Savu 1*†, Iuliana Armaș 1†, Marin Burcea 2† and Daniela Dobre 1† 1 Faculty of Geography, University of Bucharest, Bucharest, Romania, 2 Faculty of Administration and Business, University of Bucharest, Bucharest, Romania Aim: The COVID-19 pandemic represented a great disturbance for medical systems around the world, putting medical personnel on the front lines of the fight against the SARS-Cov2 virus. This fight was particularly impactful in countries with medical systems already facing various challenges, including Romania; where the pandemic unfolded in five waves that severely affected the psychological and physical well-being of medical professionals in terms of overload and continuous exposure to health threats. Against this background, our research aims to identify the mediating role of potential affecting factors of healthcare work sustainability during the change-related uncertainty conditions generated by the COVID 19 crisis. Dynamics and relations of nine carefully selected constructs were tracked along all five pandemic waves in Romania, which span from March 2020 to April 2022. The tested variables and constructs are perception of healthcare workers of their own state of health, their workplace safety, the work–family conflict, the satisfaction of basic needs, the work meaningfulness and work engagement, patient care, pandemic stress and burnout. 1. Introduction effort made through the processes of integration and regulation, which makes possible the optimal use of functional reserves, as well as their restoration during the period when the demand ceases.” At the same time, adaptation facilitates the elimination or change of conditions that create problems; perceiving the control of the meaning of the experiences in such a way as to neutralize their problematic character; keeping the emotional consequences of problems within controllable limits (21, 22). The COVID-19 pandemic caused by the SARS-CoV-2 virus appeared in China in December 2019 and spread rapidly throughout the world (1). The disease recorded 525,467,084 confirmed cases and more than 6,285,171 deaths between March 2020 and May 2022 (2), putting humanity in front of an unprecedented health policy crisis (3). The classic sanitary control measures (i.e., social distancing and wearing a gauze face mask), which were introduced with great difficulty by doctors such as Max C. Starkloff aiming to limit the spread of the Spanish flu in 1918 (4), were complemented in modern times by appropriate new treatments and vaccines, which could trigger possible adaptive behaviors and attitudes of medical personnel towards COVID-19 threats in successive pandemic waves. Kiymaz (23) stated that “our brain and body react physiologically and behaviourally to adapt to a social and physical environment that can put your life in danger” ((23), p  1163). In the process of psychosocial adaptation, the individual tends to achieve a harmony between living conditions and internal or external activity. As this harmony is achieved, the degree of adaptability of the individual increases. Psychosocial adaptation also appears as a means of protecting the individual, with the help of which one relaxes and eliminates internal psychic tension, restlessness, destabilizing states (24).h In Romania, the first case of SARS-CoV-2 infection emerged on February 26, 2020 (5), and by April 2022 the total number of deaths caused by the COVID-19 disease raised to 65,486 (6, 7) (Table 1). Being confronted with periodic increases in the number of infected people, hospitals faced both an overload, and the contamination of the medical staff. Moreover, in certain hospitals, the number of medical personnel was reduced due to redistribution to the areas most affected by COVID (e.g., Marius Nasta Hospital, Bucharest) (8). An immediate consequence was the mental and physical overload of the medical staff remaining active in the source hospital (9). OPEN ACCESS RECEIVED 04 March 2023 ACCEPTED 02 May 2023 PUBLISHED 01 June 2023 CITATION Savu C, Armaș I, Burcea M and Dobre D (2023) Behind the scenes of the healthcare COVID-19 pandemic crisis: potential affecting factors of healthcare work sustainability in Romania during 2020–2022. Savu C, Armaș I, Burcea M and Dobre D (2023) Behind the scenes of the healthcare COVID-19 pandemic crisis: potential affecting factors of healthcare work sustainability in Romania during 2020–2022. Front. Psychiatry 14:1179803. doi: 10.3389/fpsyt.2023.1179803 Front. Psychiatry 14:1179803. doi: 10.3389/fpsyt.2023.1179803 COPYRIGHT © 2023 Savu, Armaș, Burcea and Dobre. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Methods: This cross-sectional study is based on an online snowball sampling of 738 health workers from 27 hospitals. Panel research is limited to a maximum of 61 respondents for two successive waves. The analytical part is built on means comparison of analysed variables between all five pandemic waves and an in- depth model to explain the relationships between the variables. Results: The results indicate statistically significant correlations between the perception of health risks and all selected factors excluding patient care, which seems to be above the own health perception. The factors’ dynamics was followed along all five pandemic waves. The developed model identified that one’s health status satisfaction is a mediator of the family–work conflict and, together, of work engagement. In turn, work engagement plays a significant role in satisfying basic psychological needs and supporting work meaningfulness. Also, work meaningfulness influences the satisfaction of basic psychological needs. Discussion: Health workers with higher levels of positive perceived health are better at managing pandemic stress, burnout effects and work-family imbalances. Adaptive behaviors and attitudes towards COVID-19 pandemic threats could be identified in later pandemic waves due to the progress in terms of medical protocols and procedures. 01 Frontiers in Psychiatry frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 10.3389/fpsyt.2023.1179803 KEYWORDS COVID-19, medical staff, cross-sectional survey, panel study, SEM, health perception, work engagement and meaningfulness 1. Introduction The COVID 19 crisis generated a large amount of change-related uncertainty (25) most dramatically affecting the health professionals put on the front lines of the fight against the SARS-CoV-2 virus. During the COVID 19 pandemic crisis, the medical staff was constantly faced with mighty challenges related to limited resources (26), longer shifts, disturbances in the balance between professional and private life, sleep impairment, and major changes in their working environment (27–29). As changes in the working environment increased dramatically during the COVID 19 crisis, the stress levels also increased. Neuroscience studies ((30), p 384 (31)) show that under increased stress levels, both creativity and the ability to sustain high-level thinking decrease. Stressors have an impact on creative problem-solving skills in difficult situations, so that the ability to multitask is reduced. 2. Research background Our research is embedded in the behavioral adaptability theoretical background at the individual context-specific level (10, 11). Our research is embedded in the behavioral adaptability theoretical background at the individual context-specific level (10, 11). In the last decades, extensively investigations have been conducted by scholars in the field of adaptability at the individual level (12–18). In the last decades, extensively investigations have been conducted by scholars in the field of adaptability at the individual level (12–18). i Doron (19) defines adaptation as a “dynamic process of change, in order to find balance with the environment and assuming the ability to learn.” In the same way, Gorgos (20) believes that adaptation is an “active, dynamic and creative process, which requires a permanent Health workers underwent several dramatical changes in their lifestyle, living with the constant fear of contamination, sleep shortening, behavioral changes (32). As a direct consequence, an increase in mental problems has been measured in an abundance of TABLE 1  The highest daily number of COVID-19 cases per wave in Romania (source Ministry of Internal Affairs and Ministry of Health, 2020–2022). Pandemic waves Variant of SARS-CoV-2 New daily cases People hospitalized People hospitalized in (intensive care units) Deaths Wave 1 (March–May 2020) Alpha 362 – 247 – Wave 2 (September– December 2020) Alpha Beta 10,260 12,133 1,130 171 Wave 3 (March–May 2021) Gamma 6,651 14,165 1,531 237 Wave 4 (September– December 2021) Delta 18,863 20,962 1902 574 Wave 5 (January–March 2022) Omicron 40,018 11,884 1,169 215 02 02 Frontiers in Psychiatry frontiersin.org frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 10.3389/fpsyt.2023.1179803 10.3389/fpsyt.2023.1179803 Participants were recruited from the medical personnel of 27 hospitals in Romania. These professionals were involved in the fight against the pandemic along the five waves, beginning in May 2020 and ending in April 2022. We  applied the on-probability snowball sampling technique (60), sharing the questionnaire online for voluntary participation. Respondents were asked to read and agree to informed consent and the statement regarding the processing of the data collected through the survey. Upon request, additional information was made available for respondents via email. publications investigating the complex ways in which medical personnel were psychologically affected (29, 33). 3.2.2. Work–family conflictl Work–family conflict was measured with the Work Family Conflict Scale proposed by Carlson and Kacmar (61). The scale is based on six dimensions of conflict, resulting from the combination of three forms of work–family conflict (time, strain, and behavior), and two directions of work–family conflict (work-family interference and family-work interference). In the current study, only 6 items of the Work–Family Conflict Scale were used, considering only 2 dimensions of the scale: time based on work-family interference, and strain based on work-family interference. The current study aims to highlight the impact of the activities carried out by medical personnel at work on their family, as well as the state of tension and stress resulting from this interaction (e.g., “Work keeps me away from my family for too much time,” “The time I spend at work does not allow me to participate enough in family activities,” “When I get home from work, I am too tired to participate in family activities,” “It happens that the stress at work also affects me at home, so that I can no longer do what I like or what I enjoy).” All items are scored directly and involved responses on a five-point Likert scale, from 1 (never) to 5 (always). Designed against this background of pandemic induced uncertainty, our study aims to identify potential affecting factors in physicians’ work sustainability during the COVID-19 pandemic in Romania in terms of health workers’ perception-related variables (their own state of health), perceived threat of COVID-19 (pandemic stress), work–family conflict, patient care, work engagement, meaning of and commitment to work, satisfaction of basic psychological needs, as well as psychological and professional burnout of healthcare professionals during the COVID-19 pandemic, at the level of 27 hospitals in Romania. An important attempt is to identify possible relationships between the nine tested constructs and adaptive behaviors and attitudes towards COVID-19 pandemic threats due to the progress of medical protocols, from the way the tested factors fluctuated during the 5 pandemic waves, both at the level of the independent samples and among the subjects in the panel. An in-depth structural model was built to explain the relationships between the selected constructs. 2. Research background In the context of COVID-19 pandemic, the most frequently recorded consequences were anxiety, stress due to overwork, frustration, discrimination, isolation, lack of contact with family members, pressure and exhaustion due to high risk of infection and inadequate protection against contamination, post-traumatic stress disorder, psychological distress and depression, sleep disorders, and fear (28, 34–49). According to other studies, during COVID-19, the possibility of infecting family and friends traumatized medical workers (50) and diminished the level of their psychological well-being that contributes to safety, happiness, satisfaction and increased work performance (51). 3.2. Measures Therefore, the burnout increased by 25–30% compared to the period before the pandemic (52, 53), and the most prominent sources of burnout were the cumulative work tasks, uncertainties caused by the pandemic, work-family imbalance and strained relationships at work (54). Taylor (49) mentioned, referring to a study on natural disasters and making a correlation with the COVID 19 pandemic crisis, that 10% of people who have gone through traumatic events, in the context of increased emotional stress and social problems, can immediately or later develop severe psychological problems, such as post-traumatic stress disorders, anxiety disorders, restlessness, sleep disorders, mood disorders. Frontiers in Psychiatry 3.2.1. Risk perceptionh The questionnaire investigated the perception of various risks to which participants are exposed, the work–family conflict, the satisfaction of basic needs, the work meaningfulness and work engagement, patient care, pandemic stress and burnout. This section describes the operationalisation of these aspects. The measuring of risk perception focused on the level of perceived state of health, perceived safety versus workplace insecurity, the perceived level of danger, and the existence of an “unhealthy” environment (e.g., with risk of injury, death, health damage) during the SARS-CoV-2 pandemic. An example of an item is: “Describe your workplace in relation to the crisis generated by the Coronavirus pandemic: Dangerous, Safe, Risky, Unhealthy, Uncertain, Risk of death.” Answers were scored on a 5-point Likert scale, from 1 (strongly disagree) to 5 (strongly agree). On the other hand, support, job satisfaction, and an improvement in the self-esteem of medical personnel, were listed as protective factors against burnout (55), showing that the pandemic had serious implications for patient care and job satisfaction (56). The association between the doctor’s burnout scale with work involvement and the quality of care given to patients is important for highlighting the general efficiency of medical service providers (57–59). 3.2.7. Pandemic stress Pandemic stress was measured with the corresponding Stanford Acute Stress Reaction Questionnaire – SASRQ, which was developed and validated by Cardena et al. (67) to assess psychological symptoms experienced following a traumatic episode. The SASRQ instrument investigates dissociation (e.g., subjective feeling of numbness, detachment and lack of emotional responsiveness, reduced awareness of the environment, derealization, depersonalization, and dissociative amnesia - 10 items), reexperiencing trauma (6 items), avoidance (6 items), hyper anxiety (6 items), anxiety and impairment of functioning (2 items). The questionnaire was initially applied in the context of natural disasters (floods), but we  adapted it to fit the context of traumatic episodes related to the COVID-19 pandemic crisis. All the items are directly scored, on a 6-point Likert scale, from 0 (“I have not experienced/experienced the respective condition.”) to 5 (“I have experienced the respective condition very often.”). This scale was introduced in the research design starting with the second pandemic wave, to measure if new waves lead to an increase in the stress felt by medical professionals. The final stage of data collection unfolded between January and March 2022: 138 healthcare workers completed the survey and 31 were panel respondents (i.e., subjects who repeated in waves 4 and 5, respectively 45.58% of respondents from the fourth wave, and 22.46% from the fifth wave). At large, the number of people who participated in the survey in at least two successive pandemic waves (panel participants) reached 61 respondents (respectively 8.26%). 3.4. Analytics Analytical procedures were computed using the IBM® SPSS® Statistics and Jamovi v1.6.23 softwares (71). The computation procedures focused on absolute and relative frequency, means, standard deviations, and normality indicators (i.e., skewness and kurtosis). We calculated the bivariate Pearson correlations (r) across the study variables, following Cohen’s (72) benchmarks for interpretation: weak correlation (r  < 0.3), moderate correlation (0.3 < r < 0.55), strong correlations (r > 0.5). 2.4. Basic psychological needs satisfaction cynicism, and inadequacy, including feelings of overwhelming emotional exhaustion, feelings of cynicism and detachment from the workplace defined as depersonalization, and a sense of ineffectiveness and reduced personal fulfilment). Each subscale has 3 directly scored items, measured on a 7-point Likert scale, from 0 (never) to 6 (daily). This scale was introduced in the research design starting with the third pandemic wave, to test whether the persistence of the pandemic waves leads to various degrees of professional exhaustion. Basic needs satisfaction was measured using the Basic Psychological Needs Satisfaction Scale and the Frustration Scale (64). The three subscales focus on autonomy (e.g., “At work, I feel a sense of choice and freedom in the things I undertake”), competence (e.g., “I feel confident that I can do things well at work”), and relatedness/ referring to the extent to which a person feels connected with and valued by others (e.g., “I feel that people who care at work care about me too”). Each of the three subscales was assessed via satisfaction questionnaires with 4 directly scored items on a seven-point Likert scale from 1 (strongly disagree) to 7 (strongly agree). Thus, participants report the extent to which their three basic needs were met in the last weeks at work. We tested the reliability of the structure by assessing the internal consistency of all applied scales using Cronbach’s alpha. The Cronbach’s alpha of each construct was higher than the recommended level of 0.7. 3.2.5. Work meaningfulness Work meaningfulness was measured with the Work inventory and the Meaningful work scale (65), which introduces three components of meaning at work: positive meaning (e.g., “I have a good sense of what makes my work meaningful”), gaining meaning through work (e.g., “My work helps me understand myself better”) and better motivations (e.g., “I know my work makes a positive difference in the world.”). The directly scored items required answers on a five-point Likert scale, from 1 (absolutely not true) to 5 (absolutely true). The data was gathered online during the five pandemic waves using Google Forms for each of the corresponding five rounds, and mainly came from Bucharest (425) and the neighbouring counties: Ialomița (152), followed by Brăila (77), Galați (10), but also from other counties such as Bacău (6), Teleorman (6), Constanța (5), Tulcea (5), Sibiu (5), Călărași (5), Giurgiu (4), Argeș (3), Bihor (2), Neamț (2), Vaslui (2), Olt (2), Brașov (2), Timiș (2) (Figure 1). The first survey took place between March and May 2020, with a total of 216 healthcare workers. The second round of data collection unfolded in September 2020–January 2021, with 121 respondents, of which 18 subjects were on the panel (i.e., those who took part in the survey in two successive pandemic waves, namely waves 1 and 2). 3.2.6. Patient care Patient care was measured with the Patient Care scale (66), an 8-item scale that investigates suboptimal patient care practices (five items, e.g., “We did not fully discuss treatment options.”) and patient care attitudes (three items, e.g., “We paid little attention to the social or personal aspects of the illness impact.”). All the items were scored directly, on a 5-point Likert scale, from 1 (never) to 5 (weekly). The third survey took place from February to May 2021, focusing on 195 respondents, with only 6 participants in the panel between waves 2 and 3 (i.e.,9.37% of wave 2, and 3.09% of the third wave). The third survey took place from February to May 2021, focusing on 195 respondents, with only 6 participants in the panel between waves 2 and 3 (i.e.,9.37% of wave 2, and 3.09% of the third wave). The fourth round of data collection took place between September and December 2021, and 68 healthcare workers completed the survey, of whom 6 were included in the corresponding panel (i.e., those who repeated in waves 3 and 4). They accounted for 3.09% respondents from the third wave, and 8.82% of respondents from the fourth wave. Frontiers in Psychiatry 3.1. General approach Work engagement was measured with the short version of the Utrecht Work Engagement Scale (UWES-9) (62, 63), which refers to three factors of work engagement, with three items each: vigor (e.g., “When I  wake up in the morning, I  feel like going to work.”), dedication (e.g.: “I am proud of the work I do”), and absorption (e.g., “I am fully involved when I work”). All the items are scored directly, on a 7-point Likert scale, from 0 (never) to 6 (always). This study undertakes a cross-sectional approach, applying repeated surveys with different respondents in each wave on a total of 738 subjects. This is complemented by a panel-oriented approach (participants who responded in two successive pandemic waves), totalling a maximum of 61 respondents per successive waves. 03 frontiersin.org 10.3389/fpsyt.2023.1179803 10.3389/fpsyt.2023.1179803 Savu et al. frontiersin.org Frontiers in Psychiatry 3.2.8. Burnouth The Burnout Scale [22-item MBI - Maslach Burnout Inventory (68)] was originally developed to measure burnout as a specific type of response to occupational stress among human service professionals. We  used the 9-item short version of the Maslach Burnout Measurement Inventory (69, 70), with three subscales (exhaustion, The analyses relied on two non-parametric statistical hypothesis tests. The Mann–Whitney U test was applied to explore perception differences at the level of cross-sectional samples during the five 04 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 FIGURE 1 Geographical distribution of respondents. FIGURE 1 Geographical distribution of respondents. pandemic waves. The Wilcoxon signed rank test was applied for the comparative analysis of health status perception of medical staff participating in two consecutive waves. variables with mean = 0 and standard deviation = 1). The fit of the model to the field data was measured using the chi-square test (78), the comparative Tucker-Lewis Index [TLI; (79)], the comparative fit index [CFI; (80, 81)], the root mean square error of approximation related to the residual in the model [RMSEA; (82)], and the standardized root mean squared residual (SRMR; 75, 76, 83). Using nonexperimental data, causal relationships were examined with path analysis using the Jamovi open software. We developed a model of hypothesized causes in order to test the coping role during the COVID-19 pandemic of health workers’ perception regarding their own state of health in relation with work engagement, family- work relations, basic psychological needs satisfaction, and work meaningfulness. The constructs and their indicators were specified and estimated, followed by structural relationships in the model, which were obtained using the same steps. Further on, the hypothesized model of relations was statistically tested to determine the extent to which it was consistent with the data. We first applied the path analysis to test the initial hypothesized model. Next, the latent variable structural equation modelling - SEM was applied to accurately identify the relationships in the system. frontiersin.org 4.1. Sample The 738 completed questionnaires that resulted from the data collection phase showed that the sample is dominated by women (74.5% respondents, mirroring the predominantly female structure of the health personnel in Romania, with 70.5% women among doctors in 2020). Participants with ages of 40–49 years totalled 42.68% of the sample. Most of the medical staff graduated from post-secondary schools (46.61%), and only a third from college (29.40%). The other study levels were represented as follows: postgraduate studies (14.76%), secondary education (7.99). These numbers also indicate the age structure of specific occupations in the medical sector: respondents aged up to 20–29 years mainly have secondary education (post-secondary school); 10.70%, having the lowest average period of employment 0–1 year (20–29 years) (Table 2). The SEM comprises both a measurement model and a structural model. It was used to analyse the relationships between observed variables (derived directly from measurements) and latent variables (constructs that can be measured indirectly by determining their influence on the responses of the measured variables) (73–77). The applied methodological steps were: (1) model identification, (2) parameter estimation, (3) model-fitting, (4) model redefinition, and (5) interpretation of results. In the case of SEM, we followed the five step-model specification, firstly defining the independent and dependent variables; and continuing with the same next steps. We used standardized coefficients to increase comparability and to make inferences regarding the strength of identified relationships (i.e., Respondents aged 40–49 years have the highest share (24.12%) in the category of post-secondary school. The lowest weight of those with secondary school education (2.43%) is in the age category 50–59 years. Results underline that the positions that do not require specialized 05 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 TABLE 2  Frequency distribution of socio-demographic characteristics on research waves. TABLE 2  Frequency distribution of socio-demographic characteristics on research waves. Frontiers in Psychiatry 4.2. Descriptive statistics Overall, the distribution of answers by pandemic waves is relatively balanced, but there are also particularities that need to be considered: medical assistants provided the most answers in the 4th pandemic wave (75%), and doctors in the 2nd wave (23.20%).h 4.3. Inferential results Medical doctors represented only 1.47% of the participants who completed the survey during the fourth wave, they were best represented in the second wave (23.20%) (Table 2). Inferential analysis tested the importance of one’s own state of health in relation to all selected factors and the dynamics of these variables during the five pandemic waves, both at the level of independent samples and in the panel study, to identify possible adaptive behaviors and attitudes towards the COVID-19 pandemic threats. The path analysis and the latent variable structural equation modelling - SEM were applied to explain the relationships between the selected constructs. Regarding seniority at work, the most numerous were people with seniority >10 years, followed by seniority of 3–5 years, most of them being medical assistants, followed by doctors. 4.1. Sample Variables Total (738) Wave 1 (216) Wave 2 (121) Wave 3 (195) Wave 4 (68) Wave 5 (138) Gender M 25.61 30.60 29.00 24.10 25.00 21.74 F 74.39 69.40 92.00 75.90 75.00 78.26 Education Secondary education 7.99 15.30 8.20 3.60 2.94 5.07 Post-secondary studies 46.61 42.60 43.00 62.10 48.52 39.85 University studies 29.40 28.70 31.40 25.10 33.82 32.60 Postgraduate studies 14.76 13.40 17.40 9.20 14.70 22.46 Position Caregiver, nurse, stretcher bearer, registrar 21.54 9.40 9.10 8.10 23.53 28.26 Medical assistance, paramedic 73.57 71.76 67.76 74.36 75 63.04 Doctor 5.82 19.00 23.20 5.60 1.47 2.90 Age 20–29 years old 10.70 10.70 9.10 7.10 8.82 18.11 30–39 years old 26.83 30.10 45.50 15.10 17.64 26.81 40–49 years old 42.68 44.10 37.30 49.40 51.47 32.20 50–59 years old 18.43 14.40 8.30 27.80 17.64 21.74 >60 years old 1.35 1.00 – 1.00 2.94 1.44 Professional experience 0–1 years 5.01 4.20 2.50 5.60 2.94 8.69 1–3 years 10.56 9.30 18.20 8.70 5.88 10.87 3–5 years 17.47 14.81 25.60 16.49 13.23 18.11 >10 years 66.93 71.80 53.70 69.20 77.94 62.32 perception of the workplace achieved higher values in waves 2 and 3. Patient care has higher values in the first two waves, and professional burnout has higher values towards the end of the pandemic, during waves 4 and 5. On the other hand, stress associated with COVID surged during the middle of the pandemic, in waves 2 and 4. studies (unqualified) in the medical system are occupied by young people, who are in the early stages of employment, look out for new job opportunities and are less likely to fill their current position for long periods. The best represented professional categories in this study were nurses (68.29%) and doctors (18.56%), the rest of 13.13% being paramedics, stretcher bearers, and registrars. However, these percentages change from one pandemic wave to another. frontiersin.org 4.3.2. Dynamics of health perception strength) between the perception of one’s state of health and almost all other variables, with one exception (Table 4). Patient care seems to be above the own health perception, but it is significantly, moderately negatively correlated with stress reaction, and significantly, weakly negatively correlated with burnout (Table 4). An important task of our research was to capture the dynamics of health perception (“How satisfied are you with your state of health?”) during the 5 pandemic waves, both at the level of independent samples (based on the Mann–Whitney U test) and in the panel study (via the Wilcoxon signed rank test). Health workers with higher levels of positive perceived health are better at managing pandemic stress (−0.328; p = 0.001) and burnout effects (−0.265; p = 0.001). In the same acceptance, a positive health perception balances family-work conflicts (−0.283; p = 0.001) and negative workplace engagements (−0.163; p  = 0.001). Work engagement is also negatively correlated with burnout (−0.453; p = 0.001) and pandemic stress (−0.282; p = 0.001). Results summarized in Table 5 (the value of p associated with the Mann–Whitney U test is listed) show statistically significant differences in terms of health assessment scores between the first wave and the other four waves, and between wave 3 and the next two ones, as described below. Certain adaptive behaviors and attitudes towards the COVID-19 pandemic threats are visible during waves 2, 4 and 5 (Mann–Whitney U test p < 0.05). Respondents acknowledge that they have coped better with latter pandemic waves due to the progress in terms of medical protocols and procedures. At the beginning of the pandemic there was some lack of confidence and a high degree of scepticism regarding the perception of one’s own health state. Also, waves 1 and 3 are significantly different from waves 4 and 5 (Table 5A). A decrease in the satisfaction level regarding the state of health can be observed during the last two pandemic waves, compared to the onset of the COVID-19 pandemic (wave 1). However, it should be highlighted that the maximum values of health state declared in the self-assessment at the beginning of the pandemic may be  the result of a cognitive dissonance effect, which is a psychosocial phenomenon of denying a possible personal vulnerability (84). The perception of one’s own state of health fluctuates from wave to wave, maintaining lower values than in the initial state. Frontiers in Psychiatry 4.3.1. Health perceptionh The fundamental hypothesis is that, in the context of the COVID-19 pandemic, perception of one’s own state of health is pivotal for the perception of all other considered variables: the perception of the specific risks at work, the felt state of pandemic stress, satisfaction of basic psychological needs, meaning of and commitment to work, patient care, but also of the conflict between work and the family life.hi The descriptive analysis (Table 3) shows that the highest values in terms of the mean of work - family conflict scale were recorded during the 2nd and 4th pandemic waves, while the means regarding the satisfaction of basic psychological needs, the perception of the personal state of health, meaning of and commitment to work have higher values in waves 1 and 3. Workplace engagement and the This set of working hypotheses are supported by significant direct and indirect proportional correlations (with various degrees of 06 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 TABLE 3  Descriptive analysis on research waves. Scale (no respondents) Total mean (SD) Wave 1 mean (SD) 216 Wave 2 mean (SD) 121 Wave 3 mean (SD)195 Wave 4 mean (SD)68 Wave 5 mean (SD) 138 Perception of the personal state of health (738) 7.57 (1.69) 8.07(1.28) 7.09 (1.93) 7.64 (1.44) 6.75 (2.08) 6.97 (1.88) The perception of the workplace (738) 34.67 (7.12) 30.7 (6.92) 33.0 (6.42) 32.9 (6.30) 33.3 (5.79) 30.5 (6.61) Work–family conflict scale (738) 21.67 (6.09) 19.8 (6.47) 22.2 (5.69) 22.5 (5.72) 22.9 (5.78) 22.4 (5.96) Workplace engagement (738) 42.26 (8.60) 4.36 (8.52) 41.8 (8.24) 43.1 (8.28) 40.0 (8.33) 40.5 (9.18) Scale of satisfaction of basic psychological needs (738) 66.78 (12.72) 68.7 (12.7) 65.9 (13.5) 67.5 (11.4) 65.1 (12.5) 64.3 (13.5) The work and meaningful inventory (738) 42.01 (5.79) 42.4 (5.66) 41.9 (6.24) 42.4 (5.45) 41.3 (5.64) 41.3 (6.13) Patient care (591) 21.11 (5.55) 19.8 (8.71) 20.4 (8.89) 18.5 (9.09) 17.0 (9.74) 10.7 (6.04) Stanford acute stress reaction questionnaire (555) 53.67 (36.94) 7.0 (22.1) 55.4 (38.2) 48.2 (33.5) 62.5 (39.1) 57.5 (38.1) Maslach burnout inventory (415) 21.28 (12.92) – 2.18 (6.68) 19.1 (12.6) 24.2 (12.9) 23.2 (13.2) frontiersin.org 4.3.2. Dynamics of health perception Thus, although the medical personnel was confronted with a shortage of knowledge on adequate mitigation procedures and treatment protocols during the first pandemic wave, they displayed a compensatory overconfidence in their own state of health (85), which may have helped the fight against the virus. The lower values related to one’s own health state recorded during the 2nd Perceived health and work engagement are positively correlated with the satisfaction of basic psychological needs (0.271, p = 0.001 and 0.690; p = 0.001, Table 4). The perception of workplace highly positively correlates with the work versus family conflict, drawing attention to the fact that the workplace fulfils the function of a second family (0.501; 0.0001). Intuitively, the meaning of work negatively correlates with the effects of burnout (−0.335; p = 0.001). These results are best expressed among doctors, who display high levels of meaningful work and work engagement. Such positive attitudes towards work are shown by the following percentages: 50.73% of responders are highly involved in their work, 36.02% being excited when they work, 30.88% state that they want to go to work when they wake up in the morning, 28.67% are happy when they work intensively, and 11.76% of the doctors answered that they are full of energy at work. In terms of work significance, 59.55% of the medical personnel declared that they have a career full of significance, 57.35% of doctors stated that their work has a positive impact in the world. Also 50.73% know the significance of their work and 48.52% recognize the contribution of their work to the meaning of life. 4.3.2. Dynamics of health perception In the fourth wave, they already adapted to the pandemic conditions, believing that their health will not be severely impacted by the SARS CoV-2 virus. The perception of the state of health and the perceived danger at work during the pandemic was tested using the Mann–Whitney U test. The results show that there are significant differences (p < 0.05) in the scores obtained for these variables between the following waves: wave 1 and waves 2, 3; wave 2 and wave 5; wave 3 and wave 5 (Table 6A). The mean values show that the perception of workplace safety against the effects of the pandemic in waves 1 and 5 was lower than in waves 2, 3, 4. The transition from the COVID-19 Alpha variants specific to the first wave, to the Omicron variant characteristic to the last wave had a significant impact on health workers. Nonetheless, at the beginning of the pandemic, COVID-19 was something new and quite dangerous, causing the medical staff to doubt the safety of their workplace. The measures mandated to combat the pandemic were also very strict during the first wave (lockdown), whereas the Omicron variant of the virus, which emerged at the end of the pandemic, was perceived more as an easy flu, meaning that the perception of the danger to one’s health at work decreased. In waves 2, 3, and 4, the COVID-19 isolation measures were no longer very strict in Romania, which led to higher levels of health-related self-assessment. The Wilcoxon signed rank test recorded statistically significant differences between responses regarding the workplace perception of people in the panel between wave 1 and 4 (Table 6B), in the sense that the perception of dangerousness at work decreases in wave 4 compared to wave 1. These findings could be related to the new, less aggressive variants of SARS-CoV-2, but also to the advancement of knowledge, increased treatment capacities, and more effective measures to mitigate the pandemic (85, 86). 4.3.2. Dynamics of health perception 07 frontiersin.org Work– amily onflict scale Workplace engagement Scale of satisfaction of basic psychological needs The work and meaning inventory Patient care Stanford acute stress reaction questionnaire Maslach burnout inventory −0.283** 0.298** 0.271** 0.196** −0.328** −0.265** 0.000 0.000 0.000 0.000 0.117 0.000 0.000 525 525 525 525 416 384 304 0.501** −0.155** −0.139** −0.004 0.107* 0.314** 0.294** 0.000 0.000 0.001 0.921 0.03 0.000 0.000 525 525 525 525 416 384 304 1 −0.265** −0.239** −0.005 −0.054 0.370** 0.423** 0.000 0.000 0.250 0.274 0.000 0.000 525 525 525 416 384 304 1 0.690** 0.643** 0.086 −0.281** −0.453** 0.000 0.000 0.080 0.000 0.000 525 525 416 384 304 1 0.610** 0.108* −0.258** −0.299** 0.000 0.028 0.000 0.000 525 416 384 304 1 0.058 −0.168** −0.335** 0.238 0.001 0.000 416 384 304 1 −0.022 −0.105 0.694 0.105 309 241 (Continued) 10.3389/fpsyt.2023.1179803 10.3389/fpsyt.2023.1179803 Savu et al. Correlations How satisfied are you with your state of health? The perception of the work place Work– family conflict scale Workplace engagement Scale of satisfaction of basic psychological needs The work and meaning inventory Patient care Stanford acute stress reaction questionnaire Maslach burnout inventory Stan ford acute stress reaction questionnaire Pearson Correlation 1 0.559** Sig. (2-tailed) 0.000 N 304 Maslach burnout inventory Pearson Correlation 1 Sig. (2-tailed) 0.000 N 304 Correlations How satisfied are you with your state of health? The perception of the work place Work– family conflict scale Workplace engagement Scale of satisfaction of basic psychological needs The work and meaning inventory Patient care Stanford acute stress reaction questionnaire Maslach burnout inventory Stan ford acute stress reaction questionnaire Pearson Correlation 1 0.559** Sig. (2-tailed) 0.000 N 304 Maslach burnout inventory Pearson Correlation 1 Sig. (2-tailed) 0.000 N 304 wave indicate a delay in these concerns, which increased in the next wave, on the grounds that experience gained in previous waves makes us more confident in preventing disease. Lower health state-related values correspond to waves 4 and 5, suggesting an adaptation to the situation, reflected by a decrease in the concern for one’s own health. The results of the Wilcoxon signed rank test, show that statistically significant differences were recorded between the answers collected in the panel during waves 1, 2 and 4 (Table 5B). These findings are explained by the fact that people were more scared at the very beginning of the pandemic, having the feeling that their health would be seriously affected. 4.3.2. Dynamics of health perception If we compare waves 1 and 4 to waves 2, 3 and 5, we can identify a sharpening of perception regarding the state of health, but also concerns about “how risky is the workplace in the context of the pandemic” and “whenever unforeseen events can occur.” Also, between wave 1 and wave 4, it appears that the respondents in the panel sharpen their perception of job security, which may come as a result of an adaptation processes. Frontiers in Psychiatry 4.3.3. Work–family conflict, work engagement and significance, psychological needs, patient care, pandemic stress and burnout dynamicsl Another research aim was to measure the dynamics of conflict between professional and personal life, work engagement and commitment, work significance, satisfying basic psychological needs, patient care, pandemic stress and burnout during the 5 pandemic waves, both at the level of independent samples (based on the Mann– Whitney U test) and in the panel study (via the Wilcoxon signed rank test). TABLE 4  (Continued) Frontiers in Psychiatry 09 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 TABLE 5  Perception of one’s own health state. TABLE 5  Perception of one’s own health state. A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.000 0.000 0.000 0.183 Wave 1 0.039 0.378 0.009 NA Wave 2 0.506 0.092 0.027 Wave 2 1,000 0.402 NA Wave 3 0.024 0.027 Wave 3 0.138 NA Wave 4 0.438 Wave 4 NA Wave 5 Wave 5ffhi ndemic waves B. Different consecutive waves (panel samples) B. Different consecutive waves (panel samples) A. Different pandemic waves (A) Different pandemic waves; (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). *NA, no answer. The italic values are statistically significant (p<0.05). A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.015 0.002 0.058 0.970 Wave 1 0.084 0.529 0.018 0.313 Wave 2 0.970 0.925 0.024 Wave 2 0.262 0.937 0.698 Wave 3 0.829 0.005 Wave 3 0.919 0.250 Wave 4 0.060 Wave 4 0.499 Wave 5 Wave 5ffhi Different pandemic waves B. Different consecutive waves (panel samples) A. Different pandemic waves TABLE 7  The work versus family conflict scale. A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.015 0.002 0.058 0.000 Wave 1 0.569 0.889 0.352 0.522 Wave 2 0.970 0.925 0.024 Wave 2 0.107 0.724 0.844 Wave 3 0.829 0.005 Wave 3 0.755 0.625 Wave 4 0.060 Wave 4 0.213 Wave 5 Wave 5 (A) Different pandemic waves; (B) different consecutive waves (panel samples-Wilcoxon signed rank test, value of p < 0.05). The italic values are statistically significant (p<0.05). A) Different pandemic waves; (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). *NA, no answer. The italic values are statisti 4.3.3. Work–family conflict, work engagement and significance, psychological needs, patient care, pandemic stress and burnout dynamicsl Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.008 0.218 0.010 0.002 Wave 1 0.343 0.414 0.407 0.820 Wave 2 0.061 0.394 0.506 Wave 2 0.800 0.388 0.944 Wave 3 0.034 0.013 Wave 3 0.813 0.875 Wave 4 0.849 Wave 4 0.962 Wave 5 Wave 5ffhi erent pandemic waves B. Different consecutive waves (panel samples) B. Different consecutive waves (panel samples) A. Different pandemic waves A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.522 0.998 0.213 0.026 Wave 1 0.464 0.432 0.585 0.850 Wave 2 0.553 0.522 0.169 Wave 2 0.866 0.700 0.925 Wave 3 0.131 0.014 Wave 3 0.058 0.423 Wave 4 0.628 Wave 4 0.345 Wave 5 Wave 5 (A) ff d ( ) d ff ( l l d h l d k ) Th l l ll fi ( ) A. Different pandemic waves TABLE 10  Perception of satisfying basic psychological needs. A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.032 0.010 0.107 0.000 Wave 1 0.347 0.393 0.733 0.183 Wave 2 0.979 0.878 0.070 Wave 2 0.272 0.272 0.147 Wave 3 0.822 0.015 Wave 3 0.528 0.625 Wave 4 0.156 Wave 4 0.162 Wave 5 Wave 5 (A) Different pandemic waves; (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). The italic values are statistically significant (p<0.05). A. Different pandemic waves When it comes to work meaningfulness, the Wilcoxon signed rank test does not show statistically significant differences between the answers given by the same people in different pandemic waves. Thus, the meaning of work remains relatively constant for the same person, at least in 2 consecutive waves (Table 9B). The Wilcoxon signed rank test indicates no statistically significant differences in terms of scores related to work commitment for the same person from one pandemic wave to another (Table 8B). 4.3.3. Work–family conflict, work engagement and significance, psychological needs, patient care, pandemic stress and burnout dynamicsl erent pandemic waves B. Different consecutive waves (panel samples) B. Different consecutive waves (panel samples) The dynamics of the conflict between work and personal life during different pandemic waves revealed statistically significant differences (p < 0.05) between wave 1 and waves 2, 3 and 5 (Table 7A). The balance between work and family was seriously affected during the first pandemic wave, a fact that can be  explained by the involvement of all available resources in the battle against an unknown impactful virus. The conflict considerably diminished in the fifth wave, and as a result of the “habit” effect. Starting with the 2nd and 3rd waves, the pandemic limitation measures relaxed (87), increasing the contamination risk of the population and causing additional pressure on the medical system. As an immediate consequence, health workers were affected, and their work conditions were subject to various risks. This explains the statistically significant differences recorded between these waves and the last one, during which the Omicron variant no longer raised major problems for the health system.h family life and the time spent at work between people who responded in two consecutive pandemic waves (Table 7B). In order to take an in-depth look on the role of profession in the life of respondents, we used the Utrecht Work Engagement Scale (UWES). One can observe that the well-being and the pleasure of going to work decreased during the last two pandemic waves, compared to first wave, and also that wave 1 is different from waves 2, 4 and 5 in this regard (Table 8A). The Mann–Whitney U test also shows statistically significant differences in terms of work engagement. Work commitment increases with positive perception of one’s health, work safety, and the satisfaction of basic psychological needs, as well as with the efficiency of medical protocols. Thus, the raising numbers of successfully treated patients fostered positive and optimistic attitudes towards dealing with the pandemic. Also, diminishing fear and anxiety associated with possible illness/infection with SARS-CoV-2, brought about consistent increases in work engagement; a trend which is observable from one pandemic wave to another. The results of the Wilcoxon signed rank test suggest that there are no statistically significant differences regarding the conflict between 10 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 TABLE 8  The role of profession in the life of respondents. TABLE 8  The role of profession in the life of respondents. A. Different pandemic waves B. Frontiers in Psychiatry frontiersin.org fferent pandemic waves; (B) different consecutive waves (panel samples-Wilcoxon signed rank test, p < 0.05). The italic values are statistically significant (p<0.05). 4.3.3. Work–family conflict, work engagement and significance, psychological needs, patient care, pandemic stress and burnout dynamicsl Different consecutive waves (panel samples) B. Different consecutive waves (panel samples) A. Different pandemic waves (A) Different pandemic waves; (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). The italic values are statistically significant (p<0.05). A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.089 0.191 0.077 0.055 Wave 1 0.813 0.000 0.462 0.813 Wave 2 0.319 0.469 0.568 Wave 2 0.641 0.701 0.570 Wave 3 0.211 0.097 Wave 3 0.724 0.713 Wave 4 0.696 Wave 4 0.794 Wave 5 Wave 5 (A) Different pandemic waves, (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). A. Different pandemic waves (A) Different pandemic waves, (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). not show statistically significant differences in the satisfaction of basic psychological needs between responses provided by the same people in successive pandemic waves (Table 10B). of scores were obtained when assessing the state of stress across pandemic waves (Table 12A), although average scores recorded in wave 3 (wave 3 = 47.32) are much lower than those recorded in wave 2 (wave 2 = 61.96). Regarding patient care, the Mann–Whitney U test shows statistically significant differences (p < 0.05) between waves 1 and 2 compared to waves 4 and 5. Also, the fourth wave stands out when compared to waves 1, 2, 3 and 5 (Table 11A). The Wilcoxon signed rank test does not show statistically significant differences between participants’ responses specific to different pandemic waves (Table 12B). Average scores (wave 1 = 23.43, wave 4 = 22.21, wave 5 = 12.05) show statistically significant differences, indicating that the basic psychological needs were satisfied by adapting to the new pandemic- related working conditions. The Maslach Burnout Inventory scale was introduced starting with wave 3. The results of the Mann–Whitney U test point out statistically significant differences (p < 0.05) between wave 3 and waves 4, 5 and also between waves 2 and 3 (Table 13A). The mean scores recorded during certain pandemic waves (wave 3 = 17.10, wave 4 = 23.67, wave 5 = 22.49) show a stronger increase in burnout cases towards the end of the COVID-19 pandemic. Frontiers in Psychiatry 4.3.3. Work–family conflict, work engagement and significance, psychological needs, patient care, pandemic stress and burnout dynamicsl The Wilcoxon signed rank test shows statistically significant differences between waves 3 and 4 (Table 13B), meaning that professional burnout is prevalent in wave 4, compared to wave 3 (wave 4 = 22.36, wave 3 = 15.18). The Wilcoxon signed rank test also shows statistically significant differences between wave 5 and waves 1, 2 and 4 (Table 11B). Panel respondents report the same level of competence when caring for patients, across the pandemic waves. However, participants from the panel samples in wave 5 considered that the medical care provided during the last pandemic wave was of a higher level than the one specific to waves 1, 2 and 4, as shown by the following mean values: wave 1 versus wave 5: 23.92 versus 11.78; wave 2 versus wave 5: 23.63 versus 11.45; wave 4 versus wave 5: 23.88 versus 11.11.h frontiersin.org 4.3.3. Work–family conflict, work engagement and significance, psychological needs, patient care, pandemic stress and burnout dynamicsl During the COVID-19 pandemic, work had a special significance in the lives of the respondents, as shown by the positive correlation between work significance and work commitment, and between the former and the satisfaction of basic psychological needs. Most health workers feel full of energy at work (52%), proud of the work they do (59.5%) and involved in their daily activities (65.5%). The respondents in our study consider the work carried out during the pandemic as meaningful, with a positive impact on those around them (69.6% of all respondents), which gives them strength and inspire them to deal with difficult situations. A career in this field of work and the professional satisfaction of healing patients significantly contributes to the fulfilment of a meaningful personal life (e.g., “I found a profession whose purpose brings me satisfaction,” 66% answering with a maximum score).hif The Mann–Whitney U test shows statistically significant differences between the first and last pandemic waves, and between waves 3 and 5 (Table  9A). The perception of work significance increased towards the end of the pandemic (in wave 5) compared to waves 1 and 3, possibly in the optimistic context created by increasing healing rates. The Mann–Whitney U test emphasises significant differences (p < 0.05) regarding the item of satisfying basic psychological needs between wave 1 and waves 2, 3 and 5; and waves 3 and 5 (Table 10A). This means that basic psychological needs were fulfilled towards the end of the pandemic, rather than in the initial (wave 1) or middle (wave 3) stages. On the contrary, the Wilcoxon signed rank test does The average values of recorded scores (wave 1 = 42.91, wave 2 = 43.53, wave 3 = 42.96, wave 4 = 42.03, wave 5 = 40.86) reflect the degree to which people appreciate their work effort makes a positive contribution and brings benefits to others, or to the whole society. 11 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 TABLE 11  Patient care. A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 0.437 0.772 0.009 0.000 Wave 1 0.723 0.590 0.179 0.001 Wave 2 0.556 0.017 0.000 Wave 2 0.713 0.943 0.004 Wave 3 0.012 0.000 Wave 3 0.786 0.250 Wave 4 0.000 Wave 4 0.009 Wave 5 Wave 5ffhi fferent pandemic waves B. A) Different pandemic waves; (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). The italic values are statistically significa 4.3.4. Structural model testingh The medical personnel appreciated that the medical care they provided during the pandemic was adequate (82% of respondents), although at the cost of one’s own mental (14.2%) and physical exhaustion (17.1%). It should be  highlighted that deeply rooted professional convictions, i.e., caring for patients is the central element in the code of professional conduct in the medical field (88), influence job satisfaction and make medical staff focus on the physical, mental, and emotional wellbeing of patients, even when this task becomes risky or even more demanding. The developed model is based on inferential research results from the previous sections. It estimates accommodation mechanisms (causes and effects) to a continuous changing work environment. We hypothesized that the perceived state of health is reflected in the family relations, and in the work involvement. Work engagement gives psychological satisfaction and makes work meaningful which makes one feel fulfilled and valuable (Figure 2A). Even if results show statistically significant but often week relationships, the parameters for the presented model show a very good-fitting reasonably consistent with the data: SRMR = 0.027, RMSEA = 0.074, CFI = 0.985, TLI = 0.962. All the statistically significant relationships are positive in direction. The level of perceived health as a potential supporting factor in the family–work conflict has The Stanford Acute Stress Reaction Questionnaire (Stanford Acute Stress Reaction Questionnaire) was introduced into the research design starting with the second wave. Following the application of the Mann–Whitney U test, no statistically significant differences in terms 12 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 TABLE 13  Professional burnout. A. Different pandemic waves B. Different consecutive waves (panel samples) A. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 B. Wave 1 Wave 2 Wave 3 Wave 4 Wave 5 Wave 1 NA NA NA NA NA Wave 1 0.045 0.250 Wave 2 0.001 0.783 0.940 Wave 2 0.862 Wave 3 0.009 0.001 Wave 3 Wave 4 0.709 Wave 4 Wave 5 Wave 5 (A) Different pandemic waves, (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). *NA, no answer. A. Different pandemic waves A) Different pandemic waves, (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). *NA, no answer. (A) Different pandemic waves, (B) different consecutive waves (panel samples used the Wilcoxon signed rank test, p < 0.05). *NA, no ans FIGURE 2 The structural model. 4.3.4. Structural model testingh (A) Hypothesized structural model. (B) Graphical representation of the structural model. FIGURE 2 The structural model. (A) Hypothesized structural model. (B) Graphical representation of the structural model. All correlations are positive in direction with one exception: the relation between age and satisfaction level (−0.18). This suggests that youth is a moderate factor of confidentiality regarding perceived health status and satisfaction level, disregarding the sex of participants. The satisfaction level is a predictor of the family-work relation (0.58), both having a moderate direct effect on work engagement. a loading factor of 0.34. The relation between perceived heath status and work engagement has a loading factor of 0.10. Well-being at home influences work engagement (loading factor 0.12). Work engagement is an important mediator for the perceived meaningfulness of work (loading factor 0.65) and for satisfying basic psychological needs (loading factor 0.47). Work meaningfulness influences also the satisfaction of basic psychological needs (loading factor 0.32). The family-work relation is saturated by the measured variables exhaustion (“Quite often I  come back from work emotionally exhausted, not being able to participate in family life,” 0.93), stress (“The stress from work also affects me at home so that I can no longer do what I like, or makes me happy,” 0.84) and tiredness (“When I get home from work, I am too tired to participate in family activities,” 0.81). The largest direct contribution to work engagement comes from inspiration (“My work inspires me,” 0.91), followed by enthusiasm (“I am  enthusiastic about my work,” 0.85), and a general feeling of happiness (“I feel happy when I work hard,” 0.67).hf Using the structural equation modelling (SEM) procedure we  obtained the relationships presented in Figure  2B. The final structural model provides a good fit with all significant paths (SRMR = 0.072, RMSEA = 0.085, CFI = 0.928, TLI = 0.910). In Figure 2B (Graphical representation of the structural model), the measurement model has observed variables shown in rectangles, and latent variables drown as circles; the structural model tests the mediating effects between the latent variables (on the path satisfaction – family–work conflict - work engagement - work meaningfulness - psychological needs); straight lines with an arrow at the end represent the hypothesized effect one variable has on another. The most consistent effect in the model is that of work engagement on work meaningfulness (0.65). Frontiers in Psychiatry frontiersin.org 5. Discussion Although the COVID-19 pandemic was emotionally exhausting, this study shows that the medical staff had a sense of personal achievement due to meaningful work and commitment to work, which is consistent with the results obtained by other research works (89, 90). Respondents in our research advocated an enhance involvement in the activities carried out during the pandemic crisis (65.5%). According to Rana (91), job satisfaction plays an important role in the work commitment and performance of medical professionals. During pandemic conditions, job satisfaction was related to the number of consecutive shifts, occupational well-being, job security, and professional stability at work. The higher the perceived job satisfaction, the higher the job performance and productivity in healthcare (91). In Mukaihata et al. (92) study on psychiatric nurses, work engagement moderated the direct and indirect effects of patient-related stressor on job satisfaction. Key findings can be summarized as follows: • The analysis identified perceived personal health status as an important factor in the perception of the dangerousness of workplace, the felt pandemic stress, the work–family conflict, the satisfaction of basic psychological needs, the meaning of and commitment to work • Patient care seems to be above the own health perception and may be associated with the satisfaction of the basic psychological needs of the medical staff, the work-family balance, and the perception of workplace safetyh • The sense of belonging (ownership) and work commitment correlate with the quality of patient care and supports the encountered facts that the medical staff managed to find resources to cope with professional stress and burnout during the COVID 19 crisis Silvia De Simone et al. (93) found correlations between work engagement, job satisfaction, and self-efficacy. During the COVID 19 crisis, research revealed that people with high self-efficacy are more able and comfortable to take on challenging tasks, being more confident in their ability to overcome difficult situations (94); self- efficacy being negatively correlated with anxiety (95, 96). • Analysing items dynamics during the 5 pandemic waves, certain adaptive attitudes (e.g., increasing confidence, satisfaction) and behaviors towards COVID 19 pandemic threats emerged related to gained experience and the progress in terms of medical protocols and procedures Our findings highlighted that work engagement and high perceived level of work meaningfulness reduce physicians’ burnout and sustain the quality of patient care. In a systematic review of over 4,700 articles focusing on physicians’ burnout Hodkinson et al. Frontiers in Psychiatry 6. Conclusion As an important outcome, in SEM there are no correlations with patient care, measuring physicians’ own perceptions of the quality of care they provide to patients, which highlights that, regardless of unfavourable long-term conditions, work involvement provides a high level of health care professionalism. The propose of this study was to identify potential affecting factors of healthcare work sustainability during the change-related uncertainty conditions generated by the COVID 19 crisis. Dynamics and relations of nine carefully selected variables and constructs were tracked along all five pandemic waves in Romania, which span from March 2020 to April 2022. The tested variables and constructs are perception of healthcare workers of their own state of health, their workplace safety, the work–family conflict, the satisfaction of basic psychological needs, the work meaningfulness and work engagement, patient care, pandemic stress and burnout. frontiersin.org 4.3.4. Structural model testingh The general disposition that life has meaning presents the highest direct contribution to work meaningfulness (“I understand how my work contributes to the meaning of my life,” 0.97), supporting personal development (“My work contributes to my personal development,” 0.86), and becoming the purpose in life (“My work contributes to a purpose greater than myself,” 0.84). Work engagement moderately satisfies psychological In SEM, the exogenous variable is Satisfaction (mainly the perceived state of health), which has no predictor within the model. All other variables are endogenous or dependent variables (e.g., family–work conflict, work engagement, work meaningfulness, and psychological needs), their values being determined by other variables in the model. 13 frontiersin.org Savu et al. 10.3389/fpsyt.2023.1179803 10.3389/fpsyt.2023.1179803 conditions. Likewise, patient care, becoming the central priority during COVID 19 health crisis conditions, seems to have disconnected from previous influencing factors and relationships. needs (0.34), saturated by the freedom of choice (“At work, I have a sense of choice and freedom in the things I do,” 0.83), competence in achieving goals (“When I am at work, I feel that I am competent to achieve my goals,” 0.70), and social connectivity (“I feel connected to the people who care about me at work and who I care about,” 0.69). References 15. Parent JD. Individual Adaptation to the Changing Workplace: Causes, Consequences and Outcomes, PhD Dissertation. Amherst: University of Massachusetts (2006). 1. Worldometers-info. (2022). COVID-live, Coronavirus Statistics. Available at: https://www.worldometers.info/coronavirus/, (Accessed November 18, 2022) 2. World Health Organization. Publications/Overview/Weekly Epidemiological Update on COVID-19 – 1 June 2022; (2022). 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Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. 5. Discussion (59) found out that burnout related to low work engagement and meaningfulness, as well as low job satisfaction, and low patient satisfaction. • The in-depth structural model identified that the own health status satisfaction is a mediator of the family–work conflict and, together, of the work engagement. In turn, work engagement plays a significant role in satisfying basic psychological needs and supporting work meaningfulness. Work meaningfulness influences also the satisfaction of basic psychological needs. Similarly, Guerrero-Barona et al. (97) carried out correlations between the quality of family life and work conflict, psychosocial factors, burnout syndrome and emotional intelligence. Literature concerning work engagement before the COVID 19 crisis indicated that work engagement was positively correlated with the quality of care (24). Other studies examined the association of burnout with the quality of patient care, based on samples from all categories of medical personnel (98, 99). Babenko (100) investigated the role of basic psychological needs (autonomy, competence, and relatedness) in physicians’ professional well-being in terms of job satisfaction, work engagement, and burnout. This study indicated that the need for relatedness had the largest contributions to physicians’ professional life satisfaction, work-related engagement, and exhaustion, respectively. However, despite these strengths, our study has also some limitations that reduce the generalizability of results. The strongly female-dominated samples, with 74.5% women, although it mirrors the structure of the health personnel in Romania [70.5% female doctors in 2020, according to the National Institute of Statistics (101)] can be a risk of gender bias in the overall generalizability of our findings. Another drawback is that we  have limited data from multiple respondents who are part of all 5 pandemic waves and cannot have a conclusive study in terms of comparing responses between waves. Another limitation of the present research is given by the fact that we did not include in the study a section on the possible infection of medical personnel and the return to work after passing through a COVID 19 disease. Unfortunately, these limitations cannot be improved by further research. Nevertheless, these findings can help medical health systems We consider that these general factors and relationships sharpened their manifestation during pandemic conditions. The uncertainty of the pandemic triggered a mobilization of resources released by physician’s commitment to work. Further, the work engagement raised the awareness of the work meaningfulness under these extreme 14 Savu et al. 10.3389/fpsyt.2023.1179803 Acknowledgments better identify inner vulnerabilities and strengths, on which coping strategies can be developed to withstand future disturbances. better identify inner vulnerabilities and strengths, on which coping strategies can be developed to withstand future disturbances. We acknowledge the contribution of the doctoral scholarship from the Faculty of Geography, University of Bucharest, CS. We greatly acknowledged the financial support of the University of Bucharest in paying publication fee. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. 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Substantial contribution of genetic variation in the expression of transcription factors to phenotypic variation revealed by eRD-GWAS
Genome biology
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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Substantial contribution of genetic variation in the expression of transcription factors to phenotypic variation revealed by eRD-GWAS Hung-ying Lin1,2, Qiang Liu1,2, Xiao Li2,3,4, Jinliang Yang1,2,5,11, Sanzhen Liu1,2,6, Yinlian Huang7,8, Michael J. Scanlon9, Dan Nettleton10 and Patrick S. Schnable1,2,3,7* Abstract Background: There are significant limitations in existing methods for the genome-wide identification of genes whose expression patterns affect traits. Results: The transcriptomes of five tissues from 27 genetically diverse maize inbred lines were deeply sequenced to identify genes exhibiting high and low levels of expression variation across tissues or genotypes. Transcription factors are enriched among genes with the most variation in expression across tissues, as well as among genes with higher-than-median levels of variation in expression across genotypes. In contrast, transcription factors are depleted among genes whose expression is either highly stable or highly variable across genotypes. We developed a Bayesian-based method for genome-wide association studies (GWAS) in which RNA-seq-based measures of transcript accumulation are used as explanatory variables (eRD-GWAS). The ability of eRD-GWAS to identify true associations between gene expression variation and phenotypic diversity is supported by analyses of RNA co- expression networks, protein–protein interaction networks, and gene regulatory networks. Genes associated with 13 traits were identified using eRD-GWAS on a panel of 369 maize inbred lines. Predicted functions of many of the resulting trait-associated genes are consistent with the analyzed traits. Importantly, transcription factors are significantly enriched among trait-associated genes identified with eRD-GWAS. Conclusions: eRD-GWAS is a powerful tool for associating genes with traits and is complementary to SNP-based GWAS. Our eRD-GWAS results are consistent with the hypothesis that genetic variation in transcription factor expression contributes substantially to phenotypic diversity. Keywords: Transcription factors, Gene expression, GWAS, Phenotypes, Traits, Association studies * Correspondence: schnable@iastate.edu 1Department of Agronomy, Iowa State University, 2035 B Roy J Carver Co-Lab, Ames, IA 50011-3650, USA 2Interdepartmental Genetics and Genomics Graduate Program, Iowa State University, Ames, IA 50011-3650, USA Full list of author information is available at the end of the article Lin et al. Genome Biology (2017) 18:192 DOI 10.1186/s13059-017-1328-6 Lin et al. Genome Biology (2017) 18:192 DOI 10.1186/s13059-017-1328-6 Open Access RESEARCH Open Access Identification of genes that are variably or stably expressed across tissues To identify genes that exhibit extreme levels of variation in transcript accumulation across tissues, a series of model selection procedures was performed. Ultimately, we se- lected negative binomial distributions to model the distri- butions of read counts for genes, and the scaled log of over-dispersion parameters of quasi-negative binomial generalized linear models to minimize the correlation be- tween expression variation and expression levels (“Methods”). Henceforth, the scaled log10 over-dispersion parameters will be termed “variation in gene expression”. Because variation in the regulation of gene expression contributes to phenotypic diversity [20], efforts have been made to identify genetic variants associated with variation in transcript accumulation, i.e., expression quantitative trait locus (eQTL) analyses [21]. Genetic variants detected via eQTL analysis can act in cis or in trans. The relative contributions of cis- and trans-acting eQTLs on pheno- typic variation are unknown. Cis-variation is often consid- ered a key mechanism in creating phenotypic novelty [22] and contributes to adaptive evolution [23–25]. Indeed, cis- effects have played a major role on gene expression during the domestication of maize [22]. It is worth noting, how- ever, that due to limitations in statistical power it is typic- ally more difficult to detect trans-acting eQTLs than cis- acting eQTLs [26]. Even so, many trans-eQTLs have been identified in maize [26, 27] and other species [28, 29]. p g p Of 39,656 high-confidence “filtered-gene set” (FGS) genes, 29,609 have sufficient levels of transcript accumula- tion (“Methods”) to be used in subsequent analyses. The distribution of variation of gene expression across tissues was a left-skewed distribution (Additional file 2: Figure S1a). We defined the upper and lower 2.5% percentiles of this distribution as tissue variable expression (T-VE) genes (N = 741 genes) and tissue stable expression (T-SE) genes (N = 741) (Additional file 2: Figure S1a). TFs as a group were enriched among T-VE genes (P value = 0.008) and homeobox (P value = 0.03) and MADS box families of TFs (P value = 5 × 10−5) were specifically enriched among T- VE genes (“Methods”). In contrast, TFs were depleted among T-SE genes (P value = 0.0005; Additional file 2: Fig- ure S1b and Additional file 3c). p Maize is one of the most genetically and phenotypic- ally diverse species [30] and has a rich collection of gen- etic resources [31], making it an important model system. Identification of genes that are variably or stably expressed across tissues Because maize exhibits high levels of SNP diver- sity and low LD, it exhibits high statistical power and resolution in GWAS [32]. We used this model species to test the role of variation in the expression of TFs and more generally variation in transcript accumulation on phenotypic variation. Following deep RNA-seq analysis of multiple tissues from diverse inbred lines we estab- lished that TFs are depleted among genes that exhibit high levels of expression variation across genotypes. Next, we developed a Bayesian-based statistical method for using RNA-seq measurements of transcript accumu- lation as the explanatory variables in GWAS and thereby directly demonstrate an association between variation in transcript accumulation of TFs and phenotypic variation for a diverse collection of traits. Results power [6]. In contrast, Bayesian methods apply multiple variable regression models combined with prior distribu- tions and Markov chain Monte Carlo (MCMC) sampling to generate posterior distributions [7–9]. Meuwissen et al. [8] first proposed the methods of ridge-regression BLUP, BayesA, and BayesB. BayesB assumes marker effects have identical and independent univariate t-distributions and assume that a designated portion of markers have no ef- fect. BayesC is similar to BayesB, but marker effects are assumed to have a common variance [10]. power [6]. In contrast, Bayesian methods apply multiple variable regression models combined with prior distribu- tions and Markov chain Monte Carlo (MCMC) sampling to generate posterior distributions [7–9]. Meuwissen et al. [8] first proposed the methods of ridge-regression BLUP, BayesA, and BayesB. BayesB assumes marker effects have identical and independent univariate t-distributions and assume that a designated portion of markers have no ef- fect. BayesC is similar to BayesB, but marker effects are assumed to have a common variance [10]. RNA-seq was conducted on mRNA extracted from mul- tiple maize organs (seedling shoot apex, immature unpol- linated ears, immature tassels, seedling shoots and roots) collected from the 27 inbred founders of the nested asso- ciation mapping (NAM) population. Six billion raw 101- bp reads were generated, trimmed, filtered, and aligned to the B73 reference genome (“Methods”); 2.9 billion non- stacking uniquely aligned reads were used to assay tran- script accumulation levels (Additional file 1). Genes with regulatory functions often exhibit high levels of expression variation across species [11, 12] compared to metabolism-related genes [13]. Several studies have revealed that, among primates, transcription factors (TFs) can evolve rapidly in response to selection [14–16]. Within species, genes exhibit different levels of variation in expression among individuals and alterations in the regulation of the expression of TFs can contribute to novel phenotypes [17], such as branching in maize [18] or pelvic loss in three-spined stickleback fish [19]. Background (LD) between genetic variants such as SNPs and loci that directly affect traits of interest. There are two main ap- proaches for identifying such associations, mixed linear models (MLM) and Bayesian-based approaches. Many projects are underway to identify loci that contrib- ute to traits, and the methods to do so remain under de- velopment [1]. Most commonly, associations are sought between genetic variants (e.g., SNPs) and variation in trait values via genome-wide association studies (GWAS). Typ- ical approaches to GWAS exploit linkage disequilibrium MLM solutions have been developed to overcome the confounding effects of population structure and the re- latedness among individuals, and provide increased com- putational efficiency and statistical power [2–5]. Typical MLM solutions estimate effects based on single markers and require the use of covariances to account for popu- lation structure. However, these approaches for control- ling for population structure also decrease statistical * Correspondence: schnable@iastate.edu 1Department of Agronomy, Iowa State University, 2035 B Roy J Carver Co-Lab, Ames, IA 50011-3650, USA 2Interdepartmental Genetics and Genomics Graduate Program, Iowa State University, Ames, IA 50011-3650, USA Full list of author information is available at the end of the article Page 2 of 14 Page 2 of 14 Page 2 of 14 Lin et al. Genome Biology (2017) 18:192 Correlation of variation in maize gene expression across genotypes and tissues A linear trend was observed between tissue-wise and genotype-wise variation in gene expression (r2 = 0.64, P value ~ 2 × 10−16; Additional file 2: Figure S4). Based on whether a gene demonstrated stable or variable variation of gene expression across tissues and genotypes, maize genes could be classified into nine categories (Additional file 3). The 520 T-VE genes that are neither G-VE nor G-SE are significantly enriched in TFs overall (P value = 9 × 10 −5) and enriched in several specific TF families, including Homeobox/HOX (P value = 0.02), MADS (P value = 2.6 × 10−5), and Squamosa promoter binding protein (SPB; P value = 0.03) genes (Additional file 3e). Gener- ally, HOX genes function in organ identity [34] and SBPs function in phase change [35]. In contrast, the 330 genes classified as being both T-SE and G-SE are de- pleted for TFs (P value = 0.006; Additional file 4). To test the hypothesis that eRD-GWAS can identify loci that contribute to variation in traits that are not identified by traditional SNP-based GWAS, we analyzed all five SAM-related and eight other traits using both SNP genotypes and RPKM values as explanatory vari- ables (“Methods”). Typically, GWAS software that relies on MLMs is de- signed to use SNPs as the explanatory variables. We elected to use a BayesB-based approach to conduct eRD-GWAS in which RPKM values (expression data) served as the explanatory variables. Our rationale for selecting a Bayesian approach to GWAS is described in the “Methods”. The BayesB model is widely used in gen- omic selection. Instead of predicting phenotype, we used model frequency (the frequency with which a gene was included in a model) as a measure of the strength of the relationship between that gene’s expression pattern and the phenotype of interest. To validate the BayesB ap- proach we repeated Leiboff et al.’s [36] SNP-based ana- lysis of SAM volume using a MLM approach and in parallel conducted a SNP-based GWAS for SAM volume using a BayesB approach (“Methods”). As expected the results we obtained from our SNP-based GWAS using the MLM approach (Fig. 1) were very similar to those of Leiboff et al. [36]. The upper and middle panels of Fig. 1 provide results from the SNP-based MLM GWAS and the SNP-based BayesB GWAS. Identification of genes that are variably or stably expressed across genotypes A similar approach was used to identify genotype vari- able expression and genotype stable expression (G-VE and G-SE) genes. The distribution of variation in gene expression across genotypes demonstrated a left-skewed distribution (Additional file 2: Figure S2a). Although TFs were enriched among genes that exhibited higher than median levels of variation in gene expression (P value = 3 × 10−5), TFs were underrepresented among both G-VE and G-SE genes (P values = 0.002 and = 0.046, respect- ively; Additional file 2: Figure S2b). Specifically, although 46 TFs would be expected among the G-VE genes by chance, only 22 were observed (Additional file 4). Simi- lar results were obtained when the G-VE and G-SE Lin et al. Genome Biology (2017) 18:192 Page 3 of 14 Page 3 of 14 genes were defined as being the upper and lower 5 and 10% of all genes. a set of lines which had been both genotyped and phe- notyped and for which RNA-seq data were available. notyped and for which RNA-seq data were available. The SAM (shoot apical meristem) diversity panel con- sists 369 diverse inbred lines, including commercially relevant inbreds with expired plant variety protection (PVP) [36]. We have genotyped this panel with 1.28 mil- lion SNPs [36]. In addition, we conducted RNA-seq on apex tissue (which includes the SAM) from each of these inbreds [36]. Using these RNA-seq data we calculated RPKM values for each of the 39,656 FGS genes in the maize genome for each of the inbreds in the SAM panel. Each of the inbreds in the SAM diversity panel had pre- viously been phenotyped for multiple traits related to the shoot apical meristem [37], i.e., volume, height, parabola radius, arc length, and SAM surface area [36], and a var- iety of other traits, including the mean node number [36], ear height, and days to anthesis (DTA) [38]. During the current study we phenotyped these inbreds for five add- itional traits, i.e., stalk circumference, stalk cross-sectional area, maximum and minimum stalk diameter, and number of nodes with brace roots. These traits exhibit varying de- grees of correlation (Additional file 2: Figure S5), some of which have been reported previously [39]. Arabidopsis thaliana RNA-seq data generated by Kawakatsu et al. [33] (N = 727 genotypes) were analyzed using similar approaches. Identification of genes that are variably or stably expressed across genotypes Consistent with our maize re- sults, TFs were depleted among G-VE (P value = 1.0 × 10 −5) and G-SE (P value = 0.004) genes in Arabidopsis (Additional file 2: Figure S3). As was observed for maize, Arabidopsis TFs were enriched among those genes that exhibit higher than median levels of expression variation across genotypes (P value = 4 × 10−8). The SAM (shoot apical meristem) diversity panel con- sists 369 diverse inbred lines, including commercially relevant inbreds with expired plant variety protection (PVP) [36]. We have genotyped this panel with 1.28 mil- lion SNPs [36]. In addition, we conducted RNA-seq on apex tissue (which includes the SAM) from each of these inbreds [36]. Using these RNA-seq data we calculated RPKM values for each of the 39,656 FGS genes in the maize genome for each of the inbreds in the SAM panel. Each of the inbreds in the SAM diversity panel had pre- viously been phenotyped for multiple traits related to the shoot apical meristem [37], i.e., volume, height, parabola radius, arc length, and SAM surface area [36], and a var- iety of other traits, including the mean node number [36], ear height, and days to anthesis (DTA) [38]. During the current study we phenotyped these inbreds for five add- itional traits, i.e., stalk circumference, stalk cross-sectional area, maximum and minimum stalk diameter, and number of nodes with brace roots. These traits exhibit varying de- grees of correlation (Additional file 2: Figure S5), some of which have been reported previously [39]. Correlation of variation in maize gene expression across genotypes and tissues The 14 significant signals that overlap between the two approaches are indicated by vertical dashed lines on chromosomes 1, 2, 6, 7, 9, and 10. Nine of these 14 SNPs that were detected via both approaches are located in or near genes that have Expression read depth genome-wide association study Based on the findings that TFs exhibited moderate vari- ation in expression across genotypes, we were interested in testing the contribution of variation in transcript ac- cumulation levels of TFs to phenotypic diversity. To dir- ectly test this association, we developed a Bayesian- based statistical approach for using transcript accumula- tion as the explanatory variable during GWAS. Typically, a GWAS is conducted using SNP genotypes as explanatory variables. We reasoned that using tran- script accumulation as an explanatory variable for GWAS would have certain advantages in that gene ex- pression levels potentially integrate the effects from mul- tiple loci that contribute to phenotype variation. To the extent that these hidden multiple locus effects poorly ex- plained by single genotyped SNPs, expression read depth genome-wide association studies (eRD-GWAS) may bet- ter explain variation in trait values. eRD-GWAS also have the potential to integrate the effects of epigenetic variation that contributes to variation in gene expression and other traits. To test the hypothesis that variation in transcript accumulation can explain diversity in trait values that is missed by traditional GWAS, we analyzed Lin et al. Genome Biology (2017) 18:192 Page 4 of 14 Fig. 1 Manhattan plots of three types of GWAS results. The upper panel reports result from a SNP-based MLM implemented in GAPIT. Only signals with P values smaller than 1.0 × 10−7 are presented. The middle and lower panels report results from the SNP-based BayesB analysis and eRD-GWAS, respectively. The model frequency cutoffs for SNP BayesB and eRD-GWAS are 0.01 and 0.02, respectively (“Methods”). Overlapping associated SNPs in the upper two panels are indicated by dashed lines. Note not all overlapping SNPs can be distinguished in this plot. Gene IDs of some trait associated genes (“Methods”) i di d Fig. 1 Manhattan plots of three types of GWAS results. The upper panel reports result from a SNP-based MLM implemented in GAPIT. Only signals with P values smaller than 1.0 × 10−7 are presented. The middle and lower panels report results from the SNP-based BayesB analysis and eRD-GWAS, respectively. The model frequency cutoffs for SNP BayesB and eRD-GWAS are 0.01 and 0.02, respectively (“Methods”). Overlapping associated SNPs in the upper two panels are indicated by dashed lines. Note not all overlapping SNPs can be distinguished in this plot. Gene IDs of some trait associated genes (“Methods”) are indicated Fig. 1 Manhattan plots of three types of GWAS results. eRD genes in gene regulatory networks g g g y Unlike co-expression networks, a gene regulatory net- work (GRN) is composed of directed edges that indicate biological relationships between pairs of nodes. For ex- ample, regulators are predicted to activate or suppress downstream genes. We examined the characteristics of our eRD genes within maize GRNs constructed using RNA-seq (23 tissues) or proteomic (33 tissues) data [48]; eRD genes were enriched among regulators in both the RNA- and protein-based GRNs (Fig. 3 a–c). Sets of eRD-GWAS genes selected using model frequency cut- offs larger than 0.03 have enrichment test P values smaller than 0.05, indicating that the targets of eRD- GWAS regulators are themselves enriched in eRD- GWAS genes (Fig. 3a–c). These results indicate that eRD-GWAS can identify both GRN regulators and their downstream targets. eQTL for eRD-GWAS-detected genes (eRD genes) g g If eRD-GWAS is accurately identifying genes that con- tribute to variation in a trait, we would expect that eQTLs that act in trans to regulate the expression of eRD genes may also be associated with variation in that trait. Hence, we conducted an eQTL analysis using an MLM approach (“Methods”) for the five eRD genes asso- ciated with the DTA trait that had the highest model fre- quencies. The resulting eQTLs were compared to the eRD genes and also to the genes associated with the DTA trait via BayesB GWAS (Additional file 6). Hyper- geometric analyses (“Methods”) established that the eQTLs were enriched in genes associated with variation in the DTA trait. To ensure this phenomenon was ro- bust across traits, we used the same strategy on multiple traits (Additional file 7). The results were consistent with our hypothesis, i.e., that the eQTLs associated with specific eRD genes are enriched overall in eRD genes. This result provides support for the view that eRD- GWAS accurately identifies genes whose expression is associated with variation in trait values. Expression read depth genome-wide association study Genome Biology (2017) 18:192 inflorescence meristem [40] and BRANCH ANGLE DE- FECTIVE 1 (BAD1) [40] is a TCP class II gene that is expressed in inflorescence meristems and lateral organs where it functions to promote cell proliferation. inflorescence meristem [40] and BRANCH ANGLE DE- FECTIVE 1 (BAD1) [40] is a TCP class II gene that is expressed in inflorescence meristems and lateral organs where it functions to promote cell proliferation. modules that were enriched for eRD genes associated with the DTA trait were also enriched for a variety of GO categories. The “honeydew” module was enriched for the GO category “maintenance of floral meristem identity”, which would appear to be relevant to the DTA trait. Other modules were enriched for categories that the literature reported may be relevant to the DTA trait, such as “metal ion transport”, “response to nitrate”, and “NAD(P) metabolic” [43–45]. All GWAS provide lists of genes that are hypothesized to be associated with traits of interest. To assay the accur- acy of the gene–trait associations from eRD-GWAS we performed a series of analyses, including tenfold cross- validation, eQTL analyses of eRD-GWAS genes, tests for the enrichment of eRD-GWAS genes within specific nodes of RNA co-expression networks, protein–protein interaction networks, and gene regulatory networks. eRD-GWAS in protein–protein interaction networks Protein–protein interaction networks (PPINs) can be used to identify proteins (and genes) that contribute to phenotypes and thereby help elucidate complex genetic mechanisms [46]. We downloaded maize PPIN data from the maize PPIM [47], clustered proteins into net- work communities, and then tested whether eRD genes were enriched in network communities. As was the case for the enrichment tests within the RNA co-expression network, eRD genes were significantly enriched (“Methods”) in GO categories associated with the DTA trait among three of the 12 network communities that contained more than one eRD gene (Table 2 and Fig. 2). This finding provides further evidence that eRD-GWAS can identify biologically relevant gene–trait associations. Tenfold cross-validation Tenfold cross validation is a technique used for assessing the accuracy of prediction models [41]. Our tenfold cross-validation analyses of the results of eRD-GWAS (“Methods”) yielded accuracies of 0.41–0.76, indicating that eRD-GWAS accurately detects associations between variation in transcript accumulation and multiple traits (Additional file 5). Based on comparisons to similar cross-validation analyses conducted using results from SNP BayesB, the accuracies of the two approaches are similar for multiple traits (Additional file 5). Expression read depth genome-wide association study The upper panel reports result from a SNP-based MLM implemented in GAPIT. Only signals with P 7 Fig. 1 Manhattan plots of three types of GWAS results. The upper panel reports result from a SNP-based MLM implemented in GAPIT. Only signals with P values smaller than 1.0 × 10−7 are presented. The middle and lower panels report results from the SNP-based BayesB analysis and eRD-GWAS, respectively. The model frequency cutoffs for SNP BayesB and eRD-GWAS are 0.01 and 0.02, respectively (“Methods”). Overlapping associated SNPs in the upper two panels are indicated by dashed lines. Note not all overlapping SNPs can be distinguished in this plot. Gene IDs of some trait associated genes (“Methods”) are indicated exceed the arbitrarily selected model frequency cutoff of 0.02 in the eRD-GWAS. If we search for candidate genes, GRMZM2G140721 is detected by both the SNP- based BayesB and eRD-GWAS. GRMZM2G140721 is a predicted transcriptional factor in Arabidopsis, rice, and maize. In total, 120 genes identified via eRD-GWAS (i.e., eRD genes) were not located within 30-kb windows cen- tered on the chromosomal positions of SNPs identified via either SNP-based GWAS approach (MLM or Bayes- ian). Even so, some of these genes detected via eRD- GWAS but not by SNP-based GWAS have previously been demonstrated to affect the morphology of the SAM. For example, ZEA CENTRORADIALIS4 (ZCN4) functions in the maintenance of indeterminate shoot meristem, thereby affecting the transition to an been shown previously to be associated with SAM vol- ume [36]. If we consider SNPs present in the same gen- omic regions (to account for LD), 19 of the 54 SNPs detected by SNP-MLM were present in 30-kb windows centered on SNPs detected by SNP-BayesB. Similarly, 15 of 53 SNPs detected by SNP-BayesB were present in 30- kb windows centered on SNPs detected by SNP-MLM. These results established that the BayesB approach iden- tified a significant subset of those SNPs identified by MLM GWAS, but that the BayesB approach also identi- fied signals not identified by the MLM approach. Based on these results we used BayesB-based eRD- GWAS to identify genes whose variation in transcript accumulation is associated with diversity in SAM vol- ume. Approximately 500 genes (lower panel of Fig. 1) Page 5 of 14 Lin et al. TFs are enriched among trait-associated genes from eRD- GWAS As discussed earlier, TFs are enriched among genes that exhibit a higher than median level of variation in gene expression across genotypes. To test the hypothesis that the variation in expression of TFs affects phenotype, we conducted enrichment tests for TFs among eRD genes associated with 13 phenotypes using various model fre- quency cutoffs (Fig. 3d–g; Additional file 2: Figure S6). For 11/13 traits, as the stringency of model frequency Overview of eRD-GWAS To test the hypothesis that variation in the expression of TFs (and other genes) across genotypes contributes to phenotypic variation, we developed eRD-GWAS, a stat- istical method that permits gene expression level to be tested as an explanatory variable during GWAS. eRD-GWAS enriched in an RNA co-expression network ** P value of enrichment test < 0.01 cutoffs was increased, the enrichment of TFs among the eRD-GWAS genes also increased. This result demon- strates the importance of variation in the expression of TFs on phenotypic variation. Discussion We were interested in comparing the variation in ex- pression of TFs across tissues and genotypes to that of other genes. Using an RNA-seq data set derived from five tissues and 27 genotypes, we identified genes that p y g Using eRD-GWAS we detected several hundreds of trait-associated genes for each of multiple traits included in this study. The results of tenfold cross-validation indi- cated that the predicted phenotypes based on genes de- tected via eRD-GWAS are highly correlated with empirically measured phenotypes. In addition, many trait-associated genes have annotations consistent with their presumed roles in regulating the associated traits (Additional file 7). Hence, we concluded that the eRD- GWAS pipeline can successfully identify associations be- tween variation in gene expression and diversity in phenotype. eQTL analyses of eRD-GWAS genes, tests for the enrichment of eRD-GWAS genes within specific nodes of RNA co-expression networks, PPINs, and GRNs provided further support for this conclusion. Table 2 GO enrichment among protein–protein interaction network communities that contain multiple eRD genes for the DTA trait Community GO term enrichment of community Number of eRD genes within community percentage of eRD genes in community) Log2 odds ratio for eRD genes in xcommunity 10 ATP biosynthesis process; metal ion transport 8 (7.41%) 4.75** 6 MADS-gene family; floral meristem maintain 5 (8.93%) 5.96** 4 Oxidation- reduction process; nitrate assimilation; steroid 22-alpha hydroxylase activ- ity (BR) 12 (4.67%) 2.58* * P value of enrichment test < 0.05 ** P value of enrichment test < 0.01 Table 2 GO enrichment among protein–protein interaction network communities that contain multiple eRD genes for the eRD-GWAS enriched in an RNA co-expression network eRD-GWAS enriched in an RNA co-expression network To enhance the power of this analysis we first con- structed an RNA co-expression network using WGCNA [42] using the RNA-seq data from the SAM diversity panel. We then determined gene ontology (GO) terms that were enriched among the genes within specific modules of the co-expression network (Table 1). The Page 6 of 14 Page 6 of 14 Lin et al. Genome Biology (2017) 18:192 Table 1 GO enrichment tests of RNA co-expression modules containing multiple eRD genes for the DTA trait Module name GO term enrichment of module Number of eRD genes within module (percentage of eRD genes in module) Log2 odds ratio for eRD genes in module Thistle3 Metal ion transport; transferring phosphorus- containing groups; ATP binding 20 (54.1%) 9.17** Navajowhite2 NAD(P) metabolic 18 (34.6%) 8.03** Firebrick4 Nitrate transport; magnesium ion binding 15 (35.7%) 8.38** Palevioletred3 Terpene synthase; regulation of transcription; response to nitrate 6 (11.1%) 6.33** Honeydew Cell wall organization; maintenance of floral meristem 4 (11.1%) 6.92** ** P value of enrichment test < 0.01 Table 1 GO enrichment tests of RNA co-expression modules containing multiple eRD genes for the DTA trait exhibit low and high levels of expression variation across tissues (T-VE and T-SE genes) and genotypes (G-SE and G-VE). T-VE genes are enriched in TFs, and specifically enriched for Homeobox, MADS and Squamosa pro- moter binding (SPB) proteins. In contrast, T-SE genes are depleted for TFs. In contrast to what was observed across multiple tis- sues, TFs were depleted among the G-VE and G-SE genes of both maize and Arabidopsis. Even so, in both species, TFs were enriched among those genes that ex- hibit higher than median levels of variation in gene ex- pression. Interestingly, even though there is positive correlation between maize genes that exhibit high levels of expression variation across genotypes and tissues, TFs are not enriched among G-VE genes that are also T-VE. Based on these findings we hypothesize that extreme variation in expression of TFs across genotypes is con- strained by selection against the extreme phenotypic variation that would be expected to arise via the action of TFs with extreme expression levels upon multiple downstream target genes. Similarly, because the NAM founders exhibit substantial phenotypic diversity, the de- pletion of TFs among the G-SE genes is consistent with a role of TFs in contributing to phenotypic diversity. ** P value of enrichment test < 0.01 * P value of enrichment test < 0.05 ** Challenges associated with GWAS GWAS strategies identify genes that putatively contrib- ute to variation in phenotypes. However, false positive results remain a challenge in GWAS [49]. The use of other types of genomic data in combination with SNP data has the potential to decrease biases and increase the power to detect true associations in GWAS. For Lin et al. Genome Biology (2017) 18:192 Page 7 of 14 Fig. 2 Visualization of a protein–protein interaction network that contains eRD genes. Highlighted communities that contain more than one eRD gene and in which eRD genes are statistically enriched are highlighted Fig. 2 Visualization of a protein–protein interaction network that contains eRD genes. Highlighted communities that contain more than one eRD gene and in which eRD genes are statistically enriched are highlighted approach, the limitations discussed in the “Background” apply. In contrast to the method of Jin et al., eRD- GWAS does not require that lines with intermediate ex- pression levels be treated as missing data. In addition, our statistical framework is not limited to binary pheno- types as is the case for Gamazon et al. [20]. This is im- portant because most important traits exhibit quantitative variation. example, efforts have been made to make use of eQTL results to increase the accuracy of GWAS [50, 51]. Al- though including eQTL results has the potential to de- crease the rate of false positive associations, this approach can also result in elevated rates of false nega- tive calls [49]. An alternative approach which we employed in this study is to use gene expression levels directly as explana- tory variables for GWAS. This approach substantially re- duces the multiple testing problem by using as explanatory variables expression data from ~ 40,000 maize genes vs. millions of available SNPs. This reduc- tion in the number of explanatory variables also reduces the computational cost of eRD-GWAS compared to traditional SNP-based approaches. Because eRD-GWAS directly associates candidate genes with phenotypes, it eliminates the need to hunt for causative genes within windows surrounding trait- associated SNPs. One potential concern with eRD- GWAS is whether LD creates false trait associations be- tween the expression of a gene that is simply linked to the causative gene. The Bayesian framework employed by eRD-GWAS functions to distinguish the effects of LD loci; our data suggest that this is in fact true not only for SNPs but also for expression data. Challenges associated with GWAS For example, even though the expression patterns of various alleles of ZmMADS69 are correlated with the expression patterns of other genes within the adjacent 1-Mb window (as well as genes across the genome), eRD-GWAS still could de- tect ZmMADS69 as the gene with the highest model fre- quency for flowering time (Additional file 2: Figure S7). Another group has shown that RNA expression pat- terns can predict human disease [20]. However, their statistical framework was intolerant of missing data which, required that transcriptomic data be imputed based on SNP data. This imputation would be expected to decrease accuracy. Further, their approach is limited to binary phenotypes (e.g., healthy vs. diseased). Jin et al. [52] also attempted to associate phenotypes with expres- sion patterns. For a given gene, they classified lines as ei- ther being expressed or not based on RNA-seq data. Lines having intermediate levels of expression were treated as missing data. The conversion of continuous gene expression data into a binary classification scheme would be expected to decrease statistical power [53]. Be- cause the data of Jin et al. were analyzed using an MLM Before using expression data to conduct eRD-GWAS, it is necessary to align RNA-seq reads to a reference genome. The substantial amount of SNPs [36] and struc- tural polymorphism among maize haplotypes [54] may result in alignment biases that distort RPKM values and hence the power of eRD-GWAS. Although this bias did Lin et al. Genome Biology (2017) 18:192 Page 8 of 14 Fig. 3 Enrichment testing for eRD genes. a–c Enrichment of “regulators” among eRD genes associated with the DTA trait at various model frequency cutoffs. a, b Numbers of eRD genes associated with the DTA trait that are defined as regulators of RNA- and protein-based GRNs by Walley et al. [48]. c Numbers of eRD genes that are themselves downstream of eRD genes that are regulators from the RNA-based GRNs. d–g Enrichment of TFs among eRD genes for various traits at various model frequencies. The number of eRD genes above indicated model frequency cutoffs are shown within each plot. The red dashed lines indicate P values of 0.05 Fig. 3 Enrichment testing for eRD genes. a–c Enrichment of “regulators” among eRD genes associated with the DTA trait at various model frequency cutoffs. Tissue collection, library preparation, and RNA sequencing Maize shoot apex, immature, unpollinated ears, imma- ture tassels, and seedling shoots and roots of 27 NAM founders were collected for RNA extraction (Additional file 1). There exists a universal dilemma of sampling tis- sues from genotypes with different maturities. One must either sample from a common environment (same har- vest date) and accept variation in developmental stage at harvest, or harvest at a common developmental stage and accept the risk of differences in micro-environment at harvest. For the NAM RNA-Seq experiment we elected to use the second approach. Conclusions The Bayesian approach requires the selection of a model frequency cutoff which, unlike the q-value associ- ated with MLMs, is in some sense arbitrary. If our se- lected model frequency cutoff (0.02) had been too relaxed, it is unlikely that strong statistical evidence for module-specific enrichment within the co-expression and PPINs would have been observed. Nor would we have been likely to observe a statistically significant en- richment of eQTLs for eRD genes among the eRD genes. If the selected model frequency cutoff were more strin- gent (i.e., if a larger model frequency), fewer genes would have been called as being associated with a given phenotype. This relationship is explored in Fig. 3 and Additional file 2: Figure S6, which demonstrate that the enrichment of TFs among the eRD genes for multiple traits is robust across a wide range of model frequency cutoffs, but that the enrichment P value can become more significant at increasingly stringent model fre- quency cutoffs. This finding is consistent with the hy- pothesis that a more stringent cutoff would result in a higher proportion of true positives, although presumably at the cost of more false negatives. TFs are enriched among genes with the most variation in expression across tissues and among genes with higher than median levels of variation in expression across genotypes. To better understand the relationship between variation in gene expression on phenotypes, we developed eRD-GWAS, which identifies associations be- tween variation in gene expression and variation in phe- notypes or traits. The enrichment of TFs among trait- associated genes identified via eRD-GWAS highlights the impact of expression variation on phenotypes. eRD- GWAS is complementary with SNP-based GWAS. Challenges associated with GWAS a, b Numbers of eRD genes associated with the DTA trait that are defined as regulators of RNA- and protein-based GRNs by Walley et al. [48]. c Numbers of eRD genes that are themselves downstream of eRD genes that are regulators from the RNA-based GRNs. d–g Enrichment of TFs among eRD genes for various traits at various model frequencies. The number of eRD genes above indicated model frequency cutoffs are shown within each plot. The red dashed lines indicate P values of 0.05 Page 9 of 14 Lin et al. Genome Biology (2017) 18:192 Page 9 of 14 demonstrated that genes responsible for directional/di- versifying selection are often TFs [11, 12, 62, 63]. As a step towards testing the hypothesis that TFs contribute substantially to phenotypic variation in maize, we dem- onstrated that TFs exhibit elevated levels of variation in expression across genotypes. More directly, using our newly developed eRD-GWAS method we established that genes associated with phenotypic variation for mul- tiple traits are enriched in TFs, demonstrating that vari- ation in the expression of TFs contributes substantially to phenotypic diversity in maize. not interfere with our ability to detect trait-associated loci, the use of new alignment approaches that better control for polymorphisms [55] may provide additional power to eRD-GWAS. This study included a direct comparison between the use of SNPs and expression data as explanatory variables within a common statistical framework. Our results es- tablish that the two types of explanatory variables pro- vide different association signals, such that some signals are detected by only one type of explanatory variable. This result argues that eRD-GWAS are complementary with SNP-based GWAS. Transcription factors contribute significantly to phenotypic variation Variation in gene expression contributes to phenotypic variation [56] upon which natural and artificial selection can act. The mechanisms that regulate variation in gene expression can act in cis (e.g., transcription binding sites) or in trans (e.g., TFs). It has, for example, been shown that variants located upstream of maize genes are enriched in GWAS analyses of multiple morphological traits [57]. Similarly, GWAS signals are enriched near human TF binding sites [58]. These findings are at least consistent with the hypothesis that variation in TF bind- ing sites contributes to phenotypic variation. Ear and tassel were harvested from greenhouse-grown plants with the exception of Ms71 ears, which were har- vested from field-grown plants. Immature ear tips were harvested ~ 68 days after planting (depending on the maturity rate of each line). At this stage ear ranged from 0.5 to 3 inches; only the top one-third to one-fifth of each ear was collected. Tassels were harvested prior to tassel emergence, i.e., ~ 60 days after planting. Three healthy plants were sampled and pooled per genotype prior to homogenization in liquid nitrogen and RNA ex- traction. Maize shoot apexes were collected by pooling three to six 14-day-old seedlings from each NAM founder. Seedlings were grown by planting ten kernels of each line in germination paper which was rolled and placed in a tall plastic beaker filled with approximately 3 It is also likely that variation in the expression of TFs per se can contribute to phenotypic variation, and in- deed specific cases of this type have been identified [18, 59]. Previous case studies have revealed roles for TFs in phenotypic evolution [60, 61]. In addition, genome-wide comparative genomics studies among primates have Lin et al. Genome Biology (2017) 18:192 Page 10 of 14 Page 10 of 14 GLMs (some graphical display used ggplot2, version 2.2.1 [68]). Full models were fitted when comparing Poisson, negative binomial, and quasi-negative binomial GLMs, as follows: inches of tap water. Beakers were covered with “cling- wrap” and placed in a dark 28 °C incubator for approxi- mately 4–5 days, when shoots emerged from the ger- mination paper. Two to three inches of the shoot and root were cut and frozen in liquid nitrogen for immedi- ate homogenization and extraction. Samples from three plants of each inbred were pooled for homogenization. RNA-seq reads: processing, alignment, and SNP calling RNA-seq reads: processing, alignment, and SNP calling Quality trimming, alignment to the B73 reference genome, and SNP calling were as described by Leiboff et al. [36]. Φ ¼ Var Y ð Þ κE Y ð Þ2 þ E Y ð Þ where Y is fragment count for a gene, Var(Y) and E(Y) are the variance and expectation of Y, respectively, and κ is the dispersion parameter of a negative binomial GLM. Tissue-wise over-dispersion parameters were estimated with genotype as the only factor in the model, while genotype-wise over-dispersion parameters were esti- mated treating tissue as the only factor in the model. A total of 29,609 genes with mean read counts ≥5 and numbers of samples with zero read counts ≤2 were in- cluded in the analysis. Z-score normalization was per- formed against log transformed over-dispersion parameter estimates, where: Alignment coordinates of confidently (uniquely) mapped reads within the same chromosomal regions were compared for potential read stacks caused by PCR artifacts during sequencing. If a stack consisting of two or more reads with identical start and end positions were detected, only a single read with best alignment score (least number of mismatches and least number of ambiguous bases) was selected for variant detection. If the distance from the left base pair to right base pair was more than 12,000 bp, the reads/read pairs were fur- ther removed. Reads with non-canonical splice sites were also removed. Z ¼ log ^Φ   −^E log Φ ð Þ ð Þ ffiffiffiffiffiffiffiffiffi d Var p log Φ ð Þ ð Þ Discovery and annotation of expression variable/stable genes Read counts are discrete and usually exhibit correlation between mean and variance [64]. Proper models, tech- niques, and summary statistics are essential to evaluate expression variation. To reduce ascertainment bias be- tween expression level and expression variability, Pear- son correlations were computed between expression level and each of several summary statistics (Additional file 2: Figure S8), including over-dispersion parameter of the Poisson model [65], mean coefficient of variance based on the Poisson model, deviance of the negative bi- nomial model [66], and the over-dispersion parameter of the quasi-negative binomial model [65]. The R packages edgeR (version 3.14.0) [67] and QuasiSeq (version 1.0-8) [65] were used to estimate dispersion parameters and over-dispersion parameters of quasi-negative binomial Upper and lower 0.025 quantiles of transformed nor- malized distributions were used to define highly variable and highly stable genes. MAPMAN annotation of maize filter gene sets (5b.61) was used to perform functional enrichment tests [69]. Fisher exact test was performed with the Benjamini–Hochberg method controlling false discovery rates (FDRs). Transcription factors contribute significantly to phenotypic variation For the SAM diversity panel, all plants were grown and sampled according to as in Leiboff et al. [36]. log λijk   ¼ μ þ αi þ βj þ oijk Where λijk is mean fragment count for genotype i, tis- sue j, and observation k, μ is an intercept parameter, αi is an effect of genotype i, βj is an effect of tissue j, and oijk is the normalization offset for genotype i, tissue j, and observation k. All RNA extractions were performed with the Qiagen RNeasy kit according to the manufacturer’s protocol. RNA was eluted twice with 30 μl RNase free water. Indexed RNA-seq libraries were prepared using the Illu- mina protocol outlined in the “TruSeq RNA Sample Preparation Guide” (part number 15008136 rev. A, No- vember 2010). Maize shoot apex RNA was sequenced with an Illumina Genome Analyzer II instrument while ear, root, shoot, and tassel RNA were sequenced with an Illumina HiSeq 2000 instrument. Of the four measures of variation discussed above, the over-dispersion parameter of quasi-negative binomial model, which measures the deviation of a gene’s read counts from the best-fitting negative binomial distribu- tion, had the smallest correlation with expression level, and was thus used to measure expression variability (Additional file 2: Figure S8). The over-dispersion par- ameter Φ of quasi-negative binomial GLMs is: Bayesian-based GWAS y We selected a Bayesian approach for exploring the rela- tionship between gene expression and phenotype, rather than a MLM approach, for two major reasons. First, the multivariate Bayesian framework internally controls for the effects of other genes by testing whether the inclu- sion of a given marker (i.e., the expression level of a given gene) can explain more genetic variance in each MCMC (Markov chain Monte Carlo) iteration. Although it may be possible to fit all the markers (i.e., gene expres- sion levels of all genes) simultaneously by iterating an MLM approach, this would be time consuming. In con- trast, this feature is “baked into” the Bayesian approach. Equally important, population structure can be con- trolled automatically via Bayesian approaches that in- clude multiple genes in each MCMC iteration [72]. In contrast, population structure information is required to control false positives as covariances in MLM, which can decrease statistical power. Mixed linear model GWAS y ¼ Wν þ Xβ þ Zυ þ e The Bayesian-based GWAS was constructed using GenSel v4.1 [10] BayesC and BayesB methods. The model in GenSel was: where y is the phenotype value, ν and β are unknown fixed effect vectors, and υ is a vector of random effects that follows a multivariable normal distribution with a null mean and a covariance matrix of G. G = Kσ2 a, where K is the kinship matrix [2]. e follows a normal distribution with null mean and σ2 e I variance. In general W, X, and Z are the matrices containing principal component scores that account for population structure, known covariates, and SNP genotypes, respectively. In our case, W contains scores for the first three principal components, X was not used because we had no known covariates to adjust for, and Z had data on 1.28 million SNPs. Manhattan plots were generated from our in-house R scripts based on the P value from the GAPIT results. The cutoff was arbitrarily set at 10−7. Other settings followed GAPIT’s defaults. y ¼ Xβ þ Zυ þ e where X, Z, β, and υ are the same as in the MLM model, e follows a normal distribution with null mean, and covari- ance matrix σ2 e R (R is a diagonal matrix), σ2 a, and σ2 e have independent inverse Chi-square priors with degree of free- dom 4 and scale parameters set to 50% of phenotypic vari- ation as prior. For BayesB (eRD-GWAS) and BayesC (SNP- based GWAS), the fraction (f) of markers having no effect was set at 0.9996 and 0.995, respectively. We used a chain length of 41,000 and discarded 1000 iterations as a burn-in run. Significance cutoffs for SNP-BayesB and eRD-GWAS were set as model frequencies of 0.01 and 0.02, respectively. Then we used genetic variance and error variance poste- riors from BayesC as priors in BayesB; other settings were as above. The accuracy of Bayesian-based GWAS results were estimated via tenfold cross-validation. Collection of phenotypic data Phenotypic trait data were collected from a panel of 369 diverse inbreds designated as the “SAM panel” [36]. Data were collected from three plants per location in two fields grown in Ames, Iowa during the summer of 2014. Prior to data collection leaf sheaths and brace roots (if present) were removed. Measured traits included Page 11 of 14 Lin et al. Genome Biology (2017) 18:192 Lin et al. Genome Biology (2017) 18:192 Multiple genomic selection models were constructed employing different values of π (the proportion of SNPs, assumed to have no effect on phenotype). The accuracies of these various models were evaluated using tenfold cross-validation and heritability. We selected for each phenotype a value of π that yielded the maximum accur- acy based on tenfold cross validation that has a heritabil- ity that is not so high as to raise concerns of over-fitting. This had the effect of thinning the number of predictors, resulting in a more limited number of descriptors, simi- lar to the output of GWAS. Our approach differs from MLM GWAS in that rather than using a P value to re- flect the strength of the relationship between a marker and a phenotype, we used the model frequency (the fre- quency with which a gene was included in a model) to reflect the strength of the relationship between that gene’s expression and the phenotype of interest. maximum and minimum stalk diameters, stalk circumfer- ence, stalk cross-sectional area, total node number, and number of nodes with brace roots (Additional file 8). Add- itional data from the SAM panel (or members of it) were obtained from the literature. For example, several traits as- sociated with the SAM, including SAM height, radius, sur- face area, volume, and arc length from P1 notch to apex, were obtained from [36]. Ear height and DTA data were obtained from [38]. Phenotypic regression and phenotypic density distributions were conducted using the R “corr- gram” package version 1.10 [70]. Mixed linear model GWAS GAPIT version 3.35 [71] was used for MLM GWAS. The model implemented in GAPIT was: Cross-validation, enrichment tests, network visualization, and GO enrichment Tenfold-cross validations were conducted using the R “cvTools” package version 0.3.2 [73]. Enrichment test P values were based on hypergeometric distributions. Net- work visualization was conducted using “MANGO” soft- ware version 1.20 [74]. Clustering was conducted using the fastgreedy community method [75]. GO term enrichment analyses were conducted using the GOseq package version 1.20.0 [76]. Functional enrichment tests were based on MAPMAN annotations. The list of TFs used in the enrich- ment tests were obtained from the “Grassius database” [77]. A list of Arabidopsis thaliana TFs was downloaded from “AGRIS” [78]. P values for TF enrichment were obtained from single-tailed Fisher tests. Page 12 of 14 Lin et al. Genome Biology (2017) 18:192 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 1Department of Agronomy, Iowa State University, 2035 B Roy J Carver Co-Lab, Ames, IA 50011-3650, USA. 2Interdepartmental Genetics and Genomics Graduate Program, Iowa State University, Ames, IA 50011-3650, USA. 3Department of Genetics, Developmental and Cellular Biology, Iowa State University, Ames, IA 50011-3650, USA. 4The Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, MA 02142-1403, USA. 5Department of Plant Sciences, University of California, Davis, CA 95616-5270, USA. 6Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502, USA. 7Department of Plant Genetics & Breeding, China Agricultural University, Beijing 100193, China. 8DATA Biotechnology Beijing Co. Ltd, Beijing 102206, China. 9Plant Biology Section, Cornell University, Ithaca, New York 14850, USA. 10Department of Statistics, Iowa State University, Ames, IA 50011-1210, USA. 11Department of Agronomy and Horticulture, University of Nebraska, Lincoln, Nebraska 68583-0660, USA. Additional file 4: Table S3 Enrichment test for TFs among nine different gene categories. (XLSX 35 kb) Additional file 5: Table S4. Accuracy of associations from SNP-BayesB and eRD-GWAS as estimated via tenfold cross-validation. (XLSX 41 kb) Additional file 6: Table S5. Characterization of five genes associated with DTA identified via eRD-GWAS with highest model frequencies. (XLSX 39 kb) Additional file 7: Table S6. Comparison of eQTL analyses for 13 traits and eRD genes with different GWAS results. (XLSX 43 kb) Additional file 8: Table S7. Phenotypes analyzed in this study. (XLSX 38 kb) Abbreviations DTA: Days to anthesis; eQTL: Expression quantitative trait locus; eRD- GWAS: Expression read depth GWAS; FGS: Filtered-gene set; GLM: General linear model; GO: Gene ontology; GRN: Gene regulatory network; G- SE: Genotype stable expression; G-VE: Genotype variable expression; GWAS: Genome-wide association study; LD: Linkage disequilibrium; MCMC: Markov chain Monte Carlo; MLM: Mixed linear model; NAM: Nested association mapping; PPIN: Protein–protein interaction network; RPKM: Reads per kilobase of transcript per million mapped reads; SAM: Shoot apical meristem; SNP: Single-nucleotide polymorphism; SPB: Squamosa promoter binding protein; TF: Transcription factor; T-SE: Tissue stable expression; T- VE: Tissue variable expression Received: 19 April 2017 Accepted: 27 September 2017 Received: 19 April 2017 Accepted: 27 September 2017 Funding Thi g This research was supported by grants from the National Science Foundation (NSF) to PSS and colleagues (IOS-1027527) and MJS, PSS, and colleagues (IOS-1238142) and from the National Institute of General Medical Sciences of the National Institutes of Health (grant number 1R01GM109458-01) to DN. 8. 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Functional enrichment and GO term enrichment tests. a Functional enrichment tests of genes that exhibit extreme expression variation across genotypes. b GO term enrichment tests of genes that exhibit extreme expression variation across genotypes. c Functional enrichment tests of genes that exhibit extreme expression variation across tissues. d GO term enrichment tests of genes that exhibit extreme expression variation across tissues. e Functional enrichment tests of genes that exhibit extreme expression variation across genotypes and tissues. f GO term enrichment tests of genes that exhibit extreme expression variation across genotypes and tissues. (XLSX 61 kb) Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 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Genome Biology (2017) 18:192 • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
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https://link.springer.com/content/pdf/10.1007/s00429-014-0732-x.pdf
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Lsamp is implicated in the regulation of emotional and social behavior by use of alternative promoters in the brain
Brain structure & function
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Brain Struct Funct (2015) 220:1381–1393 DOI 10.1007/s00429-014-0732-x ORIGINAL ARTICLE ORIGINAL ARTICLE Lsamp is implicated in the regulation of emotional and social behavior by use of alternative promoters in the brain Mari-Anne Philips • Kersti Lilleva¨li • Indrek Heinla • Hendrik Luuk • Christian Ansgar Hundahl • Karina Kongi • Taavi Vanaveski • Triin Tekko • Ju¨rgen Innos • Eero Vasar Received: 28 June 2013 / Accepted: 7 February 2014 / Published online: 15 March 2014  The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Limbic system-associated membrane protein (LSAMP) is a neural cell adhesion molecule involved in neurite formation and outgrowth. The purpose of the present study was to characterize the distribution of alter- natively transcribed Lsamp isoforms in the mouse brain and its implications on the regulation of behavior. Limbic system-associated membrane protein 1b transcript was visualized by using a mouse strain expressing beta-galac- tosidase under the control of Lsamp 1b promoter. The distribution of Lsamp 1a transcript and summarized expression of the Lsamp transcripts was investigated by non-radioactive in situ RNA hybridization analysis. Cross- validation was performed by using radioactive in situ hybridization with oligonucleotide probes. Quantitative RT-PCR was used to study correlations between the expression of Lsamp isoforms and behavioral parameters. The expression pattern of two promoters differs remarkably from the developmental initiation at embryonic day 12.5. Limbic system-associated membrane protein 1a promoter is active in ‘‘classic’’ limbic structures where the hippo- campus and amygdaloid area display the highest expres- sion. Promoter 1b is mostly active in the thalamic sensory nuclei and cortical sensory areas, but also in areas that regulate stress and arousal. Higher levels of Lsamp 1a transcript had significant correlations with all of the mea- sures indicating higher trait anxiety in the elevated plus- maze test. Limbic system-associated membrane protein transcript levels in the hippocampus and ventral striatum correlated with behavioral parameters in the social inter- action test. The data are in line with decreased anxiety and alterations in social behavior in Lsamp-deficient mice. We propose that Lsamp is involved in emotional and social operating systems by complex regulation of two alternative promoters. Keywords Lsamp  Alternative promoters  Limbic system  Sensory pathways  Anxiety  Social behavior Electronic supplementary material The online version of this article (doi:10.1007/s00429-014-0732-x) contains supplementary material, which is available to authorized users. Introduction M.-A. Philips (&)  K. Lilleva¨li  I. Heinla  H. Luuk C. A. Hundahl  K. Kongi  T. Vanaveski  T. Tekko  J. Innos  E. Vasar Department of Physiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu 50411, Estonia e-mail: maphilips@gmail.com M.-A. Philips (&)  K. Lilleva¨li  I. Heinla  H. Luuk  C. A. Hundahl  K. Kongi  T. Vanaveski  T. Tekko  J. Innos  E. Vasar Department of Physiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu 50411, Estonia e-mail: maphilips@gmail.com Limbic system-associated membrane protein (LSAMP) is a neural cell adhesion molecule expressed on the neuronal dendrites and somata (Zacco et al. 1990) on structures known to be especially important for emotional and motivational functions (Heimer and Van Hoesen 2006; Levitt 1984). Recently, LSAMP has been linked with a spectrum of psy- chiatric disorders in humans. The levels of the LSAMP protein have been found to be approximately 20 % increased in the postmortem frontal cortex both in patients with schizophrenia and bipolar disorder (Behan et al. 2009). Polymorphisms in the LSAMP gene have been associated K. Lilleva¨li Department of Developmental Biology, University of Tartu, Tartu, Estonia K. Lilleva¨li C. A. Hundahl Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark 123 1 3 Brain Struct Funct (2015) 220:1381–1393 1382 with depression (Koido et al. 2012) and LSAMP has been suggested to have a role in the neurobiology of male com- pleted suicide (Must et al. 2008). et al. 2003; Yamamoto and Reiner 2005), but the ana- tomical distribution of the alternative transcripts has not been reported. The purpose of the present study was to characterize the distribution of the two alternatively tran- scribed Lsamp isoforms in the mouse brain and to provide an initial analysis of the developmental activation of Lsamp promoters. Additionally, we investigated the relation between Lsamp expression and the regulation of emotional and social behavior. Functional studies have shown that LSAMP can promote or inhibit neurite outgrowth depending on interactions with other members of the IgLON family (Mann et al. 1998; Gil et al. 2002), indicating its prominent role in neurite for- mation and synaptogenesis (Hashimoto et al. 2009). Before second postnatal week of development, LSAMP is tran- siently also expressed in developing axons and growth cones (Horton and Levitt 1988) indicating importance in developing of the brain structures. However, the lack of obvious deviations in brain organization in both of the two independently created Lsamp-deficient mouse strains (Catania et al. 2008; Innos et al. 2011) suggests that LSAMP is mediating finely specialized aspects of circuit formation and maturation of the limbic system. Genetic deletion of the Lsamp gene in mice induced no detectable changes in sensory and motor development, but caused increased activity in novel environments and reduced anx- iety-like behavior in both knockout models (Catania et al. 2008; Innos et al. 2011). Increased trait anxiety in rats has been shown to be related with increased level of the Lsamp transcript in the amygdaloid area, periaqueductal gray (Nelovkov et al. 2006), raphe, hippocampus and frontal cortex (Alttoa et al. 2010). Elevated levels of the Lsamp transcript in the amygdaloid area of rats have been associ- ated with acute fear reaction (Koks et al. 2004) and fear conditioning (Lamprecht et al. 2009). The amino acid sequence of LSAMP is highly conserved among species. There is 99 % sequence identity between human and rodent LSAMP (Pimenta and Levitt 2004) and 91 % identity with chicken (Bru¨mmendorf et al. 1997), indicating remarkable phylogenetic conservation of protein structure and associ- ated functional properties. K. Lilleva¨li Growing evidence indicates that LSAMP is involved in the formation of anatomical sub- strate for emotional behavior both in rodents and humans. Non-radioactive in situ RNA hybridization analysis with digoxigenin-UTP Non-radioactive in situ RNA hybridization analysis with digoxigenin-UTP Non-radioactive in situ RNA hybridization analysis with digoxigenin-UTP Mouse Lsamp cDNA fragments were cloned from a cDNA pool from C57BL/6 mouse hippocampus and inserted into pGEM7-Zf(?) vector (Promega) to create an in situ probe. cDNA fragment specific for 1a promoter (400 bp) con- sisted of 1a-specific 50UTR, exon 1a and exon 1a0. Uni- versal Lsamp probe (567 bp) consisted of a cDNA fragment consisting of exons 2–6. RNA in situ hybridiza- tion on free-floating PFA-fixed 40 lm mouse brain cryo- sections using digoxigenin-UTP (Roche) labeled Lsamp sense and antisense RNA probes was performed as described previously (Braissant and Wahli 1998). As a major modification, active DEPC treatment was avoided and 0.25 % Triton X-100 was added to the PBS to improve probe penetration. Radioactive in situ hybridization with oligonucleotides Radioactive in situ hybridization with oligonucleotides Antisense 400mer DNA oligonucleotide probes comple- mentary to mouse Lsamp gene (Accession No. uc007zfr.1, UCSC Genome Browser, genome.ucsc.edu) Lsamp1a (50- accccagcacccagacgctgtgcagccagtaggtcctcat-30), Lsamp1b (50-gaagaaggcagagcagtctcagtaggaccagcggcaactg-30) and LsampUNI (50-agagcatggcgcttctccagctcaacccgagggtcca- gag-30) were labeled with 33P-dUTP by use of terminal transferase (Sigma-Aldrich, Europa). Free-floating in situ hybridization was carried out essentially as described pre- viously (Hundahl et al. 2010). Pimenta and Levitt (2004) reported revised genomic structure of the mouse Lsamp gene, demonstrating that, besides the well-known exon 1 (now referred as exon 1b), the Lsamp gene has an alternative exon 1 (currently exon 1a) located 1.6 Mbp upstream, and both of them have separate promoter sequences. The two-promoter structure is conserved and has been described in mouse, rat, human (Pimenta and Levitt 2004) and also in chicken (Bru¨m- mendorf et al. 1997). Limbic system-associated membrane protein distribution in the whole adult mammalian brain was originally described in rat using immunohistochemis- try (Levitt 1984) and in situ hybridization (Reinoso et al. 1996). The anatomical distribution of the LSAMP transcript and protein has been extensively described in various species (Chesselet et al. 1991; Coˆte´ et al. 1995; Prensa Lsamp-deficient mice with beta-galactosidase knock-in Lsamp-deficient mice with beta-galactosidase knock-in On the first day after 150-s acclimation period animals received six trials with the following stimuli: 15 s tone (12 kHz; 70 dB) and bright light (pulsing at 200 ms) were terminated by a 2-s electric shock (0.6 mA) during which the light was constant. Inter- trial interval was 120 s (±50 %). After the last trial the animals were returned to their home cages. On the second day, animals in the ‘‘Conditioned fear’’ group were placed into the conditioning cages and exposed to similar stimuli without an electric shock for about 45 min (20 trials). The animals were killed immediately afterwards. We chose a 45-min duration as it has been shown that neuronal stim- ulation induces gene expression between timepoints 30–60 min (Lamprecht et al. 2009). ‘‘Naı¨ve’’ and ‘‘Pre- conditioning’’ groups received no treatment on the second day. The hippocampus and temporal lobe (including the temporal cortex and the lateral, basolateral, central and medial nuclei of the amygdala) were dissected from the brains. Experimental animals for behavioral studies and quantitative real-time PCR analysis Male C57BL/6 strain mice were used for all behavioral studies. In the first part of the behavioral studies we investigated the correlations between general behavioral profile and Lsamp expression that was measured 10 days after behavioral testing. All mice (n = 15) were subjected to a battery of three behavioral tests: elevated plus-maze test (day 0), locomotor activity test (day 5), and social interaction test (day 10), with less invasive tests preceding more stressful tests. Testing began at age 60 days (2 months). The motility box was carried out as described previously (Innos et al. 2013) and four behavioral param- eters were recorded: (1) time spent moving in seconds (move, s); (2) distance travelled in meters (distance, m); (3) time spent in the center (time center, s); and (4) time spent in the corners (time corner, s). The plus-maze experiment was performed as described in Philips et al. (2010) and eight behavioral parameters were recorded: (1) the number of closed arm entries; (2) the number of open arm entries; (3) the ratio between open and closed arm entries; (4) the latency to enter open arm (latency, s); (5) time spent on open arms; (6) the number of protected head-dips; (7) the number of unprotected head-dips; and (8) the number of stretch-attend postures (SAPs). The social interaction test was carried out as described previously (Innos et al. Lsamp-deficient mice with beta-galactosidase knock-in Detailed description of the generation of the Lsamp-defi- cient mice with a LacZ transgene can be found in Innos et al. (2011). Briefly, exon 1b of mouse Lsamp gene was replaced by an in-frame NLSLacZNeo cassette resulting in insertion of gene encoding beta-galactosidase immediately after Lsamp 1b promoter. X-Gal staining for detecting the 123 123 Brain Struct Funct (2015) 220:1381–1393 1383 distribution of 1b promoter specifically produced beta- galactosidase and NeuN immunostaining was performed as described previously (Luuk et al. 2008). The brains were cut into 100 lm coronal sections and transferred to glass slides. The contours in Fig. 4c, f and abbreviations in all the figures representing anatomical data have been adopted from the mouse brain atlas (Franklin and Paxinos 1997). For embryonic brains, the embryos were dissected from timed matings as in Philips et al. (2009). E13.5 (embryonic day 13.5) and E15.5 brains and/or embryos were fixed in 4 % PFA/PBS at 4 C overnight for in situ hybridization, or for 30 min for X-gal staining. For cutting 50-lm vi- bratome sections, the stained (E13.5 and E15.5) specimens were inserted into 1 ml of 0.5 % gelatine/30 % BSA/20 % sucrose/PBS, wherein 140 ll of 25 % glutaraldehyde was added immediately before insertion and incubated for 10 min. The sections were mounted into 70 % glycerol and microphotographed. social sniffing, s). The animals were decapitated 10 days after the last experiment and three parts were dissected from the brains: the ventral striatum (including nucleus accumbens and olfactory tuberculi), hippocampus and temporal lobe (including temporal cortex and lateral, cen- tral and medial nuclei of amygdala). In the second part of the behavioral studies, we inves- tigated the influence of acute fear reaction on the expres- sional activity of the Lsamp gene. In order to induce acute fear reaction, fear conditioning was carried out by means of a computer-controlled Multi Conditioning System (TSE). Sixteen mice were divided into three groups: ‘‘Naı¨ve’’ (n = 5); ‘‘Pre-conditioning’’ (n = 5) and ‘‘Conditioned fear’’ (n = 6). Training for the ‘‘Conditioned fear’’ and ‘‘Pre-conditioning’’ groups was performed in a dimly illuminated (15 lx) acrylic cage (30 9 30 9 30 cm) with stainless steel rod floor. Between subjects the cages were cleaned with isopropanol. Lsamp-deficient mice with beta-galactosidase knock-in 2012), briefly: 14 mice were matched into 7 pairs of two unfa- miliar mice according to the bodyweight and (1) the time spent sniffing the partner’s anogenital area (anogenital sniffing, s) and (2) the time spent sniffing other body regions (sniffing other body parts, s) were recorded sepa- rately. These two measures were also summarized for (3) the time of total social sniffing for each animal (time of All the experiments were performed in accordance with the EU guidelines directive 86/609/EEC) and permit (No. 59, September 5, 2006) from the Estonian National Board of Animal Experiments. qRT-PCR analysis of Lsamp expression in mouse brain areas The tissue samples were frozen in liquid nitrogen. Dis- section of the mouse brain was performed according to coordinates obtained from the mouse brain atlas (Franklin and Paxinos 1997). Limbic system-associated membrane protein mRNA level was determined by quantitative real- time PCR (qRT-PCR). Total RNA was extracted individ- ually from each brain structure by using Trizol reagent (Invitrogen, USA) according to the manufacturer’s proto- col. First strand cDNA was synthesized by using Random 12 3 Brain Struct Funct (2015) 220:1381–1393 1384 Hexamers (Applied Biosystems) and SuperScriptTM III Reverse Transcriptase (Invitrogen, USA). TaqMan Assay was designed for the detection of 1a- and 1b-specific transcripts. FAM-MGB-probe AACCGAGGCACGGA CAAC was used with universal reverse primer combined with alternative forward oligos specific for either 1a iso- form or 1b isoform. For mRNA quantification of the immediate-early gene c-fos, a Taqman assay from Applied Biosystems was employed (Mm00487425_m1). The detailed sequences of primers and probe for housekeeping gene hypoxanthine guanine phosphoribosyl transferase (Hprt-1) have been described in Areda et al. (2005). Taq- Man Universal PCR Master Mix was used in the ABI Prism 7900HT Sequence Detection System (Applied Bio- systems, USA). Reactions were carried out in 10-ll reac- tion volumes in four replicates. Hexamers (Applied Biosystems) and SuperScriptTM III Reverse Transcriptase (Invitrogen, USA). TaqMan Assay was designed for the detection of 1a- and 1b-specific transcripts. FAM-MGB-probe AACCGAGGCACGGA CAAC was used with universal reverse primer combined with alternative forward oligos specific for either 1a iso- form or 1b isoform. For mRNA quantification of the immediate-early gene c-fos, a Taqman assay from Applied Biosystems was employed (Mm00487425_m1). The detailed sequences of primers and probe for housekeeping gene hypoxanthine guanine phosphoribosyl transferase (Hprt-1) have been described in Areda et al. (2005). Taq- Man Universal PCR Master Mix was used in the ABI Prism 7900HT Sequence Detection System (Applied Bio- systems, USA). Reactions were carried out in 10-ll reac- tion volumes in four replicates. (Me, Fig. 1f) and posterolateral (PLCo, Fig. 1e–g) and posteromedial (PMCo, Fig. 1g, h) cortical amygdaloid nuclei. Transcript 1b-specific X-Gal staining and in situ signal are much weaker in these areas. However, there is moderate 1b-specific staining in the central amygdaloid nucleus (Ce, Figs. 1i, 2l, 4f) and cortical amygdaloid nuclei (PLCo/PMCo, Fig. 1j–l). Expression of 1b isoform in the hippocampal formation is moderate and homogeneous (Figs. 1i–l, 2k, l; Fig. S1m). Lsamp 1b promoter activity is prevalent in the sensory nuclei and primary cortex areas Many of the sensory systems are distinguished by 1b pro- moter-specific staining. In the major afferent pathways for somatosensory information, intense Lsamp 1b-specific staining is seen in the ventral posterior lateral thalamic nucleus (VPL; Figs. 1i–k, 2k, l) and primary somatosensory cortex (S1, Figs. 1j, k, 2c, d, g, k). The expression signal is the highest in the barrel field (S1b, f, Figs. 1i, 2h, l). There is moderate 1b transcript-specific staining in the gracile, cuneate (Cu, supplementary Fig. S1s) and spinal trigeminal (Sp5, Fig. S1s) nuclei and strong staining in the laterodorsal (Bezdudnaya and Keller 2008) thalamic nucleus (LD, Fig. 1i). In the ascending auditory pathway there is strong 1b-specific staining in the dorsal and ventral cochlear nuclei (DC and VC, respectively, Fig. S1o, p) and moderate 1b- specific staining in the superior olivary complex (SOC, Fig. S1p) and trapezoid body (Tz, Fig. S1p). Isoform 1b staining is strong in the nuclei of lateral lemniscus (LL, Fig. S1n), in the inferior colliculus (IC, Fig. S1o), in the medial genicu- late nucleus (MG, Fig. 1l) and also in the primary and secondary (Au1/Au2, Fig. 1k, l) auditory cortex. In the visual pathway there is intensive 1b transcript-specific staining in the dorsal lateral geniculate thalamic nucleus (DLG, Fig. 1j, k; Fig. S1m) and primary visual cortex (V1, Fig. 1l; Fig. S1n). Strong 1b-specific X-Gal staining is also found in other brain areas receiving major projections from the retina: the superior colliculus (SC, Fig. S1n) and sup- rachiasmatic nucleus (SCh, Fig. S1i, j, l), and weak in the ventral lateral geniculate thalamic nucleus (VLG, Fig. 1j; Fig. S1m). In the sensory areas of the cortex, Lsamp 1b staining forms two distinct lines corresponding to layers 4 and 6 of the cortex (Lein et al. 2007) as estimated by comparing X-Gal staining (Fig. S1d) with NeuN immuno- reactivity in the cortex (Fig. S1c). Staining reflecting pro- moter 1a activity is weak in the sensory areas of the cortex (Fig. S1a), and summarized expression of both isoforms reveals two moderate but distinct lines (Fig. S1b). Data analysis The analysis of qRT-PCR data was performed as described earlier (Raud et al. 2009). Briefly, qRT-PCR data in Fig. 3 is presented in linear scale, calculated as 2-DCT, where DCT is the difference in cycle threshold (CT) between the target gene (Lsamp) and housekeeper gene Hprt-1 (VIC- MGB). All data were analyzed using Statistica version 8.0 (StatSoft, Inc., USA). As the behavioral scores were not normally distributed, Spearman’s rank-order method was used for the calculation of correlation coefficients. In the fear conditioning study, one-way ANOVA (conditioning type as grouping variable) was performed. Tukey HSD post hoc analysis was used when applicable after statistically significant ANOVA. Data are presented as mean ± SEM. qRT-PCR analysis of Lsamp expression in mouse brain areas qRT-PCR results confirm the prevalent expression of 1a transcript in the hippocampal area and temporal lobe (Fig. 4e). Lsamp 1b promoter activity is prevalent in the sensory nuclei and primary cortex areas Results Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures 1 Non-radioactive in situ RNA hybridization analysis with igoxigenin-UTP representing universal Lsamp transcript (a–d) or samp 1a transcript (e–h) and X-Gal staining expressing 1b promoter ctivity (i–l). AHi amygdalohippocampal area, Arc arcuate hypotha- amic nucleus, Au1/Au2 primary/secondary auditory cortex, BL/BM asolateral/basomedial amygdaloid nucleus, CA1/CA3 CA1/CA3 field f hippocampus, Ce central nucleus of amygdala, CM central medial halamic nucleus, CPu caudate putamen, DEn dorsal endopiriform ucleus, DG dentate gyrus, DLG/VLG dorsal/ventral lateral geniculate ucleus, DM dorsomedial hypothalamic nucleus, Ect ectorhinal ortex, Ins insular cortex, La lateral amygdaloid nucleus, LHb/MHb ateral/medial habenular nucleus, LD/MD laterodorsal/mediodorsal halamic nucleus, LP lateral posterior thalamic nucleus, LH lateral hypothalamic area, MB mammillary bodies, Me medial amygdalo nucleus, MG medial geniculate nucleus, Pe periventricular hypoth lamic nucleus, Pir piriform cortex, PF parafascicular thalam nucleus, PLCo/PMCo posterolateral/posteromedial cortical amygd loid nucleus, Po posterior thalamic nuclear group, RSA/RSG retr splenial agranular/granular cortex, S1/S2 primary/seconda somatosensory cortex, S1BF S1, barrel field, SNC/SNR substant nigra, compact/reticular part, STh subthalamic nucleus, TeA tempor association cortex, V1/V2 primary/secondary visual cortex, VMH VMHDM/VMHVL ventromedial thalamic nucleus, dorsomedial pa ventrolateral part, VPM/VPL ventral posteromedial/posterolater thalamic nucleus, VTM ventral tuberomammillary nucleus. Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures Sca bar 1 mm BL- -Ce VMH VPL Pir- DEn- MD S1BF PLCo CA1 CA3 RSA -La VPM BM -Me Ect S2 CM LD In situ hybridization (1a+1b) a CA1 DG MG PMCo SNC d AHi BL MB V1 Au1 Au2 Au2 V2 TeA BL PF CA1 DG DLG Po VPL VPM Pir- PLCo CPu -Ce -La DEn- DM BM RSG Au2 b LP Me S1 S2 Ect STh CA1 DG RSG PMCo AHi PLCo BM BL DEn- LH TeA c MB Au2 VPM Po DLG Pir- V1 Au2 V2 Au1 d c BL- -Ce Pir - CM MHb- PLCo RSG -La CA1 e VMHDM CA3- BM Me Ect VMHVL In situ hybridization (1a) PF CA3 CA1 Pir- PLCo -La -Me RSG Ect DM TeA f BL- Au2 BM LH STh CPu PMCo CA1 TeA Pir- AHi h BL MB La- CA1 DG CA3 -La PLCo AHi PMCo RSG LH TeA Pir- g MB AHi BL f e g h -Ce LD DEn- IAM MHb S1BF RSA RSG S1 Pe- VMH i Pir- MD Po S2 LHb X-Gal-staining (1b) VPM VPL BL PF VPM Po DLG- VLG- VPL Pir- CPu DEn- Me S1 S2 Au2 Arc- MHb RSG RSA Ce LP j PMCo PLCo STh -MG SNC SNR Au2 V1 Au1 V2 V2 l PMCo Au2 DEn- VTM Au2 LP DLG- Au1 S1 V1 RSA VPL VPM k RSG PMCo Arc Au2 l k PMCo VTM Arc hypothalamic area, MB mammillary bodies, Me medial amygdaloid nucleus, MG medial geniculate nucleus, Pe periventricular hypotha- lamic nucleus, Pir piriform cortex, PF parafascicular thalamic nucleus, PLCo/PMCo posterolateral/posteromedial cortical amygda- loid nucleus, Po posterior thalamic nuclear group, RSA/RSG retro- splenial agranular/granular cortex, S1/S2 primary/secondary somatosensory cortex, S1BF S1, barrel field, SNC/SNR substantia nigra, compact/reticular part, STh subthalamic nucleus, TeA temporal association cortex, V1/V2 primary/secondary visual cortex, VMH, VMHDM/VMHVL ventromedial thalamic nucleus, dorsomedial part/ ventrolateral part, VPM/VPL ventral posteromedial/posterolateral thalamic nucleus, VTM ventral tuberomammillary nucleus. Scale bar 1 mm Fig. 1 Non-radioactive in situ RNA hybridization analysis with digoxigenin-UTP representing universal Lsamp transcript (a–d) or Lsamp 1a transcript (e–h) and X-Gal staining expressing 1b promoter activity (i–l). Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures AHi amygdalohippocampal area, Arc arcuate hypotha- amic nucleus, Au1/Au2 primary/secondary auditory cortex, BL/BM asolateral/basomedial amygdaloid nucleus, CA1/CA3 CA1/CA3 field f hippocampus, Ce central nucleus of amygdala, CM central medial halamic nucleus, CPu caudate putamen, DEn dorsal endopiriform ucleus, DG dentate gyrus, DLG/VLG dorsal/ventral lateral geniculate ucleus, DM dorsomedial hypothalamic nucleus, Ect ectorhinal ortex, Ins insular cortex, La lateral amygdaloid nucleus, LHb/MHb ateral/medial habenular nucleus, LD/MD laterodorsal/mediodorsal halamic nucleus, LP lateral posterior thalamic nucleus, LH lateral hypothalamic area, MB mammillary bodies, Me medial amygdalo nucleus, MG medial geniculate nucleus, Pe periventricular hypoth lamic nucleus, Pir piriform cortex, PF parafascicular thalam nucleus, PLCo/PMCo posterolateral/posteromedial cortical amygd loid nucleus, Po posterior thalamic nuclear group, RSA/RSG ret splenial agranular/granular cortex, S1/S2 primary/seconda somatosensory cortex, S1BF S1, barrel field, SNC/SNR substan nigra, compact/reticular part, STh subthalamic nucleus, TeA tempo association cortex, V1/V2 primary/secondary visual cortex, VM VMHDM/VMHVL ventromedial thalamic nucleus, dorsomedial pa ventrolateral part, VPM/VPL ventral posteromedial/posterolate thalamic nucleus, VTM ventral tuberomammillary nucleus. Sca bar 1 mm Brain Struct Funct (2015) 220:1381–1393 13 Brain Struct Funct (2015) 220:1381–1393 1385 BL- BL- BL BL -Ce -Ce PF PF PF -Ce VMH VPL LD DEn- Pir - Pir- DEn- IAM CM CA1 MD MHb- MHb S1BF S1BF PLCo PLCo RSG RSA RSG S1 Pe- VPM Po DLG- VLG- VPL CA3 CA1 DG DLG Po VPL VPM Pir- PLCo Pir- Pir- PLCo PMCo CA1 CA3 RSA -La -La CA1 CA1 CA1 CA1 CA1 DG DG DG CA3 MG -MG -La -Me CPu CPu -Ce -La DEn- DEn- RSG RSG Me DM S1 S2 Au2 Arc- MHb RSG RSA Ect DM VMH BM Ce -La DEn- PMCo AHi RSG PLCo PLCo AHi PMCo RSG PMCo BM BL DEn- LH LH VTM Au2 Au2 TeA TeA TeA TeA Pir- Pir- SNC SNR SNC LP LP DLG- Au1 Au2 S1 V1 RSA V1 Au1 V2 V2 AHi VPL VPM b c d g h e f i j k l VMHDM BL- MB MB VPM BM CA3- BM -Me Me Pir- LP MD Po S2 Ect Ect S2 CM Me S1 S2 Au2 Ect BM PMCo PLCo Au2 VPM Po DLG Pir- AHi RSG PMCo Arc AHi BL BL BL MB MB Au2 PMCo V1 V1 Au1 Au2 Au2 Au2 V2 V2 TeA Au2 LH STh STh STh LHb LD VMHVL Au1 La- X-Gal-staining (1b) In situ hybridization (1a) In situ hybridization (1a+1b) VPM VPL a CPu ig. Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures Limbic system-associated membrane protein 1a transcript is intensively and specifically expressed in the brain areas that are commonly considered to be limbic structures (Heimer and Van Hoesen 2006; Morgane et al. 2005). Transcript 1a-specific staining is pronounced in the cin- gulate cortex (Cg, Fig. 2b, f; supplementary Fig. S1e), insular cortex (Ins, Fig. 2b, f; Fig. S1e, f), prelimbic cortex (PrL, Fig. S1e) and infralimbic cortex (IL, Fig. S1e). Extensive 1a-specific staining can be seen in the hippo- campal formation (CA1, CA3 and DG; Figs. 1e–h, 2j), amygdalohippocampal area (AHi, Fig. 1g, h), lateral amygdaloid nucleus (La, Figs. 1e–g, 2j, 4c), basolateral (BL, Fig. 1e–h and BLA, Fig. 4c) and basomedial (BM, Fig. 1e, f) amygdaloid nuclei, medial amygdaloid nucleus Both 1a and 1b promoters are expressed in brain areas involved in the processing of gustatory and olfactory 123 123 BL- BL- BL BL -Ce -Ce PF PF PF -Ce VMH VPL LD DEn- Pir - Pir- DEn- IAM CM CA1 MD MHb- MHb S1BF S1BF PLCo PLCo RSG RSA RSG S1 Pe- VPM Po DLG- VLG- VPL CA3 CA1 DG DLG Po VPL VPM Pir- PLCo Pir- Pir- PLCo PMCo CA1 CA3 RSA -La -La CA1 CA1 CA1 CA1 CA1 DG DG DG CA3 MG -MG -La -Me CPu CPu -Ce -La DEn- DEn- RSG RSG Me DM S1 S2 Au2 Arc- MHb RSG RSA Ect DM VMH BM Ce -La DEn- PMCo AHi RSG PLCo PLCo AHi PMCo RSG PMCo BM BL DEn- LH LH VTM Au2 Au2 TeA TeA TeA TeA Pir- Pir- SNC SNR SNC LP LP DLG- Au1 Au2 S1 V1 RSA V1 Au1 V2 V2 AHi VPL VPM b c d g h e f i j k l VMHDM BL- MB MB VPM BM CA3- BM -Me Me Pir- LP MD Po S2 Ect Ect S2 CM Me S1 S2 Au2 Ect BM PMCo PLCo Au2 VPM Po DLG Pir- AHi RSG PMCo Arc AHi BL BL BL MB MB Au2 PMCo V1 V1 Au1 Au2 Au2 Au2 V2 V2 TeA Au2 LH STh STh STh LHb LD VMHVL Au1 La- X-Gal-staining (1b) In situ hybridization (1a) In situ hybridization (1a+1b) VPM VPL a CPu Fig. 1 Non-radioactive in situ RNA hybridization analysis with igoxigenin-UTP representing universal Lsamp transcript (a–d) or Lsamp 1a transcript (e–h) and X-Gal staining expressing 1b promoter ctivity (i–l). Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures AcbC accumbens nucleus, core, AVPe anteroventral periventricular hypothalamic nucleus, BAC bed nucleus of the anterior commissure, BST bed nucleus of stria terminalis, Ce central nucleus of amygdala, Cg cingulate cortex, DP dorsal peduncular cortex, DM dorsomedial hypothalamic nucleus, Ins insular cortex, LSD/LSV lateral septal nucleus, dorsal part/ventral part, LSS lateral stripe of striatum, LD/MD laterodorsal/mediodorsal thalamic nucleus, MPA medial preoptic area, Pir piriform cortex, PV paraventricular thalamic nucleus, S1/S2 primary/secondary somato- sensory cortex, S1BF S1, barrel field, SFi septofimbrial nucleus, Tu olfactory tubercle, VPM/VPL ventral posteromedial/posterolateral thalamic nucleus. Scale bar 1 mm Radioactive in situ hybridization with oligonucleotides Radioactive in situ hybridization with oligonucleotides -S1 DP AcbC Pir- d 1b X-Gal-staining VPM -SFi -LSD -SFi -LSD -AVPe -AVPe - BAC S2 - Pir MD -MHb -MHb -LD S1BF RS DMC- Ce- DEn- VPL -PV PLCo- -S1 -S1 - Pir des -LSV LSV- DP -LSS AcbC Pir- VMH S1BF Pir- S1BF MD d h l c g k 1b 1b X-Gal-staining peduncular cortex, DM dorsomedial hypothalamic nucleus, Ins insular cortex, LSD/LSV lateral septal nucleus, dorsal part/ventral part, LSS lateral stripe of striatum, LD/MD laterodorsal/mediodorsal thalamic nucleus, MPA medial preoptic area, Pir piriform cortex, PV paraventricular thalamic nucleus, S1/S2 primary/secondary somato- sensory cortex, S1BF S1, barrel field, SFi septofimbrial nucleus, Tu olfactory tubercle, VPM/VPL ventral posteromedial/posterolateral thalamic nucleus. Scale bar 1 mm -S1 - Pir c 1b c 1b Ins Cg AcbC b 1a - Tu 1b 1a Universal Cg Ins AcbC a b 1a a b MPA - Pir - Tu MPA- -LSD Cg -LSV Ins S2 S1- -Cg -LSD -BST -BST Ins LSV- -SFi e f -SFi -LSD -AVPe - BAC S2 - Pir LSV- -LSS S1BF h MPA- -LSD -LSV S2 S1- -Cg -BST Ins e MPA - Pir - Tu Cg Ins -LSD -BST LSV- -SFi f -LSD -SFi -AVPe -LSV Pir- S1BF g h g e VPMMD -MHb -LD S1BF RS DMC- Ce- DEn- VPL -PV PLCo- l VMH MD i -DM VMH PlCo PV La j l -MHb VMH MD k k Fig. 2 Radioactive in situ hybridization with oligonucleotides. Universal probe (a, e, i); 1a transcript-specific probe (b, f, j) or 1b specific probe (c, g, k) has been used and complementary X-Gal staining in respective brain areas representing 1b-specific staining (d, h, l) has been shown. Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures AHi amygdalohippocampal area, Arc arcuate hypotha- lamic nucleus, Au1/Au2 primary/secondary auditory cortex, BL/BM basolateral/basomedial amygdaloid nucleus, CA1/CA3 CA1/CA3 field of hippocampus, Ce central nucleus of amygdala, CM central medial thalamic nucleus, CPu caudate putamen, DEn dorsal endopiriform nucleus, DG dentate gyrus, DLG/VLG dorsal/ventral lateral geniculate nucleus, DM dorsomedial hypothalamic nucleus, Ect ectorhinal cortex, Ins insular cortex, La lateral amygdaloid nucleus, LHb/MHb lateral/medial habenular nucleus, LD/MD laterodorsal/mediodorsal thalamic nucleus, LP lateral posterior thalamic nucleus, LH lateral information. In the gustatory system, 1b-specific staining is strong in the ventral posteromedial nucleus (VPM, Fig. 1i– k) and weak in the solitary nucleus (Sol, supplementary Fig. S1s). In the insular cortex only 1a isoform is expressed (Ins, Fig. 2b, f; Fig. S1e, f). In the olfactory system, the activity of 1b promoter is remarkable in the mediodorsal Fig. S1s). In the insular cortex only 1a isoform is expressed (Ins, Fig. 2b, f; Fig. S1e, f). In the olfactory system, the activity of 1b promoter is remarkable in the mediodorsal information. In the gustatory system, 1b-specific staining is strong in the ventral posteromedial nucleus (VPM, Fig. 1i– k) and weak in the solitary nucleus (Sol, supplementary 123 12 3 Brain Struct Funct (2015) 220:1381–1393 1386 Universal MPA VPM - Pir - Tu -SFi MPA- -LSD -SFi -LSD -LSD Cg -LSV Ins -AVPe -AVPe - BAC S2 S2 S1- -Cg -LSD -BST -BST - Pir MD -MHb -MHb -LD S1BF RS DMC- Ce- DEn- VPL -PV PLCo- -DM VMH VMH -S1 Ins Cg Cg -S1 Ins Ins - Pir Radioactive in situ hybridization with oligonucleotides LSV- -LSV LSV- -SFi DP MD PlCo -LSS AcbC AcbC Pir- AcbC VMH S1BF Pir- S1BF PV MD La a d e h i l c b g f k j 1a 1b 1b X-Gal-staining - Tu Fig. 2 Radioactive in situ hybridization with oligonucleotides. Universal probe (a, e, i); 1a transcript-specific probe (b, f, j) or 1b specific probe (c, g, k) has been used and complementary X-Gal staining in respective brain areas representing 1b-specific staining (d, h, l) has been shown. Lsamp 1a promoter activity predominates in ‘‘classic’’ limbic structures AcbC accumbens nucleus, core, AVPe anteroventral periventricular hypothalamic nucleus, BAC bed nucleus of the anterior commissure, BST bed nucleus of stria terminalis, Ce central nucleus of amygdala, Cg cingulate cortex, DP dorsal peduncular cortex, DM dorsomedial hypothalamic nucleus, Ins insular cortex, LSD/LSV lateral septal nucleus, dorsal part/ventral part, LSS lateral stripe of striatum, LD/MD laterodorsal/mediodorsal thalamic nucleus, MPA medial preoptic area, Pir piriform cortex, PV paraventricular thalamic nucleus, S1/S2 primary/secondary somato- sensory cortex, S1BF S1, barrel field, SFi septofimbrial nucleus, Tu olfactory tubercle, VPM/VPL ventral posteromedial/posterolateral thalamic nucleus. Scale bar 1 mm nucleus, namely in the ventrolateral (VMHVL, Fig. 1e) and dorsomedial parts (VMHDM, Fig. 1e); weak 1b-specific staining is present in the anterior part of the VMH (Fig. 2k). Promoter 1a is active in the dorsomedial hypothalamic nucleus (DM, Fig. 1f), while the activity of 1b promoter is limited to the compact part of the DM (DMC, Fig. 2l). Strong 1a promoter-specific expression can be seen in the medial preoptic area (MPA, Fig. 2b), including medial preoptic nucleus, and also in the ventromedial preoptic nucleus (VMPO, Fig. S1f). Promoter 1a-specific staining is moderate in the anterolateral (LA, Fig. S1k) and lateral hypothalamus (LH, Fig. 1f, g; Fig. S1k, l). Strong 1b pro- moter-specific expression can be detected in the periven- tricular hypothalamic nucleus (Pe, Fig. 1i; Fig. S1l), anteroventral periventricular nucleus (AVPe, Fig. 2h), sup- rachiasmatic nucleus (SCh, Fig. S1i, j, l), supraoptic nucleus (SO, Fig. S1l) and arcuate nucleus (Arc, Fig. 1j, k). (Tham et al. 2009) thalamic nucleus (MD, Figs. 1i, 2k, l), and prevalent in the olfactory bulb (data not shown) and entorhinal cortex (Ent, Fig. S1n). The expression of 1a promoter is distinct in the nucleus of the lateral olfactory tract (LOT, Fig. S1k), and it dominates over 1b signal in the olfactory tubercle (Tu, Fig. 2b) and piriform cortex (Pir, Fig. 1e–h). Differences in the activity of Lsamp 1a and 1b promoters in adult brain Differences in the activity of Lsamp 1a and 1b promoters in adult brain Differences in the activity of Lsamp 1a and 1b promoters in adult brain Thalamic and hypothalamic nuclei are distinguished by the isoform-specific expression of Lsamp. Only two hypotha- lamic nuclei display activity for both 1a and 1b promoters: the paraventricular nucleus (Pa, supplementary Fig. S1k, l) and mammillary bodies (MB, Fig. 1c, g, k). High expression of 1a isoform is seen in the ventromedial hypothalamic 123 123 1387 Brain Struct Funct (2015) 220:1381–1393 Promoter 1a is specifically active in the anterior thala- mus: the anteroventral thalamic nucleus (AV, supplemen- tary Fig. S1g), reticular thalamic nucleus (Rt, Fig. S1k) and central medial thalamic nucleus (CM, Fig. 1e). Weak 1a- specific expression can be detected in the anteromedial thalamic nucleus (AM, Fig. S1g). Isoform 1b-specific staining can be seen in the sensory thalamic nuclei as described above, but also in the posterior thalamic nuclei (Po, Fig. 1i, j), lateral habenular nucleus (LHb, Fig. 1i), lateral posterior thalamic nucleus (LP, Fig. 1j, k), parate- nial thalamic nucleus (PT, Fig. S1h) and reuniens thalamic nucleus (Re, Fig. S1l). There are numerous thalamic nuclei where both 1a and 1b promoters are active: the antero- dorsal thalamic nucleus (AD, Fig. S1g, h), paraventricular thalamic nucleus (PV, Fig. S1k, l), paracentral thalamic nucleus (PC, Fig. S1g, h), parafascicular thalamic nucleus (PF, Fig. 1b, f, j), medial habenular nucleus (Mhb, Fig. 1a, e, i) and subthalamic nucleus (STh, Fig. 1b, f, j). midbrain, being prominent in the outer layers of the neural tube (Fig. 3a, b), in the forebrain the first signs were detected at around E13.5 also in the outer surface of the neuroepithelium. The first signs of 1b transcript expression were detected in the lateral side of the lateral ventricle (Fig. 3d, e). During later embryonic develop- ment (E15.5), strong signal is detectable also in the lining of the aqueduct and in the deepest layers of the sensory region of the neocortex (S1, Fig. 3f). At E15.5, Lsamp 1a promoter is especially active in the caudate putamen (CPu, Fig. 3c), whereas this activity shades off during the first postnatal week (data not shown) and is not detectable in adult brain (CPu, Fig. 1f). The expression of Lsamp 1b promoter in the CPu is weak during development [as shown in E15,5; CPu (Fig. 3f) and moderate in adulthood (CPu, Fig. 1j)]. Differences in the activity of Lsamp 1a and 1b promoters in adult brain Expression of Lsamp transcripts correlates with behavioral measures of trait anxiety and social interaction ) ( g j) Alternative expression of Lsamp 1a and 1b promoters can be seen throughout the brain. Both 1a and 1b promoters are active in the dorsal (LSD, Fig. 2e–h) and ventral (LSV, Fig. 2e–h) part of the lateral septal nucleus, septofimbrial nucleus (SFi, Fig. 2f–h), subfornical organ (SFO, Fig. S1g, h) and retrosplenial granular cortex (RSG, Fig. 1e–g, i–k), whereas only 1b promoter is active in the retrosplenial agranular cortex (RSA, Fig. 1i–k). On the level of anterior commissure, 1b promoter is active in the bed nucleus of anterior commissure (BAC, Fig. 2h); 1a transcript is pre- valent in the bed nuclei of stria terminalis (BST, Fig. 2f, Fig. S1f) and in the core of the nucleus accumbens (AcbC, Fig. 2b). Isoform 1b-specific staining is present in the dorsal peduncular cortex (DP, Fig. 2d), caudate putamen (CPu, Fig. 1j), dorsal endopiriform nucleus (Den, Fig. 1i– k), claustrum (Cl, Fig. 2h) and lateral stripe of striatum (LSS, Fig. 2h). The expression of the Lsamp transcript in the cerebellum is mostly initiated from promoter 1b, which is abundant in the Purkinje cell layer (Pc, Fig. S1r). There is moderate 1b expression in the molecular layer (Mc, Fig. S1r) and weak 1a expression in the granule cells (Gc, Fig. S1q). Limbic system-associated membrane protein expression is moderate in the spinal cord: both 1a (Fig. S1t) and 1b (Fig. S1u) isoforms are expressed in the ventral and dorsal horns. A detailed overview of the estimated inten- sities of Lsamp 1a, 1b and summarized transcripts in dif- ferent brain areas has been presented in supplementary Table S2. We looked for the correlations between various behavioral parameters and the activity of Lsamp transcript expression in three brain areas (ventral striatum, hippocampus and temporal lobe). Most of the significant correlations emerged between Lsamp expression in the temporal cortex and behavioral parameters in the elevated plus maze (Table 1). Both Lsamp 1a and 1b transcript levels were negatively correlated with time on open arms in the ele- vated plus maze (Fig. 4a). Furthermore, Lsamp 1a tran- script levels were negatively correlated with unprotected headdips in the elevated plus maze (Fig. 4b), with the number of open arm entries and with the ratio between open and closed arm entries. Differences in the activity of Lsamp 1a and 1b promoters in adult brain There was also a significant positive correlation between Lsamp 1a transcript levels and the latency to enter open arm. Additionally, Lsamp transcript levels in the hippocampus and ventral striatum correlated with behavioral parameters in the social inter- action test. Both Lsamp 1a and 1b transcript levels were negatively correlated with the time that the mice spent sniffing the other animal. The time of anogenital sniffing positively correlated with Lsamp 1a activity in the ventral striatum. In the experiment for acute fear response, all the mice in the ‘‘Conditioned fear’’ group displayed an obvious fear reaction as evidenced by startling response and freezing (data not shown). The ‘‘Conditioned fear’’ group had sig- nificantly higher c-Fos expression in the amygdala [F(2,13) = 11.6, p \ 0.01] and hippocampus [F(2,13) = 8.8, p \ 0.01] than the ‘‘Naı¨ve’’ or ‘‘Pre-conditioning’’ groups (Fig. 3d). Furthermore, in the ‘‘Conditioned fear’’ group, the c-Fos activation in the temporal lobe was significantly higher than in the hippocampus (p \ 0.05), indicating Differences in the activity of Lsamp 1a and 1b promoters in embryonic brain We detected the first signals for both promoters of the Lsamp gene at around E12.5. Limbic system-associated membrane protein 1a transcript is firstly activated in the 123 12 3 3 1388 Brain Struct Funct (2015) 220:1381–1393 a d E13.5 E13.5 Eye lv lv lv lv lv lv mb mb tc tc CPu GP MeA III v III v IV v III v mb GE Pir sc sc lv GP S1 S1 c b f e E15.5 E13.5 E15.5 E13.5 In situ hybridization (1a) X-Gal-staining (1b) Fig. 3 Distribution of Lsamp 1a and 1b transcripts during develop- ment. Non-radioactive in situ RNA hybridization analysis represent- ing Lsamp 1a transcript (a–c) and X-Gal staining expressing 1b promoter activity (d–f) in the embryonic mouse brain. CPu caudate putamen, GE ganglionic eminence, GP globus pallidus, lv lateral ventricle, mb midbrain, Pir piriform cortex, S1 primary somatosen- sory cortex, sc spinal cord, tc telencephalon, III v third ventricle, IV v fourth ventricle. Dashed lines in a and d represent approximate cross- sections for b and e, respectively. Scale bar 1 mm lv IV v III v mb GE b E13.5 a E13.5 mb tc sc In situ hybridization (1a) lv lv CPu GP MeA III v Pir S1 c E15.5 b b a d E13.5 mb tc sc X-Gal-staining (1b) Eye lv lv e E13.5 lv lv III v GP S1 f E15.5 e e f d d ventricle, mb midbrain, Pir piriform cortex, S1 primary somatosen- sory cortex, sc spinal cord, tc telencephalon, III v third ventricle, IV v fourth ventricle. Dashed lines in a and d represent approximate cross- sections for b and e, respectively. Scale bar 1 mm Fig. 3 Distribution of Lsamp 1a and 1b transcripts during develop- ment. Non-radioactive in situ RNA hybridization analysis represent- ing Lsamp 1a transcript (a–c) and X-Gal staining expressing 1b promoter activity (d–f) in the embryonic mouse brain. Table 1 Correlation analysis between relative expression levels (2-DCT) of Lsamp 1a and 1b transcript in three brain areas and behavioral parameters in the motility box, elevated plus- maze and social interaction test Differences in the activity of Lsamp 1a and 1b promoters in embryonic brain CPu caudate putamen, GE ganglionic eminence, GP globus pallidus, lv lateral ifi i i f h d l id i Di i Table 1 Correlation analysis between relative expression levels (2-DCT) of Lsamp 1a and 1b transcript in three brain areas and behavioral parameters in the motility box, elevated plus- maze and social interaction test The behavioral measures have been presented in either counts, seconds (s) or meters (m) * p \ 0.01, ** p \ 0.05 (Spearman’s rank-order correlation) Hippocampus Temporal lobe Ventral striatum 1A 1B 1A 1B 1A 1B Motility box Move, s -0.01 -0.06 0.11 0.06 0.12 -0.34 Distance, m -0.06 -0.16 0.19 0.08 0.05 -0.30 Time center, s -0.40 -0.36 0.43 0.36 0.05 0.03 Time corner, s 0.21 0.33 -0.38 -0.10 -0.13 0.17 Elevated plus maze Closed arm entries -0.20 -0.43 0.17 -0.02 0.08 -0.14 Open arm entries 0.16 0.16 -0.57* -0.40 0.01 -0.34 Ratio open/closed arm entries 0.08 0.13 -0.63 * -0.38 -0.11 -0.34 Latency, s 0.03 -0.01 0.53* 0.22 -0.15 0.45 Time on open arms, s 0.24 0.15 -0.65** -0.66** 0.14 -0.21 Protected headdips 0.17 0.08 -0.06 -0.06 -0.12 -0.26 Unprotected headdips 0.10 0.12 -0.61* -0.50 0.20 -0.40 SAPs -0.05 0.10 0.24 0.23 0.11 0.17 Social interaction test Anogenital sniffing, s -0.10 -0.22 -0.09 -0.26 0.54* 0.11 Sniffing other body parts, s -0.67** -0.65* 0.06 0.15 -0.19 -0.07 Time of social sniffing, s -0.51 -0.60* -0.01 -0.09 0.14 -0.06 specific activation of the amygdaloid area in response to conditioned fear. There were no statistically significant changes in the expression levels of Lsamp 1a of 1b tran- scripts at the 45 min timepoint (Fig. 3e). specific activation of the amygdaloid area in response to conditioned fear. There were no statistically significant changes in the expression levels of Lsamp 1a of 1b tran- scripts at the 45 min timepoint (Fig. 3e). Discussion Limbic system-associated membrane protein has been linked with a wide spectrum of psychiatric disorders in 123 Brain Struct Funct (2015) 220:1381–1393 1389 a Expression of c-Fos transcript Expression of Lsamp transcripts Lsamp 1b transcript in temporal lobe 0 2 4 6 8 10 12 14 16 18 20 22 24 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 r = -0.65 (p<0.01) r = -0.61 (p<0.05) Lsamp 1a transcript in temporal lobe d 0 1 2 3 4 5 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Naive Pre- Conditioned conditioning Fear Naive Pre- Conditioned conditioning Fear - 1a transcript - 1b transcript Tem Hip Tem Hip Tem Hip - Temporal lobe - Hippocampus DEn- DEn- BLA MePD MePD BLA BMA BMA Ce Ce La La Time (sec) in open arm in plus maze Unprotected headdips (count) in plus maze c b f e 1a transcript in temporal lobe Relative expression 1a transcript in temporal lobe Relative expression Fig. 4 Lsamp expression in temporal lobe correlates with measures of trait anxiety (a, b), but was not altered 45 min after acute conditioned fear experience (e) that significantly raised c-Fos transcript in the temporal lobe and hippocampal area (d). Distribution of Lsamp 1a (c) and 1b (f) in the temporal lobe. Dashed lines indicate 95 % confidence limits (a, b). The whiskers represent SEM; *p \ 0.01; **p \ 0.05 (d, e). BLA basolateral amygdaloid nucleus, anterior part, BMA basomedial amygdaloid nucleus, anterior part, Ce central amygdaloid nucleus, Den dorsal endopiriform nucleus, Hip hippocampus, La lateral amygdaloid nucleus, MePD medial amyg- daloid nucleus, posterodorsal part, Tem temporal lobe. Discussion Scale bar 500 lm (c and f) r = -0.61 (p<0.05) 0 1 2 3 4 5 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Unprotected headdips (count) in plus maze b 1a transcript in temporal lobe Lsamp 1a transcript in temporal lobe DEn- MePD BLA BMA Ce La e c c a 0 2 4 6 8 10 12 14 16 18 20 22 24 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 r = -0.65 (p<0.01) Time (sec) in open arm in plus maze 1a transcript in temporal lobe b e a Lsamp 1a transcript in temporal lobe Expression of Lsamp transcripts Naive Pre- Conditioned conditioning Fear - 1a transcript - 1b transcript Tem Hip Tem Hip Tem Hip f e Relative expression Expression of c-Fos transcript d Naive Pre- Conditioned conditioning Fear - Temporal lobe - Hippocampus Relative expression Lsamp 1b transcript in temporal lobe DEn- BLA MePD BMA Ce La f e d f Relative expression Lsamp 1b transcript in temporal lobe Expression of Lsamp transcripts Fig. 4 Lsamp expression in temporal lobe correlates with measures of trait anxiety (a, b), but was not altered 45 min after acute conditioned fear experience (e) that significantly raised c-Fos transcript in the temporal lobe and hippocampal area (d). Distribution of Lsamp 1a (c) and 1b (f) in the temporal lobe. Dashed lines indicate 95 % confidence limits (a, b). The whiskers represent SEM; *p \ 0.01; **p \ 0.05 (d, e). BLA basolateral amygdaloid nucleus, anterior part, BMA basomedial amygdaloid nucleus, anterior part, Ce central amygdaloid nucleus, Den dorsal endopiriform nucleus, Hip hippocampus, La lateral amygdaloid nucleus, MePD medial amyg- daloid nucleus, posterodorsal part, Tem temporal lobe. Scale bar 500 lm (c and f) insular cortex. Promoter 1a is active in the anterior tha- lamic nuclei that have been specified as ‘‘limbic thalamus’’ (Vogt and Gabriel 1993; Marchand et al. 2013) and in the anterior hypothalamus, including preoptic area that is a major interacting structure of the limbic system (Morgane et al. 2005). Promoter 1b of the Lsamp gene is notably active in the sensory pathways ranging from the brainstem and sensory nuclei in the thalamus and up to the primary sensory areas in the cortex. In the cerebral cortex, the signal from 1b promoter can be seen in layers 4 and 6 of the cortex (supplementary Fig. Discussion S1d) emphasizing system- atic expression in the areas involved in the processing of sensory input. Layer 4 is the primary recipient of sensory input from the thalamus (Liao and Lee 2012) and most thalamic relay neurons receive feedback from layer 6 of the same cortical column they innervate (Lam and Sherman 2010). The specific activity of 1b promoter is obvious in the sensory pathways in visual, auditory and somatosen- sory areas. However, both 1a and 1b promoters are active in the neural pathways transmitting olfactory and gustatory information. This finding can be anticipated as brain humans and with behavioral alterations in rodents. The limbic system-specific neuroanatomical distribution of the Lsamp transcript and protein has been described in various species (Chesselet et al. 1991; Coˆte´ et al. 1996; Reinoso et al. 1996; Yamamoto and Reiner 2005) and LSAMP immunostaining has been used as an anatomical marker of the limbic system (Prensa et al. 2003; Uroz et al. 2004). Here we provide first evidence that the activity profile of the two alternative promoters of the Lsamp gene has a heterogenous anatomical distribution in the developing and adult brain and the activity of these two promoters corre- lates with trait anxiety and social behavior in mice. Limbic system-associated membrane protein 1a pro- moter is transcriptionally active in the ‘‘classic’’ limbic structures known to be especially important for emotional and motivational functions (Heimer and Van Hoesen 2006). Namely, 1a promoter is specifically active in the hippocampal formation, temporal cortex and amygdaloid area and also in the ventral striatum that includes nucleus accumbens and olfactory tuberculi; furthermore, 1a tran- script is expressed specifically in the limbic, cingulate and 12 3 Brain Struct Funct (2015) 220:1381–1393 1390 promoter is specifically activated during the regulation of striatal development. The expression patterns of the Lsamp 1a and 1b transcripts during mouse embryogenesis indicate a potential role of the Lsamp gene and protein in the development all over the brain. regions associated with olfactory and gustatory perception (e.g., the piriform cortex and insular cortex) are often overlapping with brain regions that are involved in emo- tional processing (Gutman et al. 2013). While Lsamp 1b promoter is predominantly active in sensory areas, it is also highly expressed in areas that are either traditional components of the limbic system and/or actively involved in regulating stress and arousal, such as the mammillary bodies and the paraventricular nucleus of the hypothalamus. Discussion In the current study, we did not detect any expressional changes in Lsamp transcripts after acute fear reaction although there is evidence that Lsamp is also activated in reaction to acute fear in the amygdaloid area of rats (Koks et al. 2004) and in the lateral amygdaloid nucleus of rats after fear conditioning (Lamprecht et al. 2009). It is most likely that acute changes are limited to specific subnuclei in the amygdala that can be more exactly separated from rat brain. In our study, we used the temporal lobe of mice, including all the amygdaloid nuclei and also the temporal cortex. However, the expression of Lsamp transcripts of the same area was significantly correlated with trait anxiety of the mice. We provide further evidence that the Lsamp gene is implicated in the formation of fear and anxiety pro- cessing circuits in the temporal cortex/amygdaloid area (Nieh et al. 2012), but this influence seems to be mediated differentially in acute fear reaction and trait anxiety. Although related, fear and trait anxiety are distinctly dif- ferent—fear is an emotional reaction triggered by an immediate threat, while anxiety is a state of heightened apprehension in the absence of an immediate threat (Davis et al. 2010). Taken together, our results fit with previous evidence relating increased levels of Lsamp with height- ened trait anxiety (Nelovkov et al. 2006; Alttoa et al. 2010); the implication of Lsamp in acute fear reaction seems to be more complicated and might be related with p ) Developmentally, the initial activation of both 1a and 1b promoters can be detected on embryonic day E12.5, the time preceding active neurite outgrowth and path-finding, suggesting that Lsamp is important already at the time of neuroepithelial patterning. The expressional initiation is consistent with previously published data about the developing brain of the rat (Pimenta et al. 1996). However, as in the adult, the expression pattern of the two alternative promoters differs remarkably at this early stage. Limbic system-associated membrane protein 1a promoter activa- tion is initiated in the outer surface of the neuroepithelium. The first signs of 1b transcript expression, on the other hand, are detected in the lateral aspect of the lateral ven- tricle and in the lining of the aqueduct. Discussion Additionally, 1b promoter is prevalently active in the central nucleus of amygdala that is commonly referred to as the central part of the limbic structures (Heimer and Van Hoesen 2006). However, according to a recent study, the central nucleus of amygdala gets projec- tions from several sensory-related regions (Bienkowski and Rinaman 2013). 1b promoter activity is highly enriched but not strictly limited to sensory areas; however, connections with the sensory systems can be found in most of the areas expressing 1b transcript (see supplementary Table S2 for overview). The expression of Lsamp in the brain areas processing sensory information has been reported in earlier studies (Reinoso et al. 1996; Yamamoto and Reiner 2005). Yet, the discussion of whether the distribution of LSAMP is really specific for limbic structures has not been raised. Our results indicate that it is questionable to use the sum- marized LSAMP staining as a marker of the limbic regions, however, we propose that Lsamp 1a transcript is inten- sively and specifically expressed in the brain areas that are commonly considered to be limbic structures (Heimer and Van Hoesen 2006; Morgane et al. 2005; Ko¨tter and Ste- phan 1997). Increased levels of the Lsamp transcript have been associated with lower activity and higher levels of trait anxiety or acute fear reaction and the genetic deletion of the Lsamp gene in mice resulted in increased activity in novel environments and reduced anxiety (Catania et al. 2008; Innos et al. 2011). To get further insight of how LSAMP is involved in the regulation of adaptive and emotional behavior by the usage of alternative promoters, we studied behavioral correlates for Lsamp 1a and 1b transcripts in three brain areas. Most of the significant correlations appeared between Lsamp expression in the temporal lobe and behavioral parameters in the elevated plus maze. Higher levels of Lsamp 1a transcript had sig- nificant correlations with all of the measures indicating higher anxiety (Cruz et al. 1994) in the elevated plus-maze test. Higher levels of Lsamp 1b in the temporal cortex correlated significantly with the time that mice spent on open arm that is again, a common measure of anxiety. Current results are correlative in nature, but well in line with our previous loss-of-function studies with Lsamp- deficient mice displaying decreased anxiety (Innos et al. 2011). Discussion According to our primary analysis, the extensive 1a transcript-specific staining in the dorsal striatum that disappears during the first postnatal week represents the most striking difference in the distribution of Lsamp between the developing and the adult brain. Strong 1a staining in the dorsal striatum before the second postnatal week overlaps with the time period of transient expression of LSAMP on developing axons (Horton and Levitt 1988) suggesting that Lsamp 1a 123 123 1391 Brain Struct Funct (2015) 220:1381–1393 certain subnuclei in the amygdala or/and specific time points. intron flanking exon 1b of LSAMP are associated with major depressive disorder (Koido et al. 2012). Furthermore, lower expression level of LSAMP 1a transcript has been linked with the susceptibility allele for coronary artery disease (Vance 2007). These data emphasize the importance of studying 1a and 1b isoforms separately to find out relevant information that can be used in diagnostic panels in the future. Both Lsamp 1a and 1b transcript levels in the hippocam- pus correlated negatively with social sniffing and Lsamp 1a transcript in the ventral striatum was positively correlated with the time of anogenital sniffing in the social interaction test. The implication of LSAMP in the regulation of social activity is again in line with the behavioral phenotype of Lsamp-deficient mice displaying lack of inter-male domi- nance hierarchy and whisker trimming (Innos et al. 2011). The positive correlation of Lsamp 1a transcript with ano- genital sniffing fits with reduced anogenital sniffing accompanying reduced inter-male aggressiveness reported in Lsamp-deficient mice (Innos et al. 2011). General social sniffing is not altered in Lsamp-deficient mice; therefore, the correlation between higher level of Lsamp in the hippo- campal area and shorter time of social sniffing may reflect higher Lsamp levels correlating with higher trait anxiety as the social interaction test was initially designed to measure anxiogenic and anxiolytic drug effects (File and Hyde 1978). Current data are in line with our previous reports showing that Lsamp-deficient mice have decreased anxiety and alterations in social behavior. Our results provide further evidence that Lsamp is functional in brain areas processing emotional reactions, particularly those related to anxiety/ hyperactivity and social behavior. The distinct system-specific use of alternative promoters reveals highly organized transcriptional regulation of LSAMP gene/protein associated with a broad spectrum of emotional behaviors. Discussion Certain SNPs that reside in the first Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, dis- tribution, and reproduction in any medium, provided the original author(s) and the source are credited. Discussion We propose that LSAMP is involved in emotional and social operating systems by complex regulation of two alternative promoters that guide the development of neural circuits in the limbic and sensory brain areas. Acknowledgments This study was supported by a personal inves- tigation grant from the Estonian Research Council PUT129 (M.A. Philips) and an institutional investigation grant from the Estonian Research Council IUT20-41 (E.Vasar); grants from the Estonian Science Foundation 8408 (K. Lilleva¨li) and 9099 (H.Luuk), a grant from the Estonian Research Council PUT120 (C.A. Hundahl), a grant from the Ministry of Science and Education (SF0180125s08) and the European Regional Development Fund. We thank professor Luis Puelles from University of Mucia, Spain, and Professor Charles Watson from Curtin University, Australia, for their great insights and help with interpreting anatomical data. Conflict of interest The authors declare that they have no conflict of interest. Comparison of anatomical data from different species reveals high levels of conservation in the anatomical distri- bution of LSAMP transcript/protein. The summarized ana- tomical distribution from the current study is in line with the data from humans and primates: in humans LSAMP expression is intensive in the paraventricular thalamic nucleus (Uroz et al. 2004) and moderate in the nucleus ac- cumbens and claustrum (Prensa et al. 2003). In primates, the hippocampus displays the strongest immunoreactivity, amygdala has a highly heterogeneous staining pattern (Coˆte´ et al. 1996) and ventral striatum displays more intense LSAMP immunostaining than the dorsal striatum (Coˆte´ et al. 1995). Furthermore, the twin promoter structure of the LSAMP gene seems to be essential also in humans. In the human genome, the exon 1a0 has been mutated byinsertion of two nucleotides introducing a frame shift and resulting in a termination codon. Surprisingly, consequent loss of the acceptor site prevents the inclusion of the mutated exon 1a0 (Pimenta and Levitt 2004). The consequence of these two evolutionary events suggests that two promoters and alter- natively regulated expression is needed for functional emo- tional responses in humans. Altered expression of LSAMP has been demonstrated in brain areas of human psychiatric patients in the frontal cortex (Behan et al. 2009) and hippo- campus (Hyde et al. 1997), but in the previous studies 1a and 1b transcripts of the LSAMP gene in the nervous system have not been distinguished. 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A QoS-configurable failure detection service for internet applications
Journal of internet services and applications
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To cite this version: Rogério C Turchetti, Elias P Duarte Júnior, Luciana Arantes, Pierre Sens. A QoS-configurable failure detection service for internet applications. Journal of Internet Services and Applications, 2016, 7 (9), pp.14. ￿10.1186/s13174-016-0051-y￿. ￿hal-01405120￿ *Correspondence: elias@inf.ufpr.br †Equal contributors 1Department Informatics, Federal University of Parana, UFPR, Curitiba, Brazil Full list of author information is available at the end of the article 1 Introduction provides the precise information, e.g. consider that a mon- itored process that is correct suddenly crashes; until the crash is perceived, the failure detector will report that the monitored process is correct. Chen, Toueg and Aguilera [8] defined a set of criteria to evaluate the quality of the service (QoS) of failure detectors. The authors defined a set of metrics to quantify the speed (e.g. how fast a process crash is detected) as well as the accuracy (e.g. how well it avoids mistakes) of failure detectors. Consensus [1] and other equivalent problems, such as atomic broadcast and group membership are used to implement dependable distributed systems [2, 3]. How- ever, given the FLP impossibility [4], i.e., consensus can not be solved deterministically in asynchronous dis- tributed systems in which even a single process can fail by crashing, deploying high-available distributed systems on the Internet is a challenge. In order to circumvent the impossibility of solving consensus in asynchronous dis- tributed systems, Chandra and Toueg introduced failure detectors based on timeouts [5–7]. In this work we describe an Internet Failure Detection Service (IFDS) that can be used by applications that con- sist of processes running on independent autonomous systems of the Internet. IFDS reconfigures itself to provide the QoS level required by the applications. Chen et al. [8] and Bertier et al. [9] have investigated how to configure QoS parameters according to the network performance and the application needs. The parameters include the upper bounds on the detection time and mistake duration, and the lower bound on the average mistake recurrence time. Bertier et al. extended that approach by comput- ing a dynamic safety margin which is used to compute the timeout. The main contribution of the present work is that IFDS handles multiple concurrent applications, each with different QoS requirements. In this way, two or more applications with different QoS requirements can Failure detectors are used to monitor processes of any application running on a network. Failure detectors pro- vide process state information. In this way, failure detec- tors are described as distributed “oracles” that supply information about the state of processes. Each application that needs process state information accesses the failure detector as a local module. Failure detectors can make mistakes, i.e., report that a fault-free process has failed or vice-versa and, therefore are said to be unreliable. © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. A QoS-configurable failure detection service for internet applications Rogério C. Turchetti1,3†, Elias P. Duarte Jr.1*†, Luciana Arantes2 and Pierre Sens2 Abstract Unreliable failure detectors are a basic building block of reliable distributed systems. Failure detectors are used to monitor processes of any application and provide process state information. This work presents an Internet Failure Detector Service (IFDS) for processes running in the Internet on multiple autonomous systems. The failure detection service is adaptive, and can be easily integrated into applications that require configurable QoS guarantees. The service is based on monitors which are capable of providing global process state information through a SNMP MIB. Monitors at different networks communicate across the Internet using Web Services. The system was implemented and evaluated for monitored processes running both on single LAN and on PlanetLab. Experimental results are presented, showing the performance of the detector, in particular the advantages of using the self-tuning strategies to address the requirements of multiple concurrent applications running on a dynamic environment. Keywords: Internet process monitoring, Unreliable failure detectors, Distributed systems, Fault-tolerance, SNMP rds: Internet process monitoring, Unreliable failure detectors, Distributed systems, Fault-tolerance, S HAL Id: hal-01405120 https://inria.hal.science/hal-01405120v1 Submitted on 29 Nov 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Open licence - etalab Journal of Internet Services and Applications Journal of Internet Services and Applications Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 DOI 10.1186/s13174-016-0051-y Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 DOI 10.1186/s13174-016-0051-y 1 Introduction It is impossible to implement a failure detector that always *Correspondence: elias@inf.ufpr.br †Equal contributors 1Department Informatics, Federal University of Parana, UFPR, Curitiba, Brazil Full list of author information is available at the end of the article Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 2 of 14 use the detector to monitor their processes, and IFDS reconfigures itself so that all requirements are satisfied. impossibility represents a challenge for developing practi- cal fault-tolerant distributed systems. Furthermore, it has been proved that several other important problems are equivalent to consensus so that they are also impossible to solve under the same assumptions. These include atomic broadcast, leader election, and group membership, among others [14]. IFDS was implemented using SNMP (Simple Network Management Protocol) [10]. Processes of a distributed application access the failure detector through a SNMP interface. In the proposed service, monitors execute on each LAN where processes are monitored. The monitor is implemented as a SNMP agent that keeps state informa- tion of both local and remote processes. The implementa- tion of the service is based on a SNMP MIB (Management Information Base) called fdMIB (failure detector MIB), which can be easily integrated to distributed applications. The implementation also employs Web Services [11] that enable the transparent communication between processes running on different Autonomous Systems (AS). As Web Services are used as gateways for SNMP entities, they are transparent to the applications. Experimental results are presented for distributed applications with monitored processes executing both on a single LAN and in the PlanetLab [12], the worldwide research testbed. Results include an evaluation of the overhead incurred by the failure detector as the QoS parameters vary, the failure detection time, and average mistake rate. Unreliable failure detectors were proposed by Chan- dra and Toueg [5] as an abstraction that, depending on its properties, can be used to solve consensus in asyn- chronous systems with crash faults. In this sense, a failure detector is a distributed oracle that can be accessed by a process to obtain information about the state of the other processes of the distributed system. Each process accesses a local module implementing the failure detec- tor, which basically outputs a list of processes suspected of having failed. In a broad sense failure detectors can be used to monitor processes of any application running on a network. 1 Introduction Furthermore, the failure detector must have a well-known interface through which it provides process state information. The root of the FLP impossibility is that it is difficult to determine in an asynchronous system whether a pro- cess has crashed or is only slow. This problem also affects failure detectors: in asynchronous systems they can make mistakes. For example, fault-free but slow processes can be erroneously added to the list of failed processes. Thus, failure detectors are said to be unreliable, and instead of indicating which processes have failed, they indicate which processes are suspected to have failed. Chandra and Toueg in [5] give a classification of unreliable failure detectors in terms of two properties: completeness and accuracy. Completeness characterizes the ability of the failure detector to suspect faulty processes, while accuracy restricts the mistakes that the detector can make. Com- pleteness is said to be strong if every process that crashes is permanently suspected by all correct processes. Other- wise, if at least one correct process suspects every process that has crashed, then completeness is weak. Accuracy is classified as strong if no process is suspected before it crashes, and weak if some correct process is never sus- pected. Both completeness and accuracy are eventual if they only hold after a finite but unknown time interval. The rest of the paper is organized as follows. Section 2 is a short introduction to failure detector and also presents an overview of related work. In Section 3 IFSD is specified, in particular we describe how the service dynamically adapts to network conditions and multiple concurrent application requirements. The architecture of the proposed failure detector service is described in Section 4. The implementation and experimental results are described in Sections 5 and 6, respectively. Section 7 concludes the work. 2 Failure detectors and related work They define a set of metrics that quantify both the failure detector speed (how fast crashes are detected) and accuracy (how well it avoids mistakes). Three basic metrics (see Fig. 1) are defined as described below. Another work that extends the original approach of Chen et al. was [17] proposed by Dixit and Casimiro. The authors define an alternative way to configure the QoS parameters, which is based on the stochastic properties of the underlying system. Initially, the user must provide a specification of the average mistake recurrence time (TMR defined above) and the minimum coverage (CL), which corresponds to a lower bound on the probability that heartbeat messages are received before the timeout interval expires. These parameters are used by a config- urator which relies on another system called Adaptare that is a middleware that computes the timeout by esti- mating distributions based on the stochastic properties of the system on which the failure detector is running. The system was evaluated and presented sound results, espe- cially in terms of the average mistake recurrence time and coverage. • Detection time (TD): represents the time interval from the instant process p has crashed to the instant at which the failure detector starts to suspect p permanently. • Mistake recurrence time (TMR): this metric corresponds to the time interval between two consecutive mistakes, i.e., it represents how frequently the failure detector makes mistakes. q y • Mistake duration (TM): represents the time it takes for the failure detector to correct a mistake. In [16], Bondavalli and Falai present an extensive com- parison of a large number of failure detectors executed on a wide area network. They conclude that no failure detector presents at the same time high speed and high accuracy. In particular, they show that using a more accu- rate predictor to compute the timeout interval does not necessarily imply on better QoS. The authors conclude that a perfect solution for the failure detection problem does not exist. Other metrics are derived from those above; such as the Query accuracy probability (PA) which corresponds to the probability that the output of the failure detector is correct at a random instant of time. Besides defining the above metrics, Chen et al. 2 Failure detectors and related work Consensus is a basic building block of fault-tolerant dis- tributed computing [3, 13]. Informally, processes execute a consensus algorithm when they need to agree on a given value, which depends on an initial set of proposed val- ues. The problem can be easily solved if the system is synchronous, i.e., there are known bounds on the time it takes to communicate messages and on how fast (or slow) processes execute tasks. On the other hand, if those time limits are unknown, i.e., the system is asynchronous, and processes can fail by crashing, then consensus becomes impossible to solve. This is a well-known result proved by Fischer, Lynch, and Paterson in 1985 and also known as the FLP impossibility [4]. Note that this impossibility assumes the most “benign” type of fault, the crash fault, in which one process simply stops to produce any output given any input. The most common approach to implement failure detectors is to employ heartbeat messages, which are sent periodically by every monitored process. Based on the observed message arrival pattern, the failure detec- tor computes a timeout interval. If a heartbeat message is not received within this timeout interval, the monitored process is suspected to have crashed. A key decision to implement failure detectors is the choice of algorithm to compute a precise timeout interval. If the timeout interval is too short, crashes are quickly detected, but the likeliness of wrong suspicions is higher. Conversely, if the timeout As consensus is such an important problem and time limits for sending messages and executing tasks in real systems, such as the Internet, are unknown, the FLP Page 3 of 14 Page 3 of 14 Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 interval is too long, wrong suspicions will be rare, but this comes at the expense of long detection times [15]. An adaptive failure detector can automatically update the timeout intervals and for this reason it is generally chosen to implement failure detectors on real networks [16]. both network and processor overhead. While Chen’s fail- ure detector computes the timeout interval based on the expected arrival time of the next message, Berthier’s also uses a safety margin which is continuously updated according to Jacobson’s TCP (Transmission Control Pro- tocol) timeout algorithm. In [8], Chen, Toueg, and Aguilera specify criteria to evaluate quality of service (QoS) provided by failure detectors. 2 Failure detectors and related work also propose a failure detec- tor algorithm that adapts itself according to how metrics are configured and the characteristics of the network on which it is running, including the message loss probabil- ity (pL), the expected message delay (E(D)) and message delay variance (V(D)). Tomsic et al. [18] introduced a failure detection algo- rithm that provides QoS and adapts to sudden changes in unstable network scenarios. The failure detector is called Two Windows Failure Detector (2W-FD) and it uses two sliding windows: a small window (to react rapidly to changes in network conditions) and a larger window (to make better estimations when the network conditions change gradually). The authors show that their algorithm presents better QoS when compared to others running on networks under unstable conditions. Bertier et al. [9] extend the approach proposed by Chen et al. by implementing a failure detector in two layers: an adaption layer runs on top of a traditional heartbeat- based failure detector. The adaptation layer configures the Quality of Service (QoS) of the failure detector accord- ing to application needs. The purpose is also to minimize In [19] the authors design an autonomic failure detec- tor that is capable of self-configuration in order to provide the required QoS. In [20] a failure detector that is capa- ble of self-configuration is applied to a cloud computing environment. suspect T trust down up TMR T M D p Fig. 1 Basic metrics that represent the QoS of a failure detector Network monitoring systems based on the Internet Sim- ple Network Management Protocol (SNMP) have been used to feed system information to failure detectors. Lima and Macedo in [21] explore artificial neural networks in order to implement failure detectors that dynamically adapt themselves to communication load conditions. The training patterns used to feed the neural network were Fig. 1 Basic metrics that represent the QoS of a failure detector Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 4 of 14 obtained from a network monitoring system based on SNMP. processes; the second strategy is called ηGCD and it com- putes the greatest common divisor of the corresponding parameters. Wiesmann, Urban, and Défago present in [22] the SNMP-FD framework, a SNMP-based failure detector service that can be executed on a single LAN. 2 Failure detectors and related work Several MIBs are defined to keep information such as identifiers of hosts running monitors, identifiers and state of each mon- itored process, heartbeat intervals, heartbeat counters, among others. 3.1 System model We assume a distributed system S = {p1, p2, . . . , pn, } that consists of n units that are called processes but also correspond to hosts. The system is asynchronous but aug- mented with the proposed failure detector, i.e. we assume a partially synchronous system. Processes communicate by sending and receiving messages. Every pair of processes is connected by a communication channel, i.e. the sys- tem can be represented by a complete graph. Processes fail by crashing, i.e., by prematurely halting. Every process in S has access to IFDS as a local failure detector mod- ule. IFDS returns process state information: correct or suspected. Monitored processes periodically send heart- beat messages. Timeouts are used to estimate whether processes have crashed. In [23], a failure detector service for Internet-based dis- tributed systems that span multiple autonomous systems (AS) is proposed. The service is based on monitors which are capable of providing global process state information through a SNMP interface. The system is based both on SNMP and Web services, which allow the communication of processes across multiple AS’s. To the best of our knowledge, the present work is the first that proposes a SNMP-based failure detector ser- vice with self-configurable Quality of Service. Given the requirements of multiple simultaneous applications, we present two strategies that allow the detector to self- configure. With respect to previous SNMP-based imple- mentations of failure detectors, the major benefit of our proposed service is that it allows the user to specify QoS requirements for each application that is moni- tored, including: the failure detection time, mistake recur- rence time and mistake duration. Given this input, and the perceived network conditions, our service configures and continuously adapt the failure detector parameters, including the heartbeat rate. Furthermore, as mentioned above, the proposed service can take into considera- tion the QoS requirements for multiple simultaneous applications to configure the system. Other SNMP-based implementations of failure detectors do not support QoS parameters to be input to the system, thus the user must manually configure the system and, worse, keep recon- figuring the system manually as network conditions and requirements change. Furthermore, IFDS introduces a novel implementation strategy in which SNMP objects themselves execute process monitoring – other works employ a separate daemon for that purpose. 3.3.1 QoS configuration for a single application In Fig. 2, IFDS receives the QoS parameters from an appli- cation. The part of the Failure Detector devoted to the configuration of QoS parameter consists of two modules: Estimator and Configurator. Given the messages (heart- beats) received from a monitored process, the Estimator computes three parameters: the message loss probability (pL), the message delay variance (V(D)), and the estima- tion for the arrival time of the next message (EA). The message loss probability is computed as pL = L/k where error(k) = Ak −EAk −delay(k) (3) (3) In the expression (4), delay(k+1) is the estimated delay of next message which is computed in terms of the error(k). γ represents the weight of the new measure, Jacobson suggests γ = 0.1: delay(k+1) = delay(k) + γ .error(k) (4) (4) Fig. 2 Finding η. IFDS receives the QoS parameters from an application var(k+1) represents the error variation, and it computed from first iteration; initially var(k) is set to zero. var(k+1) represents the error variation, and it computed from first iteration; initially var(k) is set to zero. var(k+1) = var(k) + γ .(|error(k) −delay(k+1)|) (5) (5) The safety margin α(k+1) is then computed as shown below, where β and φ are constant weights for which we used β = 1, φ = 4 and γ = 0.1. α(k+1) = β.delay(k+1) + φ.var(k+1) (6) (6) The timeout interval τ is thus adjusted after each heart- beat message arrives. 3.2 IFDS adaptive timeout computation Journal of Internet Services and Applications (2016) 7:9 Page 5 of 14 Page 5 of 14 application. Then the strategy is extended to handle mul- tiple concurrent applications, each with different QoS requirements. Notice that even if two applications use the detector to monitor the same process, they may still have different QoS requirements. In this case, based on the different QoS requirements, a single heartbeat inter- val is chosen to monitor the process, i.e., the one that satisfies all requirements. On the other hand, the detec- tion time used by the service for each application may be different. For example, one application may need a short detection time while the other can cope with a larger inter- val. If the heartbeat message is delayed and arrives after the shorter detection time but before the larger detec- tion time, only the first application is informed that a false suspicion occurred. by dynamically estimating α (the safety margin) using Jacobson’s TCP timeout algorithm [24]. EA is computed as the weighted average of the n last message arrival inter- vals, where n is the window size for keeping historical records. IFDS thus computes the timeout interval (τ) for mon- itoring a process based on an estimation of the next heartbeat arrival time (EA) plus a dynamic safety mar- gin (α). Let’s consider two processes: p and q, p monitors q. Every η time units, q sends a heartbeat to p. Let m1, m2, . . . , mk be the k most recent heartbeat messages received by p. Let Ti be the time interval that each heart- beat took to be received by p, i.e. the time interval that elapsed since heartbeat i−1 had been received until heart- beat i is received, measured with the local clock. EA can be estimated as follows: As shown in Fig. 2, the detector receives as input from an application the following parameters: EAk+1 = 1 k ⎛ ⎝ k  i=1 Ti −η ⎞ ⎠+ (k + 1)η (1) (1) • TU D : an upper bound on the detection time; • TU M: an upper bound on the average mistake duration; • TL MR: a lower bound on the average mistake recurrence time • TU D : an upper bound on the detection time; EAk+1 is the expected time instant at which the next message (the k + 1-th message) will arrive. 3.2 IFDS adaptive timeout computation The next timeout will expire at: Thereafter, the failure detector processes the input data and seeks a suitable value for η, as shown next. τ(k+1) = EA(k+1) + α(k+1) (2) (2) The safety margin α is computed based on Jacobson’s TCP algorithm and is used to correct the error of the esti- mation for the arrival of the next message. Let Ai be the arrival time of heartbeat i, error(k) represent the error of the k-th message computed as follows: 3.2 IFDS adaptive timeout computation Arguably, the most important feature of a failure detec- tor is how precisely the timeout interval is computed. Remember that if a message is expected from a monitored process and it does not arrive before the timeout inter- val expires, then the process is suspected to have failed. A timeout interval that is too long increases the failure detection time, and a timeout interval that is too short increases the number of mistakes, i.e., correct processes can be frequently suspected to have crashed. Given the Internet characteristics in time and space, as well as differ- ent applications requirements, it is impossible to employ constant, predefined timeouts. Practical failure detectors must employ dynamic timeouts that are adaptive in the sense that they are reconfigured according to changing network conditions and application requirements. IFDS computes the timeout interval (τ) for the next expected message using information about the arrival times of recently received messages. Note that for a given monitored host, multiple processes running on that host can be monitored, each with different QoS requirements. A single message is periodically expected from the host conveying information about the status of all its processes. IFDS computes the timeout with a strategy inspired on the works of Chen et al. [8] and Bertier et al. [9]. Chen’s fail- ure detector allows the configuration of QoS parameters according to network performance and the application needs. Bertier et al. extend that approach by also consid- ering a dynamic safety margin to compute the timeout. 3 IFDS: failure detection and QoS configuration In this section, IFDS is described and specified. Initially, the system model is presented. Next, we give details on how IFDS computes adaptive timeout intervals, and then describe the configuration of IFDS parameters which are based both on network conditions and on applications QoS requirements. Besides showing how parameters are configured to monitor a single application process, two strategies are proposed to configure IFDS when multi- ple processes are monitored with different QoS require- ments. The first strategy is called ηmax and it maximizes monitoring parameters to encompass the needs of all Chen et al. [8] compute an estimation of the arrival time (EA) of the next message based on the arrival times of previously received messages. That estimation plus a constant safety margin (α) are the basis for calculat- ing the next timeout. Bertier et al. extend that approach Turchetti et al. 3.3 Configurig the failure detector service based on QoS parameters Algorithm 1: Computing the heartbeat interval 1 /* TU D : upper bound on the detection time 2 TU M: upper bound on the average mistake duration 3 TL MR: lower bound on the mistake recurrence time 4 θ: reflects the failure detection probability 5 pL: message loss probability 6 V(D): delay variance 7 ηmax: is set to min  θ∗TU M, TU D  8 η: heartbeat interval */ 9 10 Step1  TU D , TU M, TL MR  ▷input data 11 12 θ := (1 −pL)  TU D 2 / V(D) +  TU D 2 ; 13 14 if  (θ > 0) and  TU D > 0  and  TU M > 0  then 15 if ((θ * TU M) > TU D ) then 16 ηmax := TU D ; 17 else 18 ηmax := θ * TU M; 19 run Step2  TU D , TL MR, ηmax  20 else 21 return (“QoS cannot be achieved"); Algorithm 1: Computing the heartbeat interval 1 /* TU D : upper bound on the detection time 2 TU M: upper bound on the average mistake duration 3 TL MR: lower bound on the mistake recurrence time 4 θ: reflects the failure detection probability 5 pL: message loss probability 6 V(D): delay variance 7 ηmax: is set to min  θ∗TU M, TU D  8 η: heartbeat interval */ 9 10 Step1  TU D , TU M, TL MR  ▷input data 11 12 θ := (1 −pL)  TU D 2 / V(D) +  TU D 2 ; 13 14 if  (θ > 0) and  TU D > 0  and  TU M > 0  then 15 if ((θ * TU M) > TU D ) then 16 ηmax := TU D ; 17 else 18 ηmax := θ * TU M; 19 run Step2  TU D , TL MR, ηmax  20 else 21 return (“QoS cannot be achieved"); M Note that if either θ = 0 or TU D = 0 or TU M = 0 then the maximum heartbeat interval is zero; this means that there is no heartbeat interval that is able to guarantee the QoS requirements. In this case, Step 2 is not executed. 3.3 Configurig the failure detector service based on QoS parameters 22 Step2  TU D , TL MR, ηmax  23 24 η := ηmax; 25 while  f (η) < TL MR  do 26 η := η −(η ∗0.01); 27 return (η); ▷output data 28 Function f (η) 29 return η ∗ ⌈TU D /η⌉−1 j=1 V(D)+(TU D −jη)2 V(D)+(pL(TU D −jη)2 ; 22 Step2  TU D , TL MR, ηmax  23 24 η := ηmax; 25 while  f (η) < TL MR  do 26 η := η −(η ∗0.01); 27 return (η); ▷output data 28 Function f (η) 29 return η ∗ ⌈TU D /η⌉−1 j=1 V(D)+(TU D −jη)2 V(D)+(pL(TU D −jη)2 ; Actually, the final value of η found in Step2 is the largest interval that satisfies the QoS requirements. Thus, a shorter interval, 0 < η ≤14.6, can also be used for the example. Algorithm 1 consists of two steps. The main purpose of Step1 is to compute an upper bound for the heart- beat interval ηmax, given the input values provided by the application: the upper bound on the detection time (TU D ), upper bound on the average mistake duration (TU M), and lower bound on the average mistake recurrence time 3.3 Configurig the failure detector service based on QoS parameters Algorithm 1: Computing the heartbeat interval The main purpose of Step 2 of the algorithm is to find the smallest η that respects the third QoS parameter: TL MR, the mistake recurrence time. Step2 computes η as follows. η is initially equal to ηmax. Then η is reduced by 1 % at each iteration until f (η) geq TL MR. This reduction factor (1 %) was chosen experimentally as it gives precise results, close to TL MR – if we use 10 % the precision is lost we can get a value that is acceptable but too distant from TL MR. As proved in [8], for each value of η, f (η) computes the prob- ability that the required mistake recurrence time (TL MR) is satisfied. Note that if η decreases f (η) increases, the oppo- site is also true. For this reason, we always reduce the value of η until f (η) ≥TL MR. When this condition holds, the largest η that satisfies TL MR has been computed. Next we give an example of the computation of η by Algorithm 1. An application (App1) specifies the following QoS requirements: TU D = 30 s (i.e., a crash failure is detected within 30 s), TU M = 60 s (i.e., the failure detector corrects its mistakes within 60 s), and TL MR = 432000s (i.e., the failure detector makes at most one mistake each 5 days). Furthermore, the message loss probability is pL = 0.0 and the delay variance is V(D) = 0.01. The algorithm in Step1 computes γ = 0.99 and ηmax = min(30 , 59.99). In Step2, the input values are processed initializing η equal to 30.00. After that, the algorithm continuously reduces the value of η until f (η) ≥432, 000. The final value is η = 14.6 s, which is employed by monitored processes to periodically send heartbeat messages to meet the QoS requirements of App1. 3.3 Configurig the failure detector service based on QoS parameters Fig. 2 Finding η. IFDS receives the QoS parameters from an application In this subsection, we initially describe how IFDS is configured based on the QoS requirements of a single Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 6 of 14 (TL MR). First, θ is computed (line 12) which reflects the fail- ure detection probability, based on both the probability that a message is received (1−pL), the message delay vari- ance (V(D)), and the required detection time (TU D ). Now remember that ηmax must be chosen to meet the required upper bounds on the detection time and mistake dura- tion time. Thus ηmax is set to the minimum of the upper bound of the detection time (TU D ) or the detection proba- bility times the upper bound of the mistake duration rate (θ ∗TU M). k is the total number of messages that have been sent and L is the number of messages that were lost. V(D) is computed from the observations of the message arrival times. The configurator module runs Algorithm 1 adapted from [8, 9] to compute η, i.e., the heartbeat interval to be used by a monitored process in order to keep the qual- ity of service required by the application. Algorithm 1 is described next. 4 IFDS: architecture The ηmax approach to adapt η to all application requests computes ηmax = min  γ TU M1, TU D 1, γ TU M2, TU D 2, . . . , γ TU Mn, TU D n  , where i = 1 . . . n, TU Mi and TU D i are the QoS requirements of application i. Algorithm 1 can be then used after this modification is introduced. In this section we describe the architecture of the pro- posed Internet failure detection service. Figure 3 shows IFDS monitoring processes on two LAN’s: A and B. These two LANs are geographically distributed and connected through the Internet. The number of monitored networks can be greater than two. For each network, there is both a Monitor Host and Monitored Hosts. As the names denote, a Monitored Host runs the processes that are monitored. The Monitor Host runs applications that are responsible for monitoring processes. Applications and other Mon- itors can subscribe to receive state information about specific processes. The other proposed approach is called ηGCD and in this case we compute the GCD (Greatest Common Divisor) among all ηi, i = 1 . . . m, m is the number of QoS require- ments. The idea is that if a process sends heartbeats every x units of time, and if x divides y, it also sends heart- beats every y units of time. Each ηi is first transformed to a new η′ i as follows: η′ i = 2n so that 2n < ηi and η′ i ∈Z+. The final ηGCD = GCD(η′ i, . . . , η′ n). Note that ηGCD must be > 0, otherwise it is impossible to apply this approach. Initially, every application that needs to obtain informa- tion about the states of monitored processes configures the quality of service (QoS) it expects from IFDS. Then, the detection service computes the heartbeat interval (η) that must be applied in order to provide the required QoS. The Monitor Host then configures η on every Monitored Host, so that periodically at an η interval, the monitored host will send back a heartbeat message to the Monitor Host. Note that ηmax gives the largest possible heartbeat inter- val that satisfieds the QoS requirements. Thus in order to have a safety margin it is recommended to use an interval shorter than that, such as given by ηgcd. 3.3.2 QoS configuration for multiple applications When several applications are monitoring multiple pro- cesses with IFDS, each can have different QoS require- ments. IFDS computes a heartbeat interval (η) for the monitored processes that simultaneously satisfies the QoS Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 7 of 14 then the algorithm uses this value to ensure the QoS parameters for both applications (App1 and App2). requirements of all applications. In order to understand why this can save monitoring messages consider a simple example in which 100 hosts connected on a single LAN each of which executes 100 processes that are be moni- tored. To make the example as simple as possible assume that all processes employ the same heartbeat interval. If one uses a separate FD service to monitor each pro- cess than 10,000 heartbeat messages are sent per interval. Using our shared/simultaneous monitoring strategy this number reduces to only 100 messages, each host employs one heartbeat message for all its processes. On the other hand, if we use the ηGCD approach we have: App1: η1 = 14.6 →η′ 1 = 8; App2: η2 = 7.2 →η′ 2 = 4; ηGCD = GCD(4, 8) →4. Thus, ηGCD = 4 s. As mentioned above, we can notice that ηGCD is smaller than ηmax. Our implementation is capable of self-adapting to QoS violations caused by network conditions. If IFDS detects that the network cannot sustain the minimum QoS requirements, it readjusts η by increasing its value. For instance, in the example above, η can be readjusted to another value as long as it complies with the follow- ing condition: η ≤14.6. Hence, Algorithm 1 is used to find a value of η that satisfies QoS requirements of as many applications as possible under the current network conditions. In this work we assume that the different processes can need different heartbeat intervals to satisfy their QoS requirements. In order to determine a common heart- beat interval to be used by all processes, two differ- ent approaches are proposed: ηmax and ηGCD, described below. 4 IFDS: architecture ηGCD is always smaller than ηmax for a given system. For this reason, using the ηGCD approach produces a larger number of mes- sages on the network than ηmax. On the other hand, it has advantages such as a reduction of the failure detec- tion time (TD) and improvement of the query accuracy probability (PA). As we shall see in Section 5, SNMP (Simple Net- work Management Protocol) is used by the Monitor and Monitored Hosts to exchange messages on a LAN. Each Monitored Host is registered on a SNMP MIB (Manage- ment Information Base). If the Monitor and Monitored hosts are located on different networks and must com- municate across the Internet, they employ Web Services. In this case, a SNMP message is encapsulated with and transmitted using SOAP (Simple Object Access Protocol). Note that all monitoring is done locally on each domain. Monitors on different domains communicate to obtain process state information. This communication is imple- mented in two ways. Either the local Monitor queries the remote Monitor to obtain process state information, or, alternatively, the Monitor can register at a remote monitor As we shall see in Section 5, SNMP (Simple Net- work Management Protocol) is used by the Monitor and Monitored Hosts to exchange messages on a LAN. Each Monitored Host is registered on a SNMP MIB (Manage- ment Information Base). If the Monitor and Monitored hosts are located on different networks and must com- municate across the Internet, they employ Web Services. In this case, a SNMP message is encapsulated with and transmitted using SOAP (Simple Object Access Protocol). Consider an extension of the example given above for one application (App1) where a second application (App2) runs on the same host as App1. App2 QoS requirements are as follows: TU D = 15 s (i.e., a crash failure is detected within 15 seconds), TU M = 30 s (i.e., the failure detec- tor corrects its mistakes within 30 seconds) and TL MR = 864000 s (i.e., the failure detector makes at most one mis- take each 10 days). We assume that the message loss prob- ability is pL = 0.0 and the delay variance is V(D) = 0.01. First consider the ηmax approach. In Step1 of the algo- rithm ηmax = min((30, 59.99)app1, (29.99, 15.00)app2) = 15.00. 4 IFDS: architecture In Step2, the final value is computed as η=7.2 s, Note that all monitoring is done locally on each domain. Monitors on different domains communicate to obtain process state information. This communication is imple- mented in two ways. Either the local Monitor queries the remote Monitor to obtain process state information, or, alternatively, the Monitor can register at a remote monitor Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 8 of 14 Fig. 3 IFDS architecture Fig. 3 IFDS architecture 5 IFDS: implementation to receive notifications whenever the state of remote mon- itored processes changes. In this case, a SNMP message is encapsulated with and transmitted using SOAP (Simple Object Access Protocol). As mentioned in the previous section, IFDS was imple- mented with SNMP and Web Services. SNMP is used by the Monitor and Monitored Hosts to exchange mes- sages and each Monitored Host must be registered on the fdMIB, shown in Fig. 4. fdMIB consists of three groups: the Monitor Host Group, Monitored Host Group, and Application Notify Group. These three object groups are described below. Note that we propose a single MIB that maintains information about all IFDS modules. Web Services are thus used as a gateway for entities that implement SNMP to communicate remotely across the Internet. The use of Web Services is transparent to the user application, which employs only SNMP as the inter- face to access the failure detector. There are situations in which Web Services are not involved, i.e., when all the communication occurs on a local LAN. When a Moni- tor issues a query to another monitor running remotely, a virtual connection is open (using Web Services), and a remote procedure is executed that provides the required information. The Application Notify Group (appNotifyGroup) keeps information about QoS parameters. Each applica- tion that is supposed to receive state information about a process must give the following parameters to fdMIB: the upper bound on the detection time (TD_U), the mistake duration time (TM_U), and the lower bound on the aver- age mistake recurrence time (TMR_L). These parameters are described in Section 3. We also described how IFDS computes the heartbeat interval η, given the application parameters. When an application needs to communicate with a Monitor running remotely, it has an option to do that directly using Web Services. For example, consider the system in Fig. 3, suppose that the application process (App1) on Network A needs to learn information about the status of a process running on Network B. In this case, App1 invokes the corresponding Web Services that in turn executes the SNMP operation on Network B. Another object of the appNotifyGroup is receiveHB (see Fig. 4) which is used to receive heartbeat messages from a monitored process. Every time a heart- beat message arrives, fdMIB updates the corresponding process state object in the monitorHostGroup table. 5 IFDS: implementation In order to receive notifications, an application sub- scribes at the Monitor Host to receive SNMP traps (notifyTrap) with process state information. The application will be notified whenever the Monitor detects a change of the state of the monitored process. Object statusChange is also updated to store 0, if the mon- itored process is correct, and 1 otherwise. Note that besides receiving traps (alarms) the application can at any Another object of the appNotifyGroup is receiveHB (see Fig. 4) which is used to receive heartbeat messages from a monitored process. Every time a heart- beat message arrives, fdMIB updates the corresponding process state object in the monitorHostGroup table. When the Monitor Host detects that the state of a mon- itored process on the same LAN has changed, it updates the corresponding entry and sends the new state infor- mation to all applications and other Monitors that have subscribed to receive information about that particular process. In order to receive notifications, an application sub- scribes at the Monitor Host to receive SNMP traps (notifyTrap) with process state information. The application will be notified whenever the Monitor detects a change of the state of the monitored process. Object statusChange is also updated to store 0, if the mon- itored process is correct, and 1 otherwise. Note that besides receiving traps (alarms) the application can at any All information about the state of processes is stored in a MIB called fdMIB (failure detector MIB). In Section 5 we present detailed information about this MIB and its implementation. Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 9 of 14 Fig. 4 fdMIB objects. In this figure we propose a single MIB that maintains information about all IFDS modules Fig. 4 fdMIB objects. In this figure we propose a single MIB that maintains information about all IFDS modules time check the state of any monitored process by querying the corresponding MIB object. The Monitor Host Group (monitorHostGroup) stores information about monitored processes, including IP addresses (ipAddr), port numbers (portNumber), process IDs (processID), state (statusHost), number of false detections (falseDetection), heartbeat inter- val required (reqFreq), an estimation of the probabilistic behavior of message delays (V_D), message loss probabil- ity (P_L), QoS parameters (current_TD, current_TM and current_TMR) these parameters are continuously updated and are used to check whether any QoS require- ment has been broken (e.g., current_TD > TD_U), among others. 5 IFDS: implementation Objects of the monitorHostGroup trig- ger and execute process monitoring. For instance, they can start threads that receive heartbeat messages and compute timeouts. The Monitored Host Group (monitoredHost- Group) is responsible for sending heartbeat messages. It is executed on a host that is monitored. In order to communicate with the Monitor, the following objects are maintained: Monitor IP address (ipMonitor), Mon- itor port (portMonitor), monitored process OID (myOidInMonitor, an Object Identifier is a name used to identify the monitored process in the MIB), heartbeat interval (frequencyHB), and the process identifier in the local host (processID). 6 IFDS: experimental results In this section we report results of several experiments executed in order to evaluate the proposed failure detec- tion service. Experiments were conducted on both an Ethernet LAN and PlanetLab [12]. Three independent applications were used each with different QoS require- ments, as shown in Table 1. All applications run on the same host. For instance, App1 requires that the time to detect failures to be at most 8 seconds, i.e., TU D = 8 s. On average the failure detector corrects its mistakes within one minute, i.e., TU M = 60 s. Finally, the failure detector makes at most one mistake per month, i.e., TL MR = 30 days (2592000000 in ms). In the experiments, we assumed pL = 0.01 and V(D) = 0.02, the sliding window (W) size is defined individually for each experiment. IFDS configuration can be simply done using the regu- lar snmpset command; for instance the command below is executed to configure the monitorHostGroup table Table 1 QoS requirements Applications TU D (s) TU M (s) TL MR (days) App1 8 60 30 App2 14 120 30 App3 16 240 30 Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 10 of 14 Page 10 of 14 The first experiment was executed to evaluate how IFDS adapts to changes in the heartbeat frequency. How does the failure detector react and reconfigures itself? Figure 5 shows the expected arrival time (EA curve) and timeout (Timeout curve) computed by IFDS as the monitored host sends 200 heartbeat messages to the monitor host. The figure also shows the measured RTT (RTT curve). From the beginning to the 50th heartbeat message the interval between two messages (the Heartbeat Freq curve) is 1 s. Then from the 51st message to the 150th message this interval grows to 5 s, after that the frequency reduces to 1 s again. with the required QoS parameters (Fig. 4). In this exam- ple, App1 monitors a process on a host with IP address 192.168.1.1, the port is 80, and the QoS parameters are those shown in Table 1. snmpset -v1 -c private localhost .1.3.6.1.4.1.18722.1. 2.1.1.2.1 s 192.168.1.1:8:80:60:2592000000 snmpset -v1 -c private localhost .1.3.6.1.4.1.18722.1. 2.1.1.2.1 s 192.168.1.1:8:80:60:2592000000 The snmpset command shown above is used to update the value of a SNMP object. 6 IFDS: experimental results This command uses the fol- lowing parameters: host address on which the MIB is deployed (localhost), the id of the object to be updated (.1.3.6.1.4.1.18722.1.2.1.1.2.1) and the data that is to be written on the object (192.168.1.1:8:80:60:2592000000). The Figure shows that initially (first 10 messages) the timeout takes a while to stabilize. Then when the heart- beat frequency changes from 1 to 5 s, the EA and timeout curves show that after a short while IFDS could clearly adapt the timeout interval to the new heartbeat frequency. During that short while (after the 50th message) the Time- out curve remains below the Heartbeat Freq curve: during this time IFDS incurs in false suspicions. After a cou- ple of messages the timeout grows enough to correct the mistakes. The Figure also shows how IFDS reacts when the heartbeat frequency reduces back to 1 second (from the 150th message). The EA and Timeout curves show that after a few messages IFDS readapts itself to the new situation. Using Algorithm 1 (presented in Section 3.3) to com- pute the heartbeat interval for the three applications shown in Table 1, we get ηmax = min(1.954467, 3.901890, 4.694764). Thus, in this case the value of η is 1.954467 (enough to meet the QoS requirements of all three appli- cations). If instead we use the ηGCD strategy on the same data in Table 1, we get GCD = min(1, 2, 4). Thus η = 1 again enough to monitor App1, App2, and App3. 6.1 Experimental results: LAN The LAN experiments were executed on two hosts: the monitor host was based on an Intel Core i5 2.50 GHz processor, with 4 GB of RAM, running Linux Ubuntu 12.04 with kernel 3.2.0-58; the monitored host was an Intel Core i5 CPU 3.20 GHz, with 4 GB RAM running Linux Ubuntu 13.10 with kernel 3.2.0-58. The hosts were on a 100 Mbps Ethernet LAN. The fdMIB was implemented using Net-SNMP version 5.4.4. Next we show – for the same experiment – both the QoS requirements (given by the application), as well as the cur- rent value of the QoS parameters computed by IFDS from the actual monitoring. The objective of this experiment 0 2 4 6 8 10 12 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Time (s) Message number ’EA’ ’RTT’ ’Heartbeat Freq (n)’ ’Timeout’ Fig. 5 Adapting the timeout for different heartbeat intervals Time (s) Message number Fig. 5 Adapting the timeout for different heartbeat intervals Page 11 of 14 Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 duration time was 60 min. The results for this experiment are shown in Table 2. This table shows: the heartbeat interval η (in seconds), the detection time TD, the mis- take duration time TM, and the mistake recurrence time TMR, the PA metric (described below) plus the number of heartbeat messages sent during the experiment, and the number of false detections. During the experiment we simulated two false suspicions by omitting heartbeat mes- sages. The PA metric corresponds to the probability that the application will make a query about a given monitored process and that the reply is correct, in the sense that it reflects the real state of the monitored process. This met- ric was described in Section 2 and can be computed as follows: is to show that the applications’ QoS requirements are not violated. Figure 6a and b show the QoS parameters for the same experiment described in Fig. 5. Besides the EA, Heartbeat Freq and RTT curves that also appeared in Fig. 5, these Figures also show the detection time (TD curve), the mistake duration time (TM curve), the detec- tion times required by the applications: App1 TDU, App2 TDU, and App3 TDU curves. In Fig. 6.1 Experimental results: LAN 6a we can observe 6 false detections (TM curve), notice that the QoS requirements (Table 1) are never vio- lated. In the same Figure, the detection time TD is always less than App1 TDU, App2 TDU and App3 TDU. This means that the false suspicions shown in Fig. 6a remain transparent to the applications, i.e. they are not noticed by any of the applications, even when the heartbeat fre- quency grows to 5 s. Note that after message 50 the TD curve gets very close to but does not grow above App1 TDU. PA = 1 −E(TM) E(TMR) (7) (7) A similar situation can be seen in Fig. 6b, in which the mistake recurrence rate is checked. Note that the lower limit of the TMR QoS parameter required by the applications is 30 days (Table 1). Curve TMR shows the average TMR taking into account the false suspicions that occurred (TM curve). If we compute the average for the whole time the experiment was executed, it is 13, 086 ms, which is below the required value for this QoS metric. However, the real IFDS TMR does not take into account the false suspicions that are not reported to the appli- cations. As discussed, none of the false suspicions was ever reported to the applications, thus there was no mis- take and mistake recurrence time and there is no QoS violation. The results in Table 2 show that the heartbeat inter- val computed by ηmax is 1.95 s, while for ηGCD it is equal to 1 s. The next results are a consequence of these inter- vals. Remember that a higher heartbeat interval means that less messages are sent, and thus the overhead of ηmax (1754 messages) is lower than the overhead of ηGCD (3551 messages). On the hand, the opposite happens to the detection time TD: as ηGCD’s heartbeat interval is shorter, its detection time TD is also shorter (1.32 s) while for ηmax it grows to 2.46 s. The same happens to the mistake duration time (TM) and PA: the shorter the heart- beat interval, the shorter the mistake duration and the higher the probability that IFDS replies the correct state. Although the time recurrence time (TMR) also reduces for a shorter heartbeat interval, the variation is lower than for the other parameters. 6.1 Experimental results: LAN Next we describe an experiment designed to compare the two strategies proposed for monitoring multiple pro- cesses simultaneously: ηmax and ηGCD. The experiment a b Fig. 6 Global QoS is not violated. Subfigure (a) shows the TD and TM performance, and in subfigure (b) describes the TMR performance a Fi 6 Gl b l Q S i i l d S bfi ( ) h h T d T f b rmance and in subfigure (b) describes the TMR performance a b Fig. 6 Global QoS is not violated. Subfigure (a) shows the TD and TM performance, and in subfigure (b) describes the TMR performance Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 12 of 14 Table 2 Comparing the two strategies: ηmax and GCD η (s) TD (s) TM (s) TMR (s) PA Num. of HB message Num. of false detections ηmax 1.95 2.45 1.77 61.19 0.9711 1754 2 ηGCD 1.00 1.32 0.69 60.59 0.9998 3551 2 A discussion on these results leads to the conclusion that there is a trade-off. While ηmax proves is the best strategy for applications that do not require a small detec- tion time (which is the case for instance for monitoring remote processes via the Internet) ηGCD presents a shorter detection time and the best results for the PA metric. This strategy is thus the better suited to monitor processes on a single LAN, where the applications generally do not tolerate long delays. 6.2 Experimental results: PlanetLab The last experiment was executed to investigate the time overhead of using Web Services for communicating across the Internet. In particular we were interested in compar- ing the time to report an event using a pure SNMP solu- tion with one that also uses Web Services. We executed IFDS to monitor processes running on PlanetLab nodes located in the five continents: South America (Brazil), North America (Canada), Europe (Italy), Asia (Russia) and Oceania (New Zealand). PlanetLab hosts exchange infor- mation in two ways: using SNMP and Web Services. The time was computed using a monitor and a monitored pro- cess running in Brazil. After the monitor detects a failure it notifies yet another monitor. Then, we measured the time interval from the instant a fault was injected up to the instant the second monitor is notified. Authors’ contributions RCT carried out the implementation and ran the experiments; together with the other authors he also participated in writing this paper. EPD Jr. supervised the definition of the proposed architecture, supervised the implementation and experiments, and wrote the paper. PS and LA proposed the strategies for monitoring multiple processes that represents the main scientific contribution of this paper, they revised the architecture as it was proposed and also revised the written paper. All authors read and approved the final manuscript. 6.1 Experimental results: LAN 8 Time to notify a failure using SNMP and WS also increases, reaching up to approximately 2.01 s for the monitored process that runs on the New Zealand host. From these observations one can conclude that an appli- cation may take location also into account in order to determine the level of QoS that can be obtainable from each process – for example, the detection time of a pro- cess that is close by will be certainly lower than that of a process that is on another continent. link between an FD that provides QoS and consensus is certainly a relevant topic for future research. How can the QoS parameters have an influence on the execution of a consensus algorithm? How to specify the required QoS levels, taking into account features of the consensus algorithm as well as multiple process features – including location, for instance. Competing interests We confirm that I have read SpringerOpen’s guidance on competing interests and have included a statement indicating that none of the authors have any competing interests in the manuscript. 7 Conclusion In this paper, we described the architecture and imple- mentation of the Internet Failure Detection Service (IFDS). IFDS configures itself to meet the QoS require- ments of multiple simultaneous applications as well as network conditions. Two strategies (ηmax and ηGCD) were proposed to compute the interval (η) on which moni- tored processes send heartbeats. IFDS was implemented using SNMP and Web Services for enabling communica- tion among applications across the Internet. Experimental results were presented, in which the failure detector ser- vice ran both on a single LAN and on PlanetLab. In this case, monitored processes run on hosts of five conti- nents. The results show the effectiveness of the adaptive timeout with different intervals of heartbeat messages. On the one hand, the ηmax strategy is more suitable for monitoring remote processes where applications can tolerate longer delays. On the other hand, the ηGCD strat- egy is better suited for monitoring processes running on a LAN. Author details 1 1Department Informatics, Federal University of Parana, UFPR, Curitiba, Brazil. 2Sorbonne Université, UPMC Univ. Paris 06, CNRS, Inria, LIP6, Paris, France. 3CTISM, Federal University of Santa Maria, UFSM, Santa Maria, Brazil. Received: 16 April 2016 Accepted: 24 August 2016 Received: 16 April 2016 Accepted: 24 August 2016 Acknowledgements Thi k ti ll This work was partially supported by grants 309143/2012-8 and 141714/2014-0 from the Brazilian Research Agency (CNPq). 6.1 Experimental results: LAN In other words, the time to report an event is the sum of the detection time plus the time to notify the event. In Fig. 8, we com- pare the time using SNMP only and Web services. As expected, WS takes longer to report an event: on aver- age of 14.44 % more than SNMP, which we consider an acceptable overhead. Next we evaluate the amount of resources required by IFDS. Figure 7 shows CPU and memory usage. We gradu- ally increased the number of monitored objects. For each measure 10 samples were collected and the service was run for 60 min. The experiment comprises both the regis- tration of each object in the fdMIB (which corresponds to setting the information about monitored processes such as IP address and port) and the transmission of heartbeat messages. A very short heartbeat interval of 1 millisec- ond was chosen to stress the system. As shown in Fig. 7a, memory usage grows linearly but remains low: it never reaches 0.2 %. In Fig. 7b we can see that up to 100 objects, there is a consistent increase in CPU usage. After that (100 objects), although peak values do increase, the aver- age CPU usage remains stable around 7 %. CPU usage can be considered low enough. For example, the SNMP-based implementation of a failure detector by Wiesmann, Urban and Defago [22] reaches up to 11 % CPU utilization for 1 millisecond heartbeat interval. From the results in Fig. 8 it is also possible to see that the notification time respects the physical distance between hosts. The process that is the closest to the monitor (running on the Brazilian host) presents a noti- fication time of approximately 1.31 s. As the physical distance between hosts increases, the notification time a b Fig. 7 Evaluating the performance as the number of monitored objects increases. Subfigure (a) shows the Memory Usage, and in subfigure (b) shows the CPU Usage b a a b a Fig. 7 Evaluating the performance as the number of monitored objects increases. Subfigure (a) shows the Memory Usage, and in subfigure (b) shows the CPU Usage Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 Page 13 of 14 0 0.5 1 1.5 2 2.5 Brazil Canada Italy New Zealand Russia Time (s) Host’s location SNMP Web Service Fig. 8 Time to notify a failure using SNMP and WS Fig. 1. Turek J, Shasha D. The Many Faces of Consensus in Distributed Systems. IEEE Comput. 1992;25(6):8–17. 2. Guerraoui R, Rodrigues L. Introduction to Reliable Distributed Programming. Berlin: Springer; 2006. 3. Charron-Bost B, Pedone F, Schiper A, (eds). Replication: Theory and Practice. Berlin: Springer; 2010. References 1. Turek J, Shasha D. The Many Faces of Consensus in Distributed Systems. IEEE Comput. 1992;25(6):8–17. 2. Guerraoui R, Rodrigues L. Introduction to Reliable Distributed Programming. Berlin: Springer; 2006. 3. Charron-Bost B, Pedone F, Schiper A, (eds). Replication: Theory and Practice. Berlin: Springer; 2010. Future work includes developing reliable distributed applications on top of the proposed IFDS, including State Machine Replication, as well an atomic broadcast ser- vice, both of which also rely on consensus. Making the Page 14 of 14 Turchetti et al. Journal of Internet Services and Applications (2016) 7:9 4. Fischer MJ, Lynch NA, Paterson MS. Impossibility of distributed consensus with one faulty process. J ACM. 1985;32(2):374–82. 5. Chandra TD, Toueg S. Unreliable failure detectors for reliable distributed systems. J ACM. 1996;43(2):225–67. 5. Chandra TD, Toueg S. Unreliable fai systems. J ACM. 1996;43(2):225–67. systems. J ACM. 1996;43(2):225–67. 6. Freiling FC, Guerraoui R, Kuznetsov P. The failure detecto 6. Freiling FC, Guerraoui R, Kuznetsov P. T ACM Comput Surv. 2011;43(2):1–40. 6. Freiling FC, Guerraoui R, Kuznetsov P. The failure detector abstraction. ACM Comput Surv. 2011;43(2):1–40. 7. Delporte-Gallet C, Fauconnier H, Raynal M. Fair synchronization in the presence of process crashes and its weakest failure detector. In: 33rd IEEE International Symposium on Reliable Distributed Systems (SRDS’14). Japan: IEEE Computer Society, Nara; 2014. 8. Chen W, Toueg S, Aguilera MK. On the quality of service of failure detectors. In: International Conference on Dependable Systems and Networks (DSN’00). New York: IEEE Computer Society; 2000. 9. Bertier M, Marin O, Sens P. Performance analysis of a hierarchical failure detector. In: International Conference on Dependable Systems and Networks (DSN’03). San Francisco, USA: IEEE Computer Society; 2003. 10. Harrington D, Presuhn R, Wijnen B. An Architecture for Describing Simple Network Management Protocol (SNMP) Management Frameworks. In: Request for Comments 3411 (RFC3411). United States: RFC Editor; 2002. 11. Christensen E, Curbera F, Meredith G, Weerawarana S. Web Services Description Language(WSDL) Version 1.1. http://www.w3.org/TR/wsdl. Access Feb 2016. 12. An open platform for developing, deploying, and accessing planetaryscale services. http://www.planet-lab.org/. Access May 2015 13. Turek J, Shasha D. The many faces of consensus in distributed systems. IEEE Comput. 1992;25(6):8–17. 14. Cachin C, Guerraoui R, Rodrigues L. Introduction to Reliable and Secure Distributed Programming 2nd edn. Berlin: Springer; 2011. 15. Hayashibara N, Defago X, Katayama T. Two-ways adaptive failure detection with the φ-failure detector. In: International Workshop on Adaptive Distributed Systems. Italy: Sorrento; 2003. 16. References Falai L, Bondavalli A. Experimental evaluation of the qos of failure detectors on wide area network. In: International Conference on Dependable Systems and Networks (DSN’05). Yokohama: IEEE Computer Society; 2005. 17. Dixit M, Casimiro A. Adaptare-fd: A dependability-oriented adaptive failure detector. In: 29th IEEE Symposium on Reliable Distributed Systems (SRDS’10). Delhi: IEEE Symposium; 2010. 18. Tomsic A, Sens P, Garcia J, Arantes L, Sopena J. 2w-fd: A failure detector algorithm with qos. In: IEEE International Parallel and Distributed Processing Symposium (IPDPS’15). Washington: IEEE Computer Society; 2015. 19. de Sá AS, de Araújo Macêdo RJ. Qos self-configuring failure detectors for distributed systems. In: Distributed Applications and Interoperable Systems (DAIS) 2010. Amsterdam, The Netherlands: Springer Berlin Heidelberg; 2010. 20. Xiong N, Vasilakos AV, Wu J, Yang YR, Rindos AJ, ZHOU Y, Song WZ, Pan Y. A self-tuning failure detection scheme for cloud computing service. In: The 26th IEEE International Parallel & Distributed Processing Symposium (IPDPS) 2012. Shanghai: IEEE Computer Society; 2012. 21. Lima F, Macêdo R. Adapting failure detectors to communication network load fluctuations using snmp and artificial neural nets. In: Dependable Computing, Second Latin-American Symposium (LADC’05). Salvador: Springer Berlin Heidelberg; 2005. 22. Wiesmann M, Urbán P, Défago X. An SNMP based failure detection service. In: 25th IEEE Symposium on Reliable Distributed Systems (SRDS’06). Leeds: IEEE Computer Society; 2006. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com 23. Moraes DM, Duarte Jr EP. A failure detection service for internet-based multi-as distributed systems. In: 17th IEEE International Conference on Parallel and Distributed Systems (ICPADS’11). Tainan: IEEE Computer Society; 2011. 24. Jacobson V. Congestion avoidance and control. In: Symposium Proceedings on Communications Architectures and Protocols (SIGCOMM’88). USA: ACM, Stanford; 1988. 24. Jacobson V. Congestion avoidance and control. In: Symposium Proceedings on Communications Architectures and Protocols (SIGCOMM’88). USA: ACM, Stanford; 1988.
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English
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Generation of small-compound libraries via random chemistry by Fenton’s reagent
ARKIVOC
2,008
cc-by
3,308
Abstract Re-combinatorial chemistry, an auspicious method for generating small-compound libraries by means of γ-irradiation of active lead compounds, disclosed the formation of new compounds with reassembled or rearranged structures mainly to be derived from radical chemistry. Therefore, Fenton’s reagent was applied to a methanol/water solution of tacrine to mimic the irradiation. In fact, these preliminary studies revealed the generation of a small compound library with a product composition similar to those obtained by γ-irradiation. Keywords: Re-combinatorial chemistry, random chemistry, Fenton’s reagent ARKIVOC 2008 (xi) 247-255 ARKIVOC 2008 (xi) 247-255 General Papers Eva Kugelmann and Ulrike Holzgrabe* Eva Kugelmann and Ulrike Holzgrabe* Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany E-mail: u.holzgrabe@pzlc.uni-wuerzburg.de Introduction We have recently reported a new method for the serendipitous generation of a small-compound library of biologically active substances by means of γ-irradiation of active compounds or pharmacophoric fragments of active compounds or mixtures of fragments. The concept of the so- called “re-combinatorial chemistry” (random chemistry)1 proved valuable for the development of new inhibitors of the acetylcholinesterase2 as well as antiplasmoidal and antiviral compounds.3 However, most of the new compounds formed by γ-irradiation are products formed by radical chemistry. Especially solvent free radicals formed by γ-irradiation seem to be the main players in the formation of new compounds. Thus, reagents initiating the formation of radicals should be applicable in “re-combinatorial chemistry”. Since the γ-irradiation is a highly sophisticated and expensive method in this study we were aiming for the proof-of-principle. We checked whether Fenton’s reagent, one of the best known, cheapest and easiest to handle one electron transfer reagents, can serve as an initiator of the production of a small compound library of active substances. The inhibitor of the acetylcholinesterase, tacrine (1), was chosen as a model compound because the irradiation of tacrine has been already extensively studied.2 Hence, the findings of the irradiation and the ISSN 1551-7012 ©ARKAT USA, Inc. Page 247 Page 247 ARKIVOC 2008 (xi) 247-255 General Papers oxidation with Fenton’s reagent can be easily compared. Since Fenton’s reagent is not very soluble in organic solvents, mixtures of water and organic solvents had to be applied. oxidation with Fenton’s reagent can be easily compared. Since Fenton’s reagent is not very soluble in organic solvents, mixtures of water and organic solvents had to be applied. Results and Discussion The irradiation of tacrine with γ-rays derived from a 60Co source at room temperature led to the formation of a considerable library with a multitude of different compounds. The experiments were performed with solutions of tacrine hydrochloride in water and various alcohols, respectively. Comparison of the product spectra from the different solvents by means of RP- HPLC revealed methanol and water solutions to contain the highest number and concentration of new products (Fig. 1). After bioassay-guided HPLC fractionation compounds with very high inhibitory activity against AChE could be separated in both cases and their structures elucidated. Figure 1. HPLC separation of irradiated tacrine in water or methanol; 1-hydroxytacrine (2), ATAM (3), tacrine (1). (For chromatographic protocol see Experimental section).2 Figure 1. HPLC separation of irradiated tacrine in water or methanol; 1-hydroxytacrine (2), ATAM (3), tacrine (1). (For chromatographic protocol see Experimental section).2 Upon irradiation of tacrine in water, 1-hydroxytacrine (2), a known active metabolite of tacrine, was found. On irradiation of tacrine in methanol however, a new hydroxymethylated tacrine of high activity, named ATAM = (9-amino-5,6,7,8-tetrahydro-acridin-4-yl)-methanol (3), was identified.2 Upon irradiation of tacrine in water, 1-hydroxytacrine (2), a known active metabolite of tacrine, was found. On irradiation of tacrine in methanol however, a new hydroxymethylated tacrine of high activity, named ATAM = (9-amino-5,6,7,8-tetrahydro-acridin-4-yl)-methanol (3), was identified.2 Upon irradiation of tacrine in water, 1-hydroxytacrine (2), a known active metabolite of tacrine, was found. On irradiation of tacrine in methanol however, a new hydroxymethylated tacrine of high activity, named ATAM = (9-amino-5,6,7,8-tetrahydro-acridin-4-yl)-methanol (3), was identified.2 ISSN 1551-7012 ©ARKAT USA, Inc. Page 248 ©ARKAT USA, Inc. ARKIVOC 2008 (xi) 247-255 General Papers N NH2 N NH2 OH OH 2 3 N NH2 1 N NH2 N NH2 OH OH 2 3 N NH2 1 These compounds affirm the assumption that the radiolysis of water mainly results in hydrogen atoms (H), hydroxyl radicals (·OH) and hydrated electrons (e- aq) whereas in methanol accessory secondary radiolysis products are formed, e.g. a ·CH2OH radical, which is probably responsible for the formation of ATAM.4 N NH2 OH 2 N NH2 OH 2 N NH2 1 N NH2 OH 3 NH2 OH NH2 2 1 2 3 These compounds affirm the assumption that the radiolysis of water mainly results in hydrogen atoms (H), hydroxyl radicals (·OH) and hydrated electrons (e- aq) whereas in methanol accessory secondary radiolysis products are formed, e.g. Results and Discussion a ·CH2OH radical, which is probably responsible for the formation of ATAM.4 As solvent free radicals are obviously the main players in the formation of new compounds the idea arises that these radicals can be formed either by γ-irradiation or by radical forming agents such as Fenton’s reagent. To verify this assumption we ascertained if Fenton’s reagent is applicable to “re-combinatorial chemistry”. Thus, an aqueous H2O2 solution was added to a solution of ferrous sulfate and tacrine in a methanol/water mixture. The methanol/water mixture was chosen for two reasons, on the one hand due to the high solubility of tacrine hydrochloride in water, methanol and ethanol and the poor solubility of Fenton’s reagent in organic solvents and on the other hand because the results should be comparable to those obtained by γ-irradiation in water and methanol systems. The preliminary studies revealed the generation of a small compound library and in fact a product composition similar to those obtained by γ-irradiation in methanol and water (cf Fig.1 and Fig. 2). Both 1-hydroxytacrine and ATAM could be identified. Figure 2. HPLC separation of with Fenton’s reagent converted tacrine in a water/ methanol mixture; 1-hydroxytacrine (2), ATAM (3), tacrine (1). (For chromatographic protocol see Experimental section). Figure 2. HPLC separation of with Fenton’s reagent converted tacrine in a water/ methanol mixture; 1-hydroxytacrine (2), ATAM (3), tacrine (1). (For chromatographic protocol see Experimental section). Page 249 ©ARKAT USA, Inc. Page 249 ©ARKAT USA, Inc. ©ARKAT USA, Inc. ISSN 1551-7012 General Papers ARKIVOC 2008 (xi) 247-255 To separate the obtained library first an analytical RP-HPLC method with a gradient elution was used. Therefore the composition of the mobile phase was changed continuously by increasing the content of organic solvent from 10% to 40% in 30 minutes. With this procedure good separations of the compound libraries could be achieved in an acceptable period of time. By means of LC/MS analysis the main peaks could be assigned to the compounds 1- hydroxytacrine (2), ATAM (3) and tacrine (1). Additionally, the peak purity of each peak could be confirmed. The great advantage of the application of Fenton’s reagent is the fact that it is possible to vary the solvent as well as other reaction conditions in a wide range in order to influence product composition. When exploring limits and possibilities, differences in product composition were detected. Results and Discussion First the concentration of hydrogen peroxide was varied and found to have great impact. At hydrogen peroxide concentrations less than 100-fold excess in comparison to tacrine no reactions could be observed. A 200-fold excess of hydrogen peroxide however led to a notable conversion and distinct yields of ATAM (3) and 1-hydroxytacrine (2), whereas increasing the excess to 300 had no further effect. Thus, a 200-fold excess of Fenton’s reagent was found to be necessary to provide a satisfying conversion of tacrine. The raise of reaction time from 2 to 24 h gained no advantages. Thus, it can be concluded that under these conditions, reaction time is shorter than 2h. Trials to increase the methanol concentration to more than 15% failed, as ferric sulfate precipitates and the yields decreased. In order to exclude a possible influence of atmospheric oxygen on the reaction, one experiment was carried out under nitrogen atmosphere. However, no changes of the composition of the developed compound library appeared. The addition of EDTA had no effect on any yields although an increase of yields is often described in literature in similar cases. Only the combination of EDTA addition and increasing the reaction time led to a slight increase of the yields of 1-hydroxytacrine (2). The main influence on product composition however showed the variation of pH (cf Fig. 3). A decreasing pH value results in an increasing conversion of tacrine. Decreasing the pH-value from 5 to 4 and 2 increased the yields of the ATAM (3) and an unknown product (4), whereas at pH 5 the highest yields of the 1-hydroxytacrine (2) were obtained. ISSN 1551-7012 ISSN 1551-7012 ©ARKAT USA, Inc. Page 250 ARKIVOC 2008 (xi) 247-255 Papers ARKIVOC 2008 (xi) 247 General Papers 012 P 251 ©ARKA HPLC chromatogram of the treatment of tacrine with Fenton’s reagent in a nd water at pH 2 (a), pH 4 (b) and pH 5 (c). (For chromatographic p tal section). Figure 3. HPLC chromatogram of the treatment of tacrine with Fenton’s reagent in a mixture of methanol and water at pH 2 (a), pH 4 (b) and pH 5 (c). (For chromatographic protocol see Experimental section). Page 251 ©ARKAT USA, Inc. ©ARKAT USA, Inc. Page 251 ©ARKAT USA, Inc. Results and Discussion ISSN 1551-7012 ARKIVOC 2008 (xi) 247-255 General Papers To determine the structure of the unknown product (4), the experiment was performed at pH 2 followed by a semi-preparative HPLC separation combined with a solid-phase extraction (C18) with methanol as mobile phase for up-concentration and for isolation of the compound. For the semi-preparative HPLC separation a slightly modified version of the analytical HPLC method was used. The same solvents were applied, but a stepwise not a continuous change of the composition of the organic solvent had to be employed for isolation purpose. Accompanying LC/MS/MS experiments were performed in order to collect structural information. The tandem mass spectrometry experiments showed that compound 4 possesses a mass-to- charge ratio of m/z = 213 and reacted to a product ion with a m/z ratio of m/z = 197. In literature the loss of 16 amu corresponds either with the loss of an amino group NH2 + or with the loss of an oxygen atom O+. Beyond it MS3-measurements exhibit the formation of an m/z = 182 product ion (Fig. 4). This fragment is equivalent to the unsubstituted 1,2,3,4-tetrahydroacridine skeleton which is also observed in MS/MS experiments of tacrine (1) and 1-hydroxytacrine (2).2 Figure 4. MS2- und MS3-fragmentation of compound m/z = 213. Figure 4. MS2- und MS3-fragmentation of compound m/z = 213. Beside the fact, the MS measurements already indicate a tacrine skeleton with a carbonyl group, the NMR data affirmed the assumption. The 13C-NMR spectra and the four 1H-NMR signals for hydrogen H-5, H-6, H-7 and H-8 indicated an intact aromatic tacrine scaffold since they have nearly the same chemical shifts as in tacrine. However, only six aliphatic protons remained with two triplets at 3.24 and 2.67 ppm and one triplet of triplets at 2.16 ppm indicating a substitution on the cyclohexyl ring of tacrine in position 1 or 4. A 13C signal at 199.5 ppm ISSN 1551-7012 ©ARKAT USA, Inc. ©ARKAT USA, Inc. Page 252 Page 252 ARKIVOC 2008 (xi) 247-255 General Papers indicates furthermore the existence of a carbonyl keto group. The synthesis of an oxidized tacrine in position 1 and the comparison of the NMR and MS spectra finally confirmed the structure of 9-amino-3,4-dihydroacridin-l(2H)-one (4). This tacrine derivative is already known as precursor in the syntheses of 1-hydroxytacrine.5 Nevertheless no testing of this compound of the biological activity, i.e. of its possibility to inhibit acetylcholinesterase has been published so far. Results and Discussion Hence, compound 4 was subjected to a modified Ellman’s test6 in order to check the inhibitory activity towards AChE. 4 inhibited AChE half- maximally at IC50 = 319 nM which is about three times higher than for 1-hydroxytacrine (IC50 = 106 nM) and about 7 times higher than for tacrine (IC50 = 44 nM), respectively. Examination of X-ray crystal structure of tacrine7 revealed the 4-aminoquinoline portion of the tacrine molecule to be responsible for the binding of the drug to the enzyme. Tacrine is stacked near the bottom of the gorge against the rings of Trp84 and Phe330, the ring nitrogen is hydrogen bonded to the main-chain carbonyl oxygen of His440, and the amino nitrogen forms a hydrogen bond to a water molecule.8 The cyclohexyl ring however acts mostly as a block for the substrate at the active site.9 The introduction of a carbonyl group might lead to a steric hindrance worsening drug binding and thus be responsible for the readily increased IC50 values. Experimental Section General Procedures. Tacrine was synthesized according to refs 11. Solutions of tacrine were prepared in solvents of analytical grade and solvents used for LC, LC/MS and LC/MS/MS measurements were HPLC grade throughout. HPLC and analytical grade methanol were purchased from Merck (Darmstadt, Germany) and ammonium acetate, ferrous sulfate, formic acid, hydrogen peroxide, salts, and reagents for the Ellman test were from Fluka (Taufkirchen, Germany). HPLC grade water prepared by means of the Milli-Q purification system (Millipore, Eschborn, Germany) was used throughout. γ-Irradiation of tacrine. Solutions of tacrine hydrochloride (2.34 mg/mL) in water and methanol were exposed to γ-rays from a 60Co source as described in ref. 2. Conversion with Fenton’s reagent. A solution of 10-4 mol ferrous sulfate in sulfuric acid and 10-4 mol tacrine in a methanol/water (15:85) mixture was converted with a aqueous H2O2 solution (35%) and stirred for 2 h at room temperature. After evaporation the solvent the residue was purified by column chromatography on neutral aluminum oxide using methanol:water (2:1) as mobile phase and analyzed subsequently by HPLC/MS . High performance liquid chromatography. The analyses were performed using a Agilent 1100 system with SYNERGYTM MAX-RP columns (Phenomenex, 150 × 4.6 mm i.d. and 150 ×10.0 mm i.d., 4 µm particle size) at 25 °C. In order to protect the integrity of the analytical and semi- preparative column, all analyses were performed with a coupled C-18 guard column. Mobile phase were 10 mM ammonium acetate with 0.08% formic acid at pH = 3.5 and methanol. The elution rates were 10-40% methanol in 30 min at a flow rate of 1 ml/min for the analytical separation and a stepwise change of the composition of organic solvent (A/B (85:15) for 10 min; A/B (95:5) for 5 min; A/B (80:20) for 5 min) at a flow rate of 4.7 ml/min for the semi- preparative separation. Samples were monitored at 254 nm. Purification and identification. By means of solid phase extraction (Chromafix C18 ec, 900 mg/ 6 ml, Macherey-Nagel) the buffer could be eliminated and compound 4 purely isolated. Structure elucidation was accomplished by spectroscopic methods, including 2D-NMR experiments and comparison with literature data.12 LC/MS/MS Analysis. The electrospray ionization (ESI) mass spectra were measured on an Agilent 1100 LC/MSD Trap consisting of an orifice with nitrogen curtain gas and a capillary interface. Conclusions The easy, inexpensive and fast handling is the main advantage of the application of Fenton’s reagent in “re-combinatorial chemistry”. The limited solubility of Fenton’s reagent in organic solvents however is a significant disadvantage as well as the poor solubility of organic compounds and potential drugs in aqueous solutions. However, since drugs in general should exhibit a high water solubility to ensure bioavailability the limitation can be an advantage at the same time as the products formed under these conditions are likely to be water soluble. Taken together, the findings of the application of Fenton’s reagent in “re-combinatorial chemistry” confuted the assumption of Ploszynska et al.10 that reactive species generated in Fenton’s system are not the same as the species generated during water radiolysis, and revealed the method to be a pilot experiment for γ-irradiation and thus, to be a powerful tool for the generation of small-compound libraries in drug discovery. Additionally, it may serve as a screening method for the metabolic profile of a new biologically active compound because the underlying chemistry of both reactions Fenton’s one electron oxidation and cytochrome P450 oxidations, being the main metabolism enzyme, are similar. ISSN 1551-7012 ©ARKAT USA, Inc. Page 253 ©ARKAT USA, Inc. ARKIVOC 2008 (xi) 247-255 ARKIVOC 2008 (xi) 247-255 ARKIVOC 2008 (xi) 247-255 General Papers Experimental Section The conditions of the spray chamber were as follows: ion polarity, positive; capillary voltage, 3500 V; drying gas temperature, 350 °C; nebulizer pressure, 50 psi; drying gas flow, 12.00 l min-1. LC/MS/MS analyses were performed by using multiple reaction monitoring (MRM). Page 254 ©ARKAT USA, Inc. Page 254 ©ARKAT USA, Inc. ISSN 1551-7012 ARKIVOC 2008 (xi) 247-255 General Papers Analytical and spectroscopic data for ATAM (1) and 1-hydroxytacrine (2). See Ref. 3. Analytical and spectroscopic data for 9-amino-3,4-dihydroacridin-1(2H)-one (4) Formula: C13H12N2O (212.09). 1H NMR (400 MHz, CDCl3) δ: 10.53 (1H, br, NH2), 8.94 (1H, br, NH2), 8.08 (1H, d, J=8.2 Hz, 5-H/8-H), 7.80 (1H, d, J=8.1 Hz, 5-H/8-H), 7.56 (1H, t, J=7.6 Hz, 6-H/7-H), 7.35 (1H, t, J=8.0 Hz, 6-H/7-H), 3.24 (2H, t, J=7.4 Hz, 2-H), 2.67 (2H, t, J=7.2 Hz, 4-H) 2.16 (2H, m, 3-H). 13C NMR (100 MHz, CDCl3) δ: 199.5 (C-1) q, 161.7 (C-9) q, 156.8 (C- 4a) q, 139.5 (C-10a) q, 134.1 (C-6), 126.4 (C-7), 123.6 (C-8), 122.3 (C-5), 116.9 (C-9a) q, 104.7 (C-8a) q, 38.8 (C-4), 29.4 (C-2), 20.2 (C-3). ESI-MS m/z: 213 [M + H]+. MS2 m/z: 197 (213 [M + H]+ - O+). MS3 m/z: 182 (197 [M + H]+ - NH+). mp 237°C (Ref.12 mp 237-239°C). Analytical and spectroscopic data for 9-amino-3,4-dihydroacridin-1(2H)-one (4) Formula: C13H12N2O (212.09). 1H NMR (400 MHz, CDCl3) δ: 10.53 (1H, br, NH2), 8.94 (1H, br, NH2), 8.08 (1H, d, J=8.2 Hz, 5-H/8-H), 7.80 (1H, d, J=8.1 Hz, 5-H/8-H), 7.56 (1H, t, J=7.6 Hz, 6-H/7-H), 7.35 (1H, t, J=8.0 Hz, 6-H/7-H), 3.24 (2H, t, J=7.4 Hz, 2-H), 2.67 (2H, t, J=7.2 Hz, 4-H) 2.16 (2H, m, 3-H). 13C NMR (100 MHz, CDCl3) δ: 199.5 (C-1) q, 161.7 (C-9) q, 156.8 (C- 4a) q, 139.5 (C-10a) q, 134.1 (C-6), 126.4 (C-7), 123.6 (C-8), 122.3 (C-5), 116.9 (C-9a) q, 104.7 (C-8a) q, 38.8 (C-4), 29.4 (C-2), 20.2 (C-3). ESI-MS m/z: 213 [M + H]+. MS2 m/z: 197 (213 [M + H]+ - O+). MS3 m/z: 182 (197 [M + H]+ - NH+). mp 237°C (Ref.12 mp 237-239°C). 12. Drewe, J. A.; Groundwater, P. W. J. Chem. Soc., Perkin Trans. 1 1997, 5, 601. References 1. Folkers, G.; Kessler, U. Curr. Drug Discovery 2003, 1, 1. 2. Kapková, P.; Heller, E.; Unger, M.; Folkers, G.; Holzgrabe, U. J. Med. Chem. 2005, 48, 7496. 3. Kapková, P.; Heller, E.; Kugelmann, E.; Faber, J.; Bringmann, G.; Kessler, U.; Folkers, G.; Holzgrabe, U. Arch. Pharm. Chem. Life Sci. 2006, 339, 489. 4. Getoff, N.; Ritter, A.; Schwoerer, F.; Bayer, P. Radiat. Phys. Chem. 1993, 4 5. Shutske, G. M.; Pierrat, F. A.; Cornfeldt, M. L.; Szewczak, M. R.; Huger, F. P.; Bores, G. M.; Haroutunian, V.; Davis, K. L. J. Med. Chem. 1988, 31, 1278. 6. Kapková, P.; Stiefl, N.; Sürig, U.; Engels, B.; Baumann, K.; Holzgrabe, U. Arch. Pharm. Med. Chem. 2003, 336, 523. 7. Harel, M.; Schalk, I.; Ehret-Sabatier, L.; et al. Proc. Natl. Acad. Sci. USA 1993 ; Schalk, I.; Ehret-Sabatier, L.; et al. Proc. Natl. Acad. Sci. USA 1993, 90, 9031. 8. Zhao, Q.; Tang, X. C. Eur. J. Pharmacol. 2002, 455, 101. 8. Zhao, Q.; Tang, X. C. Eur. J. Pharmacol. 2002, 455, 101. 9. Silman, I.; Harel, M.; Axelsen, P.; Raves, M.; Sussman, J. L. Biochem. Soc. Trans. 1994, 22, 745. 10. Ploszynska, J.; Kowalski, J.; Sobkowiak, A. Pol. J. Chem. 1998, 72, 2514. 11. Moore, J. A.; Kornreich, L. D. Tetrahedron Lett. 1963, 20, 1277. 12. Drewe, J. A.; Groundwater, P. W. J. Chem. Soc., Perkin Trans. 1 1997, 5, 601. ISSN 1551-7012 ISSN 1551-7012 ©ARKAT USA, Inc. Page 255 Page 255
https://openalex.org/W4285141147
http://www.amha-journal.com/index.php/AMHA/article/download/599/596
Italian
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Il segnalamento del delinquente di Salvatore Ottolenghi
Acta medico-historica Adriatica
2,022
cc-by
5,052
Acta Med Hist Adriat 2022; 20(1); 139-153 https://doi.org/10.31952/amha.20.1.7 Acta Med Hist Adriat 2022; 20(1); 139-153 https://doi.org/10.31952/amha.20.1.7 Review article Pregledni članak Review article Pregledni članak IL SEGNALAMENTO DEL DELINQUENTE BY SALVATORE OTTOLENGHI: THE STUDY OF DERMATOGLYPHICS IN ANTHROPOMETRIC CABINETS Francesca Vannozzi*, Davide Orsini** *    Storia della Medicina, Dipartimento di Scienze mediche, chirurgiche e Neuroscienze, Università degli Studi di Siena, Siena, Italia. ORCID: https://orcid.org/0000-0002-7664-0202 **   Storia della Medicina, Sistema Museale Universitario Senese SIMUS, Università degli Studi di Siena, Siena, Italia. ORCID: https://orcid.org/0000-0003-1593-6656 Correspondence address: Davide Orsini, Storia della Medicina, Sistema Museale Universitario Senese SIMUS, Università degli Studi di Siena, Siena, Italia. E-mail: davide.orsini@unisi.it. RIASSUNTO I dermatoglifi, essendo completamente diversi da un essere umano all’altro, segnalano la diversità umana. Per questo motivo, e per le loro caratteristiche di unicità, classificazione e inalterabilità, le creste papillari sulla punta delle dita rappresentano elementi di una sicura differenziazione tra una persona e l’altra. Di conseguenza, le impronte digitali sono molto utili nell’identificazione di un essere umano. Salvatore Ottolenghi fu il primo ad utilizzare il sistema di impronte digitali per identificare gli individui e introdusse questo sistema nel suo “Cartellino di riconoscimento” nel 1902. Es- sendo sicuro della validità scientifica di questo metodo, lo considerava libero da potenziale interpretazione personale. Secondo lui “le impronte digitali, per loro natura, formano dise- gni speciali dalla nascita; questi non cambieranno per tutta la vita e saranno assolutamente diversi da un essere umano a un altro”. Questo metodo di identificazione delle impronte 139 digitali fu immediatamente perfezionato da Giovanni Gasti, scelto da Salvatore Ottolenghi come suo assistente personale presso la Scuola di Polizia Scientifica. Per questo motivo, il metodo è stato chiamato “Sistema Gasti (Sistema Gasti)”. Fu usato per tutto il Novecento. Parole chiave: dermatoglifi, impronte digitali, polizia scientifica, Salvatore Otto- lenghi, antropometria Introduzione Uno dei campi di applicazione della criminalistica è lo studio dell’impiego dei metodi per contrassegnare gli individui macchiatisi di particolari reati, al fine di permetterne l’immediata riconoscibilità. Tale esigenza è da far risalire al XVII secolo in Francia, dove la pratica corrente era di apporre marchi a fuoco o tatuaggi sulla pelle dei condannati – ad esempio le lettere “T” (travail - per i condannati ai lavori forzati) e “V” (voleur - per i ladri) – allo scopo di riconoscerli facilmente (Giuliano, 2005, p. 8). Nei secoli successivi, tuttavia, l’identificazione di uno specifico soggetto come criminale non bastava più: si avvertì la necessità di individuare i delin- quenti recidivi e di interpretare un determinato reato nella storia complessiva del soggetto stesso. “La polizia era continuamente mistificata da vagabondi e delinquenti che, mutando nome a ogni nuovo arresto, sfuggivano impu- nemente all’applicazione di tutte le misure amministrative che erano state adottate a loro carico in occasione di precedenti catture. […] I recidivi erano giudicati come imputati incensurati” (Rusticucci, 1927, p. 7). Il rilevamento, la descrizione e la conservazione dei caratteri di identifica- zione di una persona divennero pertanto obiettivi fondamentali per il segna- lamento di polizia. I dermatoglifi e la dattiloscopia È da notare che sono stati numerosi i sistemi di comparazione biometrica adottati nel passato. Quello più utilizzato nel corso del XX secolo è stata in- dubbiamente la dattiloscopia, basata sull’analisi dei dermatoglifi e sulle tracce lasciate dagli stessi. L’interesse per gli elementi digito-papillari nacque con l’anatomista te- desco Johann Christoph Andreas Mayer (1747-1801), il quale per primo, nel quarto volume della sua opera Anatomische Kupfertafeln: nebst dazu gehӧrig- en Erklӓrungen data alle stampe nel 1788, “gettò una vera e propria base di studio sulle conformazioni delle impronte digitali. Sono tuttora un mirabile 140 esempio le sue tavole illustrative” (Giuliano, 2005, p. 27). Numerose ricerche poi contribuirono alla messa a punto di un vero e proprio metodo scientifico inerente allo studio dei dermatoglifi e al conseguente utilizzo delle impronte digitali (Grzybowski & Pietrzak, 2015, pp. 117-121). Un contributo fondamentale fu dato da Sir Francis Galton (1822-1911), il quale negli anni Novanta del XIX secolo misurò la probabilità di possedi- mento delle medesime impronte digitali da due individui diversi. Egli dedicò numerosi articoli all’esposizione dei suoi studi (Galton, 1888, pp. 201-202) e creò un sistema per la loro classificazione (Galton, 1892). Il metodo di iden- tificazione attraverso le impronte digitali fu già stato introdotto da William James Herschel (1833-1917) negli anni Sessanta dell’Ottocento. Egli affermò di aver usato le impronte digitali per motivi di identificazione già dal 1858 docu- mentando “la rigorosa individualità e l’ostinata persistenza delle tracce sulle nostre dita” (Herschel, 1916, p. 5). Henry Faulds (1843-1930) propose il meto- do in ambito giudiziario nel 1880. Furono però le ricerche di Galton, riprese da Sir Edward Richard Henry (1850-1931), a impostare su base scientifica lo sviluppo e le applicazioni di questo metodo, favorendone quindi l’adozione nelle aule giudiziarie (Bulmer, 2003). Nei primi anni Novanta Henry iniziò un lungo carteggio con Francis Galton sull’utilizzo delle impronte digitali nell’i- dentificazione dei criminali che potesse sostituire il metodo di riconoscimen- to biometrico ideato da Alphonse Bertillon (1853-1914). Tra il luglio del 1896 e il febbraio del 1897 Henry sviluppò la classificazione delle impronte digitali che ne consentiva l’archiviazione e la comparazione, un sistema descritto nel- la sua opera Classification and uses of finger prints (Henry, 1900). Il metodo di classificazione da lui proposto, che prese il nome di Sistema di classificazione Galton-Henry, fu subito recepito e utilizzato da molte forze di polizia. I dermatoglifi e la dattiloscopia È tuttavia necessario attendere il 1907 per la prima definizione efficace di “impronte digitali” che si può far risalire a Salvatore Ottolenghi (1861-1934): “riproduzione delle linee papillari dei polpastrelli delle dita che formano per la loro disposizione speciali disegni, fino dalla nascita, immutabili per tutta la vita, assolutamente diversi da un individuo all’altro” (Ottolenghi, 1907). La ragione per un così ampio e diffuso utilizzo della dattiloscopia sta pro- prio nelle caratteristiche stesse del disegno dei dermatoglifi, i quali si contrad- distinguono per univocità, classificabilità e immutabilità. I disegni papillari digitali propriamente detti sono presenti sui polpastrelli e negli spazi interdigitali delle falangi, mentre le impronte palmari sono situa- te sul palmo della mano, plantari sul piede. Il processo formativo delle linee 141 papillari nell’essere umano è già in atto tra il terzo e il quarto mese di vita in- trauterina. Verso la dodicesima settimana di gestazione sulla superficie inter- na dell’epidermide compaiono invece delle prime ondulazioni che danno poi origine alla formazione di creste e solchi dermici. Infine, dopo il sesto mese di vita fetale, le figure sono complete e rimangono invariate nel tempo. Già Marcello Malpighi (1628-1694), con la sua definizione dei cinque diver- si strati dell’epidermide, aveva ipotizzato l’immutabilità delle creste papillari, il principio fondamentale della scienza dattiloscopica (Malpighi, 1665). Altri elementi identificativi sono quelli legati all’evento accidentale che può por- tare, ad esempio, a una cicatrice che, lasciando tracce permanenti sulle creste papillari, assume un valore probatorio ai fini dell’identificazione. Solo ma- lattie della pelle o ferite profonde che danneggiano il derma possono infatti interferire sulla loro configurazione, questo il motivo del loro ruolo di indica- tori di anomalie di sviluppo. Oltre a tale indicazione, lo studio delle creste papillari è usato nell’esame del cadavere, per il quale sono comunque fondamentali gli esami sugli stadi degenerativi. Agli studi delle creste viene aggiunta la morfologia della mano e la valuta- zione delle secrezioni delle ghiandole sudoripare e delle loro tracce che si pos- sono presentare nei reperti da esaminare. Indagini microanatomiche, grazie a sofisticate strumentazioni come un microscopio per visione stereoscopica o l’uso di fibre ottiche, consentono in modo migliore di investigare le creste pa- pillari di un polpastrello. Alle indagini ottiche vengono poi associate quelle fisiche, fisico-chimiche, chimiche e con reattivi diversi. I dermatoglifi e la dattiloscopia Di particolare rilievo è la possibilità dell’impiego delle creste papillari per l’estrazione del DNA, che concorre all’identificazione dell’autore di un determinato reato, come attesta- to dalle indagini di genetica forense (Ricci, 2001). I dermatoglifi, valore semiologico e correlazioni cromosomiche Lo studio della mano potrebbe costituire, nel corso dell’esame del malato, un momento di grande interesse per la molteplicità delle informazioni che si possono desumere dalla sua ispezione. Ciò comprende l’inquadramento nosologico del paziente e il corso di affezioni dell’apparato polmonare, car- diocircolatorio, digerente, nervoso ed endocrino. È noto come precisi quadri patologici siano desumibili anche dallo studio dell’aspetto della mano, inqua- drabili nel rilevamento dei segni risalente al tempo dalla semeiotica. Recen- 142 te invece è lo studio delle impronte digitali nella medicina clinica. A partire dalla medicina legale per l’identificazione personale, per poi estendersi all’an- tropologia e alla biologia per gli “studi sulle razze”, i quadri dermatoglifici e le anomalie delle impronte sono diventati dalla prima metà del Novecento utili per l’identificazione di alcune malattie, ma soprattutto per i rilievi di pa- tologie ereditarie. Le impronte hanno quindi acquisito un ruolo importante quale mezzo diagnostico, specie in riferimento all’individuazione di certi tipi di aberrazioni cromosomiche. I primi studi rivolti a indagare la possibilità dell’esistenza di un quadro dermatoglifico particolare in alcune forme di patologia ereditaria risalgono agli ultimi anni del XIX secolo. Nei primi quarant’anni del XX secolo furono condotte iniziali ricerche sistematiche, pervenendo alla individuazione di dati di notevole importanza per la patologia cromosomica e non. Tra le ricerche che correlano lo studio delle impronte e patologie merita menzionare quelle di inizio Novecento sui pazienti psichiatrici e le prime correlazioni in alcune malattie ereditarie come la talassemia (Dallapiccola, 1968). Il fine perseguito fu quello di redigere un attendibile elenco di segni, peculiari e costanti, re- peribili dallo studio dei disegni formati dalle creste cutanee della mano, che avrebbero assunto un interesse di primo piano in forme morbose cromosomi- che, come la trisomia 21 o morbo di Down, consentendo con lo studio delle sue caratteristiche nuove prospettive di diagnosi. Lo studio dei dermatoglifi palmari aprì così nuove strade per la moderna semeiotica medica. I dermatoglifi per il segnalamento del delinquente Tornando all’argomento principe di questo scritto, ovvero la possibilità di identificare un individuo attraverso caratteristiche e segni del corpo, va ricor- dato che prima dell’introduzione della dattiloscopia il criminologo francese Alphonse Bertillon aveva messo a punto una tecnica di segnalamento antro- pometrico, applicando per primo un metodo scientifico in un atto di polizia giudiziaria. Egli promuoveva lo studio antropologico delle caratteristiche del corpo umano quale strumento identificativo, accompagnandolo con un accu- rato segnalamento descrittivo e con la fotografia, frontale e laterale, del reo (Bertillon, 1885, 1890; Bertillon & McClaughry, 1896). Iniziò un’accurata misurazione antropometrica dei caratteri fisici delle persone “schedate”, registrati in un archivio che nel 1883 annoverava 1600 classificazioni. L’archivio gli consentì l’identificazione di numerosi criminali fino a quel momento rimasti impuniti in un contesto nel quale la recidività 143 assumeva proporzioni enormi e le tecniche a disposizione si rivelavano inef- ficaci. Lo studio antropologico condotto da Bertillon diede frutto a un sistema per identificare i criminali detto Bertillonage, rapidamente adottato in Eu- ropa. Prevedeva, oltre a una foto segnaletica mezzo busto, frontale e laterale, la descrizione e le misure fisiche dell’individuo. Tale metodo era incentrato sul postulato che vuole l’ossatura umana differente da individuo a individuo, tenendo presente che la stessa non varia dal 20° anno di età. Il metodo antropometrico trovò un forte sostenitore in Cesare Lombroso (1835-1909) che fin dagli inizi della sua carriera si fece sostenitore del nesso tra sembianze umane e delitti commessi per scoprire il delinquente ma an- che, eventualmente, per riconoscere l’innocenza di un individuo (Lombro- so, 1902). Affermò inoltre con decisione l’importanza di una “polizia tecnica” adeguatamente formata per una conoscenza psicologico-comportamentale dell’uomo (Dallapiccola, 1968, p. 9, e ssgg.). Oltre agli studi psichiatrici alimen- tati da vari suoi contatti e viaggi all’estero, certamente determinante fu anche la sua esperienza di sanitario alle carceri di Torino. Questo evento contribuì a un sapere quanto mai vasto, fatto di psichiatria e medicina legale, come ben si evince già dalla prima edizione del suo L’uomo delinquente del 1876. Nel 1914, nel corso del congresso internazionale di Polizia Scientifica di Monaco, si pose termine all’uso del metodo antropometrico. È con Salvatore Ottolenghi (1861-1934) (Fig. 1), uno dei protagonisti del- lo sviluppo della Medicina legale, che lo studio dei metodi di identificazione del delinquente ebbe una forte e decisiva accelerazione. I dermatoglifi per il segnalamento del delinquente Nel 1893 Ottolenghi ottenne la cattedra di Medicina Legale presso le Facoltà di Medicina e Chi- rurgia e Giurisprudenza dell’Università di Siena, dove lavorò per dieci anni per poi passare all’Ateneo romano. Nel 1895 organizzò a Siena il primo Corso di Polizia Scientifica, realizzato in forma di lezioni sulle metodologie connesse all’osservazione delle tracce, al rilievo delle caratteristiche e alla loro compara- zione ai fini della formulazione del giudizio (Ottolenghi, 1897, pp. 33-44 - Fig. 3). Il pensiero di Ottolenghi fu determinante nella decisione di istituire nel 1902, a Roma, la Polizia Scientifica Italiana, a cui seguì la creazione dei Gabi- netti di Polizia Scientifica, già 11 nel 1910. 144 Figura 1. Salvatore Ottolenghi (Archivio Università di Siena) Figura 1. Salvatore Ottolenghi (Archivio Università di Siena) Allievo di Cesare Lombroso all’Università di Torino, ma con prevalente formazione medico-legale, a Ottolenghi va ampiamente riconosciuto il meri- to di aver compreso il bisogno di nuovi metodi di identificazione personale, oggettivamente più validi, e di aver operato in tal senso. “Intuì la necessità di metodiche più accurate, non limitate ai dati essenziali dei connotati relativi alla corporatura, alla statura, al colore dei capelli e degli occhi, alla barba, ai segni particolari, posto che tali indicazioni si rivelavano del tutto insufficien- ti, prive come erano di aggettivi di valore universale e di comune riscontro” (Martini & Piva, 1993, p. 64). Nel suo scritto Il segnalamento del delinquente in servizio della Polizia giu- diziaria (Ottolenghi, 1898; Fig. 2), Ottolenghi evidenziava l’insufficienza dei metodi in quel tempo praticati, tanto da arrivare ad affermare: “Su questo ge- nere di segnalazioni si può far così poco assegnamento che dobbiamo stupirci 145 quando dai dati comunicati si riesce a scoprire la persona ricercata” (Ottolen- ghi, 1898, p. 15). Motivava l’affermazione nel seguente modo: “I lineamenti del viso se non sono studiati scientificamente non servono a nulla; paiono uguali […]. Occorre uno studio in cui all’impressione soggettiva venga sostituendosi più che è possibile quella oggettiva, eliminando il più possibile l’apprezza- mento dell’individuo. […] La colpa sta nel ritenere la capacità di segnalazione del delinquente come proprietà comune intuitiva di qualsiasi persona, senza bisogno di cultura speciale, mentre invece la faccia umana si può ben dire è la pagina di un libro che non si lascia leggere che da chi ne ha appreso l’alfa- beto” (Ottolenghi, 1898, pp. 15-16). I dermatoglifi per il segnalamento del delinquente Il problema fondamentale stava pertanto, a suo avviso, nella necessità di introdurre nuove metodiche ed elementi di identificazione svincolati da ogni impressione soggettiva e di una specifica preparazione dei funzionari di Polizia. Secondo lui, bisognava passare da una “polizia empirica” a una “polizia scientifica” (Ottolenghi, 1898, p. 22). Figura 2. Salvatore Ottolenghi, Il segnalamento del delinquente in serviz Polizia giudiziaria Figura 2. Salvatore Ottolenghi, Il segnalamento del delinquente in servizio della Polizia giudiziaria 146 Dall’altro lato, già nel 1879 Lombroso scriveva: “Noi abbiamo fatto finora la polizia come si faceva la guerra nei tempi eroici, tutto a casaccio, ad empiri- smo […]. Abbiamo dei questori che sono assai abili […], non ne abbiamo nes- suno, però, che […] si serva per le sue indagini delle risorse scientifiche offerte dagli studi di statistica, di antropologia criminale, che moltiplichi insomma il proprio ingegno colle forze enormi e, quel che è più, esattamente governabili della scienza” (Ottolenghi, 1897, pp. 21-22). A tal fine Ottolenghi tenne presso l’Università di Siena corsi liberi di Poli- zia giudiziaria scientifica. Con il passaggio all’Università di Roma nel 1902 tale sua attività assunse finalità ancora più importanti, facendosi egli promotore della Scuola di Polizia scientifica (D’Antonio, 2020; Ottolenghi, 1897). L’isti- tuzione della Scuola di Polizia scientifica prendeva le mosse da lontano, dal laboratorio medico-legale istituito da Ottolenghi nell’Università di Siena e dai corsi di formazione promossi in quell’Ateneo già nel 1895. Figura 3. Salvatore Ottolenghi, L’insegnamento universitario della Polizia giudiziaria scientifica Figura 3. Salvatore Ottolenghi, L’insegnamento universitario della Polizia giudiziaria scientifica 147 Sempre nel 1912 il commissario Adriano Zaiotti, Ispettore generale al Mi- nistero, decise di riformare anche la Cartella Biografica, introdotta al termine del XIX secolo. Per questo motivo si rivolse a Salvatore Ottolenghi, che così descrisse l’occasione: “Proprio mentre tenevo il 1° corso di polizia scientifica pei funzionari superiori della capitale venni incaricato, (fatto che dimostra come l’amministrazione fosse matura per le nuove idee) […], di proporre nella Cartella biografica quelle aggiunte che, secondo i criteri scientifici e le esi- genze della pratica, credeva più utili. […] Fui lusingato e nello stesso tempo spiacente dell’incarico. Lusingato, perché intravedevo di essere giunto in un momento opportuno per impiantare un forte innesto scientifico nella pratica dell’amministrazione di Pubblica Sicurezza; spiacente perché per fare quanto avrei desiderato sarebbe stato necessario compilare, ab ovo tutta una nuova cartella completamente ispirata a rigorosi criteri scientifici” (Ottolenghi, 1916, p. 106). Dopo un attento lavoro al quale contribuirono anche alcuni suoi collabo- ratori, Ottolenghi presentò una nuova cartella, “il Cartellino Segnaletico”, per mezzo della quale introdusse un metodo scientifico di segnalamento in tutti gli uffici di Pubblica Sicurezza del Regno. Il “Cartellino Segnaletico” di Ottolenghi, basato appunto su metodi scientifici di identificazione, rispondeva alla necessità di “introdurre un se- gnalamento somatico esatto” (Ottolenghi, 1916, p. 107). Con tale strumento trasformò e modernizzò interamente i metodi di indagine giudiziaria. Ciò che lo guidò in questo lavoro fu la necessità di “introdurre un segnalamen- to somatico esatto, […] che – scriveva – può essere descrittivo, fotografico, antropometrico, dattiloscopico. Col descrittivo si descrivono i connotati e i contrassegni particolari, col fotografico si fissa l’immagine dell’individuo, coll’antropometrico, ideato dal Bertillon, si raccolgono le misure di alcune parti del corpo, con il segnalamento dattiloscopico si riproducono le impron- te digitali” (Ottolenghi, 1916, p. 107). Ottolenghi metteva al primo posto il segnalamento descrittivo, accoglien- do con alcune modificazioni il ritratto parlato di Bertillon poiché con esso ven- ne introdotto il linguaggio scientifico nella funzione di identificazione. Ridimensionò ampiamente il criterio antropometrico, per anni conside- rato ottimale per il segnalamento dei pregiudicati, che divenne complemen- tare agli altri, in quanto aveva solo la possibilità di stabilire le “non identi- tà”. Privilegiò invece, oltre a quello descrittivo, il segnalamento fotografico e dattiloscopico. Figura 3. Salvatore Ottolenghi, L’insegnamento universitario della Polizia giudiziaria scientifica A completare il segnalamento somatico, inserì la fotografia, 148 per la quale si avvalse nei corsi di Polizia scientifica della professionalità del commissario Umberto Ellero. “Era mio desiderio – raccontò qualche anno più tardi – unire nella stessa pagina le “impronte digitali”, che […] molto meglio delle misure antropome- triche hanno un valore essenziale per il riconoscimento, bastando l’impronta di un dito ad identificare un individuo. Per cause da me indipendenti dovetti accontentarmi di inserire le impronte del pollice, indice, medio e anulare del- la mano destra sulla 2a pagina di un “supplemento” […]. Ma ho piena fiducia di riuscire a far accogliere nella cartella per tutti i pregiudicati tutte dieci le impronte delle dita, trattandosi di una manipolazione facilissima alla portata di tutti” (Ottolenghi, 1916, p. 110). Ciò si avverò a distanza di pochi anni, grazie anche al suo collaboratore Giovanni Gasti, che svolse un fondamentale lavo- ro sulle impronte digitali. Il “Cartellino Segnaletico” di Ottolenghi conteneva quindi tutti e quattro i metodi di segnalamento: descrittivo, fotografico, dattiloscopico e antropo- metrico. È fondamentale sottolineare come l’introduzione voluta da Ottolenghi del sistema dattiloscopico abbia rivoluzionato le metodologie e i processi di identificazione e riconoscimento. Ciò è fortemente evidente se si tiene conto del fatto che tutti gli altri Uffici di Pubblica Sicurezza iniziarono a redigere obbligatoriamente il segnalamen- to descrittivo e dattiloscopico, mezzo quest’ultimo di facile rilevazione e di grande affidabilità, che a detta di Ottolenghi erano comunque sufficienti per una corretta identificazione. Il segnalamento antropometrico veniva utilizzato solo in casi eccezionali, e cioè nel caso di soggetti per i quali veniva praticato il confronto internazio- nale con nazioni che usavano tale metodo. Il segnalamento fotografico era usato dagli Uffici di Pubblica Sicurezza del Regno di maggiore importanza, essendo solo detti Uffici muniti di mezzi ne- cessari (Ottolenghi, 1930, pp. 708-709). Operando all’interno della Scuola di Polizia, Ottolenghi si interessò an- che della trasmissione telegrafica delle impronte digitali: a Parigi il 3 giugno 1923 ottenne per la prima volta la trasmissione telegrafica di una impronta digitale da Lione. Questa informazione non fa altro che ribadire la fiducia ri- posta da Ottolenghi in questo mezzo di identificazione, che per tutto il Nove- 149 cento rappresentò il mezzo più semplice ed efficace ai fini del riconoscimento e dell’identificazione. Il sistema Gasti: l’identificazione attraverso le impronte digitali Salvatore Ottolenghi, non trovando convincenti le classificazioni per le impronte digitali diffuse in quegli anni in Europa, chiese a un suo giovane allievo, Giovanni Gasti (1869-1939), di prepararne una nuova che “fosse sem- plice e pratica allo stesso tempo” (Ottolenghi, 1912, p. 10). Fu così che Gasti, scelto da Ottolenghi come collaboratore per la selezione e l’addestramento dei futuri cadetti della Polizia scientifica, elaborò durante il servizio presso la Scuola di segnalamento e identificazione il suo nuovo metodo di identi- ficazione attraverso le impronte lasciate dai dermatoglifi (Gasti, 1912, pp. 112- 124). Modificando e adattando il sistema di classificazione di Francis Galton e Edward Henry, progettò un sistema per l’identificazione delle impronte di- gitali, noto come “Sistema Gasti” o anche come “identificazione decadattilo- scopica”. Fondato sul conteggio delle linee o disegni papillari a cui faceva cor- rispondere simboli numerici, il sistema riusciva a determinare rapidamente gli eventuali precedenti dattiloscopici di un soggetto sottoposto a controllo (Giuliano, 2007). Il suo metodo prevedeva la categorizzazione di creste e solchi, per ciascuna delle dieci dita, in tre sistemi di linee: basali, cioè parallele alla piegatura del dito; marginali, che entrano e fuoriescono dai lati del polpastrello; centrali al polpastrello stesso. Sulla base di questo sistema era possibile identificare 4 tipi principali di impronte: adelta o arco, monodelta con cappio a destra “loop right” e monodelta con cappio a sinistra “loop left”, bidelta o figura chiusa e composta o vortice “whorl”. Negli anni successivi Gasti perfezionò il sistema classificatorio identificando dieci macroaree da cui era possibile far discen- dere ulteriori sottoaree, fino ad ottenere oltre dieci milioni di combinazioni (Giuliano, 2011, pp. 29-31). Presentato al 6° Congresso Internazionale di Antropologia Criminale di Torino nel 1906, il Sistema Gasti venne adottato dalla Polizia italiana e da quelle di altre nazioni (Gasti, 1910, 68-69; Gasti, 1908, pp. 321-336). Nel 1909 fu addirittura applicato negli Stati Uniti, dove venne anche accettato come criterio di identificazione da alcuni tribunali (Giuliano, 2005, p. 32). Tale sistema di identificazione è restato in vigore in Italia fino al 2000, quando è stato introdotto il Sistema AFIS, Automated Fingerprint Identification 150 System (Sistema automatizzato di identificazione delle impronte) (Komarinski, 2005): un connubio hardware-software in grado di abbreviare i tempi di ac- quisizione, catalogazione e ricerca dei dati attraverso un algoritmo che codifi- ca le impronte e le rende disponibili ai terminali a disposizione delle forze di polizia. Il sistema Gasti: l’identificazione attraverso le impronte digitali La nuova tecnologia utilizzata oggi dalle Forze di Polizia Scientifica ha sicuramente semplificato la lettura, ma il principio fondante rimane il metodo proposto da Gasti. Per quanto riguarda l’evoluzione dei sistemi impiegati dalla sezione scien- tifica della Polizia, il contributo di Gasti non si limitò alla sola identificazione decadattiloscopica. Egli infatti contribuì al rinnovamento delle tecniche di segnalamento nella loro interezza, proponendo all’interno di un Formula- rio per il Segnalamento (Ministero degli Interni. Direzione Generale di P.S., 1908), pensato e realizzato nel 1908 presso La Scuola di Polizia, di integrare tra loro i dati fotografici, descrittivi, antropometrici oltre a quelli dattiloscopici. Tale pubblicazione costituisce il primo strumento di diffusione su tutto il ter- ritorio nazionale di una metodologia descrittiva del pregiudicato, omogenea ed uniforme per tutti gli Uffici di Polizia. Stabilisce una terminologia precisa e unica per descrivere i connotati del viso e del corpo, i segni particolari co- muni o eccezionali, ponendoli in relazione speculare al materiale fotografico, così da associare alla descrizione linguistica la sua immagine rappresentativa. L’appartenenza di Gasti ai quadri della Polizia ha certamente influito sullo spostamento di competenza nel settore dal mondo accademico a quello della Polizia Scientifica. Resta, alla base di questa storia, l’intuizione di Salvatore Ottolenghi di inserire il sistema dattiloscopico nel riconoscimento dell’individuo in quanto basato sulla scientificità e scevro da possibili interpretazioni soggettive; cui si lega la fondamentale attività svolta da Giovanni Gasti per affinare tale meto- do e renderlo il più possibile utile alle indagini di Polizia scientifica. Bibliografia 1. Bertillon, A. & McClaughry R.W. (1896). Signaletic instructions including the theory and practice of anthropometrical identification. Chicago: Werner Co.́́ 2. Bertillon, A. (1885). Identification anthropométrique. Instructions signalétiques. Melun: Imprimerie Administrative. 3. Bertillon, A. (1890). La photographie judiciaire, avec un appendice sur la classification et l’identification anthropométriques. Paris: Gauthier-Villars. 4. Bulmer, M. (2003). Francis Galton: Pioneer of Heredity and Biometry. Baltimore and London: The Johns Hopkins University Press. 151 5. D’Antonio, E. (2020) Lombroso, Ottolenghi e le origini della polizia scientifica ita- liana. In Labanca, N. & Di Giorgio, M. (Eds.), Una cultura professionale per la poli- zia dell’Italia liberale e fascista. Antologia del “Bollettino della Scuola di polizia scientifica” (1910-1939). Milano: Edizioni Unicopli. 6. Dallapiccola, B. (1968). I dermatoglifi della mano. Milano: Garzanti. 7. Galton F. (1888). Personal Identification And Description, I. Nature, 38, 173-177. 8. Galton F. (1888). Personal Identification And Description, II. Nature, 38, 201-202. 9. Galton, F. (1892). Finger. New York: Macmillan Co. n, F. (1892). Finger. New York: Macmillan Co. 10. Gasti, G. (1908). L’identification dactyloscopique et le système italien de classificati- on. In Comptes-Rendus du VI Congrès International d’anthropologie criminelle. Milano, Torino: Fratelli Bocca. 11. Gasti, G. (1910). L’identificazione dei delinquenti e la funzione di polizia nell’attuale momento giuridico e sociale. Bollettino della Scuola di Polizia scientifica e del servizio di segnalamento, 1, 68-69. 12. Gasti, G. (1912). La missione attuale dell’antropometria nelle funzioni di polizia. Bollettino della Scuola di Polizia Scientifica e del Servizio di Segnalamento, 3, 112-124. 13. Giuliano, A. (2005). Dieci e tutte diverse. Studio sui dermatoglifi umani. Torino: Tirrenia Stampatori. 14. Giuliano, A. (2007). Impronte digitali. La classificazione Gasti. Torino: Tirrenia Stam- patori. 15. Giuliano, A. (2011). Dal pensiero di Lombroso all’impronta digitale. Passato e presente del metodo più efficiente, pratico e rapido d’identificazione personale. Torino: Edizioni Libreria Cortina. 16. Giuliano, A. (2018). Salvatore Ottolenghi. Le impronte digitali in polizia scientifica e medicina legale. Torino: Edizioni Minerva Medica. 17. Grzybowski, A. & Pietrzak, K. (2015). Jan Evangelista Purkynje (1787-1869): first to describe fingerprints. Clin Dermatol, 33(1), 117-21. 10.1016/j.clinderma- tol.2014.07.011 18. Henry, E.R. (1900). Classification and uses of finger prints. London: George Routledge and Sons. 19. Herschel, W.J. (1916). The Origin of Finger-Printing. Oxford: Oxford University Press. 20. Komarinski, P. (2005). Automated Fingerprint Identification Systems (AFIS). Elsevier Academic Press. 21. Lombroso, C. (1902). Innocenza di gravissima imputazione dimostrata dalla Antro- pologia criminale. Archivio di psichiatria, scienze penali e antropologia criminale, 23(7/2). Bibliografia 22. Malpighi, M. (1665). 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SUMMARY The dermatoglyphics are signs of the human variety, as they are absolutely different from one human being to another. For this reason, and for their characteristics of uniqueness, classification, and inalterability, the papillary ridges on the fingertips represent elements of a sure differentiation between one person and another. Fingerprints are, therefore, very helpful in identifying a human being.ii classification, and inalterability, the papillary ridges on the fingertips represent elements of a sure differentiation between one person and another. Fingerprints are, therefore, very helpful in identifying a human being. Salvatore Ottolenghi was the first to utilize the fingerprinting system to identify individuals, and he introduced this system in his “Cartellino di riconoscimento (identification card)” in 1902. He was sure about the scientific validity of this method, which he considered to be free from potential personal interpretation. According to his definition, “fingerprints, by their nature, form special drawings from birth; these will not change throughout life and will be absolutely different from one human being to another”. Salvatore Ottolenghi was the first to utilize the fingerprinting system to identify individuals, and he introduced this system in his “Cartellino di riconoscimento (identification card)” in 1902. He was sure about the scientific validity of this method, which he considered to be free from potential personal interpretation. According to his definition, “fingerprints, by their nature, form special drawings from birth; these will not change throughout life and will be absolutely different from one human being to another”. f This fingerprint identification method was immediately refined by Giovanni Gasti, whom Salvatore Ottolenghi had chosen as his personal assistant at the Scuola di Polizia Scientifica (School of Forensic Science). Gasti, adapting the classification method of Francis Galton and Edward Henry, developed the “Sistema Gasti (Gasti System)”, which was in use throu- ghout the 1900s. Keywords: dermatoglyphics, fingerprints, scientific police, Salvatore Ottolenghi, anthropo- metry 153
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Effect of adenine sulphate on in vitro mass propagation of Stevia rebaudiana Bertoni
Journal of medicinal plant research
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Key words: Stevia rebaudiana, adenine sulphate, in vitro. Key words: Stevia rebaudiana, adenine sulphate, in vitro. Vol. 8(13), pp. 543-549, 3 April, 2014 DOI: 10.5897/JMPR2013.5217 ISSN 1996-0875 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR Journal of Medicinal Plant Research Vol. 8(13), pp. 543-549, 3 April, 2014 DOI: 10.5897/JMPR2013.5217 ISSN 1996-0875 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR Journal of Medicinal Plant Research Journal of Medicinal Plant Research 1Department of Molecular and Cellular Engineering, Jacob School of Biotechnology and Bioengineering, India. 2Department of Biological Sciences, School of Basic Sciences, Sam Higginbottom Institute of Agriculture, Technology and Sciences, (Formerly AAI-DU, Deemed to be University), Allahabad-211 007, Uttar Pradesh, India. Received 29 August, 2013; Accepted 19 March, 2014 Stevia rebaudiana, a medicinal plant normally used as a natural herbal sweetener, has documented properties of antimicrobial, antihypertensive and anti-hyperglycemic and hence a boon to diabetic people. An efficient protocol has been developed for the in vitro plant regeneration established from nodal explants of stevia. Best shoot proliferation was observed when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with Kinetin (9.3 µM) and adenine sulphate (Ads) 40 mg/L. Proliferated shoots were transferred to rooting media having different concentration of α-n- Naphthalene acetic (NAA) acid and Indole-3-butyric acid (IBA). Best rooting was observed with NAA 5.3 µM. The plantlets were successfully subjected to hardening media containing soil, soilrite and vermiculite (2:1:1 w/w) and then rooted plant transferred to pots and acclimatized, which showed 65% survival in the field with normal growth. *Corresponding author. E-mail: pragatimisra3@rediffmail.com. Tel: +919452517350. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License INTRODUCTION Stevia rebaudiana Bertoni (family: Asteraceae) is a perennial herb and natural sweetener plant commonly known as ‘Sweet weed’, ‘Sweet leaf’, ‘Sweet herbs’ and ‘Honey leaf’ which is estimated to be 300 times sweeter than cane sugar (Chalapathi and Thimmegowda, 1997; Liu and Li, 1995). The leaves of Stevia are the source of diterpene glycosides: stevioside and rebaudioside (Yoshida, 1986). The extracts of S. rebaudiana and purified stevioside began to be utilized commercially for sweetening and flavoring foods and beverages in Japan in the mid 1970s, in order to substitute for several synthetic sweeteners. Plant extract of Stevia have important industrial uses in beverages, energizers as well as medicinal uses such as low uric acid treatment, vasodilator cardiotonic, anesthetic and anti-inflammatory. Stevia is also helpful in treatment of hypoglycemia and diabetes because it nourishes pancreas and thereby helps to restore its normal function, and 35.2% fall in normal blood sugar levels 6 to 8 h following the ingestion of Stevia leaf extract (Soejarto et al., 1983; Oviedo, 1971). Due to difficulties of growing Stevia, tissue culture could be the only alternative for rapid mass propagation of Stevia plants because seed of Stevia show very low germination percentage and propagation by seeds does not allow the production of homogeneous population resulting in great variation in important feature like sweetening level and composition; vegetative propagation *Corresponding author. E-mail: pragatimisra3@rediffmail.com. Tel: +919452517350. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License *Corresponding author. E-mail: pragatimisra3@rediffmail.com. Tel: +919452517350. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License J. Med. Plants Res. 544 Figure 1. Shoot initiation from nodal explant of S. rebaudiana after 2 weeks. is also limited by the low number of individuals obtained from single plant (Miyazaki and Watanabe, 1974; Nakamura and Tamura, 1985; Goettemoeller and Ching, 1999; Ferreira and Handro, 1988). Adenine sulphate acts as a precursor for natural cytokinin synthesis or enhances natural cytokinin biosynthesis; the compounds produced could be more effective in causing the physiological response than the cytokinins added to the Murashig and Skoog (MS) culture medium. The benefits of adenine are often only noticed when it is associated together with cytokinins such as benzylaminopurine (BAP) or kinetin (Van Staden et al., 2008). Plant allowed to be transferred in a polyhouse for 30 days, then hardening plants were transferred to the field and plants were survived with normal growth and morphology. The twigs (about 5 to 6 cm) of pot grown S. rebaudiana plants were collected from the polyhouse at Sam Higginbottom Institute of Agriculture, Technology and Sciences (SHIATS), Allahabad. INTRODUCTION Our results suggest that kinetin, in combination with Ads, improves the process of organogenesis. The purpose of the present study was to optimize an in vitro plant regeneration system on the basis of the effect of adenine sulphate with different growth regulators: kinetin, BAP and α-n-Naphthalene acetic (NAA) for the mass propagation of Stevia which has high demand in the pharmaceutical industry and Indian System of Medicine. Figure 1. Shoot initiation from nodal explant of S. rebaudiana after 2 weeks. RESULTS Nodal explant showed emergence of shoot buds within 7 days after inoculation on MS media supplemented with MS, kinetin (9.3 µM), NAA (0.5 mg/L) and Ads (40 mg/L) (Figure 1). In the present investigation, Ads showed a marked improvement in shoot multiplication rate and emergence of shoot bud of S. rebaudiana. Proliferated nodal explants were subcultured for 25 to 30 days on MS media. To examine the effect of adenine sulphate on shoots proliferation the MS media was supplemented with or without kinetin. Profuse multiplication of shoot after 50 to 55 days, with maximum numbers of shoots (65 shoots/explant) with increased shoot length were produced on MS media supplemented with kinetin (9.3 µM) and Ads (40 mg//L) (Figure 2) whereas, MS media containing kinetin (9.3 µM) and Ads (10 mg/L) produced the minimum number of shoots (10 shoots/explant) (Table 2 and Figure 3). MS media containing kinetin alone (9.3 µM) produced 25 shoots per explant (Table 1). Adenine sulphate was also added along with kinetin and NAA, the best response (30 shoots/explant) was observ- ed on MS, kinetin (9.3 µM), NAA (5.3 µM) and adenine Explant sterilization The statistical analysis was performed using one-way analysis of variance (ANOVA) and the significance of differences among treatment means were contrasted with Tukey’s Honestly Significant Difference Test (HSD) at P < 0.05. The twigs with node explants were washed in running tap water and then washed again thoroughly by adding a few drops of Tween-20 to remove the superficial dust particles as well as fungal and bacterial spores. Explants were sterilized with 0.1% mercuric chloride for 4 min followed by rinsing for five times with sterile double distilled water. In vitro culture condition and germination were then transferred to the field and after 30 days, 65% plants were survived with normal growth and morphology. sulphate (40 mg/L) (Table 3). When adenine sulphate was added along with kinetin and BAP, the best response (8 shoots/explant) was observed on MS, kinetin (9.3 µM), BAP (22.2 µM) and adenine sulphate (40 mg/L) (Table 4). For rooting of 25 to 30 days old, proliferated shoots were transferred to rooting media having different concentration of NAA (5.3, 8.1 and 10.7 µM) and IBA (4.9, 9.8 and 14.7 µM). The best rooting with the maximum lengths of roots was observed with NAA 5.3 µM (Table 5 and Figure 4). The 60 to 65 days old plantlets were successfully subjected to hardening media containing soil, soilrite and vermiculite (2:1:1 w/w) showed the maximum survival percentage in the foam cups for 10 to 15 days (Table 6 and Figure 5) and then transferred to pots and grown at 25 to 30°C and 75 to 80% relative humidity. About 70 to 75% of plantlets were survived in the hardening procedure. These plants In vitro culture condition and germination Sterilized nodal segments (with a single axillary bud) about 0.8 to 1.0 cm were prepared aseptically and were implanted vertically on MS medium (Murashige and Skoog, 1962) fortified with specific concentrations of growth regulators 6-benzylaminopurine (BAP), kinetin and NAA singly or along with Ads as additive. The medium was gelled with 8 g/L agar and supplemented with 30 g/L sucrose. The pH was adjusted with 1 N NaOH and 1 N HCl at 5.8 before autoclaving. The cultures were incubated at 25 ± 2°C under 16 h light and 8 h dark photoperiod provided by cool white fluorescent tubes with 3000 lux intensity and relative humidity 72 to 75%. Subcultures were done every 15 days interval. Nodal segments from the proliferated shoots were subcultured again after 20 days for further multiple shoot induction. Regenerated multiple shoots were cut and individual shoots were placed on MS medium containing different concentrations of IBA and NAA for root induction. After development of sufficient roots, the plantlets were gradually pulled out from the medium and were subjected to hardening using foam cups with polythene cover. Medium for hardening methods was different combinations of soil, soilrite and vermiculite supplemented with half strength MS liquid medium (without organics) twice a week, cultures were kept under the same controlled photoperiod and temperature. Hardened plantlets were 545 Khan et al. Table 1. Effect of different concentrations of kinetin on shoot multiplication of s. rebaudiana. Table 1. Effect of different concentrations of kinetin on shoot multiplication of s. rebaudiana. Kinetin (mg/L) Average no. of shoots Average length of shoots (inches) 0.5 4 2.2 1.0 6 2.4 1.5 14 2.9 2.0 25 4.8 2.5 18 3.7 3.0 8 3.1 F-test S S SD (±) 1.139 0.177 CD at 5% 2.425 0.378 Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. Figure 3. Minimum number of shoot regeneration. Figure 2. Multiple shoot proliferation in S. rebaudiana when cultured on MS with 9.3 µM and adenine sulphate 40 mg/L. Figure 2. Multiple shoot proliferation in S. rebaudiana when cultured on MS with 9.3 µM and adenine sulphate 40 mg/L. Figure 3. Minimum number of shoot regeneration. DISCUSSION Shoot multiplication also observed MS media supplemented with kinetin, NAA and Ads whereas kinetin, BAP and Ads resulted in lower number of shoots per explant (Tables 3 and 4), respectively). BAP and Indole-3-acetic acid (IAA) combination was superior for induction of shoot bud multiplication from nodal explant of Stevia (Thyagarajan and Venkatachalam, 2012). The combination of 13.3 μM BA, 61.8 μM Ads and 8.0 μM NAA in MS basal medium showed enhanced shoot multiplication (Dubey et al., 2006). Shoot tips showed better response for shoot proliferation than nodal segments of S. rebaudiana as explants. Figure 4. Root induction from isolated shoot. p MS media in half and full strength were equally effec- tive in shoot proliferation. BA (1.0 mg/L) was superior to all other hormonal treatments for shoot proliferation (Hossain et al., 2008). The medium was supplemented with 1.0 mg/L NAA and 4.0 mg/L BA recommended for S. rebaudiana plant regeneration (Suarez and Salgado, 2008). As shown in Table 5 and Figure 4, root induction from proliferated shoot were transferred onto MS media supplemented with different concentration of NAA and IBA. Similarly, the proliferated shoots were individually separated and transferred to rooting media consisting of MS basal medium supplemented with different concen- trations of NAA (Sharma et al., 2012). Rooting of the in vitro derived shoots of Stevia could be achieved following subculture in auxin containing medium and root also optimized on MS medium with IBA 2 mg/L (Sivaram and Mukundan, 2003; Taware et al., 2010). The well rooted plants were transferred into foam cups containing diffe- rent combination of soil, soilrite and vermiculite (Table 6 and Figure 5). The rooted plantlets of Stevia were hardened in 2:1:1 ratio of sand: soil: vermicomposite Figure 4. Root induction from isolated shoot. of cytokinins by feed-back inhibition or by competing for the enzyme systems involved in cytokinin metabolism (Van Staden et al., 2008). Data presented in Table 2 and Figure 2 indicates that, the concentrations of Ads 40 mg/L improve the multiplication rate (number of shoot/explant) and length of the shoot when added in combination with kinetin. However, MS media containing kinetin alone did not improve the multiplication rate (Table 1). Adenine sulphate, adenosine and adelynic acid show cytokinin activity and therefore are applied to different culture medium in order to improve growth or to reinforce the response normally attributed to cytokinin action (Gatica et al., 2010). DISCUSSION In the present study, nodal explant from Stevia plants were inoculated on MS media supplemented with different concentrations of kinetin, NAA and Ads for shoot bud initiation. The early study reported that kinetin in combination with Ads are known to improve organo- genesis (Gatica et al., 2010). Nodal explant of Stevia placed on MS medium containing 1.0 mg/L BAP or IBA 0.05 mg/L for shoot bud initiation (Laribi et al., 2012). The natural and synthetic purine cytokinins are degraded to adenine sulphate and related nucleotides; therefore, the addition of Ads to the medium may retard the degradation In the present study, nodal explant from Stevia plants were inoculated on MS media supplemented with different concentrations of kinetin, NAA and Ads for shoot bud initiation. The early study reported that kinetin in combination with Ads are known to improve organo- genesis (Gatica et al., 2010). Nodal explant of Stevia placed on MS medium containing 1.0 mg/L BAP or IBA 0.05 mg/L for shoot bud initiation (Laribi et al., 2012). The natural and synthetic purine cytokinins are degraded to adenine sulphate and related nucleotides; therefore, the addition of Ads to the medium may retard the degradation 546 J. Med. Plants Res. Table 2. Combined effect of different concentration of adenine sulphate with Kinetin on shoot multiplication of s. rebaudiana. Table 2. Combined effect of different concentration of adenine sulphate with Kinetin on shoot multiplication of s. rebaudiana. Kinetin (mg/L) Adenine sulphate (mg/L) Average No. of shoots Average length of shoots (inches) 2.0 10 30 4.5 2.0 20 34 4.5 2.0 30 40 5.0 2.0 40 62 8.0 2.0 50 51 6.5 2.0 60 35 5.0 F-test - S S SD (±) - 1.401 0.194 CD at 5% - 2.984 0.414 Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. axillary shoots in shoot cultures and promotes adventitious shoot formation indirectly from calli or directly from explants and the benefits of Ads are often only noticed when it is associated together with ammonium nitrate or with cytokinins such as BAP or kinetin (Van Staden et al., 2008). Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. DISCUSSION Adenine sulphate also stimulates somatic embryoge- nesis and caulogenesis, induces the proliferation of 547 Khan et al. Figure 5. Hardening media containing soil, soilrite and vermiculite (2:1:1 w/w). Figure 5. Hardening media containing soil, soilrite and vermiculite (2:1:1 w/w). Table 3. Combined effect of different concentration of adenine sulphate with Kinetin and NAA on shoot multiplication of S. rebaudiana. Kinetin (mg/L) NAA (mg/L) Adenine sulphate (mg/L) Average No. of shoots Average length of shoots (inch) 2.0 1.0 40 30 5.0 2.0 1.5 40 24 4.8 2.0 2.0 40 19 4.5 2.0 2.5 40 16 4.5 2.0 3.0 40 11 3.0 2.0 3.5 40 9 2.4 F-test - S S SD (±) - 0.981 0.152 CD at 5% - 2.090 0.323 Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. Table 4. Combined effect of different concentration of adenine sulphate with Kinetin and BAP on shoot multiplication of s. rebaudiana. Table 4. Combined effect of different concentration of adenine sulphate with Kinetin and BAP on shoot multiplication of s. rebaudiana. Kinetine (mg/L) BAP (mg/L) Adenine sulphate (mg/L) Average No. of shoots Average length of shoots (inches) 2.0 1.0 40 3 1.5 2.0 2.0 40 4 2.0 2.0 3.0 40 6 2.5 2.0 4.0 40 6 2.0 2.0 5.0 40 8 3.5 2.0 6.0 40 8 2.9 F-test S S SD (±) 0.882 0.125 CD at 5% 1.878 0.266 Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. Data represent average number of shoots per explant and average length of shoots in three experiments with three replicates in each. 548 J. Med. Plants Res. Table 5. Effect of different concentration of NAA and IBA on rooting of S. rebaudiana. g Growth regulators (mg/L) Growth response S. No NAA IBA Percent rooting Average No. ACKNOWLEDGEMENT were successfully established in soil (Sairkar et al., 2009). Hardened plants showed vigorous and uniform growth in the field similar to mother plants. Authors are grateful to Vice-Chancellor and Dean, College of Biotechnology and Bioengineering, SHIATS, Allahabad for providing necessary facilities. REFERENCES The present study demonstrates a simple and efficient method for high frequency direct shoot regeneration from nodal explants of S. rebaudiana. The system is rapid and maximum of shoots (65 shoots/explant) with increased shoot length were produced on MS media supplemented with kinetin (9.3 µM) and Ads (40 mg/L). Proliferating shoot cultures were established by repeatedly subculturing the nodes on the same medium. Maximum rooting occurred in shoots cultured on half strength MS medium supplemented with 5.3 µM NAA. Chalapathi T, Thimmegowda S (1997). Natural Noncalorie Sweetener (Stevia rebaudiana Bertoni) A future crop of India. Crop Res. 14:347- 350. Dubey H, Kumar M, Haider ZA (2006). In vitro morphogenesis of Stevia: the naturally occurring sweet plant-a fast and efficient method to propagate Stevia by tissue culture. J. Res. 18:1-9. Ferreira CM, Handro W (1988). Production, Maintenance and plant regeneration from cell suspesion cultures of Stevia rebaudiana (Bert), Plant Cell Rep. 7:123-126. Gatica AAM, Muñoz VJ, Ramírez FP, Valdez MM (2010). In vitro plant regeneration system for common bean (Phaseolus vulgaris): effect of N 6-benzylaminopurine and adenine sulphate. Electronic J. Biotechnol. 13(1):0717-3458. DISCUSSION of roots Mean length of roots (cm) 1 1.0 1.0 40 5 2 2 1.5 2.0 45 8 3 3 2.0 3.0 60 10 5 4 1.0 - 80 15 7 5 1.5 - 65 12 5 6 2.0 - 55 8 3 F-test - - S - - SD (±) - - 1.674 - - CD at 5% - - 3.349 - - Data represent percent rooting, average number of roots per shoots and mean length of roots in three experiments with three replicates in each. Growth regulators (mg/L) Data represent percent rooting, average number of roots per shoots and mean length of roots in three experiments with three replicates in each. Table 6. Survival percentage of micro shoots using different substrates for hardening. Table 6. Survival percentage of micro shoots using different substrates for hardening. S. No Soil composition for hardening (w/w) Percentage of survival 1 Soil + Soilrite (1:1) 10 2 Soil + Soilrite (2:1) 30 3 Soil + Vermiculite (1:1) 45 4 Soil + Soilrite + Vermiculite (2:1:1) 75 5 Soil + Soilrite + Vermiculite (2:2:1) 50 6 Soil + Soilrite + Vermiculite (2:2:2) 30 7 Soil + Soilrite + Vermiculite (1:2:2) 10 F-test S SD (±) 1.401 CD at 5% 2.984 Data represent average percentage of survival of three experiments with three replicates in each. S. No Soil composition for hardening (w/w) Percentage of survival Data represent average percentage of survival of three experiments with three replicates in each. Khan et al. 549 Hossain MA, Shamim Kabir AHM, Jahan TA, Hasan MN (2008). Micropropagation of Stevia. Int. J. Sustain. Crop Prod. 3:1-9. Taware AS, Mukandam DS, Chavan AM, Taware SD (2010). Comparative studies of in vitro and in vivo grown plants and callus of Stevia rebaudiana (Bertoni). Int. J. Integr. Biol. 9:10-15. Laribi B, Rouatbin N, Kouki K, Bettaieb T (2012). In vitro Propagation of Stevia rebaudiana (Bert.)-A non-caloric sweetener and antidiabetic medicinal plant. Int. J. Med. Arom. Plants 2(2):333-339. Thyagarajan M, Venkatachalam P (2012). Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb. Ind. Crop Prod. 37:111-117. Liu J, Li SFY (1995). Separation and determination of stevia sweeteners by capillary electrophoresis and high performance liquid chromatography. J. Liq. Chrom. 18:1703-1719. Van Staden J, Zazimalova E, George EF (2008). Plant growth regulators II, Plant Propagation by Tissue Culture: Cytokinins, their analogues and antagonist. In George E F, Hall M, De Kleck GJ (eds) Academic press, Netherland, pp. 205-226. g p y q Miyazaki Y, Watanabe H (1974). Studies on the cultivation of Stevia rebaudiana Bertoni; on the propagation of the plant (Eng. abstr.). Jpn. J. Trop. Agric. 17:154–157. Academic press, Netherland, pp. 205-226. p g Murashige T, Skoog FA (1962). Revised medium f or rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473- 497. Yoshida S (1986). Studies on the production of sweet substances in Stevia rebaudiana: I. determination of sweet glucosides in Stevia plant by thin layer chromato-scanner and their accumulation patterns with plant growth. Jpn. J. Crop Sci. 55:189-195. Nakamura S, Tamura Y (1985). Variation in the main glycosides of Stevia (Stevia rebaudiana Bertoni). Jpn. J. Trop. Agric. 29:109-116. Oviedo CA (1971). Accion hipoglicemiante de la Stevia rebaudiana Bertoni (Kaa-he-e). Excerpta Med. 208:92-93. Sairkar P, Chandravanshi MK, Shukla NP, Mehrotra NN (2009). Mass production of an economically important medicinal plant Stevia rebaudiana using in vitro propagation techniques. J. Med. Plants Res. 3:266-270. Sharma T, Khan MK, Misra P, Shukla PK (2012). Micropropagation of Kinnow through nodal explants. Bioscan 7:295-297. Sivaram L, Mukundan U (2003). In vitro culture studies on Stevia rebaudiana. In vitro Cell Dev. Biol. Plant 39:520-523. Soejarto DD, Compadre CM, Medon PJ, Kamath SK, Kinghorn AD (1983). Potential Sweetening Agents of Plant Origin, II. Field Search for Sweet-Tasting Stevia Species. Econ. Bot. 37:71-79. for Sweet-Tasting Stevia Species. Econ. Bot. 37:71-79. Suarez IE, Salgado JA (2008). Conflict of Interests Goettemoeller J, Ching A (1999). Seed germination in Stevia rebaudiana. In Janick J (eds) Perspectives on new crops and new uses. ASHS press, Alexandria, pp. 510-511. The author(s) have not declared any conflict of interests. Khan et al. Khan et al. In vitro propagation of Stevia rebaudiana Bert. (Asteraceae Eu-patorieae) via organogénesis. Enero Junio 13:40-48.
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Neural substrates of self‐ and external‐preoccupation: A voxel‐based morphometry study
Brain and behavior
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O R I G I N A L R E S E A R C H O R I G I N A L R E S E A R C H Neural substrates of self‐ and external‐preoccupation: A voxel‐based morphometry study Shigeyuki Ikeda1  | Hikaru Takeuchi2 | Yasuyuki Taki2,3,4 | Rui Nouchi5,6 | Ryoichi Yokoyama7 | Seishu Nakagawa7 | Atsushi Sekiguchi6 | Kunio Iizuka7 | Sugiko Hanawa7 | Tsuyoshi Araki6 | Carlos Makoto Miyauchi7 | Kohei Sakaki6 | Takayuki Nozawa1 | Susumu Yokota2 | Daniele Magistro7 | Ryuta Kawashima1,2,5,6,7 Abstract Introduction: Self‐ and external‐preoccupation have been linked to psychopathologi‐ cal states. The neural substrates underlying self‐ and external‐preoccupation remain unclear. In the present study, we aim to provide insight into the information‐process‐ ing mechanisms associated with self‐ and external‐preoccupation at the structural level. 2Division of Developmental Cognitive Neuroscience, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan 3Division of Medical Neuroimaging Analysis, Department of Community Medical Supports, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan Methods: To investigate the neural substrates of self‐ and external‐preoccupation, we acquired high‐resolution T1‐weighted structural images and Preoccupation Scale scores from 1,122 young subjects. Associations between regional gray matter vol‐ ume (rGMV) and Preoccupation Scale subscores for self‐ and external‐preoccupation were estimated using voxel‐based morphometry. 4Department of Radiology and Nuclear Medicine, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Results: Significant positive associations between self‐preoccupation and rGMV were observed in widespread brain areas such as the bilateral precuneus and poste‐ rior cingulate gyri, structures known to be associated with self‐triggered self‐refer‐ ence during rest. Significant negative associations between external‐preoccupation and rGMV were observed only in the bilateral cerebellum, regions known to be as‐ sociated with behavioral addiction, sustained attention, and reward system. 7Department of Functional Brain Imaging, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Conclusion: Our results reveal distinct neural substrates for self‐ and external‐preoc‐ cupation at the structural level. Shigeyuki Ikeda1  | Hikaru Takeuchi2 | Yasuyuki Taki2,3,4 | Rui Nouchi5,6 | Ryoichi Yokoyama7 | Seishu Nakagawa7 | Atsushi Sekiguchi6 | Kunio Iizuka7 | Sugiko Hanawa7 | Tsuyoshi Araki6 | Carlos Makoto Miyauchi7 | Kohei Sakaki6 | Takayuki Nozawa1 | Susumu Yokota2 | Daniele Magistro7 | Ryuta Kawashima1,2,5,6,7 1Department of Ubiquitous Sensing, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Received: 19 August 2018  |  Revised: 20 February 2019  |  Accepted: 1 March 2019 Received: 19 August 2018  |  Revised: 20 February 2019  |  Accepted: 1 March 2019 Received: 19 August 2018  |  Revised: 20 February 2019  |  Accepted: 1 March 2019 DOI: 10.1002/brb3.1267 2 of 11  |     1 | INTRODUCTION 2 of 11 IKEDA et al. during rest preferentially engaged the precuneus and both en‐ gaged ventral MPFC (vMPFC) and PCC (Whitfield‐Gabrieli et al., 2011). Because self‐preoccupation is the disposition to focus on the self regardless of individual intention, self‐preoccupation may be associated with the precuneus, vMPFC, and PCC, which are activated by self‐triggered self‐reference. In addition, self‐related processes are driven via PCC and moderated by MPFC (Davey, Pujol, & Harrison, 2016). The functions of PCC and MPFC may be implicated in a specific aspect of self‐preoccupation that maintains attention to the self. Therefore, neural substrates of self‐preoccu‐ pation should be observed primarily in the precuneus, vMPFC, and PCC but not in the dMPFC. The disposition to focus more on the self than on others or one's environment and to maintain self‐focused attention is referred to as self‐preoccupation, a state that can be assessed quantitatively using the Self‐Preoccupation Scale (SPS) (Sakamoto, 1998). Self‐preoc‐ cupation is implicated in diverse psychopathological states such as depression (Ingram, 1990). Highly self‐preoccupied individuals tend to attribute causes of negative events to themselves by self‐focused attention after the negative event (Sakamoto, 1998). In addition to the SPS, the disposition to focus on the self can be assessed by the private self‐consciousness scale (Fenigstein, Scheier, & Buss, 1975). While similar to the SPS in that it measures self‐focusing tendency, unlike the SPS the private self‐consciousness scale does not con‐ sider the disposition to prolonged self‐focus. According to a pre‐ vious report, depression is more strongly related to the SPS than to the private self‐consciousness scale (Sakamoto, 1998), suggest‐ ing the importance of self‐focusing duration in psychopathology. Furthermore, high SPS score was found to be a vulnerability factor for depression (Sakamoto, 1999). External‐preoccupation is assumed to have three distinct at‐ tributes: propensity for behavioral addiction, sustained attention, and reward system. People with high external‐preoccupation tend to be enthusiastic about things that interest them and to become absorbed by these things. The behavior exhibited by such people is thought to be analogous to behavioral addiction, such as gambling or internet addiction. A recent neuroimaging study reported that gambling disorder patients showed reduced gray matter volume (GMV) in the left supramarginal gyrus and bilateral posterior cere‐ bellum compared to healthy controls (Takeuchi, Tsurumi et al., 2017). 2 of 11  |     1 | INTRODUCTION Similarly, subjects with Internet addiction disorder showed GMV re‐ duction in the anterior cingulate cortex, supplementary motor area, cerebellum, insula, and inferior temporal gyrus (Weinstein, Livny, & Weizman, 2017 for review). Because external‐preoccupation rep‐ resents the disposition to maintain external focus, external‐preoccu‐ pation may be associated with the ability to maintain focus on a task (i.e., sustained attention). Whole‐brain functional network strength provided a neuromarker for sustained attention to external stimuli, and nodes with the most connections in the functional networks related to sustained attention were located in the cerebellum, tem‐ poral, or occipital cortices (Rosenberg et al., 2016). Finally, people with high external‐preoccupation find it difficult to stop a specific action because they expect a reward. This suggests the existence of a relationship between external‐preoccupation and reward sys‐ tem. The cerebellum, as well as canonical reward‐related areas, has been known to be related to reward processing, and is involved in prediction of future rewards (Tanaka et al., 2004), evaluation of un‐ predicted rewards (Ramnani, Elliott, Athwal, & Passingham, 2004), and reward‐based learning (Thoma, Bellebaum, Koch, Schwarz, & Daum, 2008). Recently, a region of the cerebellum was reported to be specifically activated by verbal encouragement, suggesting that the cerebellum motivates aspects of motor performance (Belkhiria et al., 2017). Furthermore, cerebellar granule cells have been shown to encode the expectation of reward (Wagner, Kim, Savall, Schnitzer, & Luo, 2017). Taken together, the cerebellum is consistently impli‐ cated in all three attributes of external‐preoccupation. Therefore, neural substrates of external‐preoccupation should be observed pri‐ marily in the cerebellum. Th d h h ( ) l b In contrast to self‐preoccupation, the disposition to maintain external focus on a specific object is referred to as external‐pre‐ occupation, which can be assessed by the External‐Preoccupation Scale (EPS) (Sakamoto, 1998). Highly external‐preoccupied individ‐ uals tend to devote their attention to their work and exert extreme effort in achieving their goals. This disposition may lead to “burnout” (Freudenberger, 1974), which is also related to depression. Despite strong relationships to psychopathological states, the neural substrates underlying self‐ and external‐preoccupation re‐ main unclear. To our knowledge, no studies have investigated poten‐ tial neural substrates using structural magnetic resonance imaging (MRI). In the present study, we aim to provide insight into the in‐ formation‐processing mechanisms associated with self‐ and external preoccupationat the structural level. K E Y W O R D S Shigeyuki Ikeda, Department of Ubiquitous Sensing, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan. cerebellum, gray matter volume, magnetic resonance imaging, posterior cingulate cortex, precuneus, Preoccupation Scale Funding information JST/CREST; JST/RISTEX; Grant‐in‐Aid for Young Scientists (A), Grant/Award Number: KAKENHI 25700012; Grant‐in‐Aid for Young Scientists (B), Grant/Award Number: KAKENHI 23700306 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Brain and Behavior published by Wiley Periodicals, Inc. |  1 of 11 wileyonlinelibrary.com/journal/brb3 |  1 of 11 wileyonlinelibrary.com/journal/brb3 Brain and Behavior. 2019;e01267. https://doi.org/10.1002/brb3.1267 include the cerebellum. To test our hypotheses, we investigate as‐ sociations between regional gray matter volume (rGMV) and in‐ dividual differences in self‐ and external‐preoccupation among a large sample of young adults (more than one thousand). To assess the associations of rGMV to self‐ and external‐preoccupation, we employ voxel‐based morphometry (VBM). Specifically, we perform whole‐brain multiple regression analysis, a method commonly used in neuroimaging research to identify regions associated with specific behavioral outcomes or metrics such as SPS scores. external focus on a specific object. Each subscale (SPS, EPS) is calcu‐ lated by summing scores of the items. The RAPM is a task to assess nonverbal reasoning ability (Raven, 1998). Intelligence quotient (e.g., RAPM score) is associated with attention deficit hyperactivity dis‐ order (ADHD) (Rommel, Rijsdijk, Greven, Asherson, & Kuntsi, 2015). The RAPM score was, therefore, used as a nuisance covariate in sub‐ sequent second‐level analysis. To check association between SPS/EPS and personality traits (i.e., neuroticism, extraversion, openness, agreeableness, and con‐ scientiousness), the subjects were asked to complete a 60‐item Japanese version (5‐point scale) of the NEO Five‐Factor Inventory (NEO‐FFI) (Costa & MacCrae, 1992; Shimonaka, Nakazato, Gondo, & Takayama, 1999). Note that one subject did not complete the NEO‐ FFI. To establish the association between SPS/EPS and personality traits, we calculated Pearson's correlation coefficients. 2.3 | Psychological measures All subjects completed the Preoccupation Scale and Raven's Advanced Progressive Matrices (RAPM). The Preoccupation Scale consists of 19 items, each scored on a 5‐point Likert scale, of which 11 items reflect self‐preoccupation, and the remaining items reflect external‐preoccupation (Sakamoto, 1998). Self‐preoccupation is de‐ fined as the tendency to focus more on the self than on others or one's environment and to maintain self‐focused attention. In con‐ trast, external‐preoccupation is defined as the tendency to maintain 2.1 | Subjects The present study is a part of an ongoing project to investigate asso‐ ciations among brain structure and activity, cognitive function, and aging. Recent work includes studies on VBM (Takeuchi et al., 2017a) and resting‐state activity (Ikeda et al., 2017; Takeuchi, Taki, et al., 2017). Therefore, subjects in this study have received psychologi‐ cal tests and MRI scans aside from those described in the present report. 2.2 | Image acquisition Magnetic resonance images were acquired as described in our previ‐ ous work (Takeuchi et al., 2017b) using a 3‐T Philips Intera Achieva scanner equipped with an eight‐channel head coil. We collected high‐resolution T1‐weighted structural images using a magnetiza‐ tion‐prepared rapid gradient echo sequence and the following set‐ tings: 240 × 240 matrix, TR = 6.5 ms, TE = 3 ms, FOV = 240 mm, slices = 162, and slice thickness = 1.0 mm. Pads and Velcro tape were used to limit subjects’ motion during scanning. 2.4 | Preprocessing of structural data We employed the same preprocessing procedures as described pre‐ viously (Takeuchi et al., 2017b). The preprocessing was performed using Statistical Parametric Mapping software (SPM12; Wellcome Department of Cognitive Neurology, London, UK, https://www. fil.ion.ucl.ac.uk/spm/) implemented in Matlab (Mathworks Inc., Natick, MA). Individual T1‐weighted structural images were seg‐ mented into six tissues using the new segmentation algorithm in SPM12. We used default parameters for this new segmentation process except for the following: the Thorough Clean option was used for removing any odd voxels, affine regularization was per‐ formed with the International Consortium for Brain Mapping tem‐ plate for East Asian brains, and the sampling distance was set at 1 mm. Separate gray matter and white matter tissue probability maps (TPMs) were created in the segmentation process. The diffeo‐ morphic anatomical registration through exponentiated lie algebra (DARTEL) registration process was then performed on these TPMs. We created the template for the DARTEL algorithm using imaging data from 800 subjects (400 males and 400 females). The following procedures were the same as in our previous work (Takeuchi et al., 2015). The created template was then used to perform the DARTEL procedures using default parameters for all subjects. Individual images were then spatially normalized to Montreal Neurological Institute space (1.5 × 1.5 × 1.5 mm3 voxels). Furthermore, to deter‐ mine regional differences in the absolute amount of brain tissue, a volume change correction was performed by modulating each voxel with the Jacobian determinants derived from spatial normali‐ zation (Ashburner & Friston, 2000). Individual images were then smoothed by convolution with a Gaussian kernel of 8‐mm full‐width at half‐maximum. The present study included 1,122 healthy, right‐handed sub‐ jects (644 males and 478 females, age 20.7 ± 1.8 years). All subjects were university, college, or postgraduate students or subjects who had graduated from these institutions within 1 year before the ex‐ periment. All had normal vision and no history of neurological or psychiatric illness. Handedness was evaluated using the Edinburgh Handedness Inventory (Oldfield, 1971). In accordance with the Declaration of Helsinki, all subjects gave written informed consent for their participation. The present study was approved by the Ethics committee of Tohoku University. 2 of 11  |     1 | INTRODUCTION Self‐preoccupation is thought to be associated with self‐refer‐ ential mental activity in cortical midline structures, including the medial prefrontal cortex (MPFC), posterior cingulate cortex (PCC), and precuneus. Indeed, explicit self‐referential tasks have been found to activate the MPFC, PCC, and precuneus (D'Argembeau et al., 2005; Gusnard, Akbudak, Shulman, & Raichle, 2001; Huang et al., 2014; Johnson et al., 2002; Kelley et al., 2002; Moran, Macrae, Heatherton, Wyland, & Kelley, 2006; Northoff & Bermpohl, 2004; Northoff et al., 2006; Qin & Northoff, 2011). Self‐reference can also occur independently of individual intention during resting‐ state (Gusnard & Raichle, 2001; Huang et al., 2016; Lipsman et al., 2014; Wolff et al., 2018), which is hereinafter referred to as self‐ triggered self‐reference. Several previous studies have investi‐ gated similarities and differences between brain regions activated by explicit self‐reference and self‐triggered self‐reference during rest using positron emission tomography (D'Argembeau et al., 2005) and functional MRI (fMRI) (Whitfield‐Gabrieli et al., 2011). The fMRI study revealed that explicit self‐reference preferentially engaged dorsal MPFC (dMPFC) while self‐triggered self‐reference The present study tests two hypotheses: (a) neural substrates of self‐preoccupation include the precuneus, vMPFC, and PCC but not the dMPFC, and (b) neural substrates of external‐preoccupation 3 of 11 IKEDA et al. 3.3 | Association between EPS and rGMV To investigate the potential associations between EPS and rGMV, we performed whole‐brain multiple regression analysis and tested the statistical significance of individual voxels with a voxel‐wise thresh‐ old (FWE‐corrected) of p < 0.05. Significant negative associations were found between EPS and rGMV only in the bilateral cerebellum (Figure 2 and Table 3), supporting our second hypothesis. No signifi‐ cant positive associations between EPS and rGMV were observed. 3.2 | Association between SPS and rGMV To investigate the potential associations between SPS and rGMV, we performed whole‐brain multiple regression analysis and tested the statistical significance of individual voxels with a voxel‐wise (FWE‐cor‐ rected) threshold of p < 0.05. Significant positive associations between SPS and rGMV were observed in widespread brain areas including the bilateral precuneus and posterior cingulate gyri (PCG) but not the dMPFC (Figure 1 and Table 2). These results support our first hypoth‐ esis. However, contrary to our expectations, no significant associations were observed in the vMPFC. In addition to the precuneus and PCG, significant positive associations with SPS were found in the bilateral superior parietal lobules, bilateral precentral gyrus, bilateral middle cingulate gyrus (MCG), bilateral cuneus, right postcentral gyrus, right supramarginal gyrus, right middle frontal gyrus, left angular gyrus, left supplementary motor cortex, and left superior frontal gyrus. No sig‐ nificant negative associations between SPS and rGMV were observed. In order to check the influence of RAPM and age as nuisance covariates, we performed additional whole‐brain multiple regres‐ sion analyses using two models (i.e., a model without RAPM and a model without age). Furthermore, we tested whether there was an interaction effect between SPS and EPS on rGMV. In order to check the interaction effect, we performed an additional analysis using the regression model which included the interaction term between SPS and EPS. The procedures of these additional analyses were similar to the previous paragraph mentioned above except for the regression models. are shown in Table 1. Pearson's correlations were computed to test associations among age, TIV, and behavioral measures (Table S1). There were significant correlations between age and SPS, and between RAPM and SPS. These results suggest the importance of using age and RAPM as nuisance covariates. as nuisance covariates in the second‐level analysis. Sex and age are commonly used as nuisance covariates in VBM, while RAPM score is reportedly related to ADHD (Rommel et al., 2015) and so was also used as a nuisance covariate. Regressing out TIV permits compari‐ son of regional volume differences unrelated to differences in total brain volume. The SPS and EPS scores were included as simultane‐ ous covariates of interest in a multiple regression model to reveal associations unique to each Preoccupation Scale subscale. Only voxels with values exceeding an absolute threshold of 0.05 were targeted for the analysis. This threshold is widely used in VBM stud‐ ies (Beal, Gracco, Lafaille, & Nil, 2007; Focke, Thompson, & Duncan, 2008; Mueller et al., 2006; Nauchi & Sakai, 2009; Schaufelberger et al., 2007; Takeuchi et al., 2010, 2011, 2017b; White, Alkire, & Haier, 2003). To test whether the associations between rGMV and the Preoccupation Scale subscores were statistically significant, a permutation‐based voxel‐wise nonparametric test (5,000 permuta‐ tions) was performed using the threshold‐free cluster enhancement (TFCE) toolbox (http://dbm.neuro.uni-jena.de/tfce/; r95) (Smith & Nichols, 2009). The TFCE method can estimate voxel‐wise values (TFCE values) representing the amount of cluster‐like local spatial support without an arbitrary cluster‐forming threshold as commonly used in the traditional approach. The resulting TFCE maps were thresholded at a family‐wise error (FWE) corrected p < 0.05. We investigated correlations between SPS, EPS, and five per‐ sonality traits assessed by the NEO‐FFI (Table S2). SPS showed sig‐ nificant correlations with all the personality traits, and in particular, showed a strong positive correlation with neuroticism (r = 0.57). On the other hand, EPS showed significant correlations with some per‐ sonality traits, and in particular, showed a strong positive correlation with conscientiousness (r = 0.29). 2.5 | Second‐level analysis To investigate associations of rGMV with SPS and EPS, a whole‐ brain multiple regression analysis was performed using SPM12. Sex, age, RAPM score, and total intracranial volume (TIV) were included 4 of 11 IKEDA et al. SD: standard deviation; RAPM: Raven's Advanced Progressive Matrices; TIV: total intracranial volume; SPS: Self‐Pr Preoccupation Scale. 3.1 | Behavioral data Ages, RAPM scores, TIVs, SPS scores, and EPS scores of the total group (both sexes), the male‐only group, and the female‐only group TA B LE 1 Summary of subject demographics and psychometric results TA B LE 1 Summary of subject demographics and psychometric results All Male Female Mean Range SD Mean Range SD Mean Range SD Age 20.7 18–27 1.8 20.8 18–27 1.9 20.5 18–27 1.6 RAPM 28.5 13–36 3.9 28.8 13–36 3.9 28.1 15–36 3.8 TIV [cm3] 1,535 1,197–2,018 141 1,612 1,352–2,018 119 1,430 1,197–1,737 93 SPS 33.1 11–55 8.9 33.5 11–55 8.9 32.5 11–55 8.9 EPS 27.0 10–40 5.4 27.3 13–40 5.2 26.6 10–40 5.7 SD: standard deviation; RAPM: Raven's Advanced Progressive Matrices; TIV: total intracranial volume; SPS: Self‐Preoccupation Scale; EPS: External‐ Preoccupation Scale. SD: standard deviation; RAPM: Raven's Advanced Progressive Matrices; TIV: total intracranial volume; SPS: Self‐Preoccupation Scale; EPS: External‐ Preoccupation Scale. |  5 of 11 5 of 11 IKEDA et al. | FI G U R E 1 Significant positive associations between Self‐Preoccupation Scale (SPS) and regional gray matter volume (rGMV). (a) Regions demonstrating significant positive associations with SPS scores. Threshold‐free cluster enhancement (TFCE) maps thresholded with a family‐ wise error (FWE)‐corrected p < 0.05 were overlaid on the avg305T1 template using SPM12. The color bar represents the TFCE magnitude. Warm colors represent positive associations. (b) A scatter plot showing the relationship between SPS scores and rGMV at the peak voxel. The peak voxel was located in the right postcentral gyrus. For the scatter plot, the mean value was subtracted from SPS scores, and the nuisance covariates were regressed out from rGMV at the peak voxel. A black line within the scatter plot shows rGMV predicted from SPS scores FI G U R E 1 Significant positive associations between Self‐Preoccupation Scale (SPS) and regional gray matter volume (rGMV). (a) Regions demonstrating significant positive associations with SPS scores. Threshold‐free cluster enhancement (TFCE) maps thresholded with a family‐ wise error (FWE)‐corrected p < 0.05 were overlaid on the avg305T1 template using SPM12. The color bar represents the TFCE magnitude. Warm colors represent positive associations. (b) A scatter plot showing the relationship between SPS scores and rGMV at the peak voxel. The peak voxel was located in the right postcentral gyrus. For the scatter plot, the mean value was subtracted from SPS scores, and the nuisance covariates were regressed out from rGMV at the peak voxel. A black line within the scatter plot shows rGMV predicted from SPS scores significant brain regions overlapped substantially with those ob‐ served in the model with age. No other significant associations were observed. 3.4 | Influence of RAPM and age, and an interaction effect between SPS and EPS We performed the additional whole‐brain multiple regression analyses using two regression models (i.e., a model without RAPM and a model without age) to check whether our results were influ‐ enced by RAPM and age. When using the model without RAPM, we observed similar results to those observed in the model with RAPM (Table S3 and S4). When using the model without age, we observed brain regions positively associated with SPS (Table S5), and many of the significant brain regions were not observed in the model with age. Moreover, we observed brain regions negatively associated with EPS in the model without age (Table S6), and the We performed the additional analysis using the regression model which included the interaction term between SPS and EPS to check the in‐ teraction effect between SPS and EPS on rGMV. As a result, we observed no brain regions showing significant associations with the interaction term. 4 | DISCUSSION The present study investigated associations between rGMV and both SPS and EPS in a large sample of young adults. We found | IKEDA et al. IKEDA et al. TA B LE 2 Brain areas showing positive associations with Self Preoccupation Scale   Anatomical areas (number of significant voxels of each anatomical area) x y z TFCE PFWE Cluster size Cluster 1 R precuneus (1,518) 10.5 −48 63 1,766.47 0.019 14,247 R postcentral gyrus (1,216) 46.5 −21 48 1,987.07 0.011   R superior parietal lobule (1,041) 33 −37.5 45 1,869.08 0.014   R precentral gyrus (743) 43.5 −16.5 63 1,612.88 0.024   R supramarginal gyrus (732) 49.5 −25.5 49.5 1,922.06 0.013   R middle cingulate gyrus (725) 4.5 −21 36 1,738.58 0.019   R posterior cingulate gyrus (472) 4.5 −28.5 40.5 1,733.41 0.019   R middle frontal gyrus (59) 46.5 7.5 52.5 1,505.51 0.029   R precentral gyrus medial segment (24) 3 −27 48 1,575.26 0.026   R postcentral gyrus medial segment (23) 10.5 −42 63 1,637.5 0.023   R cuneus (7) 1.5 −75 36 1,453.51 0.034   L middle cingulate gyrus (1,008) 0 −18 39 1,715.65 0.02   L precuneus (851) 0 −57 42 1,644.57 0.023   L posterior cingulate gyrus (621) 0 −27 45 1,687.56 0.021   L superior parietal lobule (344) −15 −73.5 46.5 1,385.42 0.039   L precentral gyrus medial segment (206) −3 −25.5 46.5 1,652.54 0.023   L angular gyrus (103) −27 −66 34.5 1,349.39 0.043   L supplementary motor cortex (10) −4.5 −18 46.5 1,317.08 0.046   L precentral gyrus (6) −24 −13.5 52.5 1,288.44 0.05   L cuneus (1) 0 −76.5 36 1,285.12 0.05   L superior frontal gyrus (1) −22.5 −12 52.5 1,285.12 0.05   *R cerebral white matter (2,634) 45 −22.5 49.5 1,983.67 0.011   *L cerebral white matter (1,205) −9 −33 37.5 1,629.66 0.023   *Unknown (697) 51 −24 49.5 1,912.4 0.013   Cluster 2 *R cerebral white matter (3) 13.5 −42 28.5 1,285.12 0.05 3 Note. Labeling of brain areas is conducted using custom Matlab scripts and labels_Neuromorphometrics.nii in SPM12. The coordinates of the peak voxel of each brain area are shown as x, y, and z. Asterisks represent white matter and areas that could not be labeled. The TFCE magnitude and corrected p‐value (FWE) for each peak voxel were shown. Cluster size represents the number of voxels which each cluster includes. R: right; L: left; FWE: family‐wise error. Note. Labeling of brain areas is conducted using custom Matlab scripts and labels_Neuromorphometrics.nii in SPM12. 4 | DISCUSSION The coordinates of the peak voxel of each brain area are shown as x, y, and z. Asterisks represent white matter and areas that could not be labeled. The TFCE magnitude and corrected p‐value (FWE) for each peak voxel were shown. Cluster size represents the number of voxels which each cluster includes. R: right; L: left; FWE: family‐wise error. study reveals distinct neural substrates for self‐ and external‐pre‐ occupation at the structural level. significant voxel‐wise positive associations between SPS and rGMV distributed over the bilateral precuneus and PCG but not the dMPFC, supporting our first hypothesis that self‐preoccupation is supported by brain regions related to self‐triggered self‐reference. However, contrary to our expectation, no significant associations with SPS score were observed in vMPFC. Alternatively, significant positive associations between rGMV and SPS were widely ob‐ served in the bilateral superior parietal lobules, bilateral precentral gyrus, bilateral MCG, bilateral cuneus, right postcentral gyrus, right supramarginal gyrus, right middle frontal gyrus, left angular gyrus, left supplementary motor cortex, and left superior frontal gyrus, implicating these regions in self‐preoccupation. We also found significant negative associations between EPS and rGMV only in the cerebellum, supporting our second hypothesis that external‐ preoccupation is subserved by regions implicated in behavioral ad‐ diction, sustained attention, and reward system. Collectively, our The associations of SPS with the precuneus and PCG suggest that self‐preoccupation occurs independently of individual intention during resting‐state because activity of the precuneus and PCG is re‐ lated to self‐triggered self‐reference (Whitfield‐Gabrieli et al., 2011). On the other hand, distinct functions of the precuneus and PCG in different aspects of self‐preoccupation are suggested by the follow‐ ing previous findings. First, activation in PCG was associated with incidence of task‐unrelated thought, while no significant associa‐ tions were observed in the precuneus (Mckiernan, Angelo, Kaufman, & Binder, 2006). Second, PCG was not activated by episodic mem‐ ory retrieval but rather by stimuli judged as self‐referential, while episodic memory retrieval activated the precuneus (Sajonz et al., 2010). Third, self‐related processes were driven via PCC (Davey et al., 2016). These previous findings suggest that self‐preoccupation is 7 of 11 IKEDA et al. |  7 of 11 |  7 of 11 IKEDA et al. FI G U R E 2 Significant negative associations between External‐Preoccupation Scale (EPS) and regional gray matter volume (rGMV). (a) Regions demonstrating significant negative associations with EPS scores. 4 | DISCUSSION Threshold‐free cluster enhancement (TFCE) maps thresholded with a family‐wise error (FWE)‐corrected p < 0.05 were overlaid on the avg305T1 template using SPM12. The color bar represents the TFCE magnitude. Cool colors represent negative associations. (b) A scatter plot showing the relationship between EPS scores and rGMV at the peak voxel. The peak voxel was located in the right cerebellum exterior. For the scatter plot, the mean value was subtracted from EPS scores, and the nuisance covariates were regressed out from rGMV at the peak voxel. A black line within the scatter plot shows rGMV predicted from EPS scores |  7 of 11 IKEDA et al. FI G U R E 2 Significant negative associations between External‐Preoccupation Scale (EPS) and regional gray matter volume (rGMV). (a) Regions demonstrating significant negative associations with EPS scores. Threshold‐free cluster enhancement (TFCE) maps thresholded with a family‐wise error (FWE)‐corrected p < 0.05 were overlaid on the avg305T1 template using SPM12. The color bar represents the TFCE magnitude. Cool colors represent negative associations. (b) A scatter plot showing the relationship between EPS scores and rGMV at the peak voxel. The peak voxel was located in the right cerebellum exterior. For the scatter plot, the mean value was subtracted from EPS scores, and the nuisance covariates were regressed out from rGMV at the peak voxel. A black line within the scatter plot shows rGMV predicted from EPS scores FI G U R E 2 Significant negative associations between External‐Preoccupation Scale (EPS) and regional gray matter volume (rGMV). (a) Regions demonstrating significant negative associations with EPS scores. Threshold‐free cluster enhancement (TFCE) maps thresholded with a family‐wise error (FWE)‐corrected p < 0.05 were overlaid on the avg305T1 template using SPM12. The color bar represents the TFCE magnitude. Cool colors represent negative associations. (b) A scatter plot showing the relationship between EPS scores and rGMV at the peak voxel. The peak voxel was located in the right cerebellum exterior. For the scatter plot, the mean value was subtracted from EPS scores, and the nuisance covariates were regressed out from rGMV at the peak voxel. A black line within the scatter plot shows rGMV predicted from EPS scores FI G U R E 2 Significant negative associations between External‐Preoccupation Scale (EPS) and regional gray matter volume (rGMV). (a) Regions demonstrating significant negative associations with EPS scores. 4 | DISCUSSION Threshold‐free cluster enhancement (TFCE) maps thresholded with a family‐wise error (FWE)‐corrected p < 0.05 were overlaid on the avg305T1 template using SPM12. The color bar represents the TFCE magnitude. Cool colors represent negative associations. (b) A scatter plot showing the relationship between EPS scores and rGMV at the peak voxel. The peak voxel was located in the right cerebellum exterior. For the scatter plot, the mean value was subtracted from EPS scores, and the nuisance covariates were regressed out from rGMV at the peak voxel. A black line within the scatter plot shows rGMV predicted from EPS scores The bilateral precentral gyrus, the right postcentral gyrus, and the right supramarginal gyrus showed significant positive associa‐ tions with SPS, consistent with previous findings that self‐recogni‐ tion (i.e., face and body) requires activity in precentral regions (Ferri, Frassinetti, Ardizzi, Costantini, & Gallese, 2012; Morita et al., 2008; Sugiura et al., 2006, 2008), postcentral regions (Ferri et al., 2012; Platek et al., 2006; Sugiura et al., 2012), and right supramarginal gyrus (Platek et al., 2006; Sugiura et al., 2006, 2008, 2012). Self‐face is among the most observable representative aspects of the self. Highly self‐preoccupied individuals are expected to focus attention on their faces during rest and feel embarrassment when comparing it to others. This may provide an explanation for the association be‐ tween SPS and depression (Sakamoto, 1999). On the other hand, we found significant positive associations between the bilateral superior driven via PCG and that the precuneus plays a role in episodic mem‐ ory retrieval during self‐preoccupation. We speculated that SPS was associated with both vMPFC and PCG. However, SPS was associ‐ ated with PCG while not associated with vMPFC. The observations were inconsistent with our speculation. A previous study reported that vMPFC and PCC belonged to different subsystems within the default network (Andrews‐Hanna, Smallwood, & Spreng, 2014). The functional difference between vMPFC and PCC may explain the inconsistency between the obtained results and our specula‐ tion. Contrary to the previous findings, a recent study reported that vMPFC and PCC belonged to the same default network (Di Plinio & Ebisch, 2018). Therefore, further investigation is needed to deter‐ mine why a significant association with SPS was not observed in the vMPFC. IKEDA et al. IKEDA et al. 4 | DISCUSSION It is likely that the superior parietal lobule has a functional role in episodic memory retrieval during self‐preoccupation, particularly in processing information regarding own face and body image. We also found the significant positive associations of SPS with MCG. The cingulate area is divided into three regions, pre‐ and subgenual anterior cingulate cortex, su‐ pragenual anterior cingulate cortex (SACC), and PCC (Northoff et al., 2006). The MCG roughly overlaps with SACC and PCC. The SACC has been found to be recruited by both up‐ and downregulation of negative emotion (Ochsner et al., 2004), so the MCG may regulate negative emotions during self‐preoccupation. RAPM and age were treated as nuisance covariates in the sec‐ ond‐level analysis because these covariates showed significant cor‐ relations with SPS. We checked whether our results were indeed influenced by RAPM and age. As a result, the model without RAPM showed similar results to those observed in the model with RAPM, while the model without age showed different results to those ob‐ served in the model with age. Therefore, our results were influenced by age but not by RAPM. The significant negative associations between EPS and rGMV were observed only in the bilateral cerebellum. These results support the second hypothesis and suggest that external‐preoccupation has three attributes, behavioral addiction, sustained attention, and re‐ ward system, all of which engage the cerebellum. On the other hand, Internet addicts reportedly showed reduced GMV in regions other than the cerebellum (Weinstein et al., 2017). However, previous findings on GMV reduction in behavioral addiction may reflect both predisposing factors and changes due to ongoing addictive behavior. Given that our study was conducted entirely with healthy subjects, the association between EPS and rGMV in cerebellum may reflect only individual differences in vulnerability to behavioral addiction. A previous neuroimaging study also reported a strong association between the cerebellum and sustained attention (Rosenberg et al., 2016), supporting our speculation that external‐preoccupation is as‐ sociated with sustained attention. On the other hand, external‐pre‐ occupation represents the tendency to maintain focus for a long time, while capacity for sustained attention is frequently assessed using relatively brief tasks (e.g., 8 min in Esterman, Noonan, Rosenberg, & Degutis, 2013). Therefore, external‐preoccupation and sustained If there is an interaction effect between SPS and EPS, it suggests the presence of brain regions related to switching between self‐ and external‐preoccupation or to maintaining focus irrespective of di‐ rection of attention. 4 | DISCUSSION Anatomical areas (number of significant voxels of each anatomical area) x y z TFCE PFWE Cluster size Cluster 1 L cerebellum exterior (3,096) −21 −76.5 −40.5 1,685.46 0.018 4,468 *L cerebellum white matter (969) −27 −55.5 −40.5 1,713.01 0.017   *Unknown (403) −24 −57 −63 1,472.71 0.028   Cluster 2 R cerebellum exterior (5,778) 18 −79.5 −36 2,204.93 0.006 7,407 Cerebellar vermal lobules viii–x (91) 7.5 −67.5 −37.5 1,738.94 0.017   Cerebellar vermal lobules vi–vii (2) 6 −66 −31.5 1,309.33 0.045   *R cerebellum white matter (1,022) 16.5 −75 −37.5 2,166.41 0.007   *Unknown (514) 18 −91.5 −27 1,873.52 0.013   Note. Labeling of brain areas is conducted using custom Matlab scripts and labels_Neuromorphometrics.nii in SPM12. The coordinates of the peak voxel of each brain area are shown as x, y, and z. Asterisks represent white matter and areas that could not be labeled. The TFCE magnitude and corrected p‐value (FWE) for each peak voxel were shown. Cluster size represents the number of voxels which each cluster includes. R: right; L: left; FWE: family‐wise error. Note. Labeling of brain areas is conducted using custom Matlab scripts and labels_Neuromorphometrics.nii in SPM12. The coordinates of the peak voxel of each brain area are shown as x, y, and z. Asterisks represent white matter and areas that could not be labeled. The TFCE magnitude and corrected p‐value (FWE) for each peak voxel were shown. Cluster size represents the number of voxels which each cluster includes. R: right; L: left; FWE: family‐wise error. attention may involve distinct neural mechanisms. Nonetheless, our results do suggest that the cerebellum is involved in both short‐ and long‐term attention. According to classical observations, rewards are primarily mediated by the dopaminergic system, including the stria‐ tum (Kawagoe, Takikawa, & Hikosaka, 1998; Samejima, Ueda, Doya, & Kimura, 2005) and prefrontal cortex (Hikosaka & Watanabe, 2000, 2004; O'Doherty, Deichmann, Critchley, & Dolan, 2002). If external‐ preoccupation is associated with the reward system, significant asso‐ ciations with EPS ought to be observed in canonical reward‐related areas. However, such associations were not observed in the present study. The reasons for this remain uncertain. parietal lobules and SPS. A previous neuroimaging study revealed self‐specific (i.e., face and body) activation in the right superior pa‐ rietal lobule (Sugiura et al., 2006). In addition, the superior parietal lobule has been implicated in episodic memory retrieval (Sajonz et al., 2010). 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Regional gray matter volume of dopami‐ nergic system associate with creativity: Evidence from voxel‐based morphometry. Neuroimage, 51, 578–585. https://doi.org/10.1016/j. neuroimage.2010.02.078 How to cite this article: Ikeda S, Takeuchi H, Taki Y, et al. Neural substrates of self‐ and external‐preoccupation: A voxel‐based morphometry study. Brain Behav. 2019;e01267. https://doi.org/10.1002/brb3.1267 Takeuchi, H., Taki, Y., Sassa, Y., Hashizume, H., Sekiguchi, A., Fukushima, A., & Kawashima, R. (2011). Regional gray matter density asso‐ ciated with emotional intelligence: Evidence from voxel‐based
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Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes
Cell reports
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To cite this version: Iva Simeonova, Sara Jaber, Irena Draskovic, Boris Bardot, Ming Fang, et al.. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes. Cell Reports, 2013, 3 (6), pp.2046-2058. ￿10.1016/j.celrep.2013.05.028￿. ￿hal-01548788￿ Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes Iva Simeonova, Sara Jaber, Irena Draskovic, Boris Bardot, Ming Fang, Rachida Bouarich-Bourimi, Vincent Lejour, Laure Charbonnier, Claire Soudais, Jean-Christophe Bourdon, et al. To cite this version: Iva Simeonova, Sara Jaber, Irena Draskovic, Boris Bardot, Ming Fang, et al.. Mutant Mice Lacki the p53 C-Terminal Domain Model Telomere Syndromes. Cell Reports, 2013, 3 (6), pp.2046-205 ￿10.1016/j.celrep.2013.05.028￿. ￿hal-01548788￿ To cite this version: Iva Simeonova, Sara Jaber, Irena Draskovic, Boris Bardot, Ming Fang, et al.. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes. Cell Reports, 2013, 3 (6), pp.2046-2058. ￿10.1016/j.celrep.2013.05.028￿. ￿hal-01548788￿ Distributed under a Creative Commons Attribution 4.0 International License SUMMARY of stem cells and is involved in diseases including diabetes and several neurological disorders (Brady and Attardi, 2010). Mutations in p53, although frequent in human can- cers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53D31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hall- marks of syndromes caused by short telomeres. Indeed, p53D31/D31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Hetero- zygous p53+/D31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53D31/D31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism. Most p53 mutations in cancers affect the core DNA-binding domain of the protein, altering its capacity to regulate transcrip- tional target genes (Toledo and Wahl, 2006). Importantly, how- ever, p53 contains a second DNA-binding domain in its carboxyl terminus (Foord et al., 1991), whose role remains elusive (Hupp et al., 1992; McKinney et al., 2004). Early studies suggested that the p53 C-terminal domain (CTD), whose interaction with DNA is not sequence dependent, acts as a negative regulator of the core DNA-binding domain (Hupp et al., 1992). However, later reports concluded that p53 requires its CTD for efficient recognition of target gene sequences (McKinney et al., 2004; Tafvizi et al., 2011; Hamard et al., 2012; Kim et al., 2012). The phosphorylation of C-terminal serines 378 and 392 was pro- posed to increase p53 DNA binding (Hupp et al., 1992; Takenaka et al., 1995), but only the latter was mutated and analyzed in vivo (Bruins et al., 2004). Furthermore, posttranslational modifica- tions of C-terminal lysines were proposed to be essential for the regulation of p53 stability and activity, but mice with C-termi- nal lysines mutated into arginines appeared similar to wild-type (WT) mice (Feng et al., 2005; Krummel et al., 2005), except for a hypersensitivity to g-irradiation (Wang et al., 2011a). HAL Id: hal-01548788 https://hal.sorbonne-universite.fr/hal-01548788v1 Submitted on 28 Jun 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Cell Reports Article Cell Reports A ti l Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes Iva Simeonova,1,5,6 Sara Jaber,1,5,6,9 Irena Draskovic,2,5,6,9 Boris Bardot,1,5,6 Ming Fang,1,5,6 Rachida Bouarich-Bourimi,1,5,6 Vincent Lejour,1,5,6 Laure Charbonnier,1,5,6 Claire Soudais,3,7 Jean-Christophe Bourdon,8 Michel Huerre,4 Arturo Londono-Vallejo,2,5,6 and Franck Toledo1,5,6,* 1Genetics of Tumor Suppression 2Telomeres and Cancer ‘‘E´ quipe Labellise´ e Ligue’’ 3CD4+T-Lymphocytes and Anti-Tumour Response 4Pathology Service Institut Curie, Centre de Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France 5UPMC Paris 06, 26 rue d’Ulm, 75248 Paris Cedex 05, France 6CNRS UMR 3244, 26 rue d’Ulm, 75248 Paris Cedex 05, France 7INSERM U932, 26 rue d’Ulm, 75248 Paris Cedex 05, France 8Ninewells Hospital, University of Dundee, Dundee DD19SY, Scotland 9These authors contributed equally to this work *Correspondence: franck.toledo@curie.fr http://dx.doi.org/10.1016/j.celrep.2013.05.028 Iva Simeonova,1,5,6 Sara Jaber,1,5,6,9 Irena Draskovic,2,5,6,9 Boris Bardot,1,5,6 Ming Fang,1,5,6 Rachida Bouarich-Bourimi,1,5,6 Vincent Lejour,1,5,6 Laure Charbonnier,1,5,6 Claire Soudais,3,7 Jean-Christophe Bourdon,8 Michel Huerre,4 Arturo Londono-Vallejo,2,5,6 and Franck Toledo1,5,6,* 1Genetics of Tumor Suppression Iva Simeonova,1,5,6 Sara Jaber,1,5,6,9 Irena Draskovic,2,5,6,9 Boris Bardo Rachida Bouarich-Bourimi,1,5,6 Vincent Lejour,1,5,6 Laure Charbonnier,1 Jean-Christophe Bourdon,8 Michel Huerre,4 Arturo Londono-Vallejo,2,5 1Genetics of Tumor Suppression 2Telomeres and Cancer ‘‘E´ quipe Labellise´ e Ligue’’ 3CD4+T-Lymphocytes and Anti-Tumour Response 4Pathology Service Institut Curie, Centre de Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France 5UPMC Paris 06, 26 rue d’Ulm, 75248 Paris Cedex 05, France 6CNRS UMR 3244, 26 rue d’Ulm, 75248 Paris Cedex 05, France 7INSERM U932, 26 rue d’Ulm, 75248 Paris Cedex 05, France 8Ninewells Hospital, University of Dundee, Dundee DD19SY, Scotland 9These authors contributed equally to this work *Correspondence: franck.toledo@curie.fr http://dx.doi.org/10.1016/j.celrep.2013.05.028 SUMMARY Impor- tantly, in vitro data suggested that a deletion of the p53 CTD, or mutations of C-terminal lysines, might cause distinct pheno- types because the CTD is required for an optimal interaction of p53 with Mdm2, a ubiquitin ligase that regulates p53 stability (Poyurovsky et al., 2010). p53D31/D31 Mice Model Telomere Syndromes D31/D31 p y p53D31/D31 mice were born in Mendelian proportions from p53+/D31 intercrosses, but most died 14–43 days after birth (Fig- ure 3A). Because a mutation removing the last 31 residues of the CTD prevents the expression of isoforms with an AS C terminus (Figure S1B), we also generated mice with a specific deletion of exon AS. None of these mutants died within 3 months after birth (Figure S3), indicating that the premature death of p53D31/D31 mice does not result from a loss of p53AS isoforms. The p53D31/D31 mice that died within a month were significantly smaller than their littermates (Figure 3B), as reported for mice with an increased p53 activity (Mendrysa et al., 2003; Liu et al., 2007, 2010). Other evidence of increased p53 activity (Liu et al., 2007; Terzian et al., 2007; McGowan et al., 2008) in p53D31/D31 mice included darker footpads and tails (Figures 3C and S4A), increased thymocyte apoptosis (Figures 3D and S4B), cerebellar hypoplasia, and hypogonadism in males (Fig- ures S4C and S4D). p53D31/D31 mice suffered from severe pancytopenia and had hypertrophic hearts typical of anemic an- imals (Figures 3E and 3F). A dramatic decrease in marrow cellu- larity was observed in p53D31/D31 mice (Figure 3G), and mutant bone marrow cells (BMCs) lacked hematopoietic progenitors (Figures 3H, 3I, S4E, and S4F). Thus, the premature death of most p53D31/D31 animals likely results from aplastic anemia and consecutive heart failure, consistent with previous reports of hematopoietic defects correlating with spontaneous perinatal death (Liu et al., 2007, 2010), accelerated aging (Dumble et al., 2007), or increased radiosensitivity (Mendrysa et al., 2003; Her- rera-Merchan et al., 2010; Wang et al., 2011a) in mouse models with increased p53 activity. D31/D31 However, targeting a nonsense mutation at the murine Trp53 locus presented two possible obstacles. First, a mutation removing the last 30–33 residues, within the penultimate exon (exon 10), might cause nonsense-mediated mRNA decay. Sec- ond, Trp53 encodes, from mutually exclusive final exons, isoforms with two distinct CTDs: exon 11, used predominantly, encodes the ‘‘classical’’ p53 protein, whereas exon AS (for alter- native splicing) encodes mouse-specific isoforms with a shorter C terminus (Arai et al., 1986). Therefore, although a mutation in exon 11 encoding a protein with a deletion of at most 26 residues would prevent mRNA degradation, we were concerned that it might perturb mRNA splicing and lead to compensatory p53AS overexpression (Figure S1B). INTRODUCTION Here, we targeted a nonsense mutation at the mouse Trp53 lo- cus to evaluate the consequences of a deletion of the p53 CTD in vivo. The results we obtained clearly demonstrate that a dele- tion of the CTD leads to increased p53 activity. Most mice expressing a p53 lacking the CTD died rapidly after birth and ex- hibited features typical of telomere syndromes, including aplas- tic anemia, lung fibrosis, and short telomeres. Consistent with these observations, we found that p53 activation leads to the downregulation of several genes involved in telomere Mutations in TP53, the gene encoding transcription factor p53, are frequently observed in sporadic cancers (Nigro et al., 1989), and germline TP53 mutations cause the Li-Fraumeni syn- drome of cancer predisposition (Malkin et al., 1990; Srivastava et al., 1990). These and other findings established p53 as a major tumor suppressor (Lane and Levine, 2010). In recent years, how- ever, p53 emerged as a protein with a wide variety of functions: it is now thought to regulate longevity, fertility, and the production 2046 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors p53+/LSL-D31 mice were also mated with PGK-Cre mice to obtain MEFs expressing p53D31 constitutively, analyzed below. Immunofluorescence revealed that the p53D31 protein is mostly nuclear, more abundant than p53WT, and that it accumulates in response to stress (Figure 2E). Western blots confirmed the increased p53 levels in mutant cells (Figure 2F). p53D31 protein levels were regulated by the ubiquitin ligase Mdm2, as indicated by treatment with Nutlin, a specific Mdm2 inhibitor (Figure 2G). The p21 and Mdm2 protein levels were increased in p53D31/D31 MEFs (Figure 2F), and both genes were transactivated more effi- ciently in mutant cells (Figure 2H), although p53D31 did not appear to bind specific sequences at the p21 and Mdm2 promoters more efficiently than p53WT (Figure 2I). Additional ex- periments confirmed that p53 target genes are more efficiently regulated in p53D31/D31 cells (Figure S2A). Mutant MEFs pre- sented increased G1/S ratios before or after irradiation (Figures 2J and S2B) and ceased to proliferate prematurely (Figures 2K and 2L). Together, these data demonstrated that p53D31/D31 cells exhibit increased p53 activity. metabolism including Dyskerin, a gene frequently mutated in dyskeratosis congenita (DC), the archetypal telomere syndrome (Armanios and Blackburn, 2012). These data provide evidence that p53 plays an important role in the regulation of telomere metabolism. Rationale for the Targeting of a Nonsense Mutation Affecting the p53 CTD In most in vitro studies, p53 mutants lacking the last 30–33 res- idues were analyzed. These mutants lacked a region enriched in basic residues (lysines, arginines, and histidines within residues 363–382 in human p53) and most if not all the C-terminal resi- dues subject to posttranslational modifications (lysines, serines, and threonines within residues 362–392 in human p53). How- ever, they retained an intact tetramerization domain (within resi- dues 326–355 in human p53) (Figure S1A). Here, we aimed to analyze the consequences of a similar deletion of the p53 CTD in vivo and, thus, to target a nonsense mutation leading to a mouse p53 mutant that lacks the last 30–33 residues. p53D31/D31 Mice Model Telomere Syndromes D31/D31 These possibilities prompted us to perform preliminary experiments in which constructs encod- ing proteins with deletions of the last 26 (p53D26) or 31 (p53D31) residues were targeted in mouse embryonic fibroblasts (MEFs) by recombinase-mediated cassette exchange (RMCE) (Figures S1C and S1D). Neither mutation led to measurable decreases in p53 activity or p53 mRNA levels, but the mutation encoding p53D26 was associated with a 9-fold increase in p53AS mRNAs (Figures S1E–S1J). From these data, we decided to create a mouse with a nonsense mutation deleting the last 31 residues. Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2047 1 MEFs Exhibit Increased p53 Activity 31 To generate a mouse expressing p53D31 conditionally, we used a targeting vector containing the mutation in exon 10 and tran- scriptional Stops flanked by LoxP sites (LSL) upstream of coding sequences (Figures 1A–1D). F1 intercrosses then produced p53LSL-D31/LSL-D31 MEFs (Figure 1E). To analyze the effect of the targeted mutation, we first excised the LSL cassette ex vivo by adding Cre recombinase to p53LSL-D31/LSL-D31 MEFs or to p53LSL-WT/LSL-WT MEFs (Ventura et al., 2007) as controls (Figures 2A and 2B). Similar p53 mRNA levels were found in the resulting p53WT/WT and p53D31/D31 MEFs, but mutant cells contained higher mRNA levels of the p53 targets p21(Cdkn1a) and Mdm2, indicating an increased p53 activity (Figures 2C and 2D). Strikingly, p53D31/D31 mice also developed pulmonary fibrosis (Figures 4A and 4B). In humans, the association of aplastic ane- mia and pulmonary fibrosis characterizes syndromes caused by abnormally short telomeres (Parry et al., 2011b). We compared the length of telomeres in WT and p53D31/D31 cells and found shorter telomeres in mutant cells (Figures 4C and 4D). Further- more, telomere dysfunction-induced foci were more frequent in p53D31/D31 cells (Figure S5). This led us to conclude that Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2047 Figure 1. Targeting a Deletion of the CTD at the Mouse Trp53 Locus (A) Targeting strategy. The Trp53 gene is within a 17-kb-long EcoRI (RI) fragment (black boxes indicate coding sequences; white boxes show UTRs). The targeting construct (above) contains (1) a 1.5-kb-long 50 homology region; (2) a LSL cassette with a neomycin selection gene (Neo), four transcriptional Stops (STOP), and a EcoRI site, flanked by LoxP sites (arrowheads); (3) p53 exons, including the nonsense mutation in exon 10 (asterisk) and an additional Bcl I site; (4) a 2.8-kb-long 30 homology region; and (5) the diphtheria a-toxin (DTA) gene for targeting enrichment. Recombinants from the depicted crossing-overs were identified by a 2.4-kb- long band after PCR withprimersi and j, and bands of 2.1,1.1,and 0.5kb after PCR with primers g and h and BclI digestion. Recombinant cloneswere also analyzed by Southern blot with the indicated probeas containinga10.5 kbEcoRI band.The mutationwas routinelygenotyped by PCR with primerse and fand Bcl Idigestion. (B–D) Screening of recombinant clones as described in (A). 1 MEFs Exhibit Increased p53 Activity 31 ES clones were screened (asterisk [*] indicates a positive clone) by PCR with primers i and j (B), with primers g and h, then PCR products were digested or not (b or u) with BclI (C) and by Southern blot (D). (E) MEF genotyping by PCR. MEFs, prepared from embryos from an intercross of p53+/LSL-D31 mice, were genotyped by PCR with primers e and f followed by Bcl I digestion. See also Figure S1 Figure 1. Targeting a Deletion of the CTD at the Mouse Trp53 Locus g g g p (A) Targeting strategy. The Trp53 gene is within a 17-kb-long EcoRI (RI) fragment (black boxes indicate coding sequences; white boxes show UTRs). The targeting construct (above) contains (1) a 1.5-kb-long 50 homology region; (2) a LSL cassette with a neomycin selection gene (Neo), four transcriptional Stops (STOP), and a EcoRI site, flanked by LoxP sites (arrowheads); (3) p53 exons, including the nonsense mutation in exon 10 (asterisk) and an additional Bcl I site; (4) a 2.8-kb-long 30 homology region; and (5) the diphtheria a-toxin (DTA) gene for targeting enrichment. Recombinants from the depicted crossing-overs were identified by a 2.4-kb- long band after PCR withprimersi and j, and bands of 2.1,1.1,and 0.5kb after PCR with primers g and h and BclI digestion. Recombinant cloneswere also analyzed by Southern blot with the indicated probeas containinga10.5 kbEcoRI band.The mutationwas routinelygenotyped by PCR with primerse and fand Bcl Idigestion. (B–D) Screening of recombinant clones as described in (A). ES clones were screened (asterisk [*] indicates a positive clone) by PCR with primers i and j (B), with primers g and h, then PCR products were digested or not (b or u) with BclI (C) and by Southern blot (D). (E) MEF genotyping by PCR. MEFs, prepared from embryos from an intercross of p53+/LSL-D31 mice, were genotyped by PCR with primers e and f followed by Bcl I digestion. See also Figure S1. p53D31/D31 mice model DC, an archetypal telomere syndrome caused by mutations in genes encoding components of the telo- merase or shelterin complexes, or telomerase regulators (Arma- nios and Blackburn, 2012). In support of this conclusion, p53D31/D31 mice presented full-blown clinical features of DC: cutaneous hyperpigmentation, nail dystrophy, and oral leukopla- kia (Figures 4E and 4F). 1 MEFs Exhibit Increased p53 Activity 31 Furthermore, the small size, hypogonad- ism, and cerebellar hypoplasia observed in some mice (Figures 3B, S4C, and S4D) characterize patients with the Hoyeraal-Hrei- darsson syndrome (HHS), a severe variant of DC (Armanios and Blackburn, 2012). p53-negative regulators (i.e., most p53+/D31 Mdm4+/ and a few p53+/D31 Mdm2+/ mice) died within 3 months (Figure 5B). This suggested that the levels of p53 inhibitors—particularly Mdm4—impacted the severity of DC-related symptoms. Accordingly, bone marrow cellularity and telomere length were decreased in p53+/D31 Mdm4+/ mice compared to p53+/D31 mice (Figures 5C, 5D, and S6). p53 Activation Alters the Expression of Genes Mutated in Telomere Syndromes A His-tagged NLS-Cre recombinase (HTN-Cre) was added to p53LSL-D31/LSL-D31 MEFs, allowing excision of the Stop cassette within 24 hr. p53D31/D31 MEFs were then recovered and analyzed (right). As controls, p53LSL-WT/LSL-WT MEFs were treated likewise (left). p53LSL-D31/LSL-D31 MEFs were also treated with a buffer solution devoid of Cre as negative controls (center). (B) Efficiency of the Cre-mediated excision of the LSL cassette. LSL excision was determined using multiplex PCR with primers k, l, and m. Prior excision, a 278- bp-long product results from amplification with primers k and l, whereas primers m and l are too far apart to generate a PCR product. After excision, primers m and l generate a 330-bp-long product. The PCR analysis of the experiment described in (A) is shown: PCRs show 100% efficiency in LSL excision (Exc.) in lanes a, d, and e; and 0% in lanes b and c. (C and D) p53LSL-D31/LSL-D31 MEFs exhibit increased p53 activity after LSL excision. mRNAs were prepared from p53LSL-WT/LSL-WT MEFs treated with HTN-Cre (a) or p53LSL-D31/LSL-D31 MEFs treated with buffer (b and c) or HTN-Cre (d and e). p53 mRNAs (C) or p21 and Mdm2 mRNAs (D) were quantified using real-time PCR, normalized to control mRNAs, then the amount in (a) was assigned a value of 1. (E–L) Comparative analysis of WT and p53D31/D31 MEFs. (E) WT and p53D31/D31 (D31/D31) MEFs untreated (Unt) or treated with 0.5 mg/ml Adriamycin (Adr) for 24 hr were stained with a p53 antibody, and their DNA was counterstained with DAPI. Figure 2. p53 MEFs Exhibit Increased p53 Activity (A) Strategy to analyze the effects of an excision of the LSL cassette ex vivo. A His-tagged NLS-Cre recombinase (HTN-Cre) was added to p53LSL-D31/LSL-D31 MEFs, allowing excision of the Stop cassette within 24 hr. p53D31/D31 MEFs were then recovered and analyzed (right). As controls, p53LSL-WT/LSL-WT MEFs were treated likewise (left). p53LSL-D31/LSL-D31 MEFs were also treated with a buffer solution devoid of Cre as negative controls (center). (B) Efficiency of the Cre-mediated excision of the LSL cassette. LSL excision was determined using multiplex PCR with primers k, l, and m. Prior excision, a 278- bp-long product results from amplification with primers k and l, whereas primers m and l are too far apart to generate a PCR product. After excision, primers m and l generate a 330-bp-long product. p53 Activation Alters the Expression of Genes Mutated in Telomere Syndromes The PCR analysis of the experiment described in (A) is shown: PCRs show 100% efficiency in LSL excision (Exc.) in lanes a, d, and e; and 0% in lanes b and c. (C and D) p53LSL-D31/LSL-D31 MEFs exhibit increased p53 activity after LSL excision. mRNAs were prepared from p53LSL-WT/LSL-WT MEFs treated with HTN-Cre (a) or p53LSL-D31/LSL-D31 MEFs treated with buffer (b and c) or HTN-Cre (d and e). p53 mRNAs (C) or p21 and Mdm2 mRNAs (D) were quantified using real-time PCR, normalized to control mRNAs, then the amount in (a) was assigned a value of 1. (E–L) Comparative analysis of WT and p53D31/D31 MEFs. (E) WT and p53D31/D31 (D31/D31) MEFs untreated (Unt) or treated with 0.5 mg/ml Adriamycin (Adr) for 24 hr were stained with a p53 antibody, and their DNA was counterstained with DAPI. (l d ti d t ) (A) Strategy to analyze the effects of an excision of the LSL cassette ex vivo. A His-tagged NLS-Cre recombinase (HTN-Cre) was added to p53LSL-D31/LSL-D31 MEFs, allowing excision of the Stop cassette within 24 hr. p53D31/D31 MEFs were then recovered and analyzed (right). As controls, p53LSL-WT/LSL-WT MEFs were treated likewise (left). p53LSL-D31/LSL-D31 MEFs were also treated with a buffer solution devoid of Cre as negative controls (center). (B) Efficiency of the Cre-mediated excision of the LSL cassette. LSL excision was determined using multiplex PCR with primers k, l, and m. Prior excision, a 278- bp-long product results from amplification with primers k and l, whereas primers m and l are too far apart to generate a PCR product. After excision, primers m and l generate a 330-bp-long product. The PCR analysis of the experiment described in (A) is shown: PCRs show 100% efficiency in LSL excision (Exc.) in lanes a, d, and e; and 0% in lanes b and c. (C and D) p53LSL-D31/LSL-D31 MEFs exhibit increased p53 activity after LSL excision. mRNAs were prepared from p53LSL-WT/LSL-WT MEFs treated with HTN-Cre (a) or p53LSL-D31/LSL-D31 MEFs treated with buffer (b and c) or HTN-Cre (d and e). p53 mRNAs (C) or p21 and Mdm2 mRNAs (D) were quantified using real-time PCR, li d t t l RNA th th t i ( ) i d l f 1 (E L) Comparative analysis of WT and p53 MEFs. p53 Activation Alters the Expression of Genes Mutated in Telomere Syndromes Animal models recently suggested that dysfunctional telomeres (Wang et al., 2011b) or altered ribosomal RNA processing (Pere- boom et al., 2011) could lead to p53 activation and bone marrow failure. Our data suggested that a p53-truncating mutation might ) DC-related features were also detected in p53+/D31 mice: three p53+/D31 mice died within a year with a hypertrophic heart (Fig- ure 5A). Moreover, heterozygous mutants with lower levels of 2048 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors Figure 2. p53D31/D31 MEFs Exhibit Increased p53 Activity (A) Strategy to analyze the effects of an excision of the LSL cassette ex vivo. A His-tagged NLS-Cre recombinase (HTN-Cre) was added to p53LSL-D31/LSL-D31 MEFs, allowing excision of the Stop cassette within 24 hr. p53D31/D31 MEFs were then recovered and analyzed (right). As controls, p53LSL-WT/LSL-WT MEFs were treated likewise (left). p53LSL-D31/LSL-D31 MEFs were also treated with a buffer solution devoid of Cre as negative controls (center). (B) Efficiency of the Cre-mediated excision of the LSL cassette. LSL excision was determined using multiplex PCR with primers k, l, and m. Prior excision, a 278- bp-long product results from amplification with primers k and l, whereas primers m and l are too far apart to generate a PCR product. After excision, primers m and l generate a 330-bp-long product. The PCR analysis of the experiment described in (A) is shown: PCRs show 100% efficiency in LSL excision (Exc.) in lanes a, d, and e; and 0% in lanes b and c. (C and D) p53LSL-D31/LSL-D31 MEFs exhibit increased p53 activity after LSL excision. mRNAs were prepared from p53LSL-WT/LSL-WT MEFs treated with HTN-Cre (a) or p53LSL-D31/LSL-D31 MEFs treated with buffer (b and c) or HTN-Cre (d and e). p53 mRNAs (C) or p21 and Mdm2 mRNAs (D) were quantified using real-time PCR, normalized to control mRNAs, then the amount in (a) was assigned a value of 1. (E–L) Comparative analysis of WT and p53D31/D31 MEFs. (E) WT and p53D31/D31 (D31/D31) MEFs untreated (Unt) or treated with 0.5 mg/ml Adriamycin (Adr) for 24 hr were stained with a p53 antibody, and their DNA was counterstained with DAPI D31/D31 Figure 2. p53D31/D31 MEFs Exhibit Increased p53 Activity Figure 2. p53 MEFs Exhibit Increased p53 Activity (A) Strategy to analyze the effects of an excision of the LSL cassette ex vivo. p53 Activation Alters the Expression of Genes Mutated in Telomere Syndromes Consistent with this, p53 activation led to the downregulation of the four genes, with a stronger effect on Dkc1 (Figure 6D). p p ( g ) In summary, we found that a nonsense mutation in the exon 10 of Trp53, encoding the truncated protein p53D31, causes an in- crease in p53 activity and phenotypes related to human telomere syndromes. To perform a more comprehensive analysis of nonsense mutations affecting Trp53 exon 10, we also targeted in MEFs mutations encoding p53D36, p53D45, or p53D52 and found that only the latter two mutations led to measurable decreases in p53 mRNAs and activity (Figures S7A–S7G). Strikingly, the nonsense mutations affecting human TP53 exon 10 that were reported in cancers also cause the loss of at least 45 residues, suggesting that our finding in mice might be relevant to humans (Figure S7H). Given that decreased dyskerin levels may cause short telomeres (Parry et al., 2011a), that Rtel is a major determinant of telomere length (Ding et al., 2004), and that mutations in DKC1 or RTEL1 are found in a significant fraction of patients with DC or HHS (Armanios and Blackburn, 2012; Ballew et al., 2013; Walne et al., 2013), it was important to test whether or not p53 downregulates these genes in human cells. We compared human primary WT cells with p53-deficient cells and observed that the activation of human p53 also leads to the downregulation of DKC1 and RTEL1 (Figures 6L and 6M). Our finding that p53 downregulates these genes in both species strengthens the notion that p53 plays a significant role in the regulation of telomere metabolism. Interestingly, mice in this study were of mixed genetic back- ground (75% C57Bl/6J; 25% 129S2/SvPas). The coat color of inbred C57Bl/6J (B6) mice is black, whereas that of 129S2/ SvPas (129) is agouti because these strains carry nonagouti (a) or agouti (Aw) alleles, respectively. We noticed that most p53D31/D31 mice alive after 3 months (Figure 3A) had an agouti coat color and, conversely, that the few p53+/D31 mice that died in less than a year (Figure 5A) had a black coat color. These observations suggested that a gene physically linked to the Aw/a locus had an impact on the survival of p53D31 mutants and that the 129 allele for this gene correlated with a better survival. p53 Activation Alters the Expression of Genes Mutated in Telomere Syndromes (E) WT and p53D31/D31 (D31/D31) MEFs untreated (Unt) or treated with 0.5 mg/ml Adriamycin (Adr) for 24 hr were stained with a p53 antibody, and their DNA was counterstained with DAPI. (l d ti d t ) Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2049 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2049 We next aimed to determine how p53 downregulates the Dkc1, Rtel1, Tinf2, and Terf1 genes. Because p21 levels are known to contribute to the p53-mediated downregulation of many genes (Lo¨ hr et al., 2003), we tested if p53 could downregu- late these genes in p21/ MEFs. We found that p21 is required for the p53-mediated downregulation of Rtel1, Tinf2, and Terf1, but not Dkc1 (Figure 6H). This suggested a more direct mecha- nism for the downregulation of Dkc1. Consistent with this, Dkc1 was efficiently downregulated after treating WT cells with Nutlin for only 6 hr, whereas for the other genes, we observed a partial downregulation after 6–9 hr of Nutlin and a more efficient down- regulation after 24 hr of Nutlin (Figure 6I). Furthermore, chro- matin immunoprecipitation (ChIP) experiments provided direct evidence that p53 binds the Dkc1 promoter (Figures 6J–6K). be sufficient to cause bone marrow failure but also typical DC features including short telomeres and oral leukoplakia (Table 1). Laboratory mice have long telomeres, and mice that lack telo- merase exhibit short telomeres only after several generations of intracrosses (Blasco et al., 1997). However, mice with a telome- rase haploinsufficiency and a deficient shelterin complex exhibit telomere dysfunction in a single generation (G1) (Hockemeyer et al., 2008; He et al., 2009). The short telomeres in G1 p53D31/D31 mice thus suggested a pleiotropic effect of p53 on telomere metabolism. Although p53 may regulate TRF2 via the ubiquitin ligase Siah1 (Fujita et al., 2010), a link between p53 and genes mutated in DC appeared most likely for TCAB1/WRAP53 because this gene partially overlaps TP53 (Zhong et al., 2011). However, we found no evidence of decreased Tcab1 expression in p53D31/D31 cells (Figure 6A). We next quantified the mRNAs for nine other genes known to cause DC or implicated in aplastic anemia in p53/, WT, and p53D31/D31 cells and obtained evidence of modest but significant decreases in the expression of Dyskerin (Dkc1), Rtel1, Tinf2, and Terf1 in p53D31/D31 cells (Figures 6B and 6C). p53 Activation Alters the Expression of Genes Mutated in Telomere Syndromes Rtel1 appeared as a candidate gene because it is a dominant- positive regulator of telomere length (Ding et al., 2004) that maps 26 cM away from the Aw/a locus. We used a SNP within the Rtel1 intron 15 (rs33116597) that differs between the B6 and 129 strains to genotype Rtel1 alleles in a cohort of 52 p53D31/D31 mice and found a significant increase in survival for p53D31/D31 mice carrying 129 Rtel1 allele(s) (Figures 6E and 6F). Importantly, when we quantified Rtel1 mRNA levels in BMCs of the C57Bl/6J and 129S2/SvPas pure inbred strains, we found increased levels in 129 cells (Figure 6G). Together, these results suggest that basal levels of Rtel1 mRNAs may affect the severity of phenotypes caused by the p53D31 mutation. (F) Protein extracts, prepared from indicated MEFs untreated or treated as in (E), were immunoblotted with antibodies against Mdm2, p53, p21, and actin. Band quantification revealed that unstressed p53D31/D31 MEFs contained three to three and a half times more p21 or Mdm2 proteins than unstressed WT cells. (G) p53D31/D31 MEFs were untreated or treated with 10 mM Nutlin for 24 hr before protein extraction. Mean + SEM from three or more experiments are shown in all figures. ***p % 0.001; **p % 0.01; *p % 0.05; n.s., not significant by Student’s t test. See also Figure S2 A Targeted Deletion of the p53 CTD Leads to Increased p53 Activity Hemograms from p53D31/D31 and WT mice. WBC, white blood cells; RBC, red blood cells; PLT, platelets. All mice were 3–4 we as determined except for mouse D31/D31 #5 moribund at 19 days which was analyzed and euthanized on the same day was determined, except for mouse D31/D31 #5, moribund at 19 days, which was analyzed and euthanized on the same day. (F) Mutant mice present enlarged hearts upon dissection. Top view shows example of hearts from WT and p53D31/D31 P18 littermates. Bottom view illustrates heart/total body weight ratios determined for nine WT and ten p53D31/D31 age-matched animals. 31/ 31 , p / , y , y y Mutant mice present enlarged hearts upon dissection. Top view shows example of hearts from WT and p53D31/D31 P18 litterm art/total body weight ratios determined for nine WT and ten p53D31/D31 age-matched animals. (F) Mutant mice present enlarged hearts upon dissection. Top view shows example of hearts from WT and p53D31/D31 P18 littermates. Bottom view illustrates heart/total body weight ratios determined for nine WT and ten p53D31/D31 age-matched animals. D31/D31 (H) BMCs, stained with antibodies against hematopoietic lineage (Lin) markers and cell surface marks Sca1 and c-Kit, were analyzed by flow cytometry. The population of Lin Sca1+ c-Kit+ (LSK) cells, enriched in hematopoietic stem cells, were calculated as percentage (%) of total mononucleated cells (MNCs). Results are from three mice per genotype. p g yp (I) BMCs (1 or 2 3 106) from WT or p53D31/D31 donor mice expressing the leukocyte marker CD45.2 were transplanted into lethally irradiated recipient mice (initially expressing marker CD45.1). Transplanted WT cells, but not p53D31/D31 cells, rescued the irradiated animals by repopulating the bone marrow. Results are from five mice per genotype. BMT, bone marrow transplant. g y (I) BMCs (1 or 2 3 106) from WT or p53D31/D31 donor mice expressing the leukocyte marker CD45.2 were transplanted into lethally irradiated recipient mice (initially expressing marker CD45.1). Transplanted WT cells, but not p53D31/D31 cells, rescued the irradiated animals by repopulating the bone marrow. Results are from five mice per genotype. BMT, bone marrow transplant. (I) BMCs (1 or 2 3 106) from WT or p53D31/D31 donor mice expressing the leukocyte marker CD45.2 were transplanted into lethally irradiated recipient mice (initially expressing marker CD45.1). Transplanted WT cells, but not p53D31/D31 cells, rescued the irradiated animals by repopulating the bone marrow. Results are from five mice per genotype. A Targeted Deletion of the p53 CTD Leads to Increased p53 Activity In this report, we targeted a nonsense mutation at the murine Trp53 locus to analyze the consequences of a deletion of the (J) Cell-cycle control was analyzed in asynchronous cell populations 24 hr after 0, 3, or 12 Gy g-irradiation. ( ) y y y p p , , y g (K) p53D31 leads to a decreased proliferation capacity in a 3T3 protocol. The proliferation of p53/ (KO), WT, and p53D31 Each point is a mean value from two independent MEFs, the value for each MEF resulting from triplicate plates. p p , g p p (L) Senescence-associated b-galactosidase staining of p53/, WT, and p53D31/D31 MEFs at passage 8. 2050 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors Figure 3. Increased p53 Activity and Aplastic Anemia in p53D31/D31 Mice (A) Survival of WT, p53+/D31 (+/D31), and p53D31/D31 (D31/D31) mice over 90 days. Cohort sizes are in parentheses. (B) Examples of 28-day-old (P28) WT, p53+/D31, and p53D31/D31 littermates. (C) Photographs of legs and tails of the same mice. Compared to the WT, the heterozygote has a slightly darker pigmentation of the palm and tail. The homozygote mutant has a much darker pigmentation which includes footpads Figure 3. Increased p53 Activity and Aplastic Anemia in p53D31/D31 Mice (A) Survival of WT, p53+/D31 (+/D31), and p53D31/D31 (D31/D31) mice over 90 days. Cohort sizes are in parentheses. (B) Examples of 28-day-old (P28) WT, p53+/D31, and p53D31/D31 littermates. Figure 3. Increased p53 Activity and Aplastic Anemia in p53D31/D31 Mice g p y p p (A) Survival of WT, p53+/D31 (+/D31), and p53D31/D31 (D31/D31) mice over 90 days. Cohort sizes are in parentheses. (B) Examples of 28-day-old (P28) WT, p53+/D31, and p53D31/D31 littermates. ( ) p y ( ) p p (C) Photographs of legs and tails of the same mice. Compared to the WT, the heterozygote has a slightly darker pigmentation of mutant has a much darker pigmentation, which includes footpads. (D) Comparative analysis of hematoxylin and eosin staining (H&E) of the thymus of WT and mutant P23 littermates, showing an increased spontaneous apoptosis in the mutant. D31/D31 ( ) p p (E) Hemograms from p53D31/D31 and WT mice. WBC, white blood cells; RBC, red blood cells; PLT, platelets. All mice were 3–4 weeks old when their hemogram was determined, except for mouse D31/D31 #5, moribund at 19 days, which was analyzed and euthanized on the same day. A Targeted Deletion of the p53 CTD Leads to Increased p53 Activity BMT, bone marrow transplant. Mean + SEM are shown in (F) and (H). *** p % 0.001, Student’s t test. See also Figures S3 and S4. Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2051 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2051 Figure 4. p53D31/D31 Mice Exhibit Features Specific to DC (A and B) Pulmonary fibrosis in p53D31/D31 mice. Masson’s trichrome staining of lungs from WT and p53D31/D31 mice, shown at different magnifications. Interstitial fibrosis, characterized by deposits of collagen (stained in green), is increased in the mutant. (C and D) p53D31/D31 cells have short telomeres. In (C), telomere length was analyzed in BMCs using flow-FISH with a telomere-specific PNA probe. On top, typical results with P18 littermate mice are shown (>2,100 cells per sample, from a WT and a p53D31/D31 mouse). For each animal, histograms on the right show green fluorescence (Fluo.), with black histograms for cells without the probe (measuring cellular autofluorescence), and green histograms for cells with the probe. The shift in fluorescence intensity is smaller in mutant cells (b < a), indicating reduced telomere length. Histograms on the left show propidium iodide fluorescence (superposed for cells with or without the probe). Bottom row presents results from four animals per genotype. Mean + SEM are shown. ***p % 0.001, Student’s (legend continued on next page) 2052 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors Figure 4. p53D31/D31 Mice Exhibit Features Specific to DC Figure 4. p53 Mice Exhibit Features Specific to DC (A and B) Pulmonary fibrosis in p53D31/D31 mice. Masson’s trichrome staining of lungs from WT and p53D31/D31 mice, shown at different magnifications. Interstitial fibrosis, characterized by deposits of collagen (stained in green), is increased in the mutant. (C and D) p53D31/D31 cells have short telomeres. In (C), telomere length was analyzed in BMCs using flow-FISH with a telomere-specific PNA probe. On top, typical results with P18 littermate mice are shown (>2,100 cells per sample, from a WT and a p53D31/D31 mouse). For each animal, histograms on the right show green fluorescence (Fluo.), with black histograms for cells without the probe (measuring cellular autofluorescence), and green histograms for cells with the probe. The shift in fluorescence intensity is smaller in mutant cells (b < a), indicating reduced telomere length. Histograms on the left show propidium iodide fluorescence (superposed for cells with or without the probe). p Syndromes Germline missense TP53 mutations affecting the core DNA- binding domain are well known to cause the cancer-prone Li- Fraumeni syndrome in humans (Malkin et al., 1990; Srivastava et al., 1990), and mice with equivalent p53 mutations may, like patients with Li-Fraumeni, develop osteosarcomas (Lang et al., 2004; Olive et al., 2004). In striking contrast, we found here that mice homozygous for a germline nonsense mutation affecting the p53 CTD, or compound heterozygotes with a Mdm4 haploinsufficiency, are remarkable models of human telo- mere syndromes. In murine cells, p53 activation led to the down- regulation of four genes including Dyskerin (Dkc1), a gene often mutated in patients with DC (Armanios and Blackburn, 2012). Importantly, in addition to its role as a telomerase component, dyskerin acts as a pseudouridine synthase in snoRNP com- plexes. This might explain why patients with DC carrying DKC1 mutations exhibit more clinical features than patients carrying TERC mutations (Vulliamy et al., 2011). Similarly, some of the DC features observed in p53D31/D31 mice might result from p53 activation or Dkc1 downregulation, rather than telomere dysfunction per se. However, decreased telomere length likely played a role in the premature death of p53D31/D31 mice because we found strain-specific differences in Rtel1 mRNA levels to correlate with differences in the survival of mutant mice. Figure 5. p53+/D31 Mice Are Extremely Sensitive to Mdm4 Levels (A) Survival of WT (+/+) and p53+/D31 (+/D31) mice over 1 year, plotted as in Figure 3A. (B) Survival of p53+/D31, Mdm2+/ (M2+/), Mdm4+/ (M4+/), p53+/D31 Mdm2+/ (+/D31 M2+/), and p53+/D31 Mdm4+/ (+/D31 M4+/) mice over 3 months. (C) H&E of sternum sections from p53+/D31 and p53+/D31 Mdm4+/ littermates. (D) Telomere length was measured by flow-FISH as described in Figure 4C, in BMCs from WT, p53+/D31, Mdm4+/, and p53+/D31 Mdm4+/ mice. Results are from three animals per genotype. Mean + SEM are shown. ***p % 0.001, Student’s t test. See also Figure S6. p g yp Mean + SEM are shown. ***p % 0.001, Student’s t test. See also Figure S6. p53 C-terminal domain ex vivo and in vivo. The mutation led to increased p53D31 levels, consistent with in vitro studies that concluded that the p53 CTD is required for an optimal interaction between p53 and its ubiquitin ligase Mdm2 (Poyurovsky et al., 2010). A Targeted Deletion of the p53 CTD Leads to Increased p53 Activity Bottom row presents results from four animals per genotype. Mean + SEM are shown. ***p % 0.001, Student’s (legend continued on next page) 2052 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2052 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors Figure 5. p53+/D31 Mice Are Extremely Sensitive to Mdm4 Levels (A) Survival of WT (+/+) and p53+/D31 (+/D31) mice over 1 year, plotted as in Figure 3A. (B) Survival of p53+/D31, Mdm2+/ (M2+/), Mdm4+/ (M4+/), p53+/D31 Mdm2+/ (+/D31 M2+/), and p53+/D31 Mdm4+/ (+/D31 M4+/) mice over 3 months. (C) H&E of sternum sections from p53+/D31 and p53+/D31 Mdm4+/ littermates. (D) Telomere length was measured by flow-FISH as described in Figure 4C, in BMCs from WT, p53+/D31, Mdm4+/, and p53+/D31 Mdm4+/ mice. Results are from three animals per genotype. Mean + SEM are shown. ***p % 0.001, Student’s t test. See also Figure S6. sumably, the deletion of the p53 CTD might activate transcription by affecting p53-Mdm2 interactions (Poyurovsky et al., 2010) and/or by preventing the formation of p53 CTD-Mediator inactive complexes (Meyer et al., 2010). Our observation that a p53 mutant lacking the CTD exhibits increased activity may seem surprising when compared to recent studies relying on ectopically expressed human p53 CTD mutants, which concluded that the CTD mainly acts as a positive regulator of p53 functions (Hamard et al., 2012; Kim et al., 2012). Importantly, however, these studies analyzed WT and mutant p53 expressed at equal levels, whereas we showed here that a targeted CTD deletion leads to mutant protein accu- mulation. In that respect, it is worth considering that despite its increased nuclear abundance, p53D31 did not exhibit an increased binding at p53 target gene promoters. This may sug- gest that, on a per molecule basis, p53D31 is less efficient than WT p53 for sequence-specific DNA binding, consistent with other studies (McKinney et al., 2004; Tafvizi et al., 2011). There- fore, it appears likely that if p53D31 had been expressed at similar levels than WT p53 in the mutant cells, the overall increase in p53 activity would not have been observed. Our observations thus suggest that differences in expression levels might underlie much of the contradictions in the p53 CTD literature. t test. In (D), telomere length was analyzed in WT or p53D31/D31 MEFs at passage 5 by quantitative FISH with a telomere-specific Cy3 PNA probe (in red). On top, typical metaphases are shown. In the mutant cells, several chromosomes exhibit faint or no visible telomeric signals. Below, quantification results from an analysis of 28 metaphases per genotype are shown. (E and F) p53D31/D31 mice exhibit the triad of physical traits classically used to diagnose DC. (E) Cutaneous hyperpigmentation and nail dystrophy on a p53D31/D31 hindleg. Arrow points to the dystrophic nail. (F) H&E comparison of the dorsal surface of a WT and a p53D31/D31 tongue. The mutant tongue exhibits acanthosis and hyperparakeratosis, typical of oral leukoplakia. See also Figure S5. ( ) yp p g y p y p g p y p (F) H&E comparison of the dorsal surface of a WT and a p53D31/D31 tongue. The mutant tongue exhibits acanthosis and hyperparakeratosis, typical of oral leukoplakia. t test. In (D), telomere length was analyzed in WT or p53D31/D31 MEFs at passage 5 by quantitative FISH with a telomere-specific Cy3 PNA probe (in red). On top, typical metaphases are shown. In the mutant cells, several chromosomes exhibit faint or no visible telomeric signals. Below, quantification results from an analysis of 28 metaphases per genotype are shown. Quantitative RT-PCR Total RNA, extracted using NucleoSpin RNA II (Macherey-Nagel), was reverse transcribed using SuperScript III (Invitrogen). Real-time quantitative PCRs (primers available upon request) were performed on an ABI PRISM 7500 using Power SYBR Green (Applied Biosystems). For strain-specific Rtel1 expression analysis, total mRNAs were extracted from the BMCs of inbred C57Bl/6J and 129S2/SvPas mice (Charles River Laboratories), and Rtel1 mRNAs were quan- tified as above. p Syndromes Cells expressing p53D31 also exhibited increased p53 ac- tivity, despite the fact that the binding of the mutant protein to target gene promoters was not significantly increased. Thus, as earlier observations suggested (Kaeser and Iggo, 2002; Espi- nosa, 2008), the induction of a p53 target gene is not simply determined by the amount of p53 bound to its promoter. Pre- t test. In (D), telomere length was analyzed in WT or p53D31/D31 MEFs at passage 5 by quantitative FISH with a telomere-specific Cy3 PNA probe (in red). On top, typical metaphases are shown. In the mutant cells, several chromosomes exhibit faint or no visible telomeric signals. Below, quantification results from an analysis of 28 metaphases per genotype are shown. ( ) yp p g y p y p g p y p (F) H&E comparison of the dorsal surface of a WT and a p53D31/D31 tongue. The mutant tongue exhibits acanthosis and hyperparakeratosis, typical of oral leukoplakia. See also Figure S5. Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2053 Table 1. Features of DC in p53D31/D31 Mice and in Other Reported Mice with Altered p53 Regulation or Dysfunctional Telomeres Clinical Features in Patients Diagnosed with DC Observed in p53D31/D31 Mice % Animals with Feature (No. Analyzed) Observed in Other Mice with Altered p53 Regulationa Observed in Mouse Models of DCb Physical Traits (Diagnostic Triad)c Skin hyperpigmentation ✔ ✔100% (25) ✔ ✔ ✔ ✔ Nail dystrophy ✔ ✔8% (25) Not reported ✔ ✔ Oral leukoplakia ✔ ✔100% (8) Not reported Not reported Pathological Traitsd Bone marrow failure ✔ ✔100% (8) ✔ ✔ ✔ ✔ Pulmonary fibrosis ✔ ✔87% (8) Not reported Not reported Molecular Featuree Poor telomere maintenance ✔ ✔100% (4) Not reported ✔ ✔ Associated Featuresf Hypertrophic heart ✔ ✔100% (25) ✔ Not reported Short stature ✔ ✔63% (25) ✔ ✔ Testicular atrophy ✔ ✔91% (11) ✔ ✔ Cerebellar hypoplasia ✔ ✔30% (10) ✔ Not reported The features used to diagnose DC are indicated in bold. p53+/D31 Mdm4+/ mice, not included in this table, exhibited the same features as p53D31/D31 mice. Likewise, p53D31/D31 MEFs exhibited short/dysfunctional telomeres but were not included in this table. p Syndromes bPreviously reported mouse models of DC combined a Pot1b deficiency with a telomerase RNA haploinsufficiency (Hockemeyer et al., 2008; He et al., 2009). cDC is classically diagnosed when at least two of these physical features are observed. dInherited bone marrow failure is found in other syndromes (e.g., Blackfan-Diamond anemia, Fanconi anemia), but the combination of bone marrow failure and pulmonary fibrosis characterizes DC (Parry et al., 2011b). eTelomeres are often analyzed to diagnose DC. fThese features can be found in patients with DC but are not specific enough for diagnosis. atures of DC in p53D31/D31 Mice and in Other Reported Mice with Altered p53 Regulation or Dysfunctiona The features used to diagnose DC are indicated in bold. p53+/D31 Mdm4+/ mice, not included in this table, exhibited the same features as p53D31/D31 mice. Likewise, p53D31/D31 MEFs exhibited short/dysfunctional telomeres but were not included in this table. aMice with altered p53 regulation include mutants with severe decreases in Mdm2 levels (Mendrysa et al., 2003; Liu et al., 2007), combined Mdm2 and Mdm4 haploinsufficiencies (Terzian et al., 2007), complex p53 mutations (McGowan et al., 2008; Liu et al., 2010; Wang et al., 2011a), or constitutive cellular stress (McGowan et al., 2008). bPreviously reported mouse models of DC combined a Pot1b deficiency with a telomerase RNA haploinsufficiency (Hockemeyer et al., 2008; He et al., 2009). cDC is classically diagnosed when at least two of these physical features are observed. dInherited bone marrow failure is found in other syndromes (e.g., Blackfan-Diamond anemia, Fanconi anemia), but the combination of bone marrow failure and pulmonary fibrosis characterizes DC (Parry et al., 2011b). eTelomeres are often analyzed to diagnose DC. fThese features can be found in patients with DC but are not specific enough for diagnosis. Cells and Cell Culture Reagents MEFs isolated from 13.5-day embryos were cultured in a 5% CO2 and 3% O2 incubator, in DMEM GlutaMAX (Gibco), with 15% FBS (Biowest), 100 mM 2- mercaptoethanol (Millipore), 0.01 mM Non-Essential Amino-Acids, and peni- cillin/streptavidin (Gibco) for six or less passages, except for 3T3 experiments, performed in a 5% CO2 incubator for eight passages. Human lung fibroblast MRC5 and its SV40-transformed derivatives were cultured in a 5% CO2 and 3% O2-regulated incubator in MEM (Gibco), completed with 10% FBS, 2 mM L-glutamine (Gibco), 1 mM pyruvate, 10 mM Non-Essential Amino-Acids, and penicillin/streptomycin. Cells were irradiated with a Cs g-irradiator or treated with Adriamycin (Sigma-Aldrich) at 0.5 mg/ml or 10 mM Nutlin 3a (Sigma-Aldrich). An important remaining question is whether germline p53 mu- tations affecting the CTD may lead to telomere syndromes in hu- mans. The high degree of conservation between the human and murine p53 CTDs, the comparison of phenotypes caused by nonsense mutations in the exon 10 of the human and mouse p53 genes, and the fact that p53 activation also leads to the downregulation of DKC1 and RTEL1 in human cells together support this possibility. Importantly, genes presently known to cause DC account for about only half of the clinical cases, and patients with DC are now thought to represent a small fraction of persons suffering from a telomere syndrome (Armanios and Blackburn, 2012). Although one should be careful about extrap- olating mouse data to human diseases, our results lead us to propose that TP53 (and possibly MDM4) should be sequenced in patients with telomere syndromes of currently unknown mo- lecular origin. EXPERIMENTAL PROCEDURES Importantly, p53+/D31 mice were mildly affected: they pre- sented only slight alterations (if any) in HSC pools and telomere length, and most were alive for more than 12 months. Because p53D31/D31 and p53+/D31 Mdm4+/ mice developed full-blown DC features, we conclude that a %2-fold difference in p53 activ- ity was sufficient to prevent the observation of DC features in het- erozygous mice. This observation might explain why mouse models with modest increases in p53 activity were only found to present slight alterations in HSC pools (e.g., Herrera-Merchan et al., 2010). p Syndromes aMice with altered p53 regulation include mutants with severe decreases in Mdm2 levels (Mendrysa et al., 2003; Liu et al., 2007), combined Mdm2 and Mdm4 haploinsufficiencies (Terzian et al., 2007), complex p53 mutations (McGowan et al., 2008; Liu et al., 2010; Wang et al., 2011a), or constitutive cellular stress (McGowan et al., 2008). bPreviously reported mouse models of DC combined a Pot1b deficiency with a telomerase RNA haploinsufficiency (Hockemeyer et al., 2008; He et al., 2009). cDC is classically diagnosed when at least two of these physical features are observed. dInherited bone marrow failure is found in other syndromes (e.g., Blackfan-Diamond anemia, Fanconi anemia), but the combination of bone marrow failure and pulmonary fibrosis characterizes DC (Parry et al., 2011b). eTelomeres are often analyzed to diagnose DC. fThese features can be found in patients with DC but are not specific enough for diagnosis. Table 1. Features of DC in p53D31/D31 Mice and in Other Reported Mice with Altered p53 Regulation or Dysfunctional Telomeres Clinical Features in Patients Diagnosed with DC Observed in p53D31/D31 Mice % Animals with Feature (No. Analyzed) Observed in Other Mice with Altered p53 Regulationa Observed in Mouse Models of DCb Physical Traits (Diagnostic Triad)c Skin hyperpigmentation ✔ ✔100% (25) ✔ ✔ ✔ ✔ Nail dystrophy ✔ ✔8% (25) Not reported ✔ ✔ Oral leukoplakia ✔ ✔100% (8) Not reported Not reported Pathological Traitsd Bone marrow failure ✔ ✔100% (8) ✔ ✔ ✔ ✔ Pulmonary fibrosis ✔ ✔87% (8) Not reported Not reported Molecular Featuree Poor telomere maintenance ✔ ✔100% (4) Not reported ✔ ✔ Associated Featuresf Hypertrophic heart ✔ ✔100% (25) ✔ Not reported Short stature ✔ ✔63% (25) ✔ ✔ Testicular atrophy ✔ ✔91% (11) ✔ ✔ Cerebellar hypoplasia ✔ ✔30% (10) ✔ Not reported The features used to diagnose DC are indicated in bold. p53+/D31 Mdm4+/ mice, not included in this table, exhibited the same features as p53D31/D31 mice. Likewise, p53D31/D31 MEFs exhibited short/dysfunctional telomeres but were not included in this table. aMice with altered p53 regulation include mutants with severe decreases in Mdm2 levels (Mendrysa et al., 2003; Liu et al., 2007), combined Mdm2 and Mdm4 haploinsufficiencies (Terzian et al., 2007), complex p53 mutations (McGowan et al., 2008; Liu et al., 2010; Wang et al., 2011a), or constitutive cellular stress (McGowan et al., 2008). Immunofluorescence MEFs were cultured on collagen-coated coverslips, exposed to Adriamycin, and analyzed 24 hr later. Coverslips were stained with the p53 antibody CM- 5 (Novocastra) and secondary Alexa Fluor 488 anti-mouse antibody (Molecular Probes). Images were captured on an epifluorescence microscope using equal exposure times for all images for each fluor. Anatomopathology g y g val of p53D31/D31 mice taking Rtel1 genotyping into account. Cohort sizes are in parentheses. The p value is from a log rank tes i f Rt l1 RNA l l i th BMC f 129S2/S P (129) d C57Bl/6J (B6) i R lt f f i t i ( ) p g g yp g p p g (G) Comparison of Rtel1 mRNA levels in the BMCs of 129S2/SvPas (129) and C57Bl/6J (B6) mice. Results are from four mice per strain. (H) p21 is required for the downregulation of Rtel1, Tinf2, and Terf1. RNAs, prepared from WT and p21/ (p21) MEFs, untreated or treated with 10 mM Nutlin for g g y g g (G) Comparison of Rtel1 mRNA levels in the BMCs of 129S2/SvPas (129) and C57Bl/6J (B6) mice. Results are from four mice per strain. (G) Comparison of Rtel1 mRNA levels in the BMCs of 129S2/SvPas (129) and C57Bl/6J (B6) mice. Results are from four mice per strain. (H) p21 is required for the downregulation of Rtel1 Tinf2 and Terf1 RNAs prepared from WT and p21/ (p21 ) MEFs untreated or treated with 10 M Nutlin for p21 is required for the downregulation of Rtel1, Tinf2, and Terf1. RNAs, prepared from WT and p21/ (p21) MEFs, untreated or t r, were used to quantify Dkc1, Rtel1, Tinf2, and Terf1 mRNAs. Results are from three independent experiments. (I) p53 activation rapidly leads to the efficient downregulation of Dkc1. mRNAs for Dkc1, Rtel1, Tinf2, and Terf1 were quantified in WT MEFs, untreated or treated with 10 mM Nutlin for 6, 9, 12, or 24 hr. Results are from two independent experiments. (I) p53 activation rapidly leads to the efficient downregulation of Dkc1. mRNAs for Dkc1, Rtel1, Tinf2, and Terf1 were quantified in WT MEFs, untreated or treated with 10 mM Nutlin for 6, 9, 12, or 24 hr. Results are from two independent experiments. (J d K) 53 l t th Dk 1 t (J) ChIP assay was performed at ten different sites surrounding the Dkc1 mRNA transcription start site (TSS) in Adriamycin-treated WT and p53/ (KO) MEFs by using a polyclonal antibody against p53 (FL-393) or rabbit IgG as a negative control. Immunoprecipitates were quantified using real-time PCR. Fold enrichments were normalized to data over an irrelevant region. Indicated values are means from two independent ChIP experiments, each quantified in triplicates. LSL-D31 Construct We used mouse genomic p53 DNA from previous constructs (Toledo et al., 2006a, 2006b) and a portion of intron 1 containing a LoxP-Stop-LoxP (LSL) cassette (Ventura et al., 2007) in which the puromycin-resistance gene was re- placed by a neomycin gene (details available upon request). A BsrG I site (in intron 9) and a Fse I site (inserted downstream Trp53) were used to swap 2054 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors (legend on n (legend on next page) Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2055 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors 2055 the nonsense mutation from the p53D31 RMCE-ASAP construct (Extended Experimental Procedures) in the LSL-targeting construct. The resulting LSL- p53D31-targeting vector was fully sequenced before use. ChIP Assay ChIP analysis was performed as described (Simeonova et al., 2012). p53-DNA complexes were immunoprecipitated from total extracts by using 5 mg of an antibody against mouse Phospho-p53 Ser18 (Cell Signaling Technology) and 15–30 mg of sonicated chromatin, or 50 mg of a polyclonal antibody against p53 (FL-393) and 300 mg of sonicated chromatin. Rabbit IgG (Abcam) was used for control precipitation. Quantitative PCR was performed on ABI PRISM 7500. treated with HTN-Cre showed that the mutant cDNA differed from a p53WT sequence only by the engineered nonsense mutation. Primers for Trp53 and Rtel1 genotyping are available upon request. All experiments were performed according to IACUC regulations. Western Blots Protein detection by immunoblotting was performed using antibodies against p53 (CM-5, Novocastra; or FL-393, Santa Cruz Biotechnology), actin (A2066; Sigma-Aldrich), p21 (F5; Santa Cruz Biotechnology), and Mdm2 (4B2), Rtel1 (MP1), and Dyskerin (H-300; Santa Cruz Biotechnology). Chemiluminescence revelation was achieved with SuperSignal West Dura (Perbio). Bands of inter- est were quantified by using ImageJ and normalized with actin. LSL Cassette Excision Triplicates of 1.8 3 105 p53LSL-WT/LSL-WT (Ventura et al., 2007) and p53LSL-D31/LSL-D31 MEFs were seeded in wells of a 6-well plate and treated with 10 mM HTN-Cre for ex vivo excision of the LSL cassette or 105 cells with buffer only. After 24 hr, wells were washed, and cells were frozen when reaching 90% confluence. Cells were then thawed for DNA and RNA extrac- tions to determine excision efficiency and for mRNA quantifications. In vivo LSL excision was performed by breeding with PGK-Cre mice. Cell-Cycle Assays Log phase cells were irradiated at RT with a Cs g-irradiator at doses of 3 or 12 Gy, incubated for 24 hr, then pulse labeled for 1 hr with BrdU (10 mM), fixed in 70% ethanol, double stained with FITC anti-BrdU and propidium iodide, and sorted by using a BD Biosciences FACSort. Data were analyzed using FlowJo. Targeting in ES Cells and Genotyping CK-35 ES cells were electroporated with the targeting construct linearized with Not I. Two independent recombinant clones, identified by long-range PCR and confirmed by Southern blot and PCR, were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping MEFs from their offspring. RT-PCR of RNAs from p53LSL-D31/LSL-D31 MEFs treated with HTN-Cre showed that the mutant cDNA differed from a p53WT sequence only by the engineered nonsense mutation. Primers for Trp53 and Rtel1 genotyping are available upon request. All experiments were performed according to IACUC regulations. CK-35 ES cells were electroporated with the targeting construct linearized with Not I. Two independent recombinant clones, identified by long-range PCR and confirmed by Southern blot and PCR, were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping MEFs from their offspring. RT-PCR of RNAs from p53LSL-D31/LSL-D31 MEFs WT Anatomopathology Organs were fixed in formol 4% for 24 hr, then ethanol 70%, and embedded in paraffin wax. Serial sections were stained with hematoxylin and eosin or Mas- son’s trichrome using standard procedures (Prophet et al., 1992). Figure 6. p53 Activation Leads to the Downregulation of Genes Involved in Telomere Metabolism / / / (A) Tcab1 mRNA levels are not altered in p53D31/D31 cells. RNAs, prepared from p53/ (KO), WT, and p53D31/D31 (D31) MEFs, untreated or treated with 10 mM Nutlin for 24 hr, were used to quantify Tcab1/Wrap53 mRNAs. Results are from four independent experiments. (A) Tcab1 mRNA levels are not altered in p53D31/D31 cells. RNAs, prepared from p53/ (KO), WT, and p53D31/D31 (D31) ME Nutlin for 24 hr, were used to quantify Tcab1/Wrap53 mRNAs. Results are from four independent experiments. (B) Evidence of decreased mRNA levels for Dkc1, Rtel1, Tinf2, and Terf1 in unstressed p53D31/D31 cells. RNAs, prepared from unstressed p53/, WT, and p53D31/D31 MEFs, were used to compare the expression of eight genes known to cause DC (Ctc1, Dkc1, Nhp2, Nop10, Rtel1, Terc, Tert, and Tinf2) and one gene (Terf1) that has been implicated in aplastic anemia, a milder form of telomere syndrome (Armanios and Blackburn, 2012). Results from four independent ex- periments were analyzed by one-way ANOVA. (C) p53D31/D31 cells contain decreased levels of the dyskerin and Rtel proteins. Protein extracts, prepared from WT and p53D31/D31 MEFs, were immunoblotted with antibodies against Rtel, dyskerin (dysk), and actin. (D) Dkc1, Rtel1, Tinf2, and Terf1 are downregulated by murine p53. mRNAs for Dkc1, Rtel1, Tinf2, and Terf1 were quantified in p5 untreated or treated with 10 mM Nutlin for 24 hr. Results are from four independent experiments. p = 0.0545. 31/ 31 f1 are downregulated by murine p53. mRNAs for Dkc1, Rtel1, Tinf2, and Terf1 were quantified in p53/, WT, and p53D31/D31 MEF mM Nutlin for 24 hr. Results are from four independent experiments. p = 0.0545. –G) Improved survival of p53D31/D31 mice carrying 129S2/SvPas Rtel1 allele(s). ( ) p p y g ( ) (E) DNA was extracted from 52 p53D31/D31 mice of mixed genetic background, then PCR was performed with primers flanking the SNP rs33116597, and products were digested with EcoNI to genotype 129S2/SvPas (129) and C57Bl/6J (B6) Rtel1 alleles because only the B6 PCR products contained an EcoNI site. A typical analysis of four mice is shown, with Rtel1 genotypes above the gel. Telomeric Flow-FISH Armanios, M., and Blackburn, E.H. (2012). The telomere syndromes. Nat. Rev. Genet. 13, 693–704. Flow-FISH was performed as described by Baerlocher et al. (2006). For each animal, the bone marrow from two tibias and two femurs was collected, red blood cells were lysed, then 2 3 106 cells were fixed in 500 ml PNA hybridization buffer (70% deionized formamide, 20 mM Tris [pH 7.4], 0.1% Blocking re- agent; Roche) and stored at 20C. Either nothing (control) or 5 ml probe stock solution was added to cells (probe stock solution: 10 mM TelC-FAM PNA probe [PANAGENE], 70% formamide, 20 mM Tris [pH 7.4]), and samples were dena- tured for 10 min at 80C before hybridization for 2 hr at RT. After three washes, cells were resuspended in PBS 13, 0.1% BSA, RNase A 1,000 U/ml, propi- dium iodide 12.5 mg/ml, and analyzed with a FACSCalibur. Baerlocher, G.M., Vulto, I., de Jong, G., and Lansdorp, P.M. (2006). Flow cy- tometry and FISH to measure the average length of telomeres (flow FISH). Nat. Protoc. 1, 2365–2376. Ballew, B.J., Yeager, M., Jacobs, K., Giri, N., Boland, J., Burdett, L., Alter, B.P., and Savage, S.A. (2013). Germline mutations of regulator of telomere elonga- tion helicase 1, RTEL1, in Dyskeratosis congenita. Hum. Genet. 132, 473–480. Blasco, M.A., Lee, H.W., Hande, M.P., Samper, E., Lansdorp, P.M., DePinho, R.A., and Greider, C.W. (1997). Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34. Anatomopathology Results suggest p53 binding near the end of exon 1 (E1) of Dkc1. (K) Data from three independent ChIP experiments focusing on the end of Dkc1 exon 1 confirm significant p53 binding. (L and M) Dyskerin and Rtel are also downregulated by p53 in human cells. (K) Data from three independent ChIP experiments focusing on the end of Dkc1 exon 1 confirm significant p53 binding. (L and M) Dyskerin and Rtel are also downregulated by p53 in human cells. (L) Protein extracts, prepared from WT MRC5 human cells (MRC) and p53-deficient SV40-MRC5 cells (SVM), were immunoblotted with antibodies against Rtel, (K) Data from three independent ChIP experiments focusing on the end of Dkc1 exon 1 confirm significant p53 binding. (L and M) Dyskerin and Rtel are also downregulated by p53 in human cells. ( ) y g y p (L) Protein extracts, prepared from WT MRC5 human cells (MRC) and p53-deficient SV40-MRC5 cells (SVM), were immunoblott dyskerin, and actin. m WT MRC5 human cells (MRC) and p53-deficient SV40-MRC5 cells (SVM), were immunoblotted with antibodies against Rtel, (M) mRNAs were prepared from SV40-MRC5 and MRC5 human cells, untreated or treated with Nutlin, then DKC1 and RTEL1 mRNAs were quantified using real- time PCR, normalized to control mRNAs, and mRNA ratios from Nutlin-treated versus untreated cells were determined. Results from three independent ex- periments. (M) mRNAs were prepared from SV40-MRC5 and MRC5 human cells, untreated or treated with Nutlin, then DKC1 and RTEL1 mRNAs were quantified using real- time PCR, normalized to control mRNAs, and mRNA ratios from Nutlin-treated versus untreated cells were determined. Results from three independent ex- periments. Mean + SEM are shown. ***p % 0.001; **p % 0.01; *p % 0.05; n.s., not significant by Student’s t test. See also Figure S7. Mean + SEM are shown. ***p % 0.001; **p % 0.01; *p % 0.05; n.s., not significant by Student’s t test. See also Figure S7. 2056 Cell Reports 3, 2046–2058, June 27, 2013 ª2013 The Authors Statistical Analyses The Student’s t test was used in all figures to analyze differences between two groups of values. In Figure 6B, differences among three groups were analyzed by one-way ANOVA. In Figure 6F, a log rank test was used to analyze survival curves. Analyses were performed by using GraphPad Prism, and values of p % 0.05 were considered significant. 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Association between brain-derived neurotrophic factor gene polymorphisms and fibromyalgia in a Korean population: a multicenter study
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Association between brain-derived neurotrophic factor gene polymorphisms and fibromyalgia in a Korean population: a multicenter study Association between brain-derived neurotrophic factor gene polymorphisms and fibromyalgia in a Korean population: a multicenter study Dong-Jin Park1, Seong-Ho Kim2, Seong-Su Nah3, Ji Hyun Lee4, Seong-Kyu Kim5, Yeon-Ah Lee6, Seung-Jae Hong6, Hyun-Sook Kim7, Hye-Soon Lee8, Hyoun Ah Kim9, Chung-Il Joung10, Sang-Hyon Kim11 and Shin-Seok Lee1* Park et al. Arthritis Research & Therapy (2018) 20:220 https://doi.org/10.1186/s13075-018-1726-5 Park et al. Arthritis Research & Therapy (2018) 20:220 https://doi.org/10.1186/s13075-018-1726-5 Open Access Abstract Background: Several lines of evidence imply that brain-derived neurotrophic factor (BDNF) is involved in the pathophysiology of fibromyalgia (FM); in this regard, patients with FM have altered blood and cerebrospinal fluid levels of BDNF. In this study, we explored the association between BDNF gene polymorphisms and FM susceptibility and the severity of symptoms. Methods: In total, 409 patients with FM and 423 healthy controls in 10 medical centers were enrolled from the Korean nationwide FM survey. The alleles and genotypes at 10 positions in the BDNF gene were genotyped. Results: The allele and genotype frequencies of BDNF rs11030104 differed significantly between the patients with FM and the controls (P = 0.031). The GG genotype of rs11030104 had a protective effect against FM (P = 0.016), and the G allele of rs11030104 was negatively associated with the presence of FM compared with the A allele (P = 0. 013). In comparison, although the allele and genotype frequencies of BDNF rs12273539 did not differ between the two groups, the TT genotype of BDNF rs12273539 was associated with susceptibility to FM (P = 0.038). Haplotype analyses implied that some BDNF haplotypes have a protective effect against FM. Finally, several genotypes and haplotypes of the BDNF gene contributed to specific symptoms of FM. Conclusions: This study is the first to evaluate the associations between BDNF gene polymorphisms and FM. Our results imply that some BDNF single-nucleotide polymorphisms and haplotypes are associated with susceptibility to, and contribute to the symptoms of, FM. Keywords: Fibromyalgia, Brain-derived neurotrophic factor, Genetics, Polymorphism Keywords: Fibromyalgia, Brain-derived neurotrophic factor, Genetics, Polymorphism Background unclear, FM is recognized as an outcome of the interac- tions of multiple genetic, psychological, neurobiological, and environmental factors [3]. Fibromyalgia (FM) is a common rheumatic syndrome characterized by chronic widespread pain, and is often accompanied by diverse symptoms including fatigue, sleep disorders, memory loss, joint stiffness, and affective dis- tress [1]. The prevalence of FM in the general population is reportedly 1–5%, and it is more prevalent among women than men [2]. Although its pathogenesis is The familial aggregation observed among patients with FM implies that genetic factors are important contributors to the etiology of FM [4]. Recent genetic studies have advanced our understanding of the pathogenesis of FM. These studies have shown that certain gene polymor- phisms alter pain sensitivity and increase susceptibility to FM [5]. In particular, polymorphisms of genes involved in the pain transmission pathway, such as the serotoninergic, dopaminergic, and catecholaminergic systems, have received much attention as possible genetic factors in FM * Correspondence: shinseok@chonnam.ac.kr 1Division of Rheumatology, Department of Internal Medicine, Chonnam National University Medical School and Hospital, 42 Jebong-ro, Dong-gu, Gwangju 61469, Republic of Korea Full list of author information is available at the end of the article Park et al. Arthritis Research & Therapy (2018) 20:220 Page 2 of 11 Page 2 of 11 [6, 7]. However, those genetic factors do not fully account for the pathophysiology and symptoms of FM. Therefore, efforts to identify other genetic factors that contribute to FM are ongoing. time of recruitment. Exactly the same informed consult form (ICF) and study protocol were provided to the inde- pendent Institutional Review Board/Ethics Committee (IRB/EC) at each medical center, and each IRB/EC reviewed the appropriateness of the protocol and risks and benefits to the study participants. Ultimately, the IRB/EC at each medical center independently approved this study without revision of the ICF or study protocol. Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival, growth, and differentiation during devel- opment of the central and peripheral nervous systems [8]. BDNF is important in the transmission of physiologic or pathologic pain [9]. BDNF is responsible for modulation of nociceptive inputs and enhanced hyperalgesia by a N-methyl-D-aspartate (NMDA) receptor-mediated mech- anism [10]. Moreover, dysregulation of BDNF in the dorsal root ganglion (DRG) and spinal cord contributes to chronic pain hypersensitivity [11]. In addition, several lines of evi- dence have converged to imply that BDNF is involved in the pathophysiology of FM. Indeed, patients with FM have been shown to have altered serum and plasma levels of BDNF compared to healthy controls [12–14]. Procedures The patients with FM were interviewed at the time of en- rollment to determine their demographics and clinical characteristics, including age, sex, body mass index, and symptom and disease duration. In addition, at enrollment, peripheral venous blood was sampled and then stored in an ethylenediaminetetraacetate (EDTA)-coated tube. Ten- der points were assessed by thumb palpation according to the standardized tender point survey protocol [16]. The number of tender points was assessed at 18 sites on the body. The intensity at each tender point was assessed by determining the tender point score as follows: 0, no tender- ness; 1, light tenderness (confirming answer when asked); 2, moderate tenderness (spontaneous verbal response); and 3, severe tenderness (moving away). Therefore, the number of tender points ranged from 0 to 18, and the possible total scores of the tender points ranged from 0 to 54. Further- more, extensive clinical assessments of patients with FM enrolled in the cohort were undertaken using a self-report questionnaire and semi-structured questionnaires. The Ko- rean version of the Fibromyalgia Impact Questionnaire (FIQ) was used to assess the functional abilities and sever- ity of FM [17], and the Brief Fatigue Inventory (BFI) and the Beck Depression Inventory (BDI) were used to evaluate the severity of fatigue and depression, respectively [18, 19]. The 36-item Medical Outcomes Study Short-Form Health Survey (SF-36) was used to access the quality of life of the patients with FM [20]. In addition, we also evaluated the severity of anxiety using the State-Trait Anxiety Inventory (STAI)-I and STAI-II [21]. However, whether polymorphisms of the BDNF gene are associated with FM remains an open question. The objective of this study was to evaluate the associations between BDNF gene polymorphisms and FM susceptibil- ity and clinical symptoms, using a large population of ethnically homogenous Koreans. Study design and population We performed a multicenter, nationwide FM cohort study (the Korean Nationwide FM Survey) in the Korean popula- tion. In the Korean Nationwide FM Survey, we established a prospective cohort to evaluate the pathophysiology of FM, and the clinical manifestations and outcomes of Korean patients with FM. The study participants were recruited from the outpatient rheumatology clinics of 10 medical centers. In this study, a cross-sectional design was employed to evaluate the association between BDNF gene polymorphisms and susceptibility to, and symptom severity of, FM. As reported previously [15], we enrolled 409 patients with FM (382 women and 27 men) with a mean (SD) age of 48.1 (10.9) years. At the time of the initial diag- nosis, patients with FM were diagnosed according to the classification criteria for FM proposed by the American College of Rheumatology (ACR) in 1990 [1]. The mean (SD) symptom duration before diagnosis was 8.5 (8.3) years, and the mean (SD) disease duration after initial diag- nosis was 1.9 (3.0) years. Based on health surveys for chronic pain, we recruited 423 healthy controls (397 women, 25 men) with a mean (SD) age of 45.5 (12.5) years and no history of chronic pain, including FM. Healthy con- trols were recruited randomly, without matching for age or sex, among the individuals visiting the general health examination clinics at each medical center. This research complied with the Helsinki Declaration, and written in- formed consent was obtained from all participants at the The patients had been treated with standard medications for FM, based on the clinical judgment of their attending rheumatologist. Concomitant medications, used at the time of enrollment, included tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), pregabalin, gabapentin, nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, benzodiazepine, trama- dol, and muscle relaxants. BDNF genotypes and alleles and their association with clinical measurements Statistical analyses were performed using IBM SPSS statis- tics (SPSS version 21; IBM SPSS Inc., Chicago, IL, USA). P values <0.05 were considered to indicate statistical signifi- cance. Each BDNF gene polymorphism was tested for Hardy-Weinberg equilibrium. The genotype and haplo- type frequencies of the BDNF single-nucleotide polymor- phisms (SNPs) were compared between the patients with FM and controls by Fisher’s exact test or Pearson’s chi-squared test. The association between each BDNF genotype and haplotype and susceptibility to FM was de- fined by logistic regression analysis. Analysis of covariance The BDNF SNPs were successfully genotyped in all en- rolled subjects, except for 5 controls with BDNF rs2883187, 1 patient and 16 controls with BDNF rs7103873, 2 controls with BDNF rs7103411, 1 patient and 10 controls with BDNF rs10835210, 2 patients and 3 controls with BDNF rs11030104, 1 control with BDNF rs12273539, 1 patient and 1 control with BDNF rs11030102, 1 patient and 3 con- trols with BDNF rs11030101, 1 patient and 4 controls with BDNF rs6265, and 2 patients and 2 controls with BDNF rs7124442. The genotype distributions of the BDNF SNPs were consistent with Hardy-Weinberg equilibrium in both the patients and controls. Table 1 Primer sequences used for TaqMan probe genotyping of BDNF Table 1 Primer sequences used for TaqMan probe genotyping of BDNF Regions Primers Primer sequence (5′ →3′) rs 2883187 Forward GTGAGGCATCCGGCCCGGCTGGGGA Reverse CGGAGCGCGGTCTCGGCAGCTCCCC rs 7103873 Forward AGGACCTTTTACCCCCAAATGTAGA Reverse ACTAAATGAAAAACCATTCTTTAAA rs 7103411 Forward GGAGCGCACTGTAAAGATACTGATA Reverse GAACACGAATGTGAGATCAATGTTG rs 10835210 Forward CTTAACTGTAAAGCACAGGAAAGTG Reverse TCATTACTTGTAGCTTAATGCAGGA rs 11030104 Forward ATTAAAAAGCAGATAACACTACCAC Reverse TACTAACTGTCCTACAATTTCCTGT rs 12273539 Forward ACTCAATGCTTCATCACTTCTGCTC Reverse GATCAGGACAGAGTCCTTGGAGTGC rs 11030102 Forward CTACTTCTCAGTTCTGAGGCATGGA Reverse TTACAAAAAGACACATACATGCAAT rs 11030101 Forward GATACTCTATTATAGCAAAGAAGAA Reverse GATAATTTCATTGAGCCATCCTGTT rs 6265 Forward TCCTCATCCAACAGCTCTTCTATCA Reverse GTGTTCGAAAGTGTCAGCCAATGAT rs 7124442 Forward AAGGAAGCTGCATAAAGTTGACATA Reverse AGCAGATATTCCAAGCATTCCTTAC BDNF brain-derived neurotrophic factor p Among the BDNF SNPs, the allele and genotype fre- quencies of BDNF SNP rs11030104 were significantly different between the patients with FM and controls. Furthermore, patients with the GG genotype of rs11030104 were found less frequently in patients with FM after adjusting for age and sex (OR 0.619; 95% confi- dence interval (CI) 0.419–0.0913; P = 0.016). In addition, the G allele was negatively associated with the presence of FM compared to the A allele (OR = 0.781, 95% CI 0.641–0.950, P = 0.013). Genotyping of BDNF polymorphisms Genotyping of BDNF polymorphisms The assay reagents for rs2883187(C > T), rs7103873 (G > C), rs7103411(C > T), rs10835210(C > A), rs11030104 (A > G), rs12273539(C > T), rs11030102(C > G), rs11030101(A > T), rs6265(G > A) and rs7124442(C > T) in the BDNF gene Page 3 of 11 Page 3 of 11 Page 3 of 11 Park et al. Arthritis Research & Therapy (2018) 20:220 were designed by Applied Biosystems (Applied Biosystems). The reagents consisted of TaqMan MGB probes (FAM and VIC dye-labeled). Each reaction (10 μL) comprised 0.125 μL of 40X reagents, 5 μL of 2X TaqMan Genotyping Master Mix (Applied Biosystems) and 2 μL of 50 ng gen- omic DNA. The PCR conditions were 1 cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The PCR reactions were performed using an ABI plus instrument (Applied Biosystems). The samples were read and analyzed using ABI plus software (Applied Biosystems). The sequences of the primers used for Taq- Man probe genotyping of the BDNF gene are summarized in Table 1. were designed by Applied Biosystems (Applied Biosystems). The reagents consisted of TaqMan MGB probes (FAM and VIC dye-labeled). Each reaction (10 μL) comprised 0.125 μL of 40X reagents, 5 μL of 2X TaqMan Genotyping Master Mix (Applied Biosystems) and 2 μL of 50 ng gen- omic DNA. The PCR conditions were 1 cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The PCR reactions were performed using an ABI plus instrument (Applied Biosystems). The samples were read and analyzed using ABI plus software (Applied Biosystems). The sequences of the primers used for Taq- Man probe genotyping of the BDNF gene are summarized in Table 1. (ANCOVA), adjusted for age and sex, was used to explore the differences in the clinical measurements of the patients with FM according to BDNF genotype and haplo- type. Haplotype structures were constructed and their frequencies estimated by combined allele analysis using PHASE v2.1.1 software (Department of Statistics, Univer- sity of Washington, Seattle, WA, USA). We carried out a permutation test for the null hypothesis that the patients with FM and the healthy controls are random draws from a common set of haplotype frequencies (number of permutations performed = 10,000). BDNF brain-derived neurotrophic factor BDNF genotypes and alleles and their association with clinical measurements In comparison, although the al- lele and genotype frequencies of the SNPs of BDNF rs12273539 were not significantly different between the patients with FM and controls, the TT genotype of rs12273539 was found more frequently in patients with FM in the age-adjusted and sex-adjusted model (OR 2.586; 95% CI 1.052–6.360; P = 0.038) (Table 2). Within the FM cohort, patients with the CG genotype of BDNF rs11030102 had more severe fatigue symptoms (measured by the BFI) and anxiety symptoms (measured by the STAI-I) than did the other genotypes (P = 0.001 and P = 0.032, respectively). Furthermore, both rs11030101 and rs10835210 were associated with the trait of anxiety (measured by the STAI-II) in patients with FM (P = 0.029 and P = 0.033, respectively). No associations were observed between clinical measurements and the other BDNF SNPs (Table 3). Park et al. BDNF genotypes and alleles and their association with clinical measurements Arthritis Research & Therapy (2018) 20:220 Page 5 of 11 Table 2 Genotype and allele analyses of BDNF in patients with fibromyalgia and healthy controlsa (Continued) Marker Genotype/allele Contol, n (%) Fibromyalgia, n (%) Exact p valueb OR (95% CI), p valuec OR (95% CI), p value, adjusted by age, sex‡ A 442 (52.7) 445 (54.5) 1.075 (0.886–1.304), p = 0.466 1.058 (0.869–1.287), p = 0.575 rs7124442 C/C 2 (0.5) 0 (0) 0.574 1 1 C/T 51 (12.1) 47 (11.5) 718,117.521 (0-Inf), p = 0.972 683,123.831 (0-Inf), p = 0.972 T/T 368 (87.4) 360 (88.5) 762,294.038 (0-Inf), p = 0.971 682,163.974 (0-Inf), p = 0.972 C 55 (6.5) 47 (5.8) 0.590 1 T 787 (93.5) 767 (94.2) 1.140 (0.763–1.705), p = 0.521 1.078 (0.716–1.623), p = 0.72 BDNF brain-derived neurotrophic factor aMissing data were excluded from the analyses: BDNF rs2883187 (5 controls), BDNF rs7103873 (1 patient and 16 controls), BDNF rs7103411 (2 controls), BDNF rs10835210 (1 patient and 10 controls), BDNF rs11030104 (2 patients and 3 controls), BDNF rs12273539 (1 control), BDNF rs11030102 (1 patient and 1 control), BDNF rs11030101 (1 patient and 3 controls), BDNF rs6265 (4 controls and 1 patient), and BDNF rs7124442 (2 patients and 2 controls) bValue was determined by Fisher’s exact test or χ2 test cLogistic regression analyses were used to calculate the OR (95% CI; confidence interval) Table 2 Genotype and allele analyses of BDNF in patients with fibromyalgia and healthy controlsa (Continued Marker Genotype/allele Contol, n (%) Fibromyalgia, n (%) Exact p valueb OR (95% CI), p valuec O a otype and allele analyses of BDNF in patients with fibromyalgia and healthy controlsa (Continued) BDNF brain-derived neurotrophic factor p aMissing data were excluded from the analyses: BDNF rs2883187 (5 controls), BDNF rs7103873 (1 patient and 16 controls), BDNF rs7103411 (2 controls), BDNF rs10835210 (1 patient and 10 controls), BDNF rs11030104 (2 patients and 3 controls), BDNF rs12273539 (1 control), BDNF rs11030102 (1 patient and 1 control), BDNF rs11030101 (1 patient and 3 controls), BDNF rs6265 (4 controls and 1 patient), and BDNF rs7124442 (2 patients and 2 controls) bValue was determined by Fisher’s exact test or χ2 test cLogistic regression analyses were used to calculate the OR (95% CI; confidence interval) Haplotype frequencies and clinical measurements [9]. Neurotrophins comprise four structurally related factors: BDNF, nerve growth factor (NGF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5). Haplotype frequencies and clinical measurements Among the 39 haplotype structures included in the haplo- type analysis of BDNF SNPs, seven frequent haplotypes (TGACCGCTGC, TATCCAACCT, TGACCACTGC, TAACTACCCT, TATCCGACCT, TAACTGCCCT, and CAACCACCGC) had a frequency of > 1% in the patients and controls. Although not shown in Table 4, the total fre- quency of the other haplotype structures was 30 (3.8%) for patients and 46 (6%) for controls. These haplotypes showed significantly different distributions between the patients with FM and the controls (P = 0.0001; Table 4). Among the frequent haplotypes, the TGACCACTGC haplotype was found less frequently in the patients with FM after adjusting for age and sex (OR 0.004, 95% CI 0.0–0.026, P < 0.001; Table 5). Interestingly, the TATC CGACCT and TAACTGCCCT haplotypes were not detected in patients with FM (Table 5) (both P > 0.05). In the clinical measures, only anxiety, assessed using the STAI-II score, was significantly different among the patients according to BDNF haplotype (Table 6). Several clinical studies have evaluated the role of BDNF in the pathogenesis of FM. Patients with FM have in- creased levels of BDNF in blood [12, 14] and cerebrospinal fluid [27] compared to healthy controls, implying that BDNF is involved in the pathophysiology of FM. In particular, Zanette et al. reported that serum BDNF levels are inversely associated with the pressure-pain threshold in patients with FM [13]. Furthermore, increased serum BDNF mediates the disinhibition of motor cortex excitability and the function of the descending inhibitory pain modulation system in patients with FM [28]. In fact, recent studies have shown that disruptions in default mode network (DMN) connectivity may be associated with impaired pain modulation, leading to the chronic pain seen in FM [29, 30]. Furthermore, certain BDNF polymorphisms have an effect on specific aspects of brain function such as DMN connectivity, which is currently considered to be central in the pathogenesis of FM [31]. These findings could be a potential explanation that BDNF genotypes and alleles and their association with clinical measurements Arthritis Research & Therapy (2018) 20:220 Page 4 of 11 Table 2 Genotype and allele analyses of BDNF in patients with fibromyalgia Marker Genotype/allele Contol, n (%) Fibromyalgia, n (%) Exact p valueb rs2883187 C/C 115 (27.5) 100 (24.4) 0.218 C/T 220 (52.6) 208 (50.9) T/T 83 (19.9) 101 (24.7) C 450 (53.8) 408 (49.9) 0.119 T 386 (46.2) 410 (50.1) rs7103873 G/G 113 (27.8) 98 (24.0) 0.245 C/G 210 (51.6) 208 (51.0) C/C 84 (20.6) 102 (25.0) G 436 (53.6) 404 (49.5) 0.112 C 378 (46.4) 412 (50.5) rs7103411 C/C 120 (28.5) 128 (31.3) 0.638 C/T 208 (49.4) 198 (48.4) T/T 93 (22.1) 83 (20.3) C 448 (53.2) 454 (55.5) 0.374 T 394 (46.8) 364 (44.5) rs10835210 C/C 204 (49.4) 196 (48.0) 0.725 A/C 175 (42.4) 172 (42.2) A/A 34 (8.2) 40 (9.8) C 583 (70.6) 564 (69.1) 0.554 A 243 (29.4) 252 (30.9) rs11030104 A/A 101 (24.0) 126 (31.0) 0.031 A/G 205 (48.8) 196 (48.2) G/G 114 (27.1) 85 (20.9) A 407 (48.5) 448 (55.0) 0.009 G 433 (51.5) 366 (45.0) rs12273539 C/C 283 (67.1) 268 (65.4) 0.101 C/T 132 (31.3) 125 (30.5) T/T 7 (1.7) 17 (4.1) C 698 (82.7) 661 (80.6) 0.299 T 146 (17.3) 159 (19.4) rs11030102 C/C 419 (99.3) 402 (98.5) 0.334 C/G 3 (0.7) 6 (1.5) C 841 (99.6) 810 (99.3) 0.335 G 3 (0.4) 6 (0.7) rs11030101 A/A 208 (49.5) 197 (48.3) 0.752 A/T 178 (42.4) 172 (42.2) T/T 34 (8.1) 39 (9.6) A 594 (70.7) 566 (69.4) 0.585 T 246 (29.3) 250 (30.6) rs6265 G/G 96 (22.9) 87 (21.3) 0.770 A/G 204 (48.7) 197 (48.3) A/A 119 (28.4) 124 (30.4) G 396 (47.3) 371 (45.5) 0.496 Table 2 Genotype and allele analyses of BDNF in patients with fibromyalgia and healthy controlsa Marker Genotype/allele Contol, n (%) Fibromyalgia, n (%) Exact p valueb OR (95% CI), p valuec OR (95% CI), p value, adjusted by age, sex‡ rs2883187 C/C 115 (27.5) 100 (24.4) 0.218 1 1 C/T 220 (52.6) 208 (50.9) 1.087 (0.783–1.510), p = 0.617 1.044 (0.747–1.458), p = 0.802 T/T 83 (19.9) 101 (24.7) 1.399 (0.943–2.078), p = 0.096 1.340 (0.897–2.002), p = 0.152 C 450 (53.8) 408 (49.9) 0.119 1 T 386 (46.2) 410 (50.1) 1.172 (0.966–1.421), p = 0.108 1.147 (0.943–1.395), p = 0.171 rs7103873 G/G 113 (27.8) 98 (24.0) 0.245 1 1 C/G 210 (51.6) 208 (51.0) 1.142 (0.820–1.591), p = 0.432 1.110 (0.791–1.556), p = 0.546 C/C 84 (20.6) 102 (25.0) 1.400 (0.943–2.080), p = 0.095 1.345 (0.899–2.010), p = 0.149 G 436 (53.6) 404 (49.5) 0.112 1 C 378 (46.4) 412 (50.5) 1.176 (0.968–1.429), p = 0.102 1.153 (0.946–1.405), p = 0.158 rs7103411 C/C 120 (28.5) 128 (31.3) 0.638 1 1 C/T 208 (49.4) 198 (48.4) 0.892 (0.651–1.224), p = 0.48 0.884 (0.641–1.220), p = 0.454 T/T 93 (22.1) 83 (20.3) 0.837 (0.568–1.232), p = 0.366 0.865 (0.584–1.280), p = 0.468 C 448 (53.2) 454 (55.5) 0.374 1 T 394 (46.8) 364 (44.5) 0.912 (0.751–1.106), p = 0.348 0.925 (0.76–1.125), p = 0.435 rs10835210 C/C 204 (49.4) 196 (48.0) 0.725 1 1 A/C 175 (42.4) 172 (42.2) 1.023 (0.767–1.364), p = 0.877 0.991 (0.740–1.326), p = 0.949 A/A 34 (8.2) 40 (9.8) 1.224 (0.745–2.014), p = 0.425 1.183 (0.714–1.957), p = 0.514 C 583 (70.6) 564 (69.1) 0.554 1 A 243 (29.4) 252 (30.9) 1.072 (0.868–1.324), p = 0.518 1.048 (0.846–1.297), p = 0.671 rs11030104 A/A 101 (24.0) 126 (31.0) 0.031 1 1 A/G 205 (48.8) 196 (48.2) 0.766 (0.553–1.063), p = 0.111 0.758 (0.544–1.057), p = 0.102 G/G 114 (27.1) 85 (20.9) 0.598 (0.407–0.877), p = 0.009 0.619 (0.419–0.913), p = 0.016 A 407 (48.5) 448 (55.0) 0.009 1 G 433 (51.5) 366 (45.0) 0.768 (0.633–0.932), p = 0.007 0.781 (0.641–0.95), p = 0.013 rs12273539 C/C 283 (67.1) 268 (65.4) 0.101 1 1 C/T 132 (31.3) 125 (30.5) 1 (0.744–1.345), p = 1 1.009 (0.747–1.362), p = 0.955 T/T 7 (1.7) 17 (4.1) 2.564 (1.047–6.282), p = 0.039 2.586 (1.052–6.36), p = 0.038 C 698 (82.7) 661 (80.6) 0.299 1 T 146 (17.3) 159 (19.4) 1.150 (0.897–1.475), p = 0.27 1.161 (0.902–1.493), p = 0.246 rs11030102 C/C 419 (99.3) 402 (98.5) 0.334 1 1 C/G 3 (0.7) 6 (1.5) 2.085 (0.518–8.392), p = 0.301 2.129 (0.524–8.649), p = 0.291 C 841 (99.6) 810 (99.3) 0.335 1 G 3 (0.4) 6 (0.7) 2.077 (0.518–8.326), p = 0.302 2.12 (0.524–8.58), p = 0.292 rs11030101 A/A 208 (49.5) 197 (48.3) 0.752 1 1 A/T 178 (42.4) 172 (42.2) 1.020 (0.766–1.358), p = 0.891 0.985 (0.737–1.317), p = 0.92 T/T 34 (8.1) 39 (9.6) 1.211 (0.735–1.996), p = 0.452 1.152 (0.694–1.912), p = 0.583 A 594 (70.7) 566 (69.4) 0.585 1 T 246 (29.3) 250 (30.6) 1.067 (0.864–1.316), p = 0.548 1.036 (0.837–1.283), p = 0.743 rs6265 G/G 96 (22.9) 87 (21.3) 0.770 1 1 A/G 204 (48.7) 197 (48.3) 1.066 (0.751–1.512), p = 0.722 1.017 (0.712–1.451), p = 0.928 A/A 119 (28.4) 124 (30.4) 1.150 (0.783–1.688), p = 0.476 1.110 (0.752–1.638), p = 0.599 G 396 (47.3) 371 (45.5) 0.496 1 Park et al. BDNF genotypes and alleles and their association with clinical measurements Neurotrophins play important roles in the transmission of physiologic and pathologic pain [22]. In particular, BDNF plays key roles in chronic pain conditions. BDNF is synthesized in the DRG, and is transported to the central terminals of the primary afferents in the spinal dorsal horn, where it is involved in the modulation of painful stimuli [9]. BDNF contributes to central sensitization by modulating nociceptive inputs and enhancing hyperalgesia through NMDA-receptor-mediated responses [23]. For these rea- sons, researchers have been interested in the role of BDNF in chronic pain disorders, including FM [24]. In addition, BDNF plays a role in depressive disorder, which is frequently comorbid with FM; indeed, the serum level of BDNF is altered in patients with depression [25, 26]. Moreover, it can be normalized by antidepressants such as milnacipran [26], which are frequently used in the treat- ment of FM. Discussion To our knowledge, we were the first to investigate the association between BDNF SNPs and FM. We found that the allele and genotype frequencies of BDNF rs11030104 were significantly different between the patients with FM and the controls. In comparison, although the allele and genotype frequencies of BDNF rs12273539 were not significantly different between the patients with FM and the controls, the TT genotype of BDNF rs12273539 was associated with susceptibility to FM. In addition to the individual SNPs, certain BDNF haplotypes may be protective against FM or contribute to its symptoms. Therefore, our data imply that BDNF gene polymorphisms contribute to the development and symptom severity of FM in the Korean population. Neurotrophic factors are a family of closely related proteins involved in neuronal survival, growth, and dif- ferentiation during development of the nervous system Page 6 of 11 Page 6 of 11 Park et al. Discussion Arthritis Research & Therapy (2018) 20:220 Table 3 Least-squares means (95% CI) of responses in patients with fibromyalgia, according to genotype Position Genotype Numbera Tender point number Tender point count FIQ BFI PCS MCS BDI STAI-I STAI-II rs2883187 C/C 100 13.38 (12.21–14.56) 25.17 (21.2–29.14) 58.56 (53.23–63.9) 7.25 (5.28–9.22) 38.02 (35.88–40.16) 31.25 (27.83–34.67) 18.09 (15.05–21.12) 51.56 (48.06–55.06) 51.97 (48.73–55.21) C/T 208 13.80 (12.83–14.77) 25.70 (22.43–28.96) 610 (56.69–65.32) 6.27 (4.66–7.89) 37.29 (35.56–39.03) 32.62 (29.85–35.39) 18.57 (16.11–21.02) 49.73 (46.9–52.56) 50.2 (47.59–52.81) T/T 101 13.77 (12.54–15.0) 25.85 (21.68–30.03) 58.21 (52.66–63.77) 7.16 (5.11–9.22) 38.17 (35.94–40.41) 34.26 (30.69–37.83) 16.89 (13.7–20.08) 49.49 (45.82–53.15) 48.29 (44.9–51.68) p value† 0.739 0.947 0.449 0.466 0.625 0.299 0.54 0.478 0.138 rs7103873 G/G 98 13.37 (12.18–14.55) 25.01 (21.01–29.01) 57.96 (52.56–63.35) 7.34 (5.35–9.32) 37.96 (35.8–40.12) 31.39 (27.93–34.85) 17.78 (14.71–20.85) 51.52 (47.98–55.06) 51.9 (48.63–55.18) G/C 208 13.84 (12.88–14.81) 25.97 (22.73–29.21) 60.89 (56.60–65.19) 6.25 (4.65–7.85) 37.34 (35.62–39.07) 32.83 (30.07–35.6) 18.45 (16.01–20.9) 49.51 (46.7–52.32) 50.08 (47.48–52.68) C/C 102 13.66 (12.42–14.9) 25.28 (21.08–29.49) 59.04 (53.43–64.64) 7.26 (5.19–9.33) 38.07 (35.82–40.32) 33.52 (29.92–37.13) 17.4 (14.18–20.62) 50.33 (46.64–54.02) 48.72 (45.31–52.13) p valueb 0.69 0.857 0.479 0.389 0.721 0.523 0.755 0.484 0.224 rs7103411 C/C 128 13.79 (12.68–14.9) 25.97 (22.21–29.72) 60.26 (55.22–65.3) 6.97 (5.11–8.83) 37.76 (35.73–39.79) 32.88 (29.64–36.12) 18.25 (15.37–21.14) 50.48 (47.17–53.8) 49.28 (46.21–52.35) C/T 198 13.74 (12.74–14.74) 25.65 (22.27–29.02) 60.54 (56.09–64.99) 6.07 (4.41–7.73) 37.5 (35.71–39.29) 33.0 (30.14–35.87) 18.32 (15.78–20.85) 49.55 (46.62–52.47) 50.44 (47.74–53.15) T/T 83 13.46 (12.23–14.68) 25.0 (20.87–29.14) 57.81 (52.24–63.38) 7.64 (5.59–9.68) 37.79 (35.55–40.02) 31.41 (27.84–34.98) 17.5 (14.33–20.67) 50.89 (47.22–54.55) 51.09 (47.69–54.48) p valueb 0.859 0.907 0.582 0.249 0.947 0.635 0.86 0.705 0.578 rs10835210 C/C 196 13.49 (12.50–14.47) 25.67 (22.37–28.97) 59.70 (55.30–64.10) 6.91 (5.27–8.55) 37.78 (36.02–39.55) 31.77 (28.96–34.58) 18.87 (16.37–21.36) 51.04 (48.18–53.9) 51.77 (49.13–54.41) C/A 172 13.87 (12.84–14.91) 25.51 (22.04–28.99) 60.15 (55.46–64.85) 6.66 (4.93–8.4) 37.3 (35.41–39.18) 33.19 (30.18–36.2) 17.17 (14.5–19.83) 48.42 (45.35–51.48) 48.63 (45.81–51.45) A/A 40 13.96 (12.18–15.74) 24.85 (18.87–30.83) 59.67 (51.64–67.7) 5.46 (2.52–8.4) 38.45 (35.22–41.67) 35.31 (30.17–40.45) 17.78 (13.17–22.4) 51.79 (46.49–57.08) 47.51 (42.62–52.4) p valueb 0.675 0.962 0.977 0.609 0.728 0.301 0.384 0.138 0.029 rs11030104 A/A 126 13.80 (12.68–14.92) 25.99 (22.22–29.75) 60.27 (55.2–65.35) 7.0 (5.13–8.87) 37.82 (35.77–39.86) 32.86 (29.6–36.12) 18.33 (15.43–21.23) 50.46 (47.13–53.79) 49.29 (46.2–52.37) A/G 196 13.79 (12.79–14.8) 25.67 (22.3–29.03) 60.66 (56.19–65.13) 6.09 (4.42–7.76) 37.4 (35.60–39.20) 32.9 (30.02–35.77) 18.41 (15.87–20.95) 49.69 (46.76–52.61) 50.53 (47.82–53.24) G/G 85 13.31 (12.08–14.53) 24.75 (20.64–28.86) 57.55 (51.98–63.12) 7.58 (5.53–9.62) 37.86 (35.63–40.1) 31.6 (28.03–35.16) 17.31 (14.15–20.47) 50.63 (46.99–54.28) 50.88 (47.49–54.27) p valueb 0.677 0.845 0.498 0.274 0.871 0.731 0.757 0.821 0.616 rs12273539 C/C 268 13.63 (12.7–14.55) 25.24 (22.11–28.37) 59.03 (54.81–63.25) 6.51 (4.95–8.07) 37.67 (35.98–39.37) 33.14 (30.42–35.86) 17.45 (15.05–19.85) 49.9 (47.14–52.66) 49.52 (46.96–52.09) C/T 125 14.13 (13.01–15.24) 27.04 (23.26–30.81) 62.11 (57.15–67.08) 6.75 (4.89–8.61) 37.48 (35.48–39.47) 31.56 (28.36–34.76) 19.34 (16.5–22.18) 50.66 (47.4–53.92) 51.73 (48.7–54.75) T/T 17 11.90 (9.7–14.1) 21.96 (14.52–29.41) 57.64 (47.31–67.96) 8.92 (5.15–12.69) 38.13 (33.98–42.27) 31.82 (25.17–38.48) 20.22 (14.35–26.09) 49.54 (42.8–56.29) 50.87 (44.61–57.14) p valueb 0.131 0.319 0.349 0.432 0.945 0.533 0.26 0.862 0.271 rs11030102 C/C 402 13.67 (12.8–14.55) 25.57 (22.61–28.53) 59.83 (55.89–63.77) 6.62 (5.17–8.07) 37.66 (36.07–39.25) 32.67 (30.14–35.21) 18.06 (15.83–20.29) 50.02 (47.45–52.59) 50.16 (47.78–52.55) C/G 6 17.58 (13.01–22.15) 36.13 (20.73–51.54) 72.91 (51.53–94.29) 19.07 (11.35–26.79) 35.01 (26.4–43.63) 23.8 (10.04–37.56) 29.58 (17.47–41.69) 65.06 (51.13–78.99) 62.56 (49.62–75.51) p valueb 0.089 0.172 0.224 0.001 0.542 0.2 0.059 0.032 0.057 rs11030101 A/A 197 13.49 (12.51–14.47) 25.69 (22.38–29.01) 59.82 (55.43–64.22) 6.91 (5.27–8.55) 37.79 (36.02–39.55) 31.73 (28.92–34.54) 18.94 (16.45–21.43) 51.01 (48.15–53.87) 51.77 (49.13–54.42) A/T 172 13.84 (12.81–14.87) 25.38 (21.9–28.87) 60.26 (55.57–64.95) 6.69 (4.95–8.43) 37.34 (35.45–39.22) 33.14 (30.14–36.15) 17.1 (14.43–19.76) 48.41 (45.34–51.48) 48.57 (45.75–51.4) T/T 39 14.23 (12.45–16.01) 26.37 (20.36–32.37) 58.75 (50.73–66.77) 5.33 (2.39–8.27) 38.16 (34.93–41.39) 35.94 (30.8–41.08) 17.66 (13.12–22.21) 52.15 (46.94–57.37) 48.07 (43.25–52.89) p valueb 0.605 0.943 0.929 0.555 0.816 0.212 0.325 0.126 0.033 rs6265 G/G 87 13.29 (12.07–14.51) 24.66 (20.55–28.78) 57.67 (52.14–63.21) 7.55 (5.52–9.59) 37.83 (35.61–40.06) 31.56 (28.01–35.11) 17.44 (14.29–20.59) 50.62 (46.97–54.26) 50.94 (47.56–54.32) Page 7 of 11 Park et al. Discussion Arthritis Research & Therapy (2018) 20:220 Page 8 of 11 Page 8 of 11 Table 4 Estimates of haplotype frequencies in patients with fibromyalgia (n = 393) and healthy controls (n = 388)a Combined allelesa All subjects Controls Fibromyalgia p valueb TGACCGCTGC 29.6 ± 0.75 20.22 ± 0.9 38.87 ± 0.88 0.0001 TATCCAACCT 20.16 ± 0.44 12.94 ± 0.57 27.29 ± 0.52 TGACCACTGC 14.8 ± 0.75 25.26 ± 0.89 4.47 ± 0.88 TAACTACCCT 11.97 ± 0.42 6.99 ± 0.5 16.89 ± 0.53 TATCCGACCT 8.94 ± 0.45 15.76 ± 0.59 2.21 ± 0.52 TAACTGCCCT 5.74 ± 0.42 9.8 ± 0.5 1.72 ± 0.53 CAACCACCGC 3.20 ± 0.17 2.06 ± 0.22 4.33 ± 0.24 aData are percentages ± SE aMissing data were excluded (n = 51). Among 39 haplotype structures, 7 haplotypes with frequency of at least 1% in both the patients and controls are presented bp values for permutation test of the null hypothesis that cases and controls are random draws from a common set of haplotype frequencies (number of permutations = 10,000) Table 4 Estimates of haplotype frequencies in patients with fibromyalgia (n = 393) and healthy controls (n = 388)a with susceptibility to FM. However, our data demonstrate that other BDNF SNPs, such as rs11030104 and rs12273539, were associated with the risk of FM in a Korean population. Furthermore, our data imply that certain BDNF haplo- types exert a protective effect against FM. A haplotype refers to a particular set of closely linked alleles that are inherited as a unit, and haplotype analysis can reveal the pattern of genetic variation associated with certain diseases [33]. Several haplotypes of certain genes are reportedly significantly associated with FM. Diatchenko et al. [34] reported that the ACCG haplotype, which consists of four SNPs (rs6269, rs4633, rs4818, and rs4680) of the catechol-O-methyltransferase (COMT) gene, is associated with both FM susceptibility and symptom severity [35, 36]. Similarly, we also suggested that a particular haplotype of TRPV2 may be associated with susceptibility to FM [37]. In the current study, our findings imply that BDNF haplo- types may be involved in the pathophysiology of FM. supports the existence of a mechanistic link between BDNF polymorphisms and FM. However, although mul- tiple lines of evidence imply a role for BDNF in the patho- genesis of FM, BDNF polymorphisms in these patients have not been investigated extensively. Discussion y y y gy Notably, we failed to uncover a direct association be- tween BDNF gene polymorphisms and pain-related symp- tom scales such as the tender point number and count. However, those polymorphisms were related to certain psy- chological symptoms in patients with FM. In particular, certain BDNF SNPs and haplotypes were associated with anxiety symptoms. Since patients with FM have a signifi- cantly higher prevalence of anxiety disorders (13–63.8%) [38], our findings imply that BDNF gene polymorphisms may indirectly affect FM through their effect on anxiety. However, diverse factors affect the development of FM, including psychological symptoms such as anxiety, so our results should be interpreted carefully. In this study, we found that certain BDNF SNPs are as- sociated with susceptibility to FM. The GG genotype and the G allele of BDNF rs11030104 exert a protective effect against FM. In contrast, although the allele and genotype frequencies of BDNF rs12273539 did not differ between the patients with FM and controls, the TT genotype of BDNF rs12273539 was associated with susceptibility to FM. To date, only one study has evaluated associations between BDNF gene polymorphisms and FM. Xiao et al. [32] evaluated whether the BDNF Val66Met polymorph- ism was associated with FM; their results implied that the BDNF Val66 Met SNP is associated with a subgroup of patients with FM with high-sensitivity C-reactive protein and high body mass index. Nevertheless, the relative distribution of the BDNF Val66Met SNP did not differ between the patients with FM and healthy controls. Simi- larly, in our study, BDNF Val66Val Met was not associated This study had several limitations. First, it was of a case-control design. Because the purpose of this study was to evaluate the role of BDNF SNPs associated with susceptibility to FM, we adopted a target-gene-based approach. Therefore, like the majority of SNP studies, we selected candidate SNPs for a case-control analysis of their association with FM. Discussion Arthritis Research & Therapy (2018) 20:220 Table 3 Least-squares means (95% CI) of responses in patients with fibromyalgia, according to genotype (Continued) Position Genotype Numbera Tender point number Tender point count FIQ BFI PCS MCS BDI STAI-I STAI-II G/A 197 13.77 (12.77–14.77) 25.62 (22.26–28.99) 60.72 (56.27–65.18) 6.11 (4.44–7.77) 37.45 (35.65–39.24) 32.92 (30.05–35.79) 18.27 (15.74–20.81) 49.6 (46.67–52.52) 50.43 (47.71–53.14) A/A 124 13.88 (12.76–15) 26.34 (22.56–30.13) 60.17 (55.09–65.24) 6.98 (5.11–8.86) 37.83 (35.79–39.88) 32.91 (29.64–36.18) 18.37 (15.47–21.28) 50.58 (47.24–53.91) 49.37 (46.28–52.46) p valueb 0.634 0.746 0.513 0.293 0.898 0.702 0.829 0.766 0.657 rs7124442 C/T 47 13.57 (12.12–15.03) 26.18 (21.3–31.07) 62.15 (55.49–68.81) 5.60 (3.16–8.05) 37.37 (34.69–40.05) 30.74 (26.46–35.03) 19.98 (16.2–23.76) 50.67 (46.27–55.07) 51.35 (47.3–55.39) T/T 360 13.72 (12.81–14.62) 25.53 (22.48–28.57) 59.53 (55.49–63.57) 6.88 (5.37–8.39) 37.69 (36.06–39.32) 32.94 (30.34–35.54) 17.81 (15.51–20.1) 50.01 (47.36–52.66) 50.04 (47.58–52.5) p valueb 0.836 0.777 0.411 0.274 0.804 0.285 0.23 0.755 0.501 Abbreviations: CI confidence interval, FIQ Fibromyalgia Impact Questionnaire, BFI Brief Fatigue Inventory, PCS Physical Component Summary, MCS Mental Component Summary, BDI Beck Depression Inventory, STAI-I State-Trait Anxiety Inventory-I, STAI-II State-Trait Anxiety Inventory-II aMissing data were excluded from the analyses: BDNF rs2883187 (5 controls), BDNF rs7103873 (1 patient and 16 controls), BDNF rs7103411 (2 controls), BDNF rs10835210 (1 patient and 10 controls), BDNF rs11030104 (2 patients and 3 controls), BDNF rs12273539 (1 control), BDNF rs11030102 (1 patient and 1 control), BDNF rs11030101 (1 patient and 3 controls), BDNF rs6265 (4 controls and 1 patient), and BDNF rs7124442 (2 patients and 2 controls) b Table 3 Least-squares means (95% CI) of responses in patients with fibromyalgia, according to genotype (Continued) Position Genotype Numbera Tender point number Tender point count FIQ BFI PCS MCS BDI STAI-I STAI-II G/A 197 13.77 (12.77–14.77) 25.62 (22.26–28.99) 60.72 (56.27–65.18) 6.11 (4.44–7.77) 37.45 (35.65–39.24) 32.92 (30.05–35.79) 18.27 (15.74–20.81) 49.6 (46.67–52.52) 50.43 (47.71–53.14) A/A 124 13.88 (12.76–15) 26.34 (22.56–30.13) 60.17 (55.09–65.24) 6.98 (5.11–8.86) 37.83 (35.79–39.88) 32.91 (29.64–36.18) 18.37 (15.47–21.28) 50.58 (47.24–53.91) 49.37 (46.28–52.46) p valueb 0.634 0.746 0.513 0.293 0.898 0.702 0.829 0.766 0.657 rs7124442 C/T 47 13.57 (12.12–15.03) 26.18 (21.3–31.07) 62.15 (55.49–68.81) 5.60 (3.16–8.05) 37.37 (34.69–40.05) 30.74 (26.46–35.03) 19.98 (16.2–23.76) 50.67 (46.27–55.07) 51.35 (47.3–55.39) T/T 360 13.72 (12.81–14.62) 25.53 (22.48–28.57) 59.53 (55.49–63.57) 6.88 (5.37–8.39) 37.69 (36.06–39.32) 32.94 (30.34–35.54) 17.81 (15.51–20.1) 50.01 (47.36–52.66) 50.04 (47.58–52.5) p valueb 0.836 0.777 0.411 0.274 0.804 0.285 0.23 0.755 0.501 Abbreviations: CI confidence interval, FIQ Fibromyalgia Impact Questionnaire, BFI Brief Fatigue Inventory, PCS Physical Component Summary, MCS Mental Component Summary, BDI Beck Depression Inventory, STAI-I State-Trait Anxiety Inventory-I, STAI-II State-Trait Anxiety Inventory-II aMissing data were excluded from the analyses: BDNF rs2883187 (5 controls), BDNF rs7103873 (1 patient and 16 controls), BDNF rs7103411 (2 controls), BDNF rs10835210 (1 patient and 10 controls), BDNF rs11030104 (2 patients and 3 controls), BDNF rs12273539 (1 control), BDNF rs11030102 (1 patient and 1 control), BDNF rs11030101 (1 patient and 3 controls), BDNF rs6265 (4 controls and 1 patient), and BDNF rs7124442 (2 patients and 2 controls) b Park et al. bComputed for the estimated coefficient of each haplotype in the logistic regression aMissing data were excluded (n = 51). Among 39 haplotype structures, 7 haplotypes with a frequency of at least 1% in both the patie presented; the total frequency of the other haplotype structures was 46 (6%) for controls and 30 (3.8%) for patients. Logistic regressio calculate ORs xcluded (n = 51). Among 39 haplotype structures, 7 haplotypes with a frequency of at least 1% in both the patients and controls are requency of the other haplotype structures was 46 (6%) for controls and 30 (3.8%) for patients. Logistic regression models were used t ted coefficient of each haplotype in the logistic regression Discussion Second, the multiple tests Table 5 Combined allele frequencies and odds ratios in patients with fibromyalgia and healthy controlsa Combined alleles Controls, n (%) Fibromyalgia, n (%) Crude OR (95% CI) p valueb Age and sex adjusted OR (95% CI) p valueb TGACCGCTGC 193 (26.6) 340 (45) 1.0 (reference) 1.0 (reference) TATCCAACCT 119 (16.4) 232 (30.7) 1.107 (0.834–1.469) 0.483 1.106 (0.833–1.47) 0.487 TGACCACTGC 160 (22) 1 (0.1) 0.004 (0–0.026) < 0.001 0.004 (0.0–0.026) < 0.001 TAACTACCCT 66 (9.1) 146 (19.3) 1.256 (0.894–1.765) 0.19 1.248 (0.887–1.756) 0.204 TATCCGACCT 103 (14.2) 0 (0) 0 (0-Inf) 0.963 0 (0-Inf) 0.963 TAACTGCCCT 65 (9) 0 (0) 0 (0-Inf) 0.971 0 (0-Inf) 0.97 CAACCACCGC 20 (2.8) 37 (4.9) 1.05 (0.593–1.861) 0.867 1.088 (0.612–1.934) 0.774 Abbreviations: OR odds ratio, CI confidence interval aMissing data were excluded (n = 51). Among 39 haplotype structures, 7 haplotypes with a frequency of at least 1% in both the patients and controls are presented; the total frequency of the other haplotype structures was 46 (6%) for controls and 30 (3.8%) for patients. Logistic regression models were used to calculate ORs bComputed for the estimated coefficient of each haplotype in the logistic regression Page 9 of 11 Park et al. Arthritis Research & Therapy (2018) 20:220 Table 6 Numbers of haplotypes and least-squares means (95% CI) of responses in patients with fibromyalgia Combined alleles Numbera Tender point number Tender point count FIQ BFI PCS MCS BDI STAI-I STAI-II TGACCGCTGC 340 13.61 (12.89–14.33) 25.33 (22.93–27.73) 59.67 (56.48–62.87) 6.81 (5.6–8.01) 37.56 (36.25–38.87) 32.12 (30.06–34.18) 18.02 (16.22–19.83) 50.66 (48.56–52.76) 51.01 (49.07–52.95) TATCCAACCT 232 13.91 (13.1–14.72) 25.29 (22.6–27.99) 59.88 (56.26–63.5) 6.35 (4.99–7.71) 37.49 (36–38.98) 33.88 (31.54–36.21) 17.25 (15.21–19.3) 49.31 (46.92–51.69) 48.26 (46.07–50.46) TAACTACCCT 146 13.66 (12.75–14.58) 26.34 (23.29–29.38) 61.2 (57.17–65.23) 7.32 (5.8–8.85) 37.49 (35.84–39.15) 31.73 (29.13–34.33) 19.5 (17.21–21.78) 50.49 (47.83–53.14) 51.16 (48.71–53.6) CAACCACCGC 37 13.53 (12.02–15.05) 25.96 (20.93–31) 63.17 (56.22–70.11) 5.67 (3.09–8.25) 37.78 (34.92–40.63) 30.7 (26.22–35.18) 20.06 (16.15–23.97) 52.65 (48.1–57.2) 52.74 (48.54–56.93) p valueb 0.874 0.902 0.696 0.527 0.998 0.271 0.221 0.459 0.027 Abbreviations: CI confidence interval, FIQ Fibromyalgia Impact Questionnaire, BFI Brief Fatigue Inventory, PCS Physical Component Summary, MCS Mental Component Summary, BDI Beck Depression Inventory, STAI-I State-Trait Anxiety Inventory-I, STAI-II State-Trait Anxiety Inventory-II aMissing data were excluded from the analyses bp values derived by analysis of covariance adjusted for age and sex Page 10 of 11 Park et al. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP This study was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP 5. Park DJ, Lee SS. New insights into the genetics of fibromyalgia. Korean J Intern Med. 2017;32(6):984–95. (2017M3A9E8023014), and by a grant (CRI16015–1) from Chonnam National University Hospital Biomedical Research Institute. 6. Buskila D, Sarzi-Puttini P, Ablin JN. The genetics of fibromyalgia syndrome. Pharmacogenomics. 2007;8(1):67–74. 7. Park DJ, Kim SH, Nah SS, Lee JH, Kim SK, Lee YA, et al. Association between catechol-O-methyl transferase gene polymorphisms and fibromyalgia in a Korean population: a case-control study. Eur J Pain. 2016;20(7):1131–9. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References f 1. Wolfe F, Smythe HA, Yunus MB, Bennett RM, Bombardier C, Goldenberg DL, et al. The American College of Rheumatology 1990 Criteria for the classification of fibromyalgia. report of the Multicenter Criteria Committee. Arthritis Rheum. 1990;33(2):160–72. 2. Jones GT, Atzeni F, Beasley M, Fluss E, Sarzi-Puttini P, Macfarlane GJ. The prevalence of fibromyalgia in the general population: a comparison of the American College of Rheumatology 1990, 2010, and modified 2010 classification criteria. Arthritis Rheumatol. 2015;67(2):568–75. 2. Jones GT, Atzeni F, Beasley M, Fluss E, Sarzi-Puttini P, Macfarlane GJ. The prevalence of fibromyalgia in the general population: a comparison of the American College of Rheumatology 1990, 2010, and modified 2010 classification criteria. Arthritis Rheumatol. 2015;67(2):568–75. Received: 27 March 2018 Accepted: 18 September 2018 Received: 27 March 2018 Accepted: 18 September 2018 Discussion Arthritis Research & Therapy (2018) 20:220 Page 10 of 11 performed in this study may have increased the type I error. In genetics, controlling for multiple testing is im- portant in estimating thresholds of significance accurately, particularly in genome-wide association studies (GWAS) [39]. However, in this target-gene-based SNP study, we did not consider the potential effects of multiple testing in the analyses. In fact, most published FM SNP case-control studies have not considered the potential effects of mul- tiple testing. Third, we were unable to prospectively evalu- ate the associations between BDNF genetic variation and clinical outcomes. Therefore, further studies are needed to investigate the effect of those genetic polymorphisms on the long-term clinical outcomes of patients with FM. Fi- nally, to overcome the insufficient statistical power, we conducted a large-scale study involving > 800 samples. However, our findings should be replicated in a larger population comprising multiple ethnicities. revised the manuscript for important intellectual content. All authors read and approved the final manuscript. revised the manuscript for important intellectual content. All authors read and approved the final manuscript. Availability of data and materials The datasets used during the current study are available from the corresponding author on reasonable request. The datasets used during the current study are available from the corresponding author on reasonable request. 8. Wu YJ, Kruttgen A, Moller JC, Shine D, Chan JR, Shooter EM, et al. Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 are sorted to dense-core vesicles and released via the regulated pathway in primary rat cortical neurons. J Neurosci Res. 2004;75(6):825–34. 8. Wu YJ, Kruttgen A, Moller JC, Shine D, Chan JR, Shooter EM, et al. Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 are sorted to dense-core vesicles and released via the regulated pathway in primary rat cortical neurons. J Neurosci Res. 2004;75(6):825–34. Acknowledgements W ld l k h We would like to thank the patients and their families for participating in this study. 3. Maletic V, Raison CL. Neurobiology of depression, fibromyalgia and neuropathic pain. Front Biosci (Landmark Ed). 2009;14:5291–338. 4. Ablin JN, Buskila D. Update on the genetics of the fibromyalgia syndrome. Best Pract Res Clin Rheumatol. 2015;29(1):20–8. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 In this study, we evaluated the association between BDNF polymorphisms and FM in a large sample of the Korean population. We found that BDNF gene polymorphisms influenced susceptibility to FM, and contributed to the severity of certain symptoms of FM. Further evidence from large prospective studies is needed to determine the generalizability of our findings to the broader population and their impact on the clinical outcomes of FM. More- over, further work is needed to elucidate the biologic and epigenetic mechanisms underlying the complex role of the BDNF gene in FM. 1Division of Rheumatology, Department of Internal Medicine, Chonnam National University Medical School and Hospital, 42 Jebong-ro, Dong-gu, Gwangju 61469, Republic of Korea. 2Department of Internal Medicine, Inje University Haeundae Paik Hospital, Busan, Korea. 3Department of Internal Medicine, Soonchunhyang University, College of Medicine, Cheonan, Korea. 4Department of Internal Medicine, Maryknoll Medical Center, Busan, Korea. 5Department of Internal Medicine, Catholic University of Daegu, School of Medicine, Daegu, Korea. 6Department of Internal Medicine, School of Medicine, Kyung Hee University, Seoul, Korea. 7Department of Internal Medicine, Soonchunhyang University Seoul Hospital, Seoul, Korea. 8Hanyang University College of Medicine and the Hospital for Rheumatic Diseases, Seoul, Korea. 9Department of Allergy and Rheumatology, Ajou University Hospital, Ajou University School of Medicine, Suwon, Korea. 10Department of Internal Medicine, Konyang University Medical School, Daejeon, Korea. 11Departments of Internal Medicine, School of Medicine, Keimyung University Daegu Korea Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki, and written informed consent was obtained from all participants at the time of recruitment. Exactly the same informed consult form (ICF) and study protocol were provided to the independent Institutional Review Board/Ethics Committee (IRB/EC) at each medical center, and each IRB/EC reviewed the appropriateness of the protocol and risks and benefits to the study participants. Ultimately, the IRB/EC at each medical center independently approved this study without revision of the ICF or study protocol. Abbreviations BDI B k D BDI: Beck Depression Inventory; BDNF: Brain-derived neurotrophic factor; BFI: Brief Fatigue Inventory; CI: Confidence interval; DRG: Dorsal root ganglion; FIQ: Fibromyalgia Impact Questionnaire; FM: Fibromyalgia; ICF: Informed consent form; MCS: Mental Component Summary; NMDA: N- methyl-D-aspartate; NSAIDs: Nonsteroidal anti-inflammatory drugs; PCS: Physical Component Summary; SF-36: 36-Item Medical Outcomes Study Short-Form Health Survey; SNP: Single-nucleotide polymorphism; SNRI: Serotonin-norepinephrine reuptake inhibitors; SSRI: Selective serotonin reuptake inhibitor; STAI-I: State-Trait Anxiety Inventory-I; STAI-II: State-Trait Anxiety Inventory-II; TCA: Tricyclic antidepressant 2. Jones GT, Atzeni F, Beasley M, Fluss E, Sarzi Puttini P, Macfarlane GJ. The prevalence of fibromyalgia in the general population: a comparison of the American College of Rheumatology 1990, 2010, and modified 2010 classification criteria. Arthritis Rheumatol. 2015;67(2):568–75. 3. Maletic V, Raison CL. Neurobiology of depression, fibromyalgia and neuropathic pain. Front Biosci (Landmark Ed). 2009;14:5291–338. 4. Ablin JN, Buskila D. Update on the genetics of the fibromyalgia syndrome. Best Pract Res Clin Rheumatol. 2015;29(1):20–8. 5. Park DJ, Lee SS. New insights into the genetics of fibromyalgia. Korean J Intern Med. 2017;32(6):984–95. 6. Buskila D, Sarzi-Puttini P, Ablin JN. The genetics of fibromyalgia syndrome. Pharmacogenomics. 2007;8(1):67–74. 7. Park DJ, Kim SH, Nah SS, Lee JH, Kim SK, Lee YA, et al. Association between catechol-O-methyl transferase gene polymorphisms and fibromyalgia in a Korean population: a case-control study. Eur J Pain. 2016;20(7):1131–9. 8. Wu YJ, Kruttgen A, Moller JC, Shine D, Chan JR, Shooter EM, et al. Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 are sorted to dense-core vesicles and released via the regulated pathway in primary rat cortical neurons. 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Polymorphisms of the TRPV2 and TRPV3 genes associated with fibromyalgia in a Korean population. Rheumatology (Oxford). 2016;55(8):1518–27. 13. Zanette SA, Dussan-Sarria JA, Souza A, Deitos A, Torres ILS, Caumo W. Higher serum S100B and BDNF levels are correlated with a lower pressure- pain threshold in fibromyalgia. Mol Pain. 2014;10:46. 38. Arnold LM, Hudson JI, Keck PE, Auchenbach MB, Javaras KN, Hess EV. Comorbidity of fibromyalgia and psychiatric disorders. J Clin Psychiatry. 2006;67(8):1219–25. 14. Haas L, Portela LV, Bohmer AE, Oses JP, Lara DR. Increased plasma levels of brain derived neurotrophic factor (BDNF) in patients with fibromyalgia. Neurochem Res. 2010;35(5):830–4. 39. Park et al. Arthritis Research & Therapy (2018) 20:220 31. Jang JH, Yun JY, Jung WH, Shim G, Byun MS, Hwang JY, et al. The impact of genetic variation in comt and bdnf on resting-state functional connectivity. Int J Imaging Syst Technol. 2012;22(1):97–102. 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Observations of high rates of NO&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; – HONO conversion in the nocturnal atmospheric boundary layer in Kathmandu, Nepal
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Yu1,*, B. Galle1, A. Panday2,**, E. Hodson2,***, R. Prinn2, and S. Wang3 1Optical Remote Sensing, Radio and Space Science, Chalmers University of Technology, 41296 Gothenburg, Sweden 2Dept. of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3Jet Propulsion Laboratory, Pasadena, CA 91109, USA *now at: Monitoring & Laboratory Division, California Air Resources Board, 9528 Telstar Avenue, El Monte, CA 91731, USA *now at: Monitoring & Laboratory Division, California Air Resources Board, 9528 Telstar Avenue, El Monte, CA 91731, USA **now at: Atmospheric and Oceanic Program, Princeton University, Princeton, NJ 08544, USA ***now at: Ecological Process Modelling Unit, Swiss Federal Institute for Forest, Snow and Landscape Research, Birmensdorf, 8903, Switzerland **now at: Atmospheric and Oceanic Program, Princeton University, Princeton, NJ 08544, USA ***now at: Ecological Process Modelling Unit, Swiss Federal Institute for Forest, Snow and Landscape Research, Bi d f 8903 S it l d now at: Atmospheric and Oceanic Program, Princeton University, Princeton, NJ 08544, USA ***now at: Ecological Process Modelling Unit, Swiss Federal Institute for Forest, Snow and Landscape Research, Birmensdorf, 8903, Switzerland Received: 7 October 2008 – Published in Atmos. Chem. Phys. Discuss.: 6 January 2009 Revised: 24 July 2009 – Accepted: 14 August 2009 – Published: 7 September 2009 Abstract. Nitrous acid (HONO) plays a significant role in the atmosphere, especially in the polluted troposphere. Its photolysis after sunrise is an important source of hydroxyl free radicals (OH). Measurements of nitrous acid and other pollutants were carried out in the Kathmandu urban atmo- sphere during January–February 2003, contributing to the sparse knowledge of nitrous acid in South Asia. The results showed average nocturnal levels of HONO (1.7±0.8 ppbv), NO2 (17.9±10.2 ppbv), and PM10(0.18±0.11 mg m−3) in ur- ban air in Kathmandu. Surprisingly high ratios of chemi- cally formed secondary [HONO] to [NO2] (up to 30%) were found, which indicates unexpectedly efficient chemical con- version of NO2 to HONO in Kathmandu. The ratios of [HONO]/[NO2] at night were found to be much higher than previously reported values from measurements in urban air in Europe, North America and Asia. The influences of aerosol surface, ground reactive surface, and relative humidity on NO2-HONO chemical conversion were discussed. The high humidity, strong and low inversion layer at night, and high aerosol pollution burden in Kathmandu may explain the par- ticularly efficient conversion of NO2 to HONO. Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. 1 Introduction Although high HONO levels are usually observed at night- time, atmospheric measurements, laboratory studies and model calculations have shown the enhanced formation of HONO during daytime (Stemmler et al., 2007; George et al., 2005; Gustafsson et al., 2006; Bejan et al., 2006; Rohrer et al., 2005; Liao et al., 2006a; Zhou et al., 2001; Honrath et al., 2002; Domine and Shepson, 2002; Akimoto et al., 1987; Kleffmann, 2007). It has also been shown that NO2 can be efficiently reduced to form HONO under UV light on the surface of TiO2, humid acid, and other organic compounds (Reaction 6) (Kleffmann, 2007; Stemmler et al., 2007, 2006; George et al., 2005; Gustafsson et al., 2006). Although high HONO levels are usually observed at night- time, atmospheric measurements, laboratory studies and model calculations have shown the enhanced formation of HONO during daytime (Stemmler et al., 2007; George et al., 2005; Gustafsson et al., 2006; Bejan et al., 2006; Rohrer et al., 2005; Liao et al., 2006a; Zhou et al., 2001; Honrath et al., 2002; Domine and Shepson, 2002; Akimoto et al., 1987; Kleffmann, 2007). It has also been shown that NO2 can be efficiently reduced to form HONO under UV light on the surface of TiO2, humid acid, and other organic compounds (Reaction 6) (Kleffmann, 2007; Stemmler et al., 2007, 2006; George et al., 2005; Gustafsson et al., 2006). (R5) (R6) NO2 (g) + photo −activated aerosol surface →HONO(g) (R6) Laboratory research has shown that Reactions (1–3) are heterogeneous formation processes involving NOx and water on surfaces (Finlayson-Pitts et al., 2003). Past research on the mechanisms of HONO heterogeneous reactions points to the importance of reaction surfaces, and indicates that the production efficiency of HONO depends upon the amount of water absorbed by surfaces (Finlayson-Pitts et al., 2003; Kurtenbach et al., 2001; Grassian, 2001, 2002). Nitrous acid can be formed through Reaction (1) on the surface of aerosols and the ground (Trick, 2004; Harrison and Kitto, 1994; Andr´es-Hern´andez et al., 1996; Reisinger, 2000; Wang et al., 2003; Notholt et al., 1992a). Some studies have sug- gested Reaction (2) as a source of HONO in the atmosphere (Andr´es-Hern´andez et al., 1996; Calvert et al., 1994); how- ever, other laboratory studies and field observations (includ- ing our observation in this paper) have shown high HONO formation in the absence of NO (Alicke et al., 2002; Kleff- mann et al., 1998a; Svensson et al., 1987). 1 Introduction Reaction (2) is thus unlikely to play an important role in the atmosphere. Reaction (3) was only studied under conditions of high NO concentrations (Saliba et al., 2000, 2001), and thus not im- portant in real atmospheric conditions with low NO concen- trations. HONO formed in homogenous and heterogeneous reac- tions is released to the gas phase and accumulates during the night. Photolysis of HONO after sunrise provides a pulse of strong OH formation, which strongly influences atmo- spheric chemistry processes. Field measurements of HONO and its precursor NO2 at locations with various aerosol loads, aerosol compositions, relative humidity, boundary layer heights and vertical transport efficiencies are thus es- sential for improving our understanding of the HONO for- mation mechanism. HONO formed in homogenous and heterogeneous reac- tions is released to the gas phase and accumulates during the night. Photolysis of HONO after sunrise provides a pulse of strong OH formation, which strongly influences atmo- spheric chemistry processes. Field measurements of HONO and its precursor NO2 at locations with various aerosol loads, aerosol compositions, relative humidity, boundary layer heights and vertical transport efficiencies are thus es- sential for improving our understanding of the HONO for- mation mechanism. Gaseous HONO in ambient air was measured for the first time by Perner and Platt (Perner and Platt, 1979) using dif- ferential optical absorption spectroscopy (DOAS). Besides the DOAS technique, a variety of other methods have been developed for the detection of HONO in the atmosphere with high sensitivity and good time resolution (Liao et al., 2006a,b; Takenaka et al., 2004; Trebs et al., 2004; Kleffmann et al., 2002; Huang et al., 2002; Vecera and Dasgupta, 1991; Heland et al., 2001). Measurements of HONO in the atmo- sphere have been carried out in many areas of Europe and the US. Elevated atmospheric HONO concentrations have also been detected in some Asian cities, suggesting HONO to be an important OH source in these places (Table 1). However, knowledge about HONO sources, sinks, and concentrations is almost nonexistent in South Asia. Due to the different source strengths, aerosol burdens, and the composition of aerosol and ground surfaces, HONO chemistry in South Asia is expected to have different features from what has been found in Europe or North America. 1 Introduction Atmospheric nitrous acid (HONO) has been studied for a long time, and at a wide range of locations. It can be an important source of hydroxyl free radical (OH) in the atmo- sphere, especially in the morning (Lammel and Cape, 1996; Harrison et al., 1996). Despite its importance, and several decades of research, nitrous acid’s formation mechanism re- mains unclear. Further investigation of the sources of nitrous acid in the atmosphere is important for improving our under- standing of atmospheric chemistry processes. g p y p One source of atmospheric HONO is the gas phase reac- tion of NO with OH (Seinfeld and Pandis, 1998; Lammel, 1999; Kleffmann, 2007). However, gas phase reactions (in- cluding NO with OH and NO2 with peroxy or alkoxy radi- cals – Stockwell and Calvert, 1983) are too slow to explain the atmospheric HONO concentration. Direct emission from combustion engines is another source of HONO in the at- mosphere (Kurtenbach et al., 2001; Kirchstetter et al., 1996; Wormhoudt et al., 2007; Geiger et al., 2002). Ratios of [HONO] to total [NOx] ([NO]+[NO2]) ranging from 0.3%– 0.8% have been observed in direct emissions from vehicle engines. However, observations of [HONO]/[NO2] ratios of a few percent in a wide range of urban atmospheres suggest that direct emission from combustion engines is probably not a major source of HONO. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 6402 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu A number of heterogeneous formation mechanisms of HONO have been proposed (Reactions 1–6): concentrations and high and fresh soot loadings with large surface areas (Arens et al., 2001; Aubin and Abbatt, 2007; Kleffmann et al., 1999), and is thus not important in many real atmospheric conditions with low NO2 and soot concen- trations. NO2(ads) + NO2(ads) + H2O(surface) →HONO(g) + HNO3(aq)(R1) NO2 + NO + H2O(surface) →2 HONO (R2) NO + HNO3(surface) →HONO + NO2 (R3) NO2(g) + Soot →HONO(g) (R4) NO2(g) + VOC(surface) →HONO(g) (R5) NO2 (g) + photo −activated aerosol surface →HONO(g) (R6) NO2(ads) + NO2(ads) + H2O(surface) →HONO(g) + HNO3(aq)(R1) (R2) Reaction (5), the reactions of NO2 with the adsorbed VOCs (i.e. anthrarobin, aromatics) on surfaces, is also a pos- sible source of HONO (Arens et al., 2002; Gutzwiller et al., 2002; Lahoutifard et al. 2002). 1 Introduction For example, the average atmospheric aerosol burden in Kathmandu in winter is much higher than in European and US cities (Sharma et al., 2002; Sharma, 1997; Yu et al., 2008a; Giri et al., 2006). Moreover, strong nocturnal inversion layer (Kondo et al., 2002; Regmi et al., 2003; Panday, 2006; Panday and Prinn, 2009) and the Recently, the reaction of NO2 on soot particles (Reac- tion 4) has been suggested to be a source of HONO in the atmosphere (Aubin and Abbatt, 2007; Alcala-Jornod et al., 2000; Nienow and Roberts, 2006; Ammann et al., 1998; Gerecke et al., 1998; Longfellow et al., 1999; Arens et al., 2001; Al-Abadleh and Grassian, 2000; Aumont et al., 1999; Grassian, 2001, 2002; Karagulian and Rossi, 2007). Soot (or black carbon) aerosols often have a loading of 1.5 to 20 µg m−3 in urban air, mostly from anthropogenic sources, e.g., fossil fuel combustions (IPCC, 2001). The fractal and porous surface of soot provides a large contribution to the total reactive aerosol surface even when soot may have a rel- atively small contribution to the total aerosol mass. Stud- ies using black carbon and soot as a substrate have demon- strated the reactive uptake of NO2 and the formation of NO and HONO (Aubin and Abbatt, 2007; Kleffmann, 2007). Re- action (4) will contribute significantly to the production of at- mospheric HONO only under certain conditions of high NO2 Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ 6403 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu Table 1. HONO and NO2 measurements in Asia. 2 Experimental section low mixing layer height keep pollutants close to the ground, providing a large reactive ground surface for HONO forma- tion. The high aerosol concentrations, the strong inversion layer, and the low inversion height could play an important role in the heterogeneous formation of HONO. 1 Introduction Location Month/Year Type NO2/ppb HONO /ppb HONO/NO2 (Mean) (Mean) (Mean) Beijing/China May, Jun–Jul, urban 0.8–3.1 (Hu et al., 2002) Sep, Dec 2000 Beijing/China Aug–Sep 2004 urban 5–85 0.4–6.1 2%–10% (Qin et al., 2006) Guangzhou/China Jul, Nov 2000 urban 1.0–2.7 (Hu et al., 2002) Hong Kong/Hong Kong Oct–Dec 2002 urban/ ∼1–10 (Zhang et al., 2007) suburban Shanghai/China Oct 2004–Jan 2005 urban 5.5–75.4 0.3–6.8 1%–13% (Hao et al., 2006) (24) (1.1) Xinken/China Oct–Nov 2004 rural 8.2–91.4 0.4–3.8 1.4%–10.2% (Su et al., 2008a,b) (34.8) (1.2) (4.3%) Taichung/Taiwan Jan–Dec 2002 urban 10–46 0.6-4.0 (Lin et al., 2006) (1.5) Taichung/Taiwan Oct 2002 urban (39.8±12) (2.9±1.4) (Cheng et al., 2007) Ren-Ai/Taiwan Oct 2002 Forest (7.8±1.4) (0.2±0.1) (Cheng et al., 2007) Seoul/South Korea Apr 2001–Feb 2002 urban 0.5–9.7 (Kang et al., 2006) (2.8) Seoul/South Korea May 2004 urban (26±14) (1.8±1.4) (Shon et al., 2007) Jun 2004 (22±11) (1.7±1.1) Yangpyung/South Korea May 2004 rural (18±9) (0.2±0.2) (Shon et al., 2007) Jun 2004 (17±9) (0.6±0.8) Kwangju/South Korea Sep–Nov 2001 Semi-urban 0–90 0.02–3.0 2%–6% (Park et al., 2004) Nara/Japan Jun 1994–May 1995 urban 0.4–1.4 (Matsumoto and Okita, 1998) (0.75) Sakai/Japan Nov–Dec 2003 urban 0.1–1.8 (Takenaka et al., 2004) Tsukuba/Japan Feb 1990 rural 5–15 0.2–0.7 (Kanda and Taira, 1990) Kathmandu/Nepal Jan–Feb 2003 urban 2.9–51.6 0.15–7.45 1%–30% (this work) (17.9)1 (1.7)1 (9.5%)1 Table 1. HONO and NO2 measurements in Asia. 1 Only nighttime data 1 Only nighttime data 2.1 Location and instruments We carried out a field campaign in Kathmandu in winter 2003, investigating air quality and atmospheric chemical pro- cesses. NOx (NO, NO2), SO2, O3, HONO, HCHO, PM10, and meteorological parameters were measured during this period. In this study, we present measurements of HONO and NO2 using long path DOAS in Kathmandu, Nepal, over several weeks in 2003. As far as we are aware, this was the first study on HONO and NO2 in the nocturnal atmosphere in South Asia. Our observations provide insights into the par- ticular HONO chemical formation processes taking place in the Kathmandu atmosphere. A home-made long-path DOAS (Chalmers University) was used to measure HONO and NO2 in Kathmandu with a time resolution of around 5–10 min. DOAS measurements were carried out from 10 January to 11 February 2003. The measurement site (27◦43.2190′ N, 85◦21.417′ E) was about 2 km east of the Kathmandu Ring Road, along the main road www.atmos-chem-phys.net/9/6401/2009/ Atmos. Chem. Phys., 9, 6401–6415, 2009 6404 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu ) y p y revised printout. Fig.1 Fig. 1. Map of Kathmandu showing the measurement site. Fig.1 Fig. 1. Map of Kathmandu showing the measurement site. variation of the quantum yield. A digital high pass filter was used to separate narrow absorption structures from the broad- band structures caused by the lamp, Rayleigh and Mie scat- tering, and other instrument effects. Then, a digital low pass filter was applied to reduce high frequency noise. Finally, the spectra were analyzed for NO2 (cross section by Vandaele et al., 2002), HONO (cross section by Stutz et al., 2000), and HCHO (cross section by Meller and Moortgat, 2000) using a nonlinear least-squares method (Stutz and Platt, 1996) in the spectral range of 310–380 nm with a spectra resolution of 0.4 nm. The sources of errors of the measured HONO and NO2 mixing ratios include the error (5% for HONO and 3% for NO2) of the cross sections, the spectral fit error (less than 5%), and the experimental error of the DOAS system (5%). The total errors for the measured HONO and NO2 mixing ratios are estimated to be around 9% and 8%, respectively. from Kathmandu to Sankhu (Fig. 1). 2.1 Location and instruments A DOAS telescope, a NOx analyzer (Model 42S Thermo Environmental Instru- ments Inc.), an O3 monitor (2B Technologies), and a PM10 aerosol monitor (TSI Dusttrak) were installed on the 5th floor of a hotel (15 m above ground). DOAS retro-reflectors were mounted at the top of a 7-story building, 25 m above ground and 957 m away from the DOAS telescope, giving a DOAS light path of 1914 m. Low buildings, roads, and some bare fields were under the light path. An automated weather sta- tion (Rainwise), which measured temperature, wind speed, wind direction, relative humidity, dew point temperature, and solar radiation, was installed on the roof of the hotel (22 m above ground) near the DOAS telescope and other instru- ments. Details of the DOAS instrument and the experimental sites have been described in earlier papers (Yu et al., 2008a., 2004). from Kathmandu to Sankhu (Fig. 1). A DOAS telescope, a NOx analyzer (Model 42S Thermo Environmental Instru- ments Inc.), an O3 monitor (2B Technologies), and a PM10 aerosol monitor (TSI Dusttrak) were installed on the 5th floor of a hotel (15 m above ground). DOAS retro-reflectors were mounted at the top of a 7-story building, 25 m above ground and 957 m away from the DOAS telescope, giving a DOAS light path of 1914 m. Low buildings, roads, and some bare fields were under the light path. An automated weather sta- tion (Rainwise), which measured temperature, wind speed, wind direction, relative humidity, dew point temperature, and solar radiation, was installed on the roof of the hotel (22 m above ground) near the DOAS telescope and other instru- ments. Details of the DOAS instrument and the experimental sites have been described in earlier papers (Yu et al., 2008a., 2004). In our experiments, several steps were taken before the spectral retrieval to determine concentrations of HONO, NO2, and other gases. The spectra were corrected for elec- tronic offset and light from sources other than the Xe arc lamp. The spectra were divided by a recorded halogen lamp spectrum, which only contains broadband structures, to elim- inate spectral structures originating from the pixel-to-pixel 2.2 Retrieval method of aerosol parameters With such limitations, the results of aerosol parameters are only approximations of the true aerosol situation. Neverthe- less, the retrieval results of aerosol surface and volume (as- suming a refractive index n =1.5 – Yu et al., 2008b) agree well with the trends seen in our PM10 data (Fig. 2), which confirms the reliability of our calculated aerosol parameters. τ(λ) = −ln  I(λ) I0(λ)  (1) The aerosol mass in this study was calculated from a typical urban aerosol density of 1.4 ± 0.5 g cm−3 (Pitz et al., 2003) and the measured aerosol volume (∼ 1×102 µm3 cm−3, 00:00–04:00 in Kathmandu in Fig. 2). We found the average atmospheric aerosol mass concentration at midnight in Kathmandu to be 140 ± 50 µg m−3. However, light attenuation only gives the aerosol information (aerosol diameters, surface and volume) for fine particles with diam- eters smaller than 1 µm. Assuming a PM1/PM10 mass ratio of 50% in urban air (Vallius et al., 2000; Ehrlich et al., 2007; Ariola et al., 2006; Spindler et al., 2004; Labban et al., 2004; Li and Lin, 2002; Gomiscek et al., 2004; Liu et al., 2004), the calculated aerosol mass by light attenuation is in agreement with our measured PM10 values. (1) Here I(λ) is the light intensity at the end of the full path length while I0(λ) is the background light intensity at 1 m path length. Inversion of the optical depth is performed us- ing a look-up table (Box and Lo, 1976). The spectral fit at 330–390 nm region was based on an assumed exponential wavelength-dependent function for optical depth: τexp(λ) = β · e−a·λ−k (2) (2) where τexp (λ) is the optical depth, α and β are the fitting parameters, and k is the offset constant. The offset k must be considered because of instrument factors such as the reflect- ing ratio of retroreflectors. Using α, β, and the pre-calculated tables of Box and Lo (1976), values of a and b can be obtained for the calcu- lation of Deirmendjian’s particles size distribution n(r) (Box and Lo, 1976; Notholt and Raes, 1990; Notholt et al., 1992b, 1991): 2.2 Retrieval method of aerosol parameters To investigate the role of aerosols in HONO formation, mea- surements of aerosol parameters such as the aerosol surface density are required, along with the ambient concentration of HONO and NO2. Box and Lo (1976) developed a simple Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ 6405 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu al., 1996; Notholt et al., 1992a; Notholt and Raes, 1990; Box and Lo, 1976). The total error of our results was estimated to be 55%. The sources are: (1) The unknown refractive index of the aerosols (we have tested different indices from 1.33 to 1.70. Trends with different indices are similar but with different values) could contribute an uncertainty as large as 50% (Box and Lo, 1976); (2) The relatively narrow spectral range compared to the application by Notholt et al. (1992a) gave 20% uncertainty as reported by Reisinger (2000); (3) The deviation of the true aerosol size distribution from the calculated results by the model (Deirmendjian’s particle size distribution) could introduce an additional uncertainty; (4) The consistency of the lamp intensity and DOAS mechanical stability is sufficient during a time period of a few weeks but with an uncertainty of 10% (Andres-Hernandez et al., 1996; Notholt et al., 1992a). inversion method to calculate the approximate aerosol size distribution from light attenuation. This has been further ap- plied to calculate other aerosol parameters such as the surface density and the volume using DOAS measurements (Notholt et al., 1991, 1992a; Notholt et al., 1992b; Notholt and Raes, 1990; Flentje et al., 1997; Reisinger, 2000). Light transmission measurements through the atmosphere can yield bulk aerosol parameters such as diameters, num- ber density, total surface, and total volume of particles (Liv- ingston and Russell, 1989). In Kathmandu, we used the DOAS system to measure the light transmission, aerosol pa- rameters and trace gas concentrations simultaneously in the same volume of air. The spectra were recorded in the 250–390 nm range. The range of 330 nm to 390 nm was used to retrieve concentra- tions of HONO, NO2 and aerosol parameters simultaneously. A background spectrum was recorded over a 1 m optical path using retroreflectors. Given the concentrations of all gases provided by the DOAS spectra retrieval processes (SO2, O3, NO2, HONO, and HCHO), the optical depth (τ) at wave- length λ can be calculated according to Eq. (1). 3.1 Time series of HONO, other pollutants and meteorological data Behavior of [NO2], [HONO], [HONO] / [NO2], PM10, and meteorological Feb 3 Feb 4 Feb 5 Feb 6 Feb 7 0 20 40 60 0.0 0.5 1.0 0 2 4 6 0 180 360 0 200 400 NO2 NO [NOx ] ppb Local time 0 2 4 HONO [HONO] ppb 0 15 30 [HONO]/[NO2] [HONO]/[NO2] % PM10 PM10 mg m -3 Wind speed Wind Speed m s -1 Wind direction Wind direction Radiation Radiation w m -2 Figure 3. Behavior of [NO2], [HONO], [HONO] / [NO2], PM10, and meteorological ( i d d i d di i d l di i ) d i b 3 i Fig. 3. Behavior of [NO2], [HONO], [HONO]/[NO2], PM10, and meteorological parameters (wind speed, wind direction, and solar radiation) during 3–7 February in Kathmandu urban air. The error bars are 1σ standard deviation. 28 28 dard deviation. by the valley’s ventilation pattern (Panday, 2006; Panday and Prinn, 2009). The correlation of HONO with NO2 and PM10 (especially after sunset) in Fig. 3 suggests three possi- bilities: (1) the same emission source; (2) chemical conver- sion of NO2 to HONO associated with aerosols; (3) all three species have their sources near the ground (not necessarily the same sources). The HONO formation mechanism will be discussed in later sections. Kathmandu urban air. The error bars are 1σ st considerable political instability in Nepal, with frequent ve- hicle searches at night, night-time driving was quite low and thus the night-time sources of NO were limited. Peaks in NO2 appeared concurrently with the NO peaks at 07:00– 08:00 and 19:00–20:00. NO2 values were around 10–20 ppb at night, and started to increase around 03:00–04:00. HONO concentrations climbed to several ppb after sunset and then decreased to around 1–1.5 ppb. It increased again after 03:00. PM10 shows elevated values (up to 1 mg m−3) during the morning and evening. The diurnal variation of the observed wind pattern shows a long calm period from sunset (∼18:00) until around 10:00 each day. The morning and evening peaks in NOx and PM10 did not correspond to rush hour alone. In particular, the evening peaks started after the rush hour. The timing of pollutant peaks in Kathmandu is determined Table 2 shows several pollutants measured in Kath- mandu’s urban air. Elevated mixing ratios of HONO (up to ∼7.5 ppbv) were observed during the whole measurement period. 3.1 Time series of HONO, other pollutants and meteorological data 0 1 2 3 Feb 4 Feb 5 Feb 6 Feb 7 Feb 8 Feb 9 Feb 10 0.0 0.1 0.2 0.3 Local time Aerosol volume X10 4µm 3cm -3 0.0 0.5 P mg Aerosol surface X10 4µm 2cm -3 Figure 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. Figure 2. PM10, aerosol surface and volume measured in Fig. 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. Figure 2. PM10, aerosol surface and volume measure Feb 3 Feb 4 Feb 5 Feb 6 Feb 7 0 20 40 60 0.0 0.5 1.0 0 2 4 6 0 180 360 0 200 400 NO2 NO [NOx ] ppb Local time 0 2 4 HONO [HONO] ppb 0 15 30 [HONO]/[NO2] [HONO]/[NO2] % PM10 PM10 mg m -3 Wind speed Wind Speed m s -1 Wind direction Wind direction Radiation Radiation w m -2 Figure 3. Behavior of [NO2], [HONO], [HONO] / [NO2], PM10, and meteorological parameters (wind speed, wind direction, and solar radiation) during February 3-7 in Kathmandu urban air. The error bars are 1σ standard deviation. Feb 3 Feb 4 Feb 5 Feb 6 Feb 7 0 20 40 60 0.0 0.5 1.0 0 2 4 6 0 180 360 0 200 400 NO2 NO [NOx ] ppb Local time 0 2 4 HONO [HONO] ppb 0 15 30 [HONO]/[NO2] [HONO]/[NO2] % PM10 PM10 mg m -3 Wind speed Wind Speed m s -1 Wind direction Wind direction Radiation Radiation w m -2 Figure 3. Behavior of [NO2], [HONO], [HONO] / [NO2], PM10, and meteorological parameters (wind speed wind direction and solar radiation) during February 3-7 in Fig. 3. Behavior of [NO2], [HONO], [HONO]/[NO2], PM10, and meteorological parameters (wind speed, wind direction, and solar radiation) during 3–7 February in Kathmandu urban air. The error bars are 1σ standard deviation. Feb 3 Feb 4 Feb 5 Feb 6 Feb 7 0 20 40 60 0.0 0.5 1.0 0 2 4 6 0 180 360 0 200 400 NO2 NO [NOx ] ppb Local time 0 2 4 HONO [HONO] ppb 0 15 30 [HONO]/[NO2] [HONO]/[NO2] % PM10 PM10 mg m -3 Wind speed Wind Speed m s -1 Wind direction Wind direction Radiation Radiation w m -2 Figure 3. 3.1 Time series of HONO, other pollutants and meteorological data Continuous measurements were carried out during January and February, 2003. Figure 3 shows, as an example from our one-month observation, the behavior of HONO, NOx, [HONO]/[NO2], PM10, and meteorological parameters (ra- diation, wind speed and wind direction) during a four day period. The wind direction becomes unimportant as wind speeds drop to zero during our observation. NO2 and HONO measurements by the long path DOAS show gaps every morning and on some evenings due to heavy fog and smog. n(r) = 1 2 · a · b3 · r2 · e−b·r (3) (3) where r is the particle radius, and the constants a and b are specified for different particles types. By using the same method as described by Notholt et al. (1992a) but with a narrower spectral range, we calcu- lated the aerosol size distribution and further obtained the total number, surface density and the volume of aerosols in time intervals of 5–10 min. The concentration of NO had two peaks (with values as high as 60 ppb) at 07:00–08:00 and 19:00–20:00, respec- tively, and remained low (a few ppb except for short dura- tion plumes) at night. As our campaign was at a time of Our light attenuation retrieval processes give a large un- certainty of aerosol parameters. Errors in this method have been extensively discussed by others (Andres-Hernandez et Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ 6406 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu 0 1 2 3 Feb 4 Feb 5 Feb 6 Feb 7 Feb 8 Feb 9 Feb 10 0.0 0.1 0.2 0.3 Local time Aerosol volume X10 4µm 3cm -3 0.0 0.5 1.0 PM10 mg m -3 Aerosol surface X10 4µm 2cm -3 Figure 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. Feb 3 Feb 4 Feb 5 Feb 6 Feb 7 0 20 40 60 0.0 0.5 1.0 0 2 4 6 0 180 360 0 200 400 NO2 NO [NOx ] ppb Local time 0 2 4 HONO [HONO] ppb 0 15 30 [HONO]/[NO2] [HONO]/[NO2] % PM10 PM10 mg m -3 Wind speed Wind Speed m s -1 Wind direction Wind direction Radiation Radiation w m -2 Figure 3. Behavior of [NO2], [HONO], [HONO] / [NO2], PM10, and meteorological parameters (wind speed, wind direction, and solar radiation) during February 3-7 in Kathmandu urban air. The error bars are 1σ standard deviation. Fig. 2. 3.1 Time series of HONO, other pollutants and meteorological data PM10, aerosol surface and volume measured in Kathmandu, 2003. 0 1 2 3 Feb 4 Feb 5 Feb 6 Feb 7 Feb 8 Feb 9 Feb 10 0.0 0.1 0.2 0.3 Local time Aerosol volume X10 4µm 3cm -3 0.0 0.5 PM mg m Aerosol surface X10 4µm 2cm -3 Figure 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. Feb 3 Feb 4 Feb 5 Feb 6 Feb 7 0 20 40 60 0.0 0.5 1.0 0 2 4 6 0 180 360 0 200 400 NO2 NO [NOx ] ppb Local time 0 2 4 HONO [HONO] ppb 0 15 30 [HONO]/[NO2] [HONO]/[NO2] % PM10 PM10 mg m -3 Wind speed Wind Speed m s -1 Wind direction Wind direction Radiation Radiation w m -2 Figure 3. Behavior of [NO2], [HONO], [HONO] / [NO2], PM10, and meteorological parameters (wind speed, wind direction, and solar radiation) during February 3-7 in Fig. 3. Behavior of [NO2], [HONO], [HONO]/[NO2], PM10, and meteorological parameters (wind speed, wind direction, and solar radiation) during 3–7 February in Kathmandu urban air. The error bars are 1σ standard deviation. 0 1 2 3 Feb 4 Feb 5 Feb 6 Feb 7 Feb 8 Feb 9 Feb 10 0.0 0.1 0.2 0.3 Local time Aerosol volume X10 4µm 3cm -3 0.0 0.5 1.0 PM10 mg m -3 Aerosol surface X10 4µm 2cm -3 Figure 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. Fig. 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. 0 1 2 3 Feb 4 Feb 5 Feb 6 Feb 7 Feb 8 Feb 9 Feb 10 0.0 0.1 0.2 0.3 Local time Aerosol volume X10 4µm 3cm -3 0.0 0.5 PM mg Aerosol surface X10 4µm 2cm -3 Figure 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. 0 1 2 3 Feb 4 Feb 5 Feb 6 Feb 7 Feb 8 Feb 9 Feb 10 0.0 0.1 0.2 0.3 Local time Aerosol volume X10 4µm 3cm -3 0.0 0.5 1.0 PM10 mg m -3 Aerosol surface X10 4µm 2cm -3 Figure 2 PM10 aerosol surface and volume measured in Kathmandu 2003 Fig. 2. PM10, aerosol surface and volume measured in Kathmandu, 2003. Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu 6407 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu Table 2. Pollutants in Kathmandu urban air. Pollutant Mean (ppbv)1 Minimum Maximum Total SD Day2 SD Night3 SD (ppbv)1 (ppbv)1 NO2 14.14 9.77 8.59 5.65 17.94 10.23 0.692 70.11 NO 3.16 6.41 4.41 7.51 2.17 5.19 0.011 162.72 HONO 1.55 1.57 0.351 0.444 1.736 0.804 0.026 7.45 PM10 0.197 0.153 0.216 0.193 0.182 0.109 0.011 1.27 1 PM10 unit is mg m−3; 2 daytime: 07:00–18:00; 3nocturnal: 18:00–07:00 Table 2. Pollutants in Kathmandu urban air. 7:00 0 20 40 60 0 2 4 6 [HONO]=0.081*[NO2] [HONO]=0.008*[NO2] HONO (ppb) NO2 (ppb) [HONO]=0.30*[NO2] O] vs. [NO2] in the nocturnal atmospheric boundary layer error bars are 1σ standard deviation. Fig. 4. [HONO] vs. [NO2] in the nocturnal atmospheric boundary layer at an urban site of Kathmandu. The error bars are 1σ standard deviation. after sunset; (3) insufficient dispersion of polluted air mass due to reduced wind speed; (4) the ceasing of photolysis of NO2 and HONO after sunset. 0 20 40 60 0 2 4 6 [HONO]=0.081*[NO2] [HONO]=0.008*[NO2] HONO (ppb) NO2 (ppb) [HONO]=0.30*[NO2] 3.2 High atmospheric [HONO]/[NO2] values in Kathmandu at night In many field studies, the value of [HONO]/[NO2] is used as an index to estimate the efficiency of heterogeneous NO2- HONO conversion because it is less influenced by transport processes than individual concentrations. Figure 3 shows that in Kathmandu, [HONO]/ [NO2] climbed after sunset and reached its highest point around 02:00–03:00. The high- est [HONO]/[NO2] values during 3 to 7 February were from 15% to 28% between 00:00 and 03:00. The diurnally re- peating trends of [NOx], [HONO] and [HONO]/[NO2] are most likely due to diurnally repeating wind profiles, emis- sion sources, boundary layer dynamics and chemical pro- cesses. During afternoon rush hours and other plume events (around 19:00, 20:00, and 21:00), [HONO] followed the same pattern as [NO2] (Fig. 3). However, during those times a lower [HONO]/[NO2] value was observed compared to a few hours later, similar to the observation in Houston (Stutz et al., 2004). It is likely that conversion of NO2 to HONO in relatively freshly polluted air has not proceeded as far as in the aged air in this period. Figure 4. [HON Kathmandu. Th ] vs. [NO2] in the nocturnal atmospheric boundary layer error bars are 1σ standard deviation. Fig. 4. [HONO] vs. [NO2] in the nocturnal atmospheric boundary layer at an urban site of Kathmandu. The error bars are 1σ standard deviation. 3.1 Time series of HONO, other pollutants and meteorological data The elevated HONO concentrations in the presence of high [NO], [NO2] and PM10 values right after sunset ap- pear to be the result of several processes: (1) direct emission at evening rush hours; (2) decrease of boundary layer height Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ 3.3 Contribution of direct emissions to the observed HONO It is necessary to separate HONO formed by chemical re- actions from HONO that is directly emitted (mainly from vehicles; the contribution from industrial combustion pro- cesses is not clear). No previous study reported the ratio of [HONO]/[NOx] in emissions in Kathmandu. The highest re- ported average emission ratio anywhere in the recent litera- ture was (0.8±0.1) % (Kurtenbach et al., 2001). Based on the observation of HONO, NOx and [HONO]/[NO2] in this study, it is believed that direct emission was not the major source of HONO in Kathmandu. The reasons are: first, the [HONO]/[NO2] ratio was low (around a few %) after sun- set when emissions were high and high NOx concentrations were observed. Second, NOx concentrations were low in the middle night, ruling out the possibility of direct emissions as the major source of HONO. Third, as we see in Fig. 4, the A scatter plot of nocturnal [HONO] and [NO2] is shown in Fig. 4. The regression coefficient, maximum and minimum ratio of [HONO] to [NO2] were 0.081, 0.30 and 0.008, re- spectively. The maximum [HONO]/[NO2] ratio (30%) found in our observation is, to our knowledge, the highest reported atmospheric HONO to NO2 ratio, with the exception of day- time findings in and above snow (Zhou et al., 2001). The high ratios of [HONO]/[NO2] in Kathmandu at night indi- cate high HONO direct emissions, or highly efficient NO2 to HONO chemical conversion, or both. In the next sections, we discuss the contribution of direct emissions and chemical processes separately. www.atmos-chem-phys.net/9/6401/2009/ Atmos. Chem. Phys., 9, 6401–6415, 2009 6408 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu minimum value of [HONO]/[NO2] was ∼0.8%; correspond- ing to the maximum possible contribution by HONO direct emissions. mained approximately constant (120 m) from 00:00 to 06:00. It has to be noted that mixing layer height measurements by SODAR are less accurate at night than during the day. The high value of [HONO]/[NO2] (upto 20% during most nights, exceeding 25% during some nights, and reaching a maximum of 30% during one night) is thus mostly due to secondary HONO. The porosity of aerosols and the roughness of the ground surface were not considered in this study. 3.3 Contribution of direct emissions to the observed HONO The area densi- ties of aerosol surfaces and ground reactive surface might be higher (up to 10 times) (Lammel and Cape, 1996; Andr´es- Hern´andez et al., 1996) than the values calculated from light attenuation and estimation by mixing layer height, respec- tively. This maximum ratio of 30% is substantially higher than the previously observed ratios at other urban and suburban sites in Europe, North America and Asia (i.e., 1–12% re- ported by Lammel and Cape (1996), 19% reported by Wang et al. (2003) during dust storms, and 1–13% around Asia – Qin et al., 2006; Park et al., 2004; Hao et al., 2006). Figure 5a shows the behaviors of [HONO]/[NO2], and during a selected night. The black line in Fig. 5a is the com- bination of the two surface area densities. [HONO]/[NO2] increased after sunset and remained relatively constant af- ter 00:00, then decreased after 03:00. S V  aerosol increased quickly after sunset for a short period (17:00–19:00), and then stayed nearly constant until 03:00. S V  aerosol, as well as PM10, dramatically increased after 03:00. The change of S V  aerosol has a positive correlation with [HONO]/[NO2] right after sunset. However, for the period of 17:00 to 03:00, S V  aerosol has a poor correlation (R2= 0.33) to [HONO]/[NO2] (see Fig. 5b), which disagree with obser- vations in some field measurements elsewhere (Notholt et al., 1992a; Reisinger, 2000). It is interesting that S V  ground shows good correlation with [HONO]/[NO2] (R2= 0.93) for the whole night except between 03:00 and 06:00 in Fig. 5b. The good correlation between [HONO]/[NO2] and S V  ground indicates that the NO2 heterogeneous reaction on the ground reactive surface was a major source of HONO in Kathmandu. Our observation of ground reactive surface as a source of at- mospheric nitrous acid is consistent with other field observa- tions (Harrison and Kitto, 1994; Harrison et al., 1996; Kleff- mann et al., 2003; Vogel et al., 2003). 3.4 NO2-HONO chemical conversion and correlation studies The contribution by NO to the formation of HONO through Reactions (2–3) could be ruled out for Kathmandu late at night because of the low NO concentrations. According to HONO formation mechanisms (Reactions 1 to 6 in the in- troduction), the major source of HONO in the nocturnal air in Kathmandu was the NO2 to HONO chemical conversion through heterogeneous reactions (i.e. Reactions 1, 4, and 5). HONO formation has been found to be a first order reac- tion in NO2 (Finlayson-Pitts et al., 2003; Kleffmann et al., 1998a). It depends on the surface absorbed water, and in- creases as the reactive surface to air volume ratio (S/V) in- creases (Stutz et al., 2004; Trick, 2004). The value of S/V is determined by a combination of the aerosol surface, ground reactive surface and the influence of vertical mixing (Febo et al., 1996; Trick, 2004). Here we investigate S/V ratio and relative humidity in the nocturnal atmosphere in Kathmandu and their influence on HONO formation. The correlation of HONO with other pollutants in Fig. 3 indicates the following. (1) the presence of all the com- pounds in the same air mass; (2) a chemical conversion of NO2 to HONO associated with aerosols; (3) all three species have their sources near the ground (not necessarily the same sources). In order to investigate the efficiency of NO2 to HONO chemical conversion without the influence of other processes (i.e. transport), we study the correlation of [HONO]/ [NO2] instead of [HONO] to the other parameters. Although the general correlation of [HONO]/[NO2] with S V  aerosol was poor, the positive correlation in the early hours after sunset (17:00-19:00) points to the possible contribution of aerosols to HONO formation under certain conditions. Recently many researchers have reported the fast conversion of NO2 on fresh soot surface to form HONO (Reaction 4). Since the soot surface is shown to rapidly deactivate as the reaction proceeds, it is believed to be an insignificant source of HONO in the real atmosphere. However, the aerosol con- centrations in Kathmandu air are much higher than in those cities, where research on this topic was carried out. It is thus worthy to investigate the potential impact of soot on HONO formation in Kathmandu atmosphere. 3.4.2 Relative humidity (RH) The RH dependent HONO formation has been studied in lab- oratory research (Harrison and Collins, 1998) and in field observations (Stutz et al., 2004). Syomin and Finlayson- Pitts (2003) have studied the decomposition of HONO un- der different RH conditions. The RH in the winter of Kath- mandu increased from a sunset value of ∼60% to ∼100% by 03:00 and remained close to 100% until sunrise during most nights. Figure 6 (panels A, B) shows the time series of [HONO]/[NO2], RH, and temperature in Kathmandu during the entire observation period. The red lines indicate the mean values. While the increasing RH (from 60% to 95%) ac- companied increasing [HONO]/[NO2], [HONO]/[NO2] val- ues decreased after RH reached ∼100%. Fig. 5. (a) Observed [HONO]/[NO2] (square), aerosol surface den- sity (open circle), calculated [HONO]/[NO2] due to Reaction (4) (blue triangle), ground reactive surface density (red line) and total surface density or (S/V)aerosol + (S/V)ground (black line) at 21–22 January 2003. (b) Linear correlations of [HONO]/[NO2] to aerosol surface density and ground reactive surface density. However, the correlation of [HONO]/[NO2] with RH in Fig. 6 might not be a proof for the humidity dependence of HONO formation in Kathmandu atmosphere. The in- creasing RH after sunset was the result of the decreasing temperature (panel C in Fig. 6). Meanwhile, the increasing [HONO]/[NO2] during the night was due to the combination of ceasing photochemistry, heterogeneous formation and a decreasing boundary layer height. Simultaneous increase of RH and [HONO]/[NO2] as shown in Fig. 6 might be two in- dependent processes. In order to test the influence of RH on the NO2-HONO conversion reactions, we study the average HONO formation rate constant during the night against the average humidity. Kathmandu valley, and found that the aerosol featured a dominant contribution from carbon, likely from fuel combus- tion and biomass burning processes in the Kathmandu valley (Carrico et al., 2003). More recent research shows elevated levels of black carbon and organic carbon in the Kathmandu urban air from September 2004 to August 2005 (Adhikary et al., 2007; Ramanathan et al., 2007). It has been esti- mated that brick-kilns are responsible for about 28% of an- nual PM10 emissions in the valley, almost all of which is con- centrated during the winter months (World Bank, 1997). 3.4.1 Aerosol surface and ground reactive surface Long path DOAS measurements can provide information about aerosol surface-to-air volume ratios (see Sect. 2). The ground reactive surface area density is defined in this paper as the inverse value of the mixing layer height (H−1) (Harri- son et al., 1996). We use average mixing layer heights from Sonic Detection & Ranging (SODAR ) measurements (Pan- day, 2006). The average measured mixing layer height de- ceased from 12:00 (800 m) until 00:00 (120 m), and then re- Although aerosol chemical properties in the Kathmandu urban area were not provided by our measurements, liter- ature and many reports in local newspapers suggest that large amounts of soot are often present in the winter Kath- mandu urban air. Some researchers have attributed the dark haze over Kathmandu valley to the presence of soot particles (Sapkota and Dhaubhadel, 2002). Others investigated the aerosol chemical properties in Nagarkot, at the rim of the Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu 6409 Fig. 5. (a) Observed [HONO]/[NO2] (square), aerosol surface den- sity (open circle), calculated [HONO]/[NO2] due to Reaction (4) (blue triangle), ground reactive surface density (red line) and total surface density or (S/V)aerosol + (S/V)ground (black line) at 21–22 January 2003. (b) Linear correlations of [HONO]/[NO2] to aerosol surface density and ground reactive surface density. cles were soot, we got an upper estimate of the average soot particle surface. The calculated [HONO]/[NO2] values cor- responding to Reaction (4) are plotted in Fig. 5a (triangles). The maximum HONO formed by NO2 conversion on soot is found to be ∼(0.3 ± 0.2) ppb, which corresponds to a [HONO]/[NO2] ratio of 3 ± 2%. Compared to our observa- tions during most parts of the nights, this is a very small con- tribution. However, for the early hours after sunset (17:00– 19:00), the majority of the observed [HONO]/[NO2] ratio (below 5%) could come from Reaction (4) on soot particles. Despite the fact that soot may play a role in NO2-HONO con- version in Kathmandu right after sunset based on our calcula- tion, we should note that: (1) there is inadequate information about aerosol chemical composition; (2) the contribution of this process to total nocturnal atmospheric HONO formation is most likely limited. 3.4.2 Relative humidity (RH) Normalized HONO formation rate constants decrease with the increasin open circles are individual HONO formation values of each night. Black dots are values of HONO formation in RH range of 65-75%, 75-85%, 85-95% and 95%-1 bars are the standard deviations. Fig. 7. Normalized HONO formation rate constants decrease with the increasing RH. Red open circles are individual HONO forma- tion values of each night. Black dots are average values of HONO formation in RH range of 65–75%, 75–85%, 85–95% and 95– 100%. Error bars are the standard deviations. 60 70 80 90 100 0.5 1.0 1.5 2.0 2.5 d([HONO]/[NO2])/dt (a.u.) RH (%) alized HONO formation rate constants decrease with the incre individual HONO formation values of each night. Black dots O formation in RH range of 65-75%, 75-85%, 85-95% and 95% ndard deviations. Fig. 7. Normalized HONO formation rate constants decrease with the increasing RH. Red open circles are individual HONO forma- tion values of each night. Black dots are average values of HONO formation in RH range of 65–75%, 75–85%, 85–95% and 95– 100%. Error bars are the standard deviations. 10 20 30 10 20 60 80 100 [HONO]/[NO2] (%) A Temperature ( oC) Local time C Relative humidity (%) 18:00 21:00 0:00 03:00 06:00 B 6 Fi 7 N 60 70 80 90 100 0.5 1.0 1.5 2.0 2.5 d([HONO]/[NO2])/dt (a.u.) RH (%) alized HONO formation rate constants decrease with the increa individual HONO formation values of each night. Black dots O formation in RH range of 65-75%, 75-85%, 85-95% and 95% ndard deviations. Fig. 7. Normalized HONO formation rate constants decrease with the increasing RH. Red open circles are individual HONO forma- tion values of each night. Black dots are average values of HONO formation in RH range of 65–75%, 75–85%, 85–95% and 95– 100%. Error bars are the standard deviations. Fig. 6. [HONO]/[NO2], relative humidity, and temperature in the nocturnal Kathmandu atmosphere during the whole observation pe- riod. Lines with plus marks show average values. Figure 7. No open circles values of HO bars are the s an “aqueous” surface affects the reaction efficiency of NO2- HONO conversion (Stutz et al., 2004; Lammel, 1999), which is in accordance with our observation. kRH is an assumed RH dependant HONO formation rate con- stant. The water uptake processes can take place on the ground surface (major HONO formation source in this paper) as well as aerosol surfaces. 3.4.2 Relative humidity (RH) The operation of 125 brick kilns in the Kathmandu valley during the dry season from December to April evidently contributes to the aerosol pollution, including black carbon emissions (Dhakal, 2003; Adhikary et al., 2007). HONO formation has been found to be first order in NO2, dependent on the surface absorbed water, and increasing with aerosol surface to air volume ratio (Kleffmann et al., 1998b; Sumner et al., 2004; Finlayson-Pitts et al., 2003). We can thus use Eq. (4) to describe the HONO formation at night: In this study, we used an integrated HONO for- mation rate for unit surface area of soot particles, 1.3×1014 molecules cm−2 (average of the values provided by Aubin and Abbatt, 2007; Kleffmann and Wiesen, 2005; Kirchner et al., 2000). By assuming that all observed parti- d[HONO] dt = kRH × [NO2] × S V (4) d[HONO] (4) Atmos. Chem. Phys., 9, 6401–6415, 2009 Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu p g p p 6410 10 20 30 10 20 60 80 100 [HONO]/[NO2] (%) A Temperature ( oC) Local time C Relative humidity (%) 18:00 21:00 0:00 03:00 06:00 B g.6 Fig. 6. [HONO]/[NO2], relative humidity, and temperature in the nocturnal Kathmandu atmosphere during the whole observation pe- riod. Lines with plus marks show average values. Figure 7. No open circles a values of HO bars are the s 60 70 80 90 100 0.5 1.0 1.5 2.0 2.5 d([HONO]/[NO2])/dt (a.u.) RH (%) malized HONO formation rate constants decrease with the increa e individual HONO formation values of each night. Black dots a O formation in RH range of 65-75%, 75-85%, 85-95% and 95% ndard deviations. Fig. 7. Normalized HONO formation rate constants decrease with the increasing RH. Red open circles are individual HONO forma- tion values of each night. Black dots are average values of HONO formation in RH range of 65–75%, 75–85%, 85–95% and 95– 100%. Error bars are the standard deviations. 10 20 30 10 20 60 80 100 [HONO]/[NO2] (%) A Temperature ( oC) Local time C Relative humidity (%) 18:00 21:00 0:00 03:00 06:00 B ig.6 Fig. 6. [HONO]/[NO2], relative humidity, and temperature in the nocturnal Kathmandu atmosphere during the whole observation pe- riod. Lines with plus marks show average values. 60 70 80 90 100 0.5 1.0 1.5 2.0 2.5 d([HONO]/[NO2])/dt (a.u.) RH (%) Figure 7. 3.4.2 Relative humidity (RH) As many earlier papers reported, water droplets (on ground or aerosol) at a RH of over 96% play a role in the HONO sink, leading to lower [HONO]/[NO2] values, which is in agreement with our observation (Lammel and Perner, 1988; Zhou et al., 2007; He et al., 2006; Acker et al., 2005, 2001; Lammel, 1999). Here we determine the HONO formation by assuming a linear increase of its mixing ratio during the time period of 18:00–00:00 in Kathmandu air. The estimation of the HONO formation rate constant in each individual night is based on the equation introduced by Alicke et al. (2002): kRH = [HONO]t2 −[HONO]t1 (t2 −t1)[NO2]night S V (5) (5) The average nighttime (18:00–00:00) HONO formation rate is (0.014 ± 0.004) (ppb HONO per hour per ppb NO2) during our observation period, which is slightly higher than the ob- servations in Milan (Alicke et al. 2002). The HONO forma- tion rate constant is normalized by the S/V, and then plotted against the average RH value of each individual night (see Fig. 7). The HONO formation evidently decreases as RH in- creases from 65–100% during the night in Kathmandu. The HONO formation in RH range of 95–100% is only about half of the value in RH range of 65–75%. References Beine, H. J., Amoroso, A., Domin, F., King, M. D., Nardino, M., Ianniello, A., and France, J. L.: Surprisingly small HONO emis- sions from snow surfaces at Browning Pass, Antarctica, Atmos. Chem. Phys., 6, 2569–2580, 2006, http://www.atmos-chem-phys.net/6/2569/2006/. Acker, K., M¨oller, D., Wieprecht, W., Auel, R., Kalass, D., and Tscherwenka, W.: Nitrous and nitric acid measurements inside and outside of clouds at Mt. Brocken, Water Air Soil Poll., 130, 331–336, 2001. Bejan, I., Abd El Aal, Y., Barnes, I., Benter, T., Bohn, B., Wiesen, P., and Kleffmann, J.: The photolysis of ortho-nitrophenols: a new gas phase source of HONO, Phys. Chem. Chem. Phys., 8, 2028–2035, 2006. Acker, K., M¨oller, D., Auel, R., Wieprecht, W., and Kalass, D.: Concentrations of nitrous acid, nitric acid, nitrite and nitrate in the gas and aerosol phase at a site in the emission zone during ESCOMPTE 2001 experiment, Atmos. Res., 74, 507–524, 2005. Box, M. A. and Lo, S. Y.: Approximate determination of aerosol size distributions, J. Appl. Meteorol., 15, 1068–1076, 1976. Adhikary, B., Carmichael, G. R., Tang, Y., Leung, L. R., Qian, Y., Schauer, J. J., Stone, E. A., Ramanathan, V., and Ramana, M. V.: Characterization of the seasonal cycle of south Asian aerosols: A regional-scale modeling analysis, J. Geophys. Res., 112, D22S22, doi:10.1029/2006JD008143, 2007. Carrico, C. M., Bergin, M. H., Shrestha, A. B., Dibb, J. E., Gomes, L., and Harris, J. M.: The importance of carbon and mineral dust to seasonal aerosol properties in the Nepal Himalaya, Atmos. En- viron., 37, 2811–2824, 2003. 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To our knowledge, this was the first time that HONO was measured in South Asia. Very high values of chemically formed HONO to NO2 ra- tios were found (up to 30%), which is higher than any other previous atmospheric observations that we are aware of. This negative RH dependence of HONO formation in our observations is most likely due to HONO lost in the grow- ing aqueous layer on the ground. The water adsorption on the ground surface can be determined by BET isotherm. The number of monolayers formed by absorbed water on vari- ous surfaces increases rapidly when RH reaches 60% (Sum- ner et al., 2004; Lammel, 1999). Lammel (1999) had re- ported that the water coverage of various materials (stone, soil, and vegetation) increases dramatically (equivalent to up to 100 monolayers) when RH is over 95%. A surface with a RH of over 95% is classified as an “aqueous” surface. Such We believe that direct emissions and heterogeneous re- actions involving NO are not the major sources of HONO in Kathmandu’s nocturnal atmosphere. NO2 heterogeneous chemical processes appear to be responsible for the high and periodic HONO levels under the conditions of high aerosol pollution burden, strong inversion, and high humidity. Kath- mandu’s very low nocturnal mixing layer height provides a large reactive surface density for HONO formation through NO2 heterogeneous reaction, which is confirmed by the good correlation of ground reactive surface area density and [HONO]/[NO2] values. Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/ 6411 Y. Yu et al.: Observations of enhanced NO2-HONO conversion in Kathmandu The high relative humidity found in Kathmandu at night provides efficient water uptake on reactive surfaces. 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K.: Measurement of ambient nitrous acid and a reliable calibration source for gaseous nitrous-acid, Environ. Sci. Technol., 25, 255–260, 1991. Zhang, J., Wang, T., Chameides, W. L., Cardelino, C., Kwok, J., Blake, D. www.atmos-chem-phys.net/9/6401/2009/ References R., Ding, A., and So, K. L.: Ozone production and hydrocarbon reactivity in Hong Kong, Southern China, Atmos. Chem. Phys., 7, 557–573, 2007, http://www.atmos-chem-phys.net/7/557/2007/. Vandaele, A.C., Hermans, C., Fally, S., Carleer, M., Colin, R., M´erienne, M.-F., and Jenouvrier, A.: High-resolution Fourier transform measurement of the NO2 visible and near-infrared ab- sorption cross-section: Temperature and pressure effects, J. Geo- phys. Res. 107, 4348, doi:10.1029/2001JD000971, 2002. Zhou, X., Civerolo, K., Dai, H., Huang, G., Schwab, J., and Demer- jian, K.: Summertime nitrous acid chemistry in the atmospheric boundary layer at a rural site in New York State, J. Geophys. Res., 107, 4590, doi:10.1029/2001JD001539, 2002. Vogel, B., Vogel, H., Kleffmann, J., and Kurtenbach, R.: Measured and simulated vertical profiles of nitrous acid – Part II. model simulations and indications for a photolytic source, Atmos. Env- iron., 37, 2957–2966, 2003. Zhou, X., Beine, H. J., Honrath, R. E., Fuentes, J. D., Simpson, W., Shepson, P. B., and Bottenheim, J. W.: Snowpack photochem- ical production of HONO: a major source of OH in the Arctic boundary layer in springtime, Geophys. Res. Lett., 28, 4087– 4090, 2001. Wang, S., Ackermann, R., Spicer, C. W., Fast, J. D., Schmel- ing, M., and Stutz, J.: Atmospheric observations of enhanced NO2-HONO conversion on mineral dust particles, Geophys. Res. Lett., 30, 1595, doi:10.1029/2003GL017014, 2003. World Bank: Urban air quality management strategy in Asia (UR- BAIR), Kathmandu valley., Technical paper 378, 157 pp, 1997. Zhou, X., Huang, G., Civerolo, K., Roychowdhury, U., and Demer- jian, K. L.: Summertime observations of HONO, HCHO, and O3 at the summit of Whiteface Mountain, New York, J. Geophys. Res., 112, D08311, doi:10.1029/2006JD007256, 2007. Wormhoudt, J., Herndon, S. C., Yelvington, P. E., Miake-Lye, R. C., and Wey, C.: Nitrogen oxide (NO/NO2/HONO) emissions measurements in aircraft exhausts, J. Propul. Power, 23, 906– 911, 2007. Atmos. Chem. Phys., 9, 6401–6415, 2009 www.atmos-chem-phys.net/9/6401/2009/
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Geographical Conditions Affecting the Development of Canada: Discussion
Geographical journal
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7,335
Geographical Conditions Affecting the Development of Canada: Discussion Author(s): G. R. Parkin, H. J. Mackinder and L. S. Amery Source: The Geographical Journal, Vol. 38, No. 4 (Oct., 1911), pp. 374-381 Published by: geographicalj Stable URL: http://www.jstor.org/stable/1778541 Accessed: 27-06-2016 04:14 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. The Royal Geographical Society (with the Institute of British Geographers), Wiley are collaborating with JSTOR to digitize, preserve and extend access to The Geographical Journal The Royal Geographical Society (with the Institute of British Geographers), Wiley are collaborating with JSTOR to digitize, preserve and extend access to The Geographical Journal This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms RAPHICAL CONDITIONS AFFECTING 374 and autumn. Our loss has been greatest in the unsettled forests north and north-west of Lake Superior. To quote from the address of Prof. J. W. Eobertson before the Commission of Conservation, " This wilderness of rock and lake and forest is Canada's great regulator of climate for ensuring regular and dependable rainfalls in summer, and if that vast area be burned over and left bare, the winds sweeping over it will go where they list, licking up the moisture instead of dropping down refreshing showers" (Report for 1910, p. 44). ( p , p ) Fortunately much of the land in question, and especially that at the headwaters of our great rivers, is still in the possession of the Crown, whereas in the United States many of the Appalachian streams are endangered by private ownership of the forests at their headwaters. Taking warning from her neighbour, Canada has, before irremediable damage had been done, established a Commission of Conservation, in whose work the Dominion, the Provinces, and the Universities are co- operating. The first report, published in 1910, reveals at once the great- ness of the loss already incurred, the imminence of greater danger, and the vastness of the heritage remaining. This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms Perhaps its most striking feature is the fragmentary state of our inventory. Much is known, but more remains conjectural. " To realize the unprospected nature of the country," says Mr. G. A. Young, of the Geological Survey, " it is only necessary to remember that the greatest asbestos deposits of the world were brought to notice by blasting the Quebec Central railway through them; that the greatest corundum deposits, extending in a belt a hundred miles long, were found in a settled district by an officer of the Survey only twelve years ago; that the Sudbury nickel deposits were discovered by putting a rail? way through them; that Cobalt, now the premier silver camp, although only a few miles from a silver-lead deposit known a hundred and fifty years ago, was discovered less than six years ago, and then only by means of a railway cutting through a rich vein " (op. eit., p. 19). railway cutting through a rich vein (op. eit., p. 19). But though much remains to do, much has been done. We have built railways, and dug canals, and conserved water-power, in the resolve to make a nation out of these fragments, geographically so discrepant. When Euglishmen ask us wherein we differ from the Americans, or when genial Presidents of the United States tell us that we are at the parting of the ways, do you wonder if we feel like telling them something of this. So far we have come, and to complete nationality we intend to go; within this British Empire, unless you east us off; but within or without it, as Canadians. Our boundaries may be artificial, our outlook often at first sight provincial; but go below the surface, and you will find that the spirit which urges us, on to-day in our work of conquering geography is the spirit that declares that Canada shall be one, one from ocean to ocean. The President (before the paper): We are met here to hear a paper on the Geographical Conditions aft'ecting the Development of Canada, by Prof. W. L. 375 THE DEVELOPMENT OF CANADA?DISCUS Grant, Professor of Colonial History, Queen's University, Kingston, Canada. It is the privilege of the President to introduce the lecturer of the evening. This duty is not only an agreeable, but a very easy one, because Prof. Grant comes to us with claims both personal and hereditary. He is the son of Prineipal George Grant, who accompanied Sandford Fleming on the first railway survey across the Rockies, and wrote a book, ' Ocean to Ocean,' in 1873, nearly forty years ago, which first revealed to the east of Canada the possibilities of the great prairie country lying to the west. Prof. Grant was educated himself at Queen's University, Kingston, Canada, and then he took one of the most prudent steps in his career?he went to Balliol College, Oxford, where he achieved the highest honours in the Final Schools that it was iri the power of the University to bestow. Then he passed under the admirable tutelage of our friend Dr. George Parkin, who has so often spoken to us in this place, and whose eloquent speeches all the world over are a source of delight to those who listen to them. A little later Prof. Grant was appointed Beit lecturer in Colonial History at Oxford, one of those foundations which, as you know, we owe to the great liberality of the late Mr. Beit. Prof. Grant has done literary work known to many of you ; he is at present editing, for the Champlain Society, one of the earliest accounts of travels in Canada, and in 1908 he addressed the British Association on the north-west expansion of Canada. He is at present Professor of Colonial History at the University of Kingston, Canada, and you will agree with me that it is impossible to imagine a man better qualified to address us on the subject which is to be the theme of his paper to-night his paper to night. Dr. G. R. Pabkin (after the paper): When any Canadian looks at the map of Canada, he feels as if he had so many things to say that it is difficult to know where to begin. When Prof. Grant was speaking of the great distances of Canada, and of the toil with which the country has been built up, two or three pictures came to my mind. I was staying some years ago with that grand old man of our country, Lord Strathcona, and we got talking about his early life?a thing he does not always do?and he told us a story. This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms He told us that when he was a young man he was asked by the chief factor of the Hudson Bay Company if he would take the Ungava district. After three days of consideration he made up his mind that he would. Then he told us how he went 1200 miles on a sledge and 700 on snowshoes before he got to his post at Ungava. He added, " And I never came back again for thirteen years." That is the kind of effort that has built up Canada. Your young emigrant who comes out now, if he were told he had to go 1200 miles on a sledge and 700 miles on snowshoes to get to a point, where he would be out of touch with civilization for thirteen years, would probably find his hair curling with fear. Another picture comes to my mind in connection with the building of the Canadian Pacific over those great ranges of mountains you have seen pictured. My friend Mr. James Ross told me that when he was asked by the Canadian Pacific Company to take the construction over the Rockie3, he had never been West; he did not know what the Rockies were, and simply went in the faith of what had been told him. He started out from Winnipeg, and travelled day after day over those prairies, and as he went along his engineering mind was impressed by the ease with which a railway could be built there. Then he said there came on a period when mist hung over all the prairies, and they advanced without seeing what was ahead of them. After some days of this travelling he was sitting one morning at his tent door, waiting to make his start, when suddenly the mist lifted. He had approached the Rocky mountains, and there before him were 37 RAPHICAL CONDITIONS AFFECTING those enormous snow-eovered peaks, and he said he never had such a task in his life as he had to get up from his chair, when he reflected that it was to put a railroad over those mountains he had come out there! Those are simply two things. Let me give you another touch. Mr. Grant was speaking to you about the extraordinary undeveloped resources of Canada. Let me give you an illustration. Last Oetober I happened to go back to the part of Canada in which I was born, and with which I was very familiar. I had heard of some extraordinary discoveries being made there, and I came to a farm which I had known something about when I was a boy. It was a place where the old farmers for the last hundred years had cut the forests in order to get wood to keep their houses warm, and the women made their tallow candles to light their homes. When I went there, some scientific man had bored a hole down in the ground, and right under where these men got their wood and the women made their candles, was found a natural gas, where a single pipe would deliver a million or more cubic feet in a day, and besides, there were great tanks of oil which had come out from the same openings. They were working machinery; they were preparing to light a large town?to drive all kinds of factories with this gas. This is merely one example of the natural resources that are constantly being developed. It is no wonder that people are enthusiastic about Canada. Every Canadian feels that way. The question of the climate was referred to, and Mr. Grant is right when he said that the Canadian climate is the greatest asset we have. It squeezes out that colour question which afnicts the Southern States in America, and which is the shadow which hangs over Africa. It puts a check upon that flood of Mediterranean population which is pouring over the centre of America. More than that, the climate takes the inefficient man of our own race, and says if you have not prudence and industry and foresight, and get a root over your head and food in your larder, and fuel, you are going to die. It trains such men to industry and foresight. The country is a relent- less country, and the men who settle there must brace themselves up, and if you let that process go on for a hundred years, it will make Canada one of the great and powerful nations of the world. Mr. Grant said we never shall be in the same sense as England a naval people. But Canada must think about things at the ends of the world. This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms At the present time we are flooding the North Atlantic with commerce. We have establtshed lines to the West Indies and South America; we are throwing Hnes across to Japan and China; others go south to Australia and New Zealand. Now, a nation that is flinging out its commerce to the ends of the Earth has got to think of naval questions, whether it is mainly inland or not. If it is sending all this commerce over the world, then it must think in the terms of a great naval future. I have diverted some? what from the geographical question, but I want to say that the geographical side of it, the fact that gives us the great northern climate, this greatest wheat area of the world, is going necessarily to place Canada in a foremost position in this Empire. We are all enthusiastic about Canada, and when we look at these maps and these great spaces, it gives us much inspiration about the future of the country. M H J M k d I ll h l h h those enormous snow-eovered peaks, and he said he never had such a task in his life as he had to get up from his chair, when he reflected that it was to put a railroad over those mountains he had come out there! Those are simply two things. Let me give you another touch. Mr. Grant was speaking to you about the extraordinary undeveloped resources of Canada. Let me give you an illustration. Last Oetober I happened to go back to the part of Canada in which I was born, and with which I was very familiar. I had heard of some extraordinary discoveries being made there, and I came to a farm which I had known something about when I was a boy. It was a place where the old farmers for the last hundred years had cut the forests in order to get wood to keep their houses warm, and the women made their tallow candles to light their homes. When I went there, some scientific man had bored a hole down in the ground, and right under where these men got their wood and the women made their candles, was found a natural gas, where a single pipe would deliver a million or more cubic feet in a day, and besides, there were great tanks of oil which had come out from the same openings. They were working machinery; they were preparing to light a large town?to drive all kinds of factories with this gas. This is merely one example of the natural resources that are constantly being developed. It is no wonder that people are enthusiastic about Canada. Every Canadian feels that way. The question of the climate was referred to, and Mr. Grant is right when he said that the Canadian climate is the greatest asset we have. It squeezes out that colour question which afnicts the Southern States in America, and which is the shadow which hangs over Africa. It puts a check upon that flood of Mediterranean population which is pouring over the centre of America. More than that, the climate takes the inefficient man of our own race, and says if you have not prudence and industry and foresight, and get a root over your head and food in your larder, and fuel, you are going to die. It trains such men to industry and foresight. The country is a relent- less country, and the men who settle there must brace themselves up, and if you let that process go on for a hundred years, it will make Canada one of the great and powerful nations of the world. Mr. Grant said we never shall be in the same sense as England a naval people. But Canada must think about things at the ends of the world. At the present time we are flooding the North Atlantic with commerce. We have establtshed lines to the West Indies and South America; we are throwing Hnes across to Japan and China; others go south to Australia and New Zealand. Now, a nation that is flinging out its commerce to the ends of the Earth has got to think of naval questions, whether it is mainly inland or not. If it is sending all this commerce over the world, then it must think in the terms of a great naval future. I have diverted some? what from the geographical question, but I want to say that the geographical side of it, the fact that gives us the great northern climate, this greatest wheat area of the world, is going necessarily to place Canada in a foremost position in this Empire. We are all enthusiastic about Canada, and when we look at these maps and these great spaces, it gives us much inspiration about the future of the country of the country. Mr. H. J. Mackinder : I will not at this late hour occupy your time with remarks of any length. I will mention only two or three general ideas which have occurred to me in the course of the evening. While Dr. Parkin was holding us fascinated on the borderland between geography and imperial politics, my mind went back to a scene which has remained firmly impressed on my memory, It was on a very hot afternoon when I sat in the observation car at the end of a train travelling over the Canadian plain along the edge of 377 THE DEVELOPMENT OF CANADA?MSCUS the sub-arid district. We were on a single railway, and the two lines of steel ran away over the level to the horizon, where they seemed to be dancing in the heat. It was just after the wheat harvest, when the crop had been mostly cut and removed. A sea-gull from the lakes followed the train, sailing behind us and occasionally diving down to pick up some fragment from our kitchen. I was bound for Winnipeg to speak before the Canadian Club, and the idea occurred to me to take that gull as my text and to speak there in the heart of the continent on the significance of the British Navy. Winnipeg can never forget the ocean, so long as wheat is the chief product of the West. g , g p I sometimes think that this strange straight line which forms the boundary between Canada and the United States is the most remarkable feature on the map of the world. Drawn by man, artificial in the highest degree, it is natural that many minds should conclude that it cannot endure. But let us not forget that men in olden times could not draw straight lines upon the map simply because they could not measure latitude and longitude. They did none the less make artificial frontiers, and those frontiers have lasted in some cases at any rate for many centuries. Look at the frontier between the counties of Leicestershire and Warwickshire. See it running as a bee-line but for a single kink in it. It follows a Roman road, which doubtless fell into disrepair and ceased to be a road, becoming a mere green alley through the forest. This alley was adopted for a frontier by the Danes and the Saxons, and has remained to this day as a boundary between counties. You will find a similar frontier following an abandoned Roman road along the north of Hampshire, except that the single parish of Silchester beyond the road is attached to that county. Or look to the map of Germany, and note the frontier between the Prussian Provinces of Westphalia and the Rhine. That frontier, which is purely con- ventional, has lasted from the time when it separated the Duchies of Saxony and Franconia in the time of the Ottos. The frontier between Canada and the United States may very well last for a long time owing to what I have called geographical momentum. You have two societies, two national societies, organized one on either side of the frontier. In each of these societies you have an organization of men such that they look towards widely separated centres, the national hearts and brains. You have also railway systems, and systems for the distribution of power which have been organized apart. In a word, much capital has been fixed on two national principles, and in fixing capital you have also fixed organization. "To him that hath shall be given." The mere fact that these two societies exist with vested interests and fixed capital leads to this, that all fresh growth tends to accrete to the separate centres. Thus, what was at first merely an imaginary line becomes in fact a frontier of the most marked character. It is as important as any natural feature. It could not be destroyed without a vast positive efiort, any more than you can destroy the momentum of an engine without a great expenditure of friction or a shock in collision. g p There is a point which I would like to put to Prof. Grant. This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms He spoke of the waste region, measuring roughly 1000 miles from east to west, which lies between the old Canada of the east and the new Canada of the west. Now, I well remember driving in the neighbourhood of Lake Temiscaming, north of the mining camp of Cobalt, and passing on to what is known as the clay beit. We drove through flourishing settlements where the forest had been cleared by the Freneh, who love the bush just as the British settlers love the open prairie. I understand that there is a good deal of this clay beit, probably extending away westward to the neighbourhood of Lake Nipigom It is a fertile region although westward to the neighbourhood NO. IV.?OCT westward to the neighbourhood NO. IV.?OCT westward to the neighbourhood NO. IV.?OCT ion, al G ion, a G 37 lArillCAL CONDITIONS AFFECTING bush covered, which will be opened up by the new Grand Trunk Pacific Railway. If still more mining camps are established, there will be a local market for agricultural produce. Let us not forget that you have to-day a well-developed agriculture in the mining north of England, in regions which were practically vacant 150 years ago, just as are these spaces in Canada. y g , j p I think that as geographers we must be rather cautious before we accept the easy generalization which would write off the region north of Lake Superior as a permanent markland between Eastern and Western Canada. I am not at all convinced that that will be the case. Prof. Grant laid stress on the fact that the summer isotherms extend in a north-westerly direction in Western Canada, but I do not think that he drew your attention to the further fact that the southernmost point of these isotherms is in the neighbourhood of Winnipeg, and that from Winnipeg they trend north-eastward. In other words, they have a V shape, and thus you have a milder climate, at any rate in summer, as you go eastward through the clay belt. It is true that the sub-soil is for the most part granitic and gneissic, but there are glacial clays on the tops, and alluvial clays in the valley-bottoms. You are bound to have a railway from the West to Hudson bay, and if the Hudson bay route is a success you will have a line to the Hudson bay port running from the clay belt of the intermediate region. You will then have a through railway communicating from the St. Lawrence round the north of Lake Winnipeg, and the breadth of Canada will not be narrowed as at present almost to a point at Winnipeg. Do not let us forget that the great West itself was not so long ago written off as impossible of settlement. One last point. The meridian 100? W. of Greenwich is a very significant line within the United States. The greater part of the space west of this line is sub-arid, if not arid. True that there is fertility in California along the Pacific coast, and a fringe of fertility running westward just south of the international frontier. But the west of the United States can never, whatever may be done in the way of irrigation, equal in importance the east. Those who look at the southward position of the United States and argue as to the inevitable inferiority of Canada, must not forget that to balance the f rozen and sub-frozen areas of Canada are the arid and sub-arid regions of the United States. The full weight of American power can be brought to bear in the Pacific in the main only through the Panama canal, for the mass of American population and wealth must, as it seems to me, remain in the east. population and wealth must, as it seems to me, remain in the east. But the meridian 100? W. has little or no significance in Canada, and in the west of Canada there are probably productive areas greater than in the west of the United States. Thus it may be that Canada and the United States a genera- tion or two hence may be more or less equal rivals in the Pacific, Canada acting directly from her western area, the United States efTectively only through the Panama canal. If that should happen, we may picture to ourselves that Canada will not merely be an important part of the British Empire, but the very centre of that empire. This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms I am by no means convinced that the engineers are not right, and that Nelson is not going to be the harbour of the future. After all, as far as the shoals are concerned, the mouth of the Thames, if you put it in the primitive condition the mouth of the Nelson is in, and brought ships to it that had never navigated these regions, would be also regarded as impossible. Of course, the great problem for oversea shipping is the length of time during which the straits are open from the Arctic ice. I would put the opening of the straits about a week later than Mr. Grant, and the closing somewhat later? possibly even to the end of the year. Mr. Grant added that the coast of Labrador ought to be included in the Dominion. I think he is mistaken unless he means to include Newfoundland as well. To separate Labrador from New- f oundland at the present stage would be impossible. Except a few Eskimo, almost the whole population is a summer population who come from Newfoundland with their wives and families. If you put that coast under Canada, you will be subjecting them to customs and police regulations with which they are not familiar, and to authorities with whom they might not be inclined to get on well. Mr. Mackinder referred just now to the country south of Hudson bay? country with a deep, rich, glacial clay soil covered with fine forest and with a good climate. Apart from mining, there is likely to be a big development of the pulp industry round the southern shores of James bay, both of which would give the necessary start to agricultural development, and I believe that even within twenty years we shall see the beginning of considerable agricultural development along that southern region by James bay. But to return to my theme. Looking back over history, we see that the geography of Canada is based on a real principle of union. And now in the present day she is being made united by a great system of railways, which system, both through the great east and west lines and through Hudson bay, also brings her closer to the old Mother country. But there is another point for the future. Mr. Those who ask if Canada is to be loyal to the empire are f orgetf ul of the fact, which I believe Canadians are beginning to realize, that Canada is probably to be the centre of the Empire probably to be the centre of the Empire. Mr. L. S. Amery : I should like to say a word or two on a remark that fell more than once from Mr. Grant's lips. He kept on referring to Canada as an " artificially created country"?as a country existing in spite of the forces of geography. Now, looking at Canada by herself that seems obvious enough, but it is not so obvious, and I do not think it is even true, when we look at Canada in relation to England and to British sea-power in the past. Canada THE DEVELOPMENT OF CANADA?DISCUS 379 exists to-day?is what she is?because of British sea-power. The Maritime Provinces are the provinces which were most easily defended at the time of the American revolution by the power commanding the sea. Quebec and Upper Canada, again, were then held as they had been originally won through sea- power. Mr. Grant referred to the extraordinary importance of Hudson bay as a feature in Canada's geography. When we go back to the period before railways, we can understand its significance even more. Canada's north-west represents the region which up to about 1860 was more accessible from England by sea through Hudson bay and thence by canoe than it was accessible from New York, or from any of the centres of American civilization. British Columbia is the region which was first of all explored from Hudson bay by men like Mackenzie and Fraser; and again in the period of the gold rush in the f orties British Columbia was more accessible from England by sea and under the protection of British ships than it was across the continent from America. I agree with Mr. Grant that the construction of the railway to Hudson bay is quite an easy matter. As to the question of the ports, the situation is this: any sailor who has ever navigated in Hudson bay will tell you that Churchill is the only port, and that, from his point of view, Nelson, with its immense area of shoals, seems almost impossible in its present unbuoyed and uncharted condition. But Churchill is a very small port, and the only use of the Hudson bay route is, if it can really clear a substantial portion of the western crops during the f our months which it is open. That necessitates a harbour capable of holding a great mass of shipping, and from that point of view the engineers look to Nelson. I am by no means convinced that the engineers are not right, and that Nelson is not going to be the harbour of the future. After all, as far as the shoals are concerned, the mouth of the Thames, if you put it in the primitive condition the mouth of the Nelson is in, and brought ships to it that had never navigated these regions, would be also regarded as impossible. Of course, the great problem for oversea shipping is the length of time during which the straits are open from the Arctic ice. I would put the opening of the straits about a week later than Mr. Grant, and the closing somewhat later? possibly even to the end of the year. Mr. Grant added that the coast of Labrador ought to be included in the Dominion. I think he is mistaken unless he means to include Newfoundland as well. To separate Labrador from New- f oundland at the present stage would be impossible. Except a few Eskimo, almost the whole population is a summer population who come from Newfoundland with their wives and families. If you put that coast under Canada, you will be subjecting them to customs and police regulations with which they are not familiar, and to authorities with whom they might not be inclined to get on well. Mr. Mackinder referred just now to the country south of Hudson bay? country with a deep, rich, glacial clay soil covered with fine forest and with a good climate. Apart from mining, there is likely to be a big development of the pulp industry round the southern shores of James bay, both of which would give the necessary start to agricultural development, and I believe that even within twenty years we shall see the beginning of considerable agricultural development along that southern region by James bay. But to return to my theme. Looking back over history, we see that the geography of Canada is based on a real principle of union. And now in the present day she is being made united by a great system of railways, which system, both through the great east and west lines and through Hudson bay, also brings her closer to the old Mother country. But there is another point for the future. Mr. Grant suggested that it required the vision of Lord Durham to see what effect railways 2 c 2 exists to-day?is what she is?because of British sea-power. The Maritime Provinces are the provinces which were most easily defended at the time of the American revolution by the power commanding the sea. Quebec and Upper Canada, again, were then held as they had been originally won through sea- power. Mr. Grant referred to the extraordinary importance of Hudson bay as a feature in Canada's geography. When we go back to the period before railways, we can understand its significance even more. Canada's north-west represents the region which up to about 1860 was more accessible from England by sea through Hudson bay and thence by canoe than it was accessible from New York, or from any of the centres of American civilization. British Columbia is the region which was first of all explored from Hudson bay by men like Mackenzie and Fraser; and again in the period of the gold rush in the f orties British Columbia was more accessible from England by sea and under the protection of British ships than it was across the continent from America. I agree with Mr. Grant that the construction of the railway to Hudson bay is quite an easy matter. As to the question of the ports, the situation is this: any sailor who has ever navigated in Hudson bay will tell you that Churchill is the only port, and that, from his point of view, Nelson, with its immense area of shoals, seems almost impossible in its present unbuoyed and uncharted condition. But Churchill is a very small port, and the only use of the Hudson bay route is, if it can really clear a substantial portion of the western crops during the f our months which it is open. That necessitates a harbour capable of holding a great mass of shipping, and from that point of view the engineers look to Nelson. Grant suggested that it required the vision of Lord Durham to see what effect railways 2 c 2 effect r 2 c 2 DEVELOPMENT OF CANADA?DISCUSSION. 380 might have upon the future of Canada. I wonder if I dare be even bolder than Lord Durham, and suggest that there also may be surprising political and economic effects following from the navigation of the air ? It is interesting to keep in mind that, as the airship flies, every part of Canada east of the Rockies is nearer to England than New York is, and that every part of Canada right away to Vancouver is nearer than New Orleans. It is possible that if long- distance aerial navigation ever comes into existence, we shall begin to realize how much closer the northern half of the American continent is to this country than the southern half, and that therefore the old historical forces connected with the command of the sea which have attached Canada to the Empire, and the new forces created by railways and fast steamships, will be supplemented by yet another newforce, strengthening Canada's future position as the centre of economic power and the centre of population in the British Empire might have upon the future of Canada. I wonder if I dare be even bolder than Lord Durham, and suggest that there also may be surprising political and economic effects following from the navigation of the air ? It is interesting to keep in mind that, as the airship flies, every part of Canada east of the Rockies is nearer to England than New York is, and that every part of Canada right away to Vancouver is nearer than New Orleans. It is possible that if long- distance aerial navigation ever comes into existence, we shall begin to realize how much closer the northern half of the American continent is to this country than the southern half, and that therefore the old historical forces connected with the command of the sea which have attached Canada to the Empire, and the new forces created by railways and fast steamships, will be supplemented by yet another newforce, strengthening Canada's future position as the centre of economic power and the centre of population in the British Empire economic power and the centre of population in the British Empire. The Peesident : I am sure you will agree that we have had an extremely interesting evening. In this country I believe the expert is looked upon with some suspicion, but for my part I have always reposed great faith in the opinions of experts, more especially when I have claimed to be one myself. This evening we have had a discussion conducted exclusively by experts. The reader of the paper described himself as a buccaneer in the sphere of geography. If so, I can only say that he has brought back a very considerable spoil. Then we had Dr. Parkin speaking about the land in which he was born, and which he knows so well, with that eloquent enthusiasm which distinguishes his accept- able oratory. He was followed by Mr. Mackinder, who always appears to me to draw large and apparently logical deductions from ingenious premises with a skill unequalled by any speaker of the day. In so far as he was dilating not merely upon the present wealth resources and conditions of Canada, but upon the considerable future that lies before that Dominion, I \ and we all are entirely with him. But I noticed a sort of chill fell upon the audience when he told us that it is to be the centre of the British Empire at some unnamed date in the future, when, as I understood from him, even London is to be replaced by Winnipeg. However, it is perhaps unnecessary to project our vision quite so far. It is enough that Canada is what it is now, and what it is certain to become. I think the only phrase in which I disagreed with the lecturer was the sentence in which he said that Canada would, in all probability, be a mighty nation. It is so already, as a matter of fact. When I interrupted him and asked, "What is the population of Canada?" he replied, " Eight millions." Now, eight millions is the population of the kingdom of Persia, which has existed for over two thousand years; it is more than the population of Afghanistan. This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms This content downloaded from 128.197.26.12 on Mon, 27 Jun 2016 04:14:46 UTC All use subject to http://about.jstor.org/terms * Map, p. 456. But in Canada you have advantages which neither of those countries have ever enjoyed; it is one of the greatest un- occupied, or only partially occupied, spaces of the world; and while some rather sharp things have at times been said about the climate of Canada, and while its obviously utilitarian advantages were dwelt upon by the reader of the paper, I have never myself found in the Canadian climate anything but what was prophetic of good in the future. I have visited Canada more than once, and every man of sensibility who goes there feels the very air throbbing with vitality and redolent of future success. But then Dr. Parkin says, " We are loyal in Canada; are you going to be loyal in England ?" Well, we are no more foolish here than you are in Canada. We are not going to see go out of the British Empire that which is one of its most splendid assets. We lost America. I will not enter into that question now; I shall be told it was the folly of our statesmen. However that may be, having made the mistake once, BANGWEULU SWAMPS AND THE WA- 381 we shall not repeat it. We desire to see grow up in the north of that continent a race, a country, a nation equal in stature to the United States, so that even though it be said that we gave birth to one child that left us, it will be con- ceded that in process of time we atoned for that error by giving birth to another of equal stature, which remained in perpetual accord with the family household. It has been a pleasure to me to have heard so good a paper, and I hope you will allow me to convey your congratulations and thanks to Prof. Grant. BANGWEULU SWAMPS AND THE WA-UNGA.* By FRANK H. MELLAND, F.R.A.I., F.Z.S., Native Commissioner, South Awemba Division, North-East Rhodesia. Although my knowledge of the Wa-Unga is, owing to many causes, limited, I have visited them at intervals for nine years, and have passed through every corner of their country, and have seen a good deal of them in one way and another, so that I think it may be of interest, and perhaps of some value, to put on record what I know of this small tribe and their home. It is, I confess, with considerable diffidence that I do so, for I cannot help realizing how very limited is my knowledge. Living among the Awemba and Wa-Wisa, I naturally know far more of them than I do of the Wa-Unga, whom I am only able to visit once or twice a year. But so much relatively is known of the two former tribes, and practically nothing of the Wa-Unga, that even a sketchy account may be of some value. The tribe is probably a branch of the Wisa Lala family, but has been for a long time settled in one corner (the south-east) of the Bangweulu swamps, and has developed characteristics of its own; they apparently came from a country some way to the west of the Luapula about the same time that the Awemba, Wa-Wisa, and others were migrating east- wards. This migration was, as is well known, a general one, and occupied a considerable time, so that one cannot fix a date for the arrival of any particular branch. Being fishermen, they chose this country, and settled here, probably driving out into the denser swamps the original inhabitants, the Wa-Twa. The name given to the northern part of the Wa-Unga country, Lunga, may be a contraction for Lu-Unga. y, g , y g Less civilized than the Wa-Wisa, they are far ahead of their neighbonrs and predecessors, the Wa-Twa, who are simply nomad fishermen. For some time it was cnstomary in this country to associate the two tribes (they have both, among certain sections of the community, been referred to as being web-footed!), but as a matter of fact they are quite separate, and, so far as I can trace, have, since the Wa-Unga entered the country, always lived in a state of war with each other. Nowadays they never meet except in trading, and on an occasional raid or abduction. The
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The Influence of Content Meaningfulness on Eye Movements across Tasks: Evidence from Scene Viewing and Reading
Frontiers in psychology
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ORIGINAL RESEARCH published: 01 March 2016 doi: 10.3389/fpsyg.2016.00257 Edited by: Edited by: Guillaume A. Rousselet, University of Glasgow, UK Guillaume A. Rousselet, University of Glasgow, UK Reviewed by: Sébastien M. Crouzet, Centre de Recherche Cerveau et Cognition, France Françoise Vitu, Aix-Marseille Université, France Keywords: eye movements, cognitive control, meaning, reading, scene perception, eye tracking, eye movement control *Correspondence: Steven G. Luke steven_luke@byu.edu The Influence of Content Meaningfulness on Eye Movements across Tasks: Evidence from Scene Viewing and Reading Steven G. Luke1* and John M. Henderson2,3 1 Department of Psychology and Neuroscience Center, Brigham Young University, Provo, UT, USA, 2 Department of Psychology, University of California Davis, Davis, CA, USA, 3 Center for Mind and Brain, University of California, Davis, Davis, CA, USA The present study investigated the influence of content meaningfulness on eye- movement control in reading and scene viewing. Texts and scenes were manipulated to make them uninterpretable, and then eye-movements in reading and scene-viewing were compared to those in pseudo-reading and pseudo-scene viewing. Fixation durations and saccade amplitudes were greater for pseudo-stimuli. The effect of the removal of meaning was seen exclusively in the tail of the fixation duration distribution in both tasks, and the size of this effect was the same across tasks. These findings suggest that eye movements are controlled by a common mechanism in reading and scene viewing. They also indicate that not all eye movements are responsive to the meaningfulness of stimulus content. Implications for models of eye movement control are discussed. INTRODUCTION When we are performing a visual task, such as reading, searching for an object, or just looking around at the world, our eyes make rapid movements, called saccades, several times a second. In between these saccades, the eyes make pauses (called fixations) to take in visual information. Researchers who study eye movements are interested in why we look where we do and why we move our eyes when we do. It is thought by many that eye movements are under cognitive control, meaning that the where and when of eye movements are influenced by cognitive processes related to perception, memory, and language (Rayner, 2009; Rayner and Reingold, 2015; but see Yang and McConkie, 2001; Vitu, 2003 for an alternate viewpoint). In short, cognitive control implies that our eye movements respond quickly to the nature of what we are looking at in any given moment. h d f d h b f l d f h fl f Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Psychology Received: 19 October 2015 Accepted: 09 February 2016 Published: 01 March 2016 Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Perception Science, a section of the journal Frontiers in Psychology Received: 19 October 2015 Accepted: 09 February 2016 Published: 01 March 2016 Citation: Luke SG and Henderson JM (2016) The Influence of Content Meaningfulness on Eye Movements across Tasks: Evidence from Scene Viewing and Reading. Front. Psychol. 7:257. doi: 10.3389/fpsyg.2016.00257 Received: 19 October 2015 Accepted: 09 February 2016 Published: 01 March 2016 Citation: Luke SG and Henderson JM (2016) The Influence of Content Meaningfulness on Eye Movements across Tasks: Evidence from Scene Viewing and Reading. Front. Psychol. 7:257. doi: 10.3389/fpsyg.2016.00257 The study of reading has proven to be fertile ground for investigating the influence of cognitive factors on eye movement control. This is primarily because words have multiple quantifiable cognitive properties such as frequency and predictability, properties that influence the processing of those words in multiple ways. Studies exploring the influence of these cognitive properties have shown that cognitive processing can influence eye movements on a moment-by-moment basis (Just and Carpenter, 1980; Rayner, 1998, 2009; Kliegl et al., 2006). March 2016 | Volume 7 | Article 257 1 Frontiers in Psychology | www.frontiersin.org Meaningfulness and Eye Movement Control Luke and Henderson Studying the influence of cognitive processing in other visual tasks, such as scene viewing, has proven more difficult, because the units of processing (i.e., objects) are not so clearly differentiated in visual scenes (they overlap) and their cognitive properties are not so easily defined or quantified. As a result, the influence of cognitive factors in scene viewing is less clearly understood (Henderson et al., 1999; Henderson, 2003; Võ and Henderson, 2009; Wang et al., 2010). Researchers have therefore been forced to rely more on manipulations of global image properties in order to investigate cognitive control of eye movements in these scene viewing tasks. These global manipulations, which are described below, reduce the possibility of cognitive control by removing or denying access to information needed to interpret and understand the image. paradigm thus simulates processing difficulty in a manner that is precisely controlled and easily applied to a variety of different visual tasks. This paradigm has been employed to study eye movement control in both reading (Rayner and Pollatsek, 1981; Morrison, 1984; Dambacher et al., 2013) and scene processing (Henderson and Pierce, 2008; Henderson and Smith, 2009). The SOD paradigm has also been used to compare reading and scene viewing directly (Nuthmann and Henderson, 2012; Luke et al., 2013), and equivalent effects of delay duration on fixation durations were observed in both tasks, strongly suggesting that reading and scene viewing share a common mechanism for the control of fixation duration. The SOD paradigm typically reveals two populations of fixations: some that outlast the delay and others that do not (Henderson and Pierce, 2008; Henderson and Smith, 2009; Luke et al., 2013). Citation: The first population of fixations appear to be under cognitive control, increasing linearly with the duration of the delay, while the second population of fixations does not lengthen in response to the presence of the mask, suggesting that these fixations are not under cognitive control. Thus, direct comparisons of eye movement control in different tasks are important not only because they tell us that these tasks share a common mechanism, but also because they reveal something about the nature of that mechanism. In some models, such as E–Z reader (Reichle et al., 1998), most or all reading saccades are initiated by successful completion of some stage of lexical access, while other models such as SWIFT (Engbert et al., 2002, 2005), CRISP (Nuthmann et al., 2010; Nuthmann and Henderson, 2012), and the competition-interaction model (Yang and McConkie, 2001) delay some saccades when cognitive processing difficulty is encountered. The existence of two different populations of fixations is more consistent with the latter class of models. Exploring eye movement control across multiple tasks can help to further adjudicate between these different proposals. These global image manipulations are also useful because they can also be applied to reading, permitting direct cross-task comparisons. This is important because reading is measurably different from other visual tasks, such as scene viewing. For example, fixation durations are significantly shorter in reading (Henderson and Hollingworth, 1998; Rayner, 2009; Luke et al., 2013). Saccade amplitudes tend to be shorter in reading as well (Rayner, 2009; Henderson and Luke, 2014). On the other hand, in reading the eyes are presumably guided by the same neural systems as in other visual tasks, and eye movements are made for the purpose of gathering information regardless of task. In support of this is the observation that aggregate measures of eye movement behavior correlate across tasks (Henderson and Luke, 2014), although not all researchers have found this relationship with regard to reading (Rayner et al., 2007). At the same time, while many core visual and eye movement control areas appear to be common to both tasks (Choi and Henderson, 2015), cognitive control in reading and scene viewing could be exerted by different cortical centers. Citation: For example, the parahippocampal place area (PPA; Epstein and Kanwisher, 1998; Epstein et al., 1999) appears to be involved in scene viewing but not in reading (Choi and Henderson, 2015; Henderson and Choi, 2015), while the visual word form area (VWFA; Cohen et al., 2000; McCandliss et al., 2003) and language areas in the left hemisphere contribute to eye movement control in reading but not in scene viewing (Choi and Henderson, 2015; Henderson et al., 2015). So, the underlying processes that control eye movements, making them sensitive to the meaning of words and objects, might differ in significant ways in reading compared to other tasks. Identifying which processes are common to all tasks and which are task-specific is an important goal of eye-movement research. Another global method for exploring eye movement control that has been employed exclusively in reading is the pseudo- reading paradigm, in which all letters in a text are replaced with block shapes or a single letter such as Z. This manipulation removes all meaning from the text, but preserves the visual- spatial layout of the words, sentences, and paragraphs. A finding from this technique is that fixations are typically longer in pseudo-reading than in reading. This finding seems rather paradoxical at first, as one might expect longer fixations when cognitive processing is engaged, not when it is absent, as no processing difficulty should occur when there is nothing to process; nevertheless this finding has been consistently shown across studies (Vitu et al., 1995; Rayner and Fischer, 1996). If we define processing difficulty simply as an inability to identify a stimulus, such as a word, then it makes sense that fixation durations should be lengthened when meaning is removed and identification is not possible. Or it could be that having meaning facilitates processing, thereby shortening fixations relative to the meaningless text condition (Reichle et al., 2012). Usually, the global image manipulations used to study eye movement control involve obscuring or removing the stimulus for an extended period of time. Sometimes participants are given a brief view of part of the text or of the scene before it is removed from view (Rayner et al., 2003, 2009). The results of these studies suggest that saccades are under cognitive control in both tasks. Frontiers in Psychology | www.frontiersin.org Materials The present study uses the principle behind the pseudo- reading paradigm, the removal of meaningfulness, to directly compare the cognitive control of eye movements in reading and in scene viewing. We manipulated text and scenes to create a pseudo-reading and a pseudo-scene viewing condition, and we then compared participants’ eye movements in the two tasks and in their pseudo-variants in a within-subjects design. Based on previous research on reading, we expected increased fixation durations for meaningless stimuli (Vitu et al., 1995; Rayner and Fischer, 1996; Luke and Henderson, 2013). Filtering scenes to remove high-frequency visual information, which makes object identification difficult, has also been shown to increase fixation durations (Mannan et al., 1997; Henderson et al., 2014b). We predict, therefore, that fixation durations will be longer for our pseudo-stimuli, indicating that at least some fixations are under cognitive control. Fifty-six short paragraphs (40–60 words) were taken from online news articles. These texts were used in Luke and Henderson (2013) and included a total of 1415 unique words: three one- letter words, 30 two-letter words, 93 three-letter words, 197 four-letter words, 247 five-letter words, 227 six-letter words, 218 seven-letter words, 159 eight-letter words, 110 nine-letter words, and 131 words 10 letters or longer. Two different versions of each text were created, a Normal Reading version, in which the text appeared on the screen in Courier New 16pt font, and a Mindless Reading version, in which the text was displayed in a custom font (also 16pt). This font transformed letters into block shapes (see Figure 1) while preserving overall word shape. Both fonts were monospace, and all letters, words, and lines of text appeared in exactly the same location regardless of font. g This manipulation permits us to test two more specific hypotheses about eye-movement control as well. The first is that the same systems control eye movements in reading and in scene viewing: If eye movements are controlled by the same systems across tasks, then eye movements should be influenced similarly by the removal of meaningfulness in both tasks. We note here that we use the term ‘meaningfulness’ because of the global nature of the manipulation; the pseudo-stimuli differ from the original text and scenes on many levels, but what is important is that they are not interpretable, meaning that cognitive control has little opportunity to influence eye movements for these stimuli (see Figures 1 and 2 below). Materials The second hypothesis is that, consistent with findings from the SOD paradigm and from pseudo-reading that not all fixations are under cognitive control (Henderson and Pierce, 2008; Henderson and Smith, 2009; Luke and Henderson, 2013; Luke et al., 2013; Henderson and Luke, 2014), the removal of meaningfulness will only affect some, and, not all, fixations. For the scene stimuli, pseudo-scenes were created that were analogous to the pseudo-texts in that they had a complex visual structure similar to that of their real scene counterparts but were not meaningful. These pseudo-scenes did not contain any identifiable objects and were not easily assigned to a particular scene category. In order to create a set of pseudo-scenes, we began with a large set of 840 images. The images depicted scenes from seven different categories, five outdoor and two indoor. The outdoor scenes were images of beaches, forests, mountains, cityscapes, and highways (Walther et al., 2009), while the indoor scenes were images of bedrooms and kitchens. g Our goal was to find a manipulation for scenes that was similar to the manipulation that we and others had previously used for text (see Figure 1), which still looked like text but was not interpretable. Specifically, we wanted to (1) preserve the spatial layout of the scene as much as possible, while (2) removing meaning. This proved to be a difficult task for visual scenes, and we tried and rejected several different methods. The manipulation we ultimately chose removed the meaning from these images via an extensive filtering process that extracted the edges from the images, expanded and distorted these edges, and then filled the empty areas within these newly warped edges with color, a process which also erased the edge lines. Then both the scenes and pseudo-scenes were transformed to grayscale. Citation: Another technique, called the stimulus onset delay (SOD) paradigm, involves covering all or part of the visual stimulus with a mask during predefined saccades, so that when the next fixation begins the stimulus is not visible. The mask remains on screen for a predetermined and varied delay and is then removed so that the stimulus again becomes visible. The SOD Like the SOD paradigm, the pseudo-reading technique has also shown that some eye movement behaviors in reading are under the influence of cognitive control, while others appear not March 2016 | Volume 7 | Article 257 2 Meaningfulness and Eye Movement Control Luke and Henderson to be (Nuthmann et al., 2007; Henderson and Luke, 2012; Luke and Henderson, 2013). The pseudo-reading technique has also been used in combination with EEG or MRI to explore the neural bases of cognitive control in reading (Henderson et al., 2013, 2014a, 2015). minimized with a chin and head rest. Although viewing was binocular, eye movements were recorded from the right eye. The experiment was controlled with SR Research Experiment Builder software. Participants Forty participants recruited from Brigham Young University completed the experiment. All participants were native English speakers with 20/20 corrected or uncorrected vision. Prior to participant recruitment, the institutional review board that Brigham Young University approved the study. This manipulation disguised the identities of the objects in the image and made the scene categories difficult to identify. The pseudo-scenes were then normed to see which ones were the most difficult to identify. All 840 meaningless images were posted on Amazon’s Mechanical Turk (Buhrmester et al., 2011). Participants were told that each image was created by altering a photograph of a scene, and asked to provide a short label identifying the category of the scene that the image was created from (or “Don’t Know” if they were unable to identify it) and to rate their confidence in the label they had provided. Each image was labeled by ten different participants. Apparatus Eye movements were recorded via an SR Research Eyelink 1000 plus tower mount eye tracker (spatial resolution of 0.01◦) sampling at 1000 Hz. Subjects were seated 60 cm away from a 24′′ LCD monitor with display resolution set to 1600 × 900, so that approximately three characters subtended 1◦of visual angle. Scenes (800 × 600 pixel images) subtended 21 by 16◦of visual angle. Head movements were March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 3 Luke and Henderson Meaningfulness and Eye Movement Control FIGURE 1 | Examples of text and pseudo-text. Normal text is above, with the corresponding pseudo-text below. Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 1 | Examples of text and pseudo-text. Normal text is above, with the corresponding pseudo-text below. Based on these norming data, 56 pseudo-scenes were selected, eight from each scene category. Overall, participants in the norming study gave the “Don’t Know” response for 73% of the images, and for the minority of images that they did attempt to provide a label for, they provided a correct label only 10% of the time. Confidence ratings were very low (M = 1.34 on a 5- point scale). Therefore, these images were extremely difficult to identify, even for participants who knew that the images had been derived from actual scenes. Examples of the images used can be seen in Figure 2. were further told that they should view each image in preparation for a memory test that would be administered at the end of the experiment. Each trial involved the following sequence. Each trial began with a gaze trigger, which consisted of a black circle presented in the center of the screen. Once a stable fixation had been detected on the gaze trigger, the image was presented for 10 s. At the end of 10 s, a new gaze trigger appeared and the next trial began. Stimulus condition (Meaningful vs. Pseudo-Stimulus) was counterbalanced across two stimulus lists, separately for each task (Reading vs. Scene Viewing), and each participant saw only one of the lists. Thus, each participant saw 28 normal texts, 28 pseudo- texts, 28 normal scenes, and 28 pseudo-scenes, and no participant saw the same text or scene twice. The order of stimulus presentation was counterbalanced across participants, with half of the participants completing the scene viewing task first, and half the reading task. Apparatus Within each task, stimuli were presented in a random order for each participant. For the memory test, participants were presented with a random selection of novel and previously viewed texts, scenes, and pseudo-scenes, and asked to indicate via button-press if they had seen the stimulus before. The memory test was administered solely to ensure that participants attended carefully to the experimental tasks, and so the data from the memory task were not analyzed. Frontiers in Psychology | www.frontiersin.org Procedure For the reading task, participants were told that they would be reading short texts on a computer screen while their eye movements were recorded. Participants were also told that some of the texts would appear with blocks in place of letters, and that in those cases they should move their eyes as if they were reading. These are the standard instructions given in pseudo- reading experiments (Vitu et al., 1995; Rayner and Fischer, 1996; Nuthmann et al., 2007; Luke and Henderson, 2013). Participants were informed that their memory for the texts and pseudo-texts would be tested at the end of the experimental session. Each trial involved the following sequence. The trial began with a gaze trigger, a black circle presented in the position of the first character in the text. Once a stable fixation was detected on the gaze trigger, the text was presented. The participant read the text and pressed a button when finished. Then a new gaze trigger appeared and the next trial began. Saccade Amplitude Thus, although the findings of the present study with regard to mean saccade amplitudes may appear to contradict previous findings that saccades are shorter is pseudo-reading, the pattern of changes in the distribution of saccade amplitudes is the same. A by-participant 2 (TASK: Reading vs. Scene Viewing) × 2 (STIMULUS TYPE: Meaningful vs. Pseudo-Stimulus) ANOVA was conducted on saccade amplitude. In this analysis, both main effects were significant, as was the interaction (all Fs > 9.12, all ps < 0.0061). Follow-up t-tests showed that the significant interaction indicated that the effect of STIMULUS TYPE was present for reading but was not significant for scene viewing (Reading t(39) = −2.82, p = 0.0082, difference = 0.44; Scene Viewing t(39) = −0.63, p = 0.53, difference = 0.1). Interestingly, the mean saccade amplitude was shorter in reading than in pseudo-reading (see Table 1). This contradicts findings from previous studies using similar pseudo-reading tasks, where longer mean saccades were observed in normal reading than in pseudo- reading (Vitu et al., 1995; Rayner and Fischer, 1996; Luke and Henderson, 2013). Luke and Henderson (2013) observed that these mean differences were due to a greater proportion of very short and long fixations in pseudo-reading. Figure 3 shows that the same pattern of results was obtained for reading in the current study. A similar increase in the proportion of longer saccades is observable for pseudo-scene viewing, although this shift was not large enough to significantly influence the means. RESULTS Fixations shorter than 50 ms or longer than 1200 ms were removed as outliers, and saccades larger than 22◦were removed to exclude return sweeps in reading. Summary statistics for all dependent variables can be found in Table 1. For the scene task, participants were told that they would be viewing both photographs and patterns of blobs and shapes on the screen as their eye movements were monitored. Participants March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 4 Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 2 | Examples of the scene and pseudo-scene stimuli. Normal scenes are on the left, with the corresponding pseudo-scenes on the right. of the scene and pseudo-scene stimuli. Normal scenes are on the left, with the corresponding pseudo-scenes on the right. FIGURE 2 | Examples of the scene and pseudo-scene stimuli. Normal scenes are on the left, with the corresponding pseu Frontiers in Psychology | www.frontiersin.org March 2016 | Volume 7 | Article 257 Fixation Duration Figure 4 shows the distribution of fixation durations for both scenes and text in the meaningful and pseudo-stimulus conditions. This figure illustrates that fixation duration distributions are not normal but are skewed to the right. Any difference in means between the meaningful and pseudo-stimuli might reflect a difference in the center of the two distributions, which occurs when most fixations in one condition are longer. However, since means are strongly influenced by outliers and TABLE 1 | Means (and standard deviations) for the dependent variables. Reading Scene viewing Meaningful Pseudo-text Meaningful Pseudo-scenes Fixation duration 206 (89) 255 (126) 284 (143) 307 (169) Saccade amplitude 3.22 (2.09) 3.66 (2.92) 4.26 (3.47) 4.36 (3.55) March 2016 | Volume 7 | Article 257 TABLE 1 | Means (and standard deviations) for the dependent variables. TABLE 1 | Means (and standard deviations) for the dependent variables. Frontiers in Psychology | www.frontiersin.org 5 Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 3 | Global Distribution of Saccade Amplitudes. There was a greater proportion of very short and long saccades for the pseudo-stimuli (dashed lines) than for the meaningful stimuli, especially for the reading task. FIGURE 3 | Global Distribution of Saccade Amplitudes. There was a greater proportion of very short and long saccades for the pseudo-stimuli (dashed lines) than for the meaningful stimuli, especially for the reading task. Previous research comparing fixation duration distributions in scene viewing and reading has observed that the distribution in scene viewing is both shifted to the right and more skewed to the right compared to reading (Luke et al. (2013); see also Henderson and Hollingworth (1998)). The removal of meaningfulness from a text stimulus has been shown to result in a “fatter tail”, skewing the distribution to the right compared to the normal, meaningful stimulus condition, but does not appear to influence µ or σ (Luke and Henderson, 2013). Figure 4 and Table 2 suggests that the primary difference between the Meaningful and Pseudo-Stimulus conditions in both tasks is indeed an increase in skew in the Pseudo-Stimulus condition, with no large shifts in the center of the distribution apparent. To look for any interactions between task and stimulus type that might indicate task-based differences in fixation duration control, especially in the analysis of the skew of the distribution (τ), by-participant 2 (TASK: Reading vs. Scene Viewing) × 2 (STIMULUS TYPE: Meaningful vs. Frontiers in Psychology | www.frontiersin.org Fixation Duration FIGURE 4 | Global Distribution of Fixation Durations. The solid vertical lines represent overall means, while the dotted vertical lines represent the values for µ derived from the response time distribution analysis. (Meaning: both ts > 5.61, both ps < 0.0001; No meaning: both ts > 2.28, both ps < 0.025). When the effect of STIMULUS TYPE was considered separately for each task, no significant differences were found in the analysis of µ (Reading: t(39) < 1.78, p = 0.072; Scene Viewing: t(39) < 0.57, p > 0.57). The effect was significant (although numerically tiny, only 4 ms) for reading only in the analysis of σ (t(39) < 2.03, p = 0.046; Scene Viewing: t(39) < 0.07, p > 0.94). These results indicate that if semantic content has an influence on the center or spread of the fixation duration distributions, such influences are quite small (<10 ms; see Table 2) and mostly non-significant. Accordingly, between- task differences in the influence of semantic content on µ or σ, if they exist at all, are on the order of a few milliseconds. Thus, these results are consistent with previous studies (Luke and Henderson, 2013). globally manipulated the meaningfulness of both texts and scenes in a within-subjects design, enabling us to explore potential differences in eye movement control across two visual tasks. Saccade amplitudes were found to increase significantly in reading for pseudo-text. This difference in mean saccade amplitude across conditions appears to result from an increase in very short and long saccade amplitudes when meaning is removed from text. This shift in the distribution has been observed in other studies (Luke and Henderson, 2013), although in these studies means decreased because the proportion of short saccades increased more than was observed here. In scene viewing there was a numeric trend toward an increase (see Table 1) but it was small and far from significant. There was some TABLE 2 | Parameters from the response time distributional analysis of fixation durations. Reading Scene viewing µ σ T µ σ τ Meaningful 139 38 66 164 52 122 Pseudo-stimulus 148 42 101 161 52 152 Effect 9 4 35 −3 0 30 The three parameters µ, σ, and τ are from the response time distributional analysis of the fixation duration distributions shown in Figure 3. Fixation Duration The first two parameters represent the normal component of the distribution (µ, the mean, and σ, the standard deviation), and τ is the exponential parameter representing the skew of the distribution. Mean log likelihoods for the four different conditions are: Reading, Meaningful: −2460.22; Reading, Pseudo-Stimulus: −2015.7; Scenes, Meaningful: −1345.4; Scenes, Pseudo-Stimulus: −1173.18. TABLE 2 | Parameters from the response time distributional analysis of fixation durations. In the analysis of τ, both main effects (TASK and STIMULUS TYPE) were significant (both Fs > 99.06, both ps < 0.0001). The interaction of the two was not significant (p > 0.32). These findings indicate that while all three parameters of the fixation duration distribution were larger in scene viewing than in reading, consistent with previous research (Luke et al., 2013), meaningfulness only had a significant effect on the skew of the distribution (Luke and Henderson, 2013). The size of this effect was statistically the same in the two tasks. The three parameters µ, σ, and τ are from the response time distributional analysis of the fixation duration distributions shown in Figure 3. The first two parameters represent the normal component of the distribution (µ, the mean, and σ, the standard deviation), and τ is the exponential parameter representing the skew of the distribution. Mean log likelihoods for the four different conditions are: Reading, Meaningful: −2460.22; Reading, Pseudo-Stimulus: −2015.7; Scenes, Meaningful: −1345.4; Scenes, Pseudo-Stimulus: −1173.18. Fixation Duration Pseudo-Stimulus) ANOVAs were conducted on all three of the ex-Gaussian parameters. In the analyses of µ and σ, there were main effects of TASK (both Fs > 43.75, all ps < 0.0046), indicating that both parameters were larger for scenes than for text. The main effects of STIMULUS TYPE were not significant (both Fs < 3.9, both ps > 0.056). The interaction of the two factors was significant in both analyses (both Fs > 6.9, all ps < 0.012), indicating that the effect of TASK was somewhat smaller in the Pseudo-Stimulus condition extreme scores, a difference between means can also occur because one distribution is more skewed than another, which occurs when some subset (but not all) of the fixations are longer. Mean differences can reflect either of these differences or both in combination (Balota and Yap, 2011). Since the center and skew of fixation duration distributions vary independently of each other and often reflect different processes (Staub and Benatar, 2013), it is important to consider them separately. To test whether the removal of meaningfulness had similar effects in the different tasks, the fixation duration distributions for each participant in each condition were analyzed using a response time distributional analysis (Balota and Yap, 2011). This analysis fits participants’ response time data with an ex- Gaussian distribution (Ratcliff, 1979), which is the convolution of a normal (Gaussian) distribution and an exponential distribution, with two parameters representing the normal component (µ, the mean, and σ, the SD), and a single exponential parameter (τ). Any changes in µ and σ indicate changes in the distribution’s normal component (i.e., the center), whereas increases in τ indicate increased skew to the right. Ex-Gaussian distributions fit eye-movement data quite well (Staub et al., 2010; Staub, 2011; White and Staub, 2012; Luke and Henderson, 2013; Luke et al., 2013). The ex-Gaussian distribution was fitted to the data from each participant in each task in each meaning condition using QMPE software (Heathcote et al., 2004). The mean ex-Gaussian parameters are found in Table 2. March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 6 Meaningfulness and Eye Movement Control Luke and Henderson FIGURE 4 | Global Distribution of Fixation Durations. The solid vertical lines represent overall means, while the dotted vertical lines represent the values for µ derived from the response time distribution analysis. DISCUSSION The present study investigated how the meaningfulness of a visual stimulus influences how our eyes move. More specifically, we March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 7 Meaningfulness and Eye Movement Control Luke and Henderson fixated stimulus (e.g., the competition-inhibition theory; Yang and McConkie (2001). suggestion of an increase in the proportion of longer fixations for pseudo-scenes as well. The absence of a significant effect of semantic content on saccades amplitudes in scene viewing may simply reflect a ceiling effect; saccades are larger by default in scene viewing, and it is probably not possible to increase them much more and still keep the eyes within the bounds of the stimulus. Regardless, these observed changes in saccade amplitude likely reflect a reduced need for foveal processing when the stimulus is not being processed for meaning. While the global manipulation employed here is useful for cross-task comparisons, it of course has certain limitations. Since our manipulation altered the stimuli in multiple ways, removing or changing some low-level visual features as well as obscuring the identity of words and objects, it is not possible to determine which cognitive processes (or which stage in processing) has the most influence on eye movements. This technique cannot therefore adjudicate cleanly between different proposals about the nature and source of cognitive control. The present study does, however, provide additional evidence, first, that reading, and scene viewing share a common control mechanism, and, second, that only some fixations are under the direct influence of the visual stimulus. Most models of eye-movement control apply to reading only (Reichle et al., 1998; Engbert et al., 2002, 2005), and so may not generalize to other tasks (but see Reichle et al. (2012) for an example of how E–Z reader can generalize to non-reading tasks). One model of eye-movement control that has been shown to successfully predict eye movements in both reading and scene viewing is the CRISP model (Nuthmann et al., 2010; Nuthmann and Henderson, 2012). CRISP also predicts that some eye movements will not be under cognitive control; fixation duration is determined by a random walk timer, after which a new saccade program is initiated. Prior to saccade program initiation, cognitive intervention can occur via inhibition of the saccade program when processing difficulty is encountered, but after the timer expires no cognitive intervention is possible. DISCUSSION Thus, CRISP is consistent with the findings of this and other studies. The results of the current study suggest that current and future models of eye movement control should, first, be able to account for eye movements across multiple tasks, and second, incorporate a mechanism for cognitive control that exempts some subset of fixations. One goal of the present study was to investigate whether the influence of stimulus meaningfulness differs across tasks. A close look at the distribution parameters from the fixation duration distribution analysis (Table 2) shows that the removal of meaningfulness affected the fixation duration distributions in the same way in both tasks, influencing the skew (τ) but not consistently influencing the center (µ, σ) of the distributions. That is, the distribution analysis showed no consistent evidence of any significant effects of meaning on either µ or σ. There was, however, a main effect of stimulus type in the analysis of τ. Further, the interaction of stimulus type and task was not significant in the analysis of τ, indicating that the removal of meaningfulness from the stimulus had a statistically identical influence in reading and in scene viewing. Thus, it appears that the influence of cognitive control on eye movements in reading and scene viewing is both qualitatively and quantitatively similar; not only did the removal of meaningfulness influence the same component of the distribution in both tasks, the magnitude of that influence was nearly identical. This observation is highly consistent with other research with the SOD paradigm showing that eye movements respond similarly to processing difficulty in reading and in scene viewing (Luke et al., 2013). Increases in τ like those observed here occur when some, but not all, of the fixations are longer, which elongates the tail of the distribution but does not significantly shift its center. Thus, the fact that for the pseudo-stimuli τ was increased but the other parameters of the ex-Gaussian distribution were not indicates that not all fixations were affected by the removal of meaningfulness. This finding fits nicely with research using the SOD paradigm that reveals two populations of eye movements, providing converging evidence that longer duration fixations are under cognitive control and shorter duration fixations are not (Henderson and Pierce, 2008; Henderson and Smith, 2009; Nuthmann and Henderson, 2012; Luke et al., 2013). AUTHOR CONTRIBUTIONS Both authors (SL, JH) made substantial contributions to the conception or design of the work. SL was primarly responsible for the acquisition, analysis, or interpretation of data for the work. SL Drafted the work and JH revised it critically for important intellectual content. Both SL and JH gave final approval of the version to be published, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. DISCUSSION This finding is most consistent with models of eye movement control in which only longer fixations are influenced by the currently Choi, W., and Henderson, J. M. (2015). Neural correlates of active vision: an fMRI comparison of natural reading and scene viewing. Neuropsychologia 75, 109–118. doi: 10.1016/j.neuropsychologia.2015.05.027 Cohen, L., Dehaene, S., Naccache, L., Lehéricy, S., Dehaene-Lambertz, G., Hénaff, M.-A., et al. (2000). The visual word form area. Brain 123, 291–307. doi: 10.1093/brain/123.2.291 Dambacher, M., Slattery, T. J., Yang, J., Kliegl, R., and Rayner, K. (2013). Evidence for direct control of eye movements during reading. J. Exp. Psychol. Hum. Percept. Perform. 39, 1468–1484. doi: 10.1037/a0031647 Dambacher, M., Slattery, T. J., Yang, J., Kliegl, R., and Rayner, K. (2013). Evidence for direct control of eye movements during reading. J. Exp. Psychol. Hum. Percept. 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Natural scene categories revealed in distributed patterns of activity in the human March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 9 Luke and Henderson Meaningfulness and Eye Movement Control brain. J. Neurosci. 29, 10573–10581. doi: 10.1523/JNEUROSCI.0559-09. 2009 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Wang, H.-C., Hwang, A. D., and Pomplun, M. (2010). Object frequency and predictability effects on eye fixation durations in real-world scene viewing. J. Eye Move. Res. 3, 3.1–3.10. Copyright © 2016 Luke and Henderson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. White, S. J., and Staub, A. (2012). The distribution of fixation durations during reading: effects of stimulus quality. J. Exp. Psychol. Hum. Percept. Perform. 38, 603–617. doi: 10.1037/a0025338 Yang, S.-N., and McConkie, G. W. (2001). Eye movements during reading: a theory of saccade initiation times. Vision Res. 41, 3567–3585. doi: 10.1016/S0042- 6989(01)00025-6 March 2016 | Volume 7 | Article 257 Frontiers in Psychology | www.frontiersin.org 10
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Right but not left angular gyrus modulates the metric component of the mental body representation: a tDCS study
Experimental brain research
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Introduction We are constantly requested to process information in the environment regarding the position of all objects sur­ rounding our body and our body itself. A theoretical frame­ work that well describe the relations between the sense of touch and the body has been proposed by Serino and Hag­ gard (2010). The authors describe an analytical model in which tactile afferent information can either be influenced by, or have influence upon a mental body representations (MBRs). Following the authors’ model, four separate pro­ cesses are necessary to link the physical body, the sense of touch, and the mental representation of one’s own body: (1) The physical body organizes tactile sensations, (2) Tactile sensations contribute to a mental MBR, (3) MBRs recip­ rocally influence primary tactile processing, and (4) MBRs mediate the formation of object representation from pri­ mary tactile sensations. G. F. Spitoni (*) · G. Pireddu · R. L. Cimmino · G. Galati · L. Pizzamiglio  Department of Psychology, The Sapienza University of Rome, Rome, Italy e-mail: grazia.spitoni@uniroma1.it G. F. Spitoni · G. Pireddu · R. L. Cimmino · G. Galati · L. Pizzamiglio  Neuropsychology Laboratory, IRCCS Santa Lucia Foundation, Rome, Italy A. Priori  Dipartimento di Scienze Neurologiche, Università degli Studi di Milano, Fondazione IRCCS Ospedale Maggiore Policlinico, Milan, Italy M. Lavidor · L. Jacobson  The Leslie and Susan Gonda Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat Gan, Israel M. Lavidor · L. Jacobson  Department of Psychology, Bar-Ilan University, Ramat Gan, Israel G. F. Spitoni (*) · G. Pireddu · R. L. Cimmino · G. Galati · L. Pizzamiglio  Department of Psychology, The Sapienza University of Rome, Rome, Italy e-mail: grazia.spitoni@uniroma1.it G. F. Spitoni · G. Pireddu · R. L. Cimmino · G. Galati · L. Pizzamiglio  Neuropsychology Laboratory, IRCCS Santa Lucia Foundation, Rome, Italy G. F. Spitoni · G. Pireddu · R. L. Cimmino · G. Galati · L. Pizzamiglio  Neuropsychology Laboratory, IRCCS Santa Lucia Foundation, Rome, Italy This latter process seems particularly important when­ ever one is asked to consider the intrinsic properties of a given object touching the skin. For example, it has been shown that altering the visual experience of the body alters perceived tactile distances (Cardini et  al. 2012; Longo et  al. 2008; Taylor-Clarke et  al. Right but not left angular gyrus modulates the metric component of the mental body representation: a tDCS study Grazia Fernanda Spitoni · Giorgio Pireddu · Rocco Luca Cimmino · Gaspare Galati · Alberto Priori · Michal Lavidor · Liron Jacobson · Luigi Pizzamiglio Received: 18 February 2013 / Accepted: 21 April 2013 / Published online: 12 May 2013 © The Author(s) 2013. This article is published with open access at Springerlink.com Abstract  The parietal lobes contribute to body-space representation. The present work aims at characterizing the functional role of the inferior parietal lobe in body- space representation and at studying the different roles of the angular gyrus in the right and left hemisphere. We con­ ducted three separate transcranial direct current stimula­ tion (tDCS) experiments using “tactile distance task” as an implicit measure of body representation. Whereas anodal tDCS on the right angular gyrus influences vocal reaction times (vRT) for stimuli delivered on the ipsilateral body parts without changes of accuracy, right tDCS improved both vRT and accuracy for tactile stimuli on the contralat­ eral limbs. Sham or left parietal anodal tDCS had no effect. These evidences support the view that right parietal areas have a crucial role in the metric component of the body representation. Keywords  Body representation · Touch · tDCS · Angular gyrus Keywords  Body representation · Touch · tDCS · Angular gyrus Exp Brain Res (2013) 228:63–72 DOI 10.1007/s00221-013-3538-9 Exp Brain Res (2013) 228:63–72 DOI 10.1007/s00221-013-3538-9 RESEARCH ARTICLE Introduction 2004): This evidence is interpreted as the brain attempt to preserve tactile size con­ stancy by rescaling the primary, distorted body-surface rep­ resentation into object-centered space according to visual experience of the body. A further study by de Vignemont A. Priori  Dipartimento di Scienze Neurologiche, Università degli Studi di Milano, Fondazione IRCCS Ospedale Maggiore Policlinico, Milan, Italy A. Priori  Dipartimento di Scienze Neurologiche, Università degli Studi di Milano, Fondazione IRCCS Ospedale Maggiore Policlinico, Milan, Italy A. Priori M. Lavidor · L. Jacobson  The Leslie and Susan Gonda Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat Gan, Israel M. Lavidor · L. Jacobson  Department of Psychology, Bar-Ilan University, Ramat Gan, Israel 1 3 Exp Brain Res (2013) 228:63–72 64 shape (see Longo et  al. 2010). Moreover, in a previous work (Spitoni et al. 2010), we argued that the right angular gyrus may house the process of body referencing of tactile inputs that is required to compare the tactile distance across different body part. Among this frame of evidences, we used the term metric component of body representation to describe a selective component of the body representation used to process tactile distances discrimination. et al. (2005) developed this issue showing that propriocep­ tion, as well as vision, plays a role in the formation of a MBR mediating tactile distance judgement. The authors also showed that the MBR used for body referencing of touch is not only a memorized body template stored in long-term memory, but also is updated online to integrate current sensory information. From this perspective, Serino and Haggard (2010) claimed that tactile perceptions are always referenced to the body, even if the content of the perception is an external object. Despite a rich description of the functional properties of the parietal areas in dealing with body representations, little is known about the neural basis of the specific metric com­ ponent of body representation. In a previous work (Spitoni et al. 2010), we pointed out the importance of parietal struc­ tures in dealing with the perception of the metric compo­ nent of MBR. In that study, subjects were stimulated with pairs of wooden cylinders separated by a variable distance and attached to nylon filaments of variable length and had to evaluate either the distance between the two cylinders (distance task) or the amount of skin deformation generated by the touch (contact task). The distance but not the contact task implicitly required to access a stored metric representa­ tion of the touched body part. Results showed that, while both tasks bilaterally activated parietal and frontal areas, only the evaluation of distances on the body surface acti­ vated the angular gyrus and the temporo-parieto-occipital junction in the right hemisphere. A. Priori Our conclusion was that the right angular gyrus, together with other cortical sensory modalities such as proprioception, plays a key role in the metric tactile perception by linking touched locations on the skin to an internal representation of body parts. The right parietal lobe contributes to body representation in several ways. Tsakiris et al. (2008) used TMS to show that the right temporal parietal junction (rTPJ) supports an internal model of the body. They suggested that the internal model of the body acts as a stored template against which novel stimuli are compared, allowing the maintenance of a basic sense of embodied self. Various functional magnetic resonance imaging (fMRI) studies investigated the proper­ ties of the parietal lobe in processing body information (see Corradi-Dell’Acqua et al. 2008; Pellijeff et al. 2006). Par­ kinson et al. (2010) showed that the superior parietal lobule (SPL) maintains an up-to-date estimate of the current pos­ tural configuration of the part of the body that is used dur­ ing the planning and the execution of a reaching movement. Dijkerman and de Haan (2007) described a model that specifies separable functional entities for somatosensory processing subserving perception and action. The authors propose that action-related processing occurs mainly in the posterior parietal cortex (PPC), whereas recognition and perception involve the insula, as well as the PPC. Relevant to our issue, this model also distinguishes between soma­ tosensory processing about the body (where you have been touched) and about external stimuli (e.g., surface features of objects). On the basis of neurophysiological and human lesions studies, Dijkerman and de Haan suggested a key role of PPC in the metric aspects of the body, such as its spatial configuration and size, whereas the insula is more concerned with higher order somatosensory processing of the body that is either related to a sense of ownership or to emotional experience. The specific role of the inferior and posterior part of the parietal lobe and the asymmetry between the left and right areas in controlling the metric component of body percep­ tion have not been reported by other studies; it is important to verify this finding using a different technique. Transcra­ nial direct current stimulation (tDCS) allows a direct com­ parison of the effect of the stimulating areas of interest, while the subjects are performing a tactile distance task on the skin of their body. 1 3 A. Priori The third dealt with the effect of anodal tDCS on the right side tested over the ipsilateral versus the contralateral arms. Since several studies agreed on the facilitative effect of anodal tDCS on cognitive tasks but not on the regu­ lar suppressive effect of cathodal stimulation (see Val­ lar and Bolognini 2011), we decided to focus on anodal stimulation. Fig. 1   Stimulus used in the tactile distance task for Experiment 1 (manual stimulation) Finally, we investigated the possible change of the effects in the time window of stimulations. To this aim, dif­ ferent epochs of stimulations session of Experiments 2 and 3 were considered. other one was either longer or shorter. There were 48 trials in total, in a pseudo-randomized and pre-ordered sequence, balanced for the location of the standard stimulus (forearm or thigh), the location of the longer stimulus (forearm or thigh), and difficulty (easy or difficult). Easy trials were those where the two stimuli differed by 2 cm (i.e., the nonstandard stimu­ lus was 5 or 9 cm long); difficult trials were those where the two stimuli differed by 1 cm (i.e., the nonstandard stimulus was 6 or 8  cm long). Subjects were instructed to respond vocally, by saying “arm” or “thigh” aloud, as fast and as accurately as possible. Vocal reaction times (vRTs) and accu­ racy were recorded by a custom made script. Participants There were 13 subjects (8 males) in the anodal group (mean age 26.2, mean handedness score 95), and 9 subjects (4 males) in the sham group (mean age 27, mean handed­ ness 95—Edinburgh Inventory, Oldfield 1971; Salmaso and Longoni 1985). The experiment was approved by the Research Ethics Committee of Bar-Ilan University. tDCS protocol tDCS protocol A direct current of 1  mA (10  min) was induced by two saline-soaked surface sponge electrodes (7 × 4.5 cm) and delivered by a battery-driven, constant-current stimulator (Rolf Schneider Electronics, Germany). Previous studies have reported that this stimulation intensity is safe to use in healthy volunteers (Brunoni et al. 2012). In the stimulation sessions, current increased in ramp-like fashion from 0 to 1 mA at the onset of stimulation, eliciting a transient tin­ gling sensation on the scalp (Hummel et al. 2005). Fifteen minutes after the onset of tDCS anodal or sham sessions the current was turned off slowly over a few seconds. In the sham group, the current turned off after 30 s. Experiment 1 The experimental paradigm was an adaptation of the fMRI study by Spitoni et al. (2010). Two pairs of tactile stimuli were rapidly delivered in close succession on two distinct body locations. Subjects concentrated on the body dis­ tance subtended by the two items in each pair and judged in which pair the distance was longer. We used a mixed design with stimulation type (anodal or sham) as a between-subject factor, and time (before or after stimulation) and difficulty (easy or difficult) as within-sub­ ject factors. A. Priori We delivered tactile stimulations to the left and right limbs and hypothesized that anodal stimulation of the crucial right parietal areas would improve behavioral responses in the same metric distance task that we used in the fMRI study previously described (Spitoni et al. 2010), while anodal stimulation of the same parietal regions in left hemisphere would not enhance subjects’ performance in the same way. The importance of the parietal lobe in dealing with the localization of somatic stimuli on the skin was previously highlighted by Van Boven et al. (2005). Authors described two areas selectively involved in a grating orientation task (stimulus orientation) and in a simple tactile localization task (stimulus location). In contrast to grating orientation which specifically activates IPL, tactile localization prefer­ entially engages the TPJ. Three separate and consecutive experiments were planned. The first aimed at testing the effectiveness of tDCS on a tactile distance discrimination task. It has been known from the previous literature that anodal tDCS applied to the primary somatosensory cortex (S1) improves performance in tactile discriminative tasks only in the parts of the body In this study, we will focus on the metric component of the MBR. Specifically, we refer to the concept of metric compo­ nent of body representation as one of the functions that the somatoperception uses to form a model of body size and 1 3 Exp Brain Res (2013) 228:63–72 65 Fig. 1   Stimulus used in the tactile distance task for Experiment 1 (manual stimulation) contralateral to the site of stimulation (Ragert et al. 2008). To our knowledge, no data are available on the actual effi­ cacy of tDCS on higher order tactile tasks such as distance/ metric discrimination tasks. The second study tested the lateralization hypothesis, that is, the effect of anodal tDCS on the right versus the left angular gyrus. The third dealt with the effect of anodal tDCS on the right side tested over the ipsilateral versus the contralateral arms. contralateral to the site of stimulation (Ragert et al. 2008). To our knowledge, no data are available on the actual effi­ cacy of tDCS on higher order tactile tasks such as distance/ metric discrimination tasks. The second study tested the lateralization hypothesis, that is, the effect of anodal tDCS on the right versus the left angular gyrus. Results Accuracy and vRTs are shown in Fig. 2a, b. A mixed- design analysis of variance (ANOVA) was conducted, with stimulation type (anodal and sham) as the between-subject factor and time (pre-stimulation, post-stimulation) and dif­ ficulty (short or long distance) as the within-subjects fac­ tors. Bonferroni post hoc tests corrected for multiple com­ parisons (p < 0.05) were conducted. Accuracy A significant main effect of difficulty emerged [F(1, 29)  =  23.3; p  <  0.001], with more accurate responses (77.6 %) to easy stimuli than difficult ones (63.8 %), in agreement with the well-documented distance effect in magnitude processing (Moyer and Bayer 1976). A sig­ nificant stimulation type by time interaction also emerged [F(2, 29)  =  4.69; p  <  0.05]. Accuracy increased fol­ lowing stimulation in the anodal group (from 70.3 to 77.6  %), but not in the sham group (pre 68.8  %, post 68.0 %). As significant facilitative effects were found for both difficult and easy comparisons, there was no interac­ tion (Fig. 2a). Experiment 1 employed anodal and sham stimulation of the right angular gyrus, while subjects performed the tactile dis­ tance task with stimuli delivered on left limbs. The results showed that anodal stimulation improved responses and speed, whereas sham was ineffective. The aim of this first study was to test the effectiveness of tDCS on higher order tactile tasks. As far these preliminary results were in line to our expectations, we carried on Experiments 2 and 3. Stimuli and task Each participant performed the distance task once (pre- stimulation); then, he was stimulated over the right angular gyrus in one of the two stimulation conditions (anodal or sham) and then performed the distance task again (post- stimulation). The anodal electrode was placed over the right angular gyrus and the cathodal electrode over the left orbitofrontal cortex. Localization was established using the 10–20 EEG technique, and the right angular gyrus was marked over P4 (Fuggetta et al. 2006). The protocol was similar to that used in fMRI study by Spi­ toni et al. (2010). Stimuli were two sets of 5 perspex inverted U-shaped (see Fig. 1) objects, in which the distance between the two touching ends varied from 5 to 9 cm. In each trial, participants were manually stimulated first on the left forearm and 1 s later on the left thigh and judged which tactile stimu­ lus was longer (distance task). Either the first or the second stimulus was a standard stimulus (distance = 7 cm), while the 1 3 Exp Brain Res (2013) 228:63–72 66 easy than difficult judgments (891 ms). A significant effect of time also emerged [F(1, 29)  =  9.94; p  <  0.01], with a reduction of vRTs after stimulation (from 914 to 796 ms). Importantly, a significant stimulation type by time interaction emerged [F(2, 29) = 3.32; p < 0.05]. Following stimulation, vRT decreased in the anodal group (from 1,004 to 821 ms), whereas in the sham group, vRTs slightly increased between the two experimental sessions (from 833 to 857  ms). The 3-way interaction was not significant (Fig. 2b). vRTs A significant main effect of difficulty emerged [F(1, 29) = 21.3; p < 0.001], with faster responses (819 ms) to Fig. 2   Results of Experiment 1 (right tDCS stimulation, tactile stim­ uli on the left arm). a Accuracy (percentage of correct responses). b Vocal response times. Error bars represent standard deviations We used a completely within-subjects design with hemi­ sphere (left or right), stimulation type (anodal or sham), time (before or during stimulation), and difficulty (easy or difficult) as within-subject factors. All subjects received tactile stimuli in the same “hemi­ body” of the tDCS stimulation, that is, that if the tDCS was given in the right hemisphere, the tactile stimuli were given to right forearm and to the right thigh. Similarly, when the tDCS was given in the left hemisphere, the tactile stimuli were given to left forearm and to the left thigh. We predicted that anodal stimulation on the right, but not on the left, angular gyrus would improve performances on the metric distance task. Experiment 2 The second experiment tested the lateralization hypothesis, that is, the effect of anodal tDCS on the right versus the left angular gyrus. This study is strongly linked to that con­ ducted by Spitoni et al. (2010), who found activation in the right angular gyrus after stimulation of the right hemibody; from this standpoint, we aimed at testing the fMRI results not only in the right but also in the left hemisphere. tDCS protocol In the first two sessions, anodal electrode was placed over the right angular gyrus (P4 in the 10–20 EEG system) and the cathode over the right shoulder. In the last two sessions, anodal electrode was place on the left angular gyrus (P3) and cathode still on the right shoulder. Experiment details are reported in Table 1. Figure 3a, b shows the accuracy and vRTs results after stimulation of the right hemisphere. Results We conducted repeated-measures ANOVAs with hemi­ spheres (left and right), stimulation type (anodal or sham), time (before or during stimulation), and difficulty (easy or difficult) as within-subject factors. Participants A total of 12 healthy volunteers (6 females) participated in this experiment. All were right-handed, as assessed by a modified version of the Edinburgh Inventory (Oldfield 1971; Salmaso and Longoni 1985). None had a history of Fig. 2   Results of Experiment 1 (right tDCS stimulation, tactile stim­ uli on the left arm). a Accuracy (percentage of correct responses). b Vocal response times. Error bars represent standard deviations 1 3 Exp Brain Res (2013) 228:63–72 67 In order to balance the administration across par­ ticipants, half of them started with anodal tDCS and the remaining half with sham. neurological or psychiatric diseases or were taking medica­ tion; all were in good health. Participants’ ages ranged from 21 to 34 years (mean age 24.2; SD = 2.9). Each partici­ pant was tested in four sessions (with a three-day interval between sessions). All subjects provided written informed consent. The experimental protocol was approved by the Research Ethics Committee of the IRCCS Santa Lucia Foundation of Rome. neurological or psychiatric diseases or were taking medica­ tion; all were in good health. Participants’ ages ranged from 21 to 34 years (mean age 24.2; SD = 2.9). Each partici­ pant was tested in four sessions (with a three-day interval between sessions). All subjects provided written informed consent. The experimental protocol was approved by the Research Ethics Committee of the IRCCS Santa Lucia Foundation of Rome. Stimuli and task A repeated-measures ANOVA on vRTs revealed an highly significant difference among stimulated hemi­ spheres [F(1, 103) = 11.740; p < 0.001] with a right-later­ alized hemispheric preference. The same repeated-measure ANOVA on accuracy showed that hemispheres did not affect subjects performances. Bonferroni post hoc tests cor­ rected for multiple comparisons (p < 0.05) were conducted. We applied tactile stimuli to the forearm and thigh. Test and reference stimuli consisted of two simultaneous con­ tacts from a line of four miniature solenoids. The active solenoids were selected randomly in each trial. Thus, par­ ticipants experienced tactile distances of one (difficult) or two (easy) steps. We asked them to judge which of the two tactile distances felt longer (arm or thigh). Anodal stimulation of right parietal area and tactile stimuli on right limbs Anodal stimulation of right parietal area and tactile stimuli on right limbs In 80 % of the trials, the tactile distance on the arm dif­ fered from that on the thigh by one or two steps; in the remaining 20  %, there was no difference. Before admin­ istering the experiment, we calculated the two-point dis­ crimination threshold (defined as the level at which 75 % of the responses were correct) of each participant. In order to make the tactile stimulation above the subjective threshold, we added 0.5  cm to the two-point discrimination thresh­ old and then used this measure to place the closest pair of solenoids. Accuracy  Here, we found a significant effect for diffi­ culty, with subjects more accurate in judging easier stim­ uli (79.4 %) then more difficult ones (72.5 %) (F = 21.5; p < 0.001). vRTs  Repeated-measure ANOVA showed a significant stim­ ulation type by time interaction [F(1, 103) = 17.6; p < 0.001], with faster vRTs during anodal than sham stimulation. Moreo­ ver, a significant effect of difficulty emerged [F(1, 103) = 48.4; p < 0.001], with faster responses to easy stimuli. Importantly, a significant stimulation by difficulty interaction emerged [F(1, 103) = 4.21; p < 0.05], with faster vRTs in the anodal-easy (633 ms) than the anodal-difficult stimulation (791 ms). vRTs  Repeated-measure ANOVA showed a significant stim­ ulation type by time interaction [F(1, 103) = 17.6; p < 0.001], with faster vRTs during anodal than sham stimulation. Moreo­ ver, a significant effect of difficulty emerged [F(1, 103) = 48.4; p < 0.001], with faster responses to easy stimuli. Stimuli and task Importantly, a significant stimulation by difficulty interaction emerged [F(1, 103) = 4.21; p < 0.05], with faster vRTs in the anodal-easy (633 ms) than the anodal-difficult stimulation (791 ms). Before the experiment, subjects were familiarized with the task in a warm-up session consisting of 10 comparisons. tDCS protocol vRT  We found a significant effect for difficulty [F(1, 174) = 6.4; p < 0.01], with faster vRTs in judging easier (619 ms) stimuli than more difficult ones (722 ms). Participants In this experiment, we tested 6 participants (3 males) ranged from 19 to 27 (mean age 23.7, SD = 2.1). Each par­ ticipant was tested in four sessions (with 2–3 days between each session). Anodal stimulation on left parietal area and tactile stimuli on left limbs Again, we performed a 3-way repeated-measures ANOVA with stimulation condition (anodal or sham), time (pre- stimulation or during stimulation), and difficulty (easy or difficult) as within factors. Table 1   tDCS protocol of Experiment 2 Pretest Stimulation Electrode Limb I session Task 15 min Sham Right AG Right II session Task 15 min Anodal Right AG Right III session Task 15 min Sham Left AG Left IV session Task 15 min Anodal Left AG Left AG Angular gyrus Table 1   tDCS protocol of Experiment 2 Accuracy  Also in this case, repeated-measures ANOVA showed that subjects were more accurate in judging easier (77.7 %) than difficult stimuli (72.1 %) [F(1, 172) = 7.7; p < 0.01]. vRT  We found a significant effect for difficulty [F(1, 174) = 6.4; p < 0.01], with faster vRTs in judging easier (619 ms) stimuli than more difficult ones (722 ms). 1 3 Exp Brain Res (2013) 228:63–72 68 Fig. 4   a, b Experiment 2: accuracy and vocal reaction times when anodal stimulation is given on the LH and the tactile stimuli on the left limbs Fig. 3   a, b Experiment 2: accuracy and vocal reaction times when anodal stimulation is given on the RH and the tactile stimuli on the right limbs Fig. 4   a, b Experiment 2: accuracy and vocal reaction times when anodal stimulation is given on the LH and the tactile stimuli on the left limbs Fig. 3   a, b Experiment 2: accuracy and vocal reaction times when anodal stimulation is given on the RH and the tactile stimuli on the right limbs Fig. 4   a, b Experiment 2: accuracy and vocal reaction times when anodal stimulation is given on the LH and the tactile stimuli on the left limbs Fig. 4   a, b Experiment 2: accuracy and vocal reaction times when anodal stimulation is given on the LH and the tactile stimuli on the left limbs Fig. 3   a, b Experiment 2: accuracy and vocal reaction times when anodal stimulation is given on the RH and the tactile stimuli on the right limbs Stimuli and task We used the same set of stimuli and the same apparatus of Experiment 2. Experiment 3 details are reported in Table 2. The order of administration was balanced across subjects. We used the same set of stimuli and the same apparatus of Experiment 2. Experiment 3 details are reported in Table 2. The order of administration was balanced across subjects. Discussion In Experiment 2, anodal and sham stimulation were given on the right and left angular gyrus, while subjects per­ formed the tactile distance task with stimuli delivered on the same side of DC stimulation. Results showed that anodal on right AG significantly improved vRTs and leave unaffected accuracy. No significant effect of anodal stim­ ulation was found when the stimulation was given in the left AG. Methods Figure 4a, b shows accuracy and vRTs after stimulation of the left hemisphere. vRTs Also, in the case of anodal on the right AG and tactile task on the right limb, we observed a reduction of vRTs consistent with results of Experiment 2. A significant stimulus side by stimulation type interaction was found with faster vRTs after anodal stimulation and tactile stimuli on the left limb [F(1, 31) = 12.6; p < 0.001; ηp 2 = 0.63]. A limitation of this third study is the employment of a small sample. For this reason, we believe that the conclu­ sion inferred from the study should be confirmed by further investigations. p We also found a significant stimulus side by stimulation type by difficulty interaction [F(1, 31) = 8.04; p < 0.05; ηp 2 = 0.491], with faster vRTs in difficult comparisons after anodal stimulation when stimuli were administered on right limbs (Fig. 5b). Effect of anodal stimulation on the time window of 15 min Figure 5a, b shows the accuracy and vRTs results after stimulation of the right hemisphere and tactile stimuli on left or right limbs. In several studies, the duration of the effects of tDCS spreads from the time period of the stimulation up to about 60 min (Dieckhöfer et al. 2006; Matsunaga et al. 2004). A systematic summary of the main features of the three experiments is given in Table 3. On the basis of a possible summative effect of time, a collateral interest of our study was to test whether or not anodal tDCS affects tactile performance in different epochs of stimulation. To this aim, we fractionated the 15 min of the online stimulation in three separate time windows: from 0 to 5, from 6 to 10, and from 11 to 15 min. Experiment 3 As shown in Table 2, the tDCS protocol was quite similar to that used in Experiment 2, but in this case, anodal tDCS was always given over the right angular gyrus, and subjects received the tactile stimuli on both contralateral and ipsi­ lateral body parts. Also, in this experiment, we contrasted sham versus anodal stimulation without a pre-stimulation session. Experiment 3 dealt with the possible differences in perfor­ mances when tDCS is given to contra or ipsilateral body parts in respect to the brain stimulation. We conducted repeated-measures ANOVAs with tactile stimulus side (ipsi—contra), stimulation type (anodal or sham), and difficulty (easy or difficult) as factors. 1 3 Exp Brain Res (2013) 228:63–72 69 Fig. 5   a, b Accuracy and vocal reaction times when anodal stimula­ tion is given on the RH and the tactile stimuli on the left or the right limbs Bonferroni post hoc tests corrected for multiple compar­ isons (p < 0.05) were conducted. Results Accuracy A main effect of difficulty emerged [F(1, 9)  =  22.9; p < 0.001; ηp 2 = 0.763], with more accurate responses to easy (79.1 %) stimuli than difficult ones (70.7 %). Moreover, a significant stimulus side by stimulation type by difficulty interaction [F(1, 9)  =  7.55; p  <  0.05; ηp 2 = 0.404] emerged: during anodal stimulation and tactile i Table 2   tDCS protocol of Experiment 3 Pretest Stimulation Electrode Limb I session Task 15 min Sham Right AG Left II session Task 15 min Anodal Right AG Left III session Task 15 min Sham Right AG Right IV session Task 15 min Anodal Right AG Right AG Angular gyrus Fig. 5   a, b Accuracy and vocal reaction times when anodal stimula­ tion is given on the RH and the tactile stimuli on the left or the right limbs Table 2   tDCS protocol of Experiment 3 Pretest Stimulation Electrode Limb I session Task 15 min Sham Right AG Left II session Task 15 min Anodal Right AG Left III session Task 15 min Sham Right AG Right IV session Task 15 min Anodal Right AG Right AG Angular gyrus Table 2   tDCS protocol of Experiment 3 Bonferroni post hoc tests corrected for multiple compar­ isons (p < 0.05) were conducted. Results Accuracy A main effect of difficulty emerged [F(1, 9)  =  22.9; p < 0.001; ηp 2 = 0.763], with more accurate responses to easy (79.1 %) stimuli than difficult ones (70.7 %). Fig. 5   a, b Accuracy and vocal reaction times when anodal stimula­ tion is given on the RH and the tactile stimuli on the left or the right limbs Moreover, a significant stimulus side by stimulation type by difficulty interaction [F(1, 9)  =  7.55; p  <  0.05; ηp 2 = 0.404] emerged: during anodal stimulation and tactile stimuli on the left limbs accuracy increased in the difficult comparisons (from 71.4 to 79.5 %), but did not change in the easy comparisons (81.4 and 80.9 %) (Fig. 5a). on the contralateral and to the ipsilateral hemibody. Here, we found that in case of anodal stimulation on the right AG and tactile task on the left limb, accuracy improved in diffi­ cult comparisons. Moreover, vRTs decreased during anodal stimulation. vRTs General discussion The involvement of parietal structures in the represen­ tations of the body has been widely investigated. Accord­ ing to Longo et al. (2010), parietal lobes play crucial roles in cognitive processes such as (1) the representation of the superficial body schema, (2) the localization of somatic stimuli on the body surface (Porro et al. 2007; Van Boven et  al. 2005), (3) the perception of the current posture of the body (Pellijeff et al. 2006; Wolpert et al. 1998; Fasold et al. 2008), and (4) the structural/topological knowledge of one’s own body (Felician et al. 2004; Corradi-Dell’Acqua et al. 2008). Specifically, the posterior parietal cortex (PPC) is thought to integrate different classes of sensory informa­ tion (e.g., somatosensory, auditory, and visual) to gener­ ate multiple representations of space. Through combining these spatial representations, we can produce a variety of actions such as saccadic eye movements and reaching or grasping movements of the upper limb. Moreover, a recent TMS study by Azañón et al. (2010) provided clear evidence for a causal role of posterior parietal cortex (PPC) from a somatotopic to an external spatial frame of reference, sug­ gesting a crucial role of this area in the remapping of touch. The main results can be summarized as follows. In the first study, we demonstrated the efficacy of tDCS in modulat­ ing subjects’ performance in a distance discrimination task. This initial result seems to be of particular interest, since no previous studies have ever focused on the effectiveness of tDCS on higher order tactile functions such as those required in a tactile metric discrimination task. Experiments 2 and 3 directly pointed at the functional properties of the angular gyrus (AG) of both hemispheres in dealing with tactile distance discrimination and, as a consequence, with the metric component of the mental body representations. Evidence from these two studies indicates that anodal stimulation on the right angular gyrus systematically reduces vocal reaction times (vRT), thus improving discrimination speed, independent of whether the stimuli are delivered to contralateral or ipsilateral body parts. However, accuracy improves only when the tactile stimuli are given on contralateral (left) limbs. Both speed and accuracy effects are totally absent when anodal stimu­ lation is delivered to left AG. Given these results, it seems reasonable that right, but not left, angular gyrus is involved in the metric component of the MBR. Lesion studies in monkeys and in humans (Rushworth et al. Discussion In the third experiment, anodal and sham stimulation were given only on the right angular gyrus, while subjects per­ formed the tactile distance task with stimuli delivered first 1 3 70 Exp Brain Res (2013) 228:63–72 Table 3   Methodological characteristics and main results of the three experiments Experiment 1 (manual stimulation) Experiment 2 (computerized stimulation) Experiment 3 (computerized stimulation) Montage Bicephalic Monocephalic Monocephalic Task Off-line Online Online tDCS stimulation side Right Right–Left Right Tactile stimulation Contralateral Ipsilateral Contralateral–Ipsilateral Effect on accuracy Yes No Yes (contra) No (ipsi) Effect on vRTs Yes Yes (only right) Yes We then performed ANOVA for repeated measures on accuracy and on vRTs from data of Experiments 2 and 3. Analyses showed no trend. Neither accuracy nor vRT sig­ nificantly changed between epochs. perception: primary tactile sensation (such as an external object pressing on the skin) and secondary tactile percep­ tion that includes the metric/spatial information need to be rescale. We suggested that the extraction of the metric information from the skin surface involves additional com­ putational processing stages over perceiving simple contact to the skin and that these rescaling processes are hosted by right AG. According to our knowledge, no systematic studies have focused on the duration of the tDCS effect over time in the posterior parietal areas, and indeed, this could be very important for those who use tDCS to rehabilitate neuropsy­ chological patients. Evidence from the present study seems to confirm the key role played by the right AG in the rescaling processing of metrical features of MBR. General discussion 1997, 1998; Sirigu et al. 1995) suggest that cerebral areas within the PPC play an important role in maintain­ ing an accurate and up-to-date representation of the current postural state of the body (the body schema). What seems of particular interest in this context is that the region selec­ tively involved in the representations of the limbs postures is the superior parietal cortex (SPC), particularly the medial location (precuneus) (Pellijeff et al. 2006; Parkinson et al. The involvement of parietal structures in the metric processing of MBR Table 3   Methodological characteristics and main results of the three experiments Accuracy and speed in the metrical processing of MBR In our study, we focused on accuracy and speed, and we found an effect of tDCS on vRTs when electrical modula­ tion was delivered to right AG, and tactile stimulation was given to ipsilateral arms. Moreover, an effect of tDCS was seen on both vRTs and accuracy when anodal stimulation was delivered over the right AG, but tactile stimuli were given to the contralateral arms. These results seem to sup­ port an aspecific effect of tDCS on speed when tactile stimuli are delivered on the ipsilateral side of the electri­ cal stimulation, together with a specific effect on accuracy when stimuli are given to the contralateral side. The rea­ sons for such dissociation can only be hypothesized. On the one hand, we can assume the existence of two differ­ ent mechanisms; the first could rely on the simple excita­ bility changes in the somatosensory cortex contralateral to the side of tactile stimulation, whereas the observed vRTs reduction (on the body part ipsilateral to the stimulation) might simply reflect a more specific arousal effect of the anodal stimulation. What we know from previous stud­ ies is that in the tDCS setup, the current flows following routes that are influenced by the impedance of the tissue crossed by the current. This means that tissue impedance and cell orientation can strongly affect the flow of the cur­ rent, resulting in different polarization patterns. Many studies in the literature assume that the maximum effect of tDCS occurs in areas perpendicular to the stimulating electrode and that cell polarization–depolarization is pre­ dominantly localized in these underlying brain structures A limit of our experimental paradigm is the small amount of different distances on the skin that it was pos­ sible to stimulate through the mechanical devices used for Experiments 2 and 3. The solenoid device allowed for 8 miniature magnets only (4 on the thigh–4 on the forearm). With just 4 stimuli, we obtained a maximum of 3 sizes, and thus just 2 distance differences, that we labeled as “easy” and “difficult,” respectively. This technical limita­ tion prevented us to fit psychophysical functions to the raw data and analyze them in terms of parameters such as bias and precision, as we did in our previous study (Spitoni et al. 2010). Such analyses may bring to a clearer explana­ tion on the nature of the improvement in accuracy found after tDCS. The involvement of parietal structures in the metric processing of MBR In a previous fMRI study (Spitoni et al. 2010), we proposed that in human brain, we can distinguish two forms of touch 3 3 Exp Brain Res (2013) 228:63–72 71 2010). Moreover, a recent fMRI study with tactile stimula­ tion over body surface (Huang et al. 2012) revealed soma­ totopic areas of the face and multiple body parts forming a higher level homunculus in the superior posterior parietal cortex. (Datta et al. 2010; Nitsche and Paulus 2011). But it has also been documented that a variable amount of current can also spread to contiguous areas (Datta et  al. 2009, 2010; Sadleir et al. 2010). This latter evidence could par­ tially give reason for the improvement of accuracy after contralateral stimulation; from this perspective, we can consider the possibility that an amount of current under right AG may spread to close somatosensory areas result­ ing in a known effect of improvement of tactile acuity of the contralateral side of stimulation (Ragert et al. 2008; Song et al. 2011). 2010). Moreover, a recent fMRI study with tactile stimula­ tion over body surface (Huang et al. 2012) revealed soma­ totopic areas of the face and multiple body parts forming a higher level homunculus in the superior posterior parietal cortex. In the present study, we stimulated the inferior parietal cortex (IPC), and we observed a modulation of the metrical component of MBR. Given these evidence, we can specu­ late that in order to build an accurate postural configura­ tion, SPC may not need metrical rescaling and that this lat­ ter process could be selectively linked to IPC. The inferior and posterior parietal structures are also involved in local­ izing parts of one’s own or others’ bodies and in compar­ ing the size of body parts. It should be noted that both the localization of parts and the comparison of distance over the body require activation of an internal body representa­ tion. In distance comparisons, however, the representation must include metric properties. Methodological comments In our study, we used both mono-and bicephalic montages to test whether electrode disposition influenced perfor­ mance on the tactile task. We found that anodal stimula­ tion provided significant results in various conditions, that is, when the electrode montage was bicephalic or when the anodal electrode was placed on the brain and the cathode on the right shoulder. We are aware that tDCS has a low spatial resolution; that is why we believe that in order to claim to a clearer distinc­ tion between the functional roles of IPC and SPC, fMRI studies need to be implemented. Moreover, results did not change when tDCS was applied before the tactile discrimination task or during the stimulation. At variance with studies that measured other cognitive tasks (Andrews et al. 2011; Cohen Kadosh et al. 2010; Ross et  al. 2010; Cerruti and Schlaug 2009), the effect was significant in terms of speeding responses and improving accuracy. References References Andrews SC, Hoy KE, Enticott PG, Daskalakis ZJ, Fitzgerald PB (2011) Improving working memory: the effect of combining cog­ nitive activity and anodal transcranial direct current stimulation to the left dorsolateral prefrontal cortex. Brain Stimul 4:84–89 Moyer RS, Bayer RH (1976) Mental comparison and the symbolic distance effect. Cogn Psychol 8:228–246 Nitsche MA, Paulus W (2011) Transcranial direct current stimula­ tion—update 2011. Restor Neurol Neurosci 29:463–492 Azañón E, Longo MR, Soto-Faraco S, Haggard P (2010) The poste­ rior parietal cortex remaps touch into external space. Curr Biol 27:1304–1309 tion—update 2011. Restor Neurol Neurosci 29:463–492 p Oldfield RC (1971) The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9:97–113 Brunoni AR, Nitsche MA, Bolognini N, Bikson M, Wagner T, Mera­ bet L, Edwards DJ, Valero-Cabre A, Rotenberg A, Pascual-Leone A, Ferrucci R, Priori A, Boggio PS, Fregni F (2012) Clinical research with transcranial direct current stimulation (tDCS): chal­ lenges and future directions. Brain Stimul 5:175–195 Parkinson A, Condon L, Jackson SR (2010) Parietal cortex coding of limb posture: in search of the body-schema. Neuropsychologia 48:3228–3234 Pellijeff A, Bonilha L, Morgan PS, McKenzie K, Jackson SR (2006) Parietal updating of limb posture: an event-related fMRI study. Neuropsychologia 44:2685–2690 Cardini F, Longo MR, Driver J, Haggard P (2012) Rapid enhance­ ment of touch from non-informative vision of the hand. Neu­ ropsychologia 50:1954–1960 Porro CA, Martinig M, Facchin P, Maieron M, Jones AK, Fadiga L (2007) Parietal cortex involvement in the localization of tactile and noxious mechanical stimuli: a transcranial magnetic stimula­ tion study. Behav Brain Res 28:183–189 Cerruti C, Schlaug G (2009) Anodal transcranial direct current stimu­ lation of the prefrontal cortex enhances complex verbal associa­ tive thought. J Cogn Neurosci 21:1980–1987 Ragert P, Vandermeeren Y, Camus M, Cohen LG (2008) Improvement of spatial tactile acuity by transcranial direct current stimulation. Clin Neurophysiol 119:805–811 Cohen Kadosh R, Soskic S, Iuculano T, Kanai R, Walsh V (2010) Modulating neuronal activity produces specific and long-lasting changes in numerical competence. Curr Biol 23:2016–2020 Ross LA, McCoy D, Wolk DA, Coslett HB, Olson IR (2010) Improved proper name recall by electrical stimulation of the ante­ rior temporal lobes. Neuropsychologia 48:3671–3674 Corradi-Dell’Acqua C, Hesse MD, Rumiati RI, Fink GR (2008) Where is a nose with respect to a foot? The left posterior parietal cortex processes spatial relationships among body parts. Cereb Cortex 18:2879–2890 Rushworth MFS, Nixon PD, Passingham RE (1997) Parietal cortex and movement. 1. References Movement selection and reaching. Exp Brain Res 117:292–310 Datta A, Bansal V, Diaz J, Patel J, Reato D, Bikson M (2009) Gyri- precise head model of transcranial direct current stimulation: improved spatial focality using a ring electrode versus conven­ tional rectangular pad. Brain Stimul 2:201–207 Rushworth MFS, Johansen-Berg H, Young SA (1998) Parietal cor­ tex and spatial-postural transformation during arm movements. J Neurophysiol 79:478–482 Datta A, Bikson M, Fregni F (2010) Transcranial direct current stimu­ lation in patients with skull defects and skull plates: high-resolu­ tion computational FEM study of factors altering cortical current flow. Neuroimage 52:1268–1278 Sadleir RJ, Vannorsdall TD, Schretlen DJ, Gordon B (2010) Transcra­ nial direct current stimulation (tDCS) in a realistic head model. Neuroimage 51:1310–1318 Salmaso D, Longoni AM (1985) Problems in the assessment of hand preference. Cortex 21:533–554 de Vignemont F, Ehrsson HH, Haggard P (2005) Bodily illusions modulate tactile perception. Curr Biol 26:1286–1290 p Serino A, Haggard P (2010) Touch and the body. Neurosci Biobehav Rev 34:224–236 Dieckhöfer A, Waberski TD, Nitsche M, Paulus W, Buchner H, Gob­ belé R (2006) Transcranial direct current stimulation applied over the somatosensory cortex—differential effect on low and high frequency SEPs. Clin Neurophysiol 117:2221–2227 Sirigu A, Cohen L, Duhamel JR, Pillon B, Dubois B, Agid Y (1995) A selective impairment of hand posture for object utilization in apraxia. Cortex 31:41–56 Dijkerman HC, de Haan EH (2007) Somatosensory processes sub­ serving perception and action. Behav Brain Science 30:189–201 Song S, Sandrini M, Cohen LG (2011) Modifying somatosensory processing with non-invasive brain stimulation. Restor Neurol Neurosci 29:427–437 Fasold O, Heinau J, Trenner MU, Villringer A, Wenzel R (2008) Pro­ prioceptive head posture-related processing in human polysen­ sory cortical areas. Neuroimage 15:1232–1242 Spitoni GF, Galati G, Antonucci G, Haggard P, Pizzamiglio L (2010) Two forms of touch perception in the human brain. Exp Brain Res 207:185–195 Felician O, Romaiguère P, Anton JL, Nazarian B, Roth M, Poncet M, Roll JP (2004) The role of human left superior parietal lobule in body part localization. Ann Neurol 55:749–751 Taylor-Clarke M, Jacobsen P, Haggard P (2004) Keeping the world a constant size: object constancy in human touch. Nat Neurosci 7:219–220 Fuggetta G, Pavone EF, Walsh V, Kiss M, Eimer M (2006) Cortico- cortical interactions in spatial attention: a combined ERP/TMS study. Accuracy and speed in the metrical processing of MBR To sum up, the present data suggest that anodal tDCS is effective in modifying the metric component of the MBR in a tactile discrimination tasks. Monocephalic montage of the electrodes and online stimulation produce consistent results, and in the specific task, these modifications have to be tested on parts of the body contralateral to the cortical stimulation. Open Access  This article is distributed under the terms of the Crea­ tive Commons Attribution License which permits any use, distribu­ tion, and reproduction in any medium, provided the original author(s) and the source are credited. 1 3 72 Exp Brain Res (2013) 228:63–72 Matsunaga K, Nitsche MA, Tsuji S, Rothwell JC (2004) Effect of transcranial DC sensorimotor cortex stimulation on soma­ tosensory evoked potentials in humans. Clin Neurophysiol 115:456–460 References J Neurophysiol 95:3277–3280 Tsakiris M, Costantini M, Haggard P (2008) The role of the right tem­ poro-parietal junction in maintaining a coherent sense of one’s body. Neuropsychologia 46:3014–3018 Huang RS, Chen CF, Tran AT, Holstein KL, Sereno MI (2012) Map­ ping multisensory parietal face and body areas in humans. Proc Natl Acad Sci USA 109(44):18114–18119 Vallar G, Bolognini N (2011) Behavioural facilitation following brain stimulation: implications for neurorehabilitation. Neuropsychol Rehabil 21:618–649 Hummel F, Celnik P, Giraux P, Floel A, Wu WH, Gerloff C, Cohen LG (2005) Effects of non-invasive cortical stimulation on skilled motor function in chronic stroke. Brain 128:490–499 Van Boven RW, Ingeholm JE, Beauchamp MS, Bikle PC, Ungerlei­ der LG (2005) Tactile form and location processing in the human brain. Proc Natl Acad Sci USA 30:12601–12605 Longo MR, Cardozo S, Haggard P (2008) Visual enhancement of touch and the bodily self. Conscious Cogn 17:1181–1191 Wolpert DM, Goodbody SJ, Husain M (1998) Maintaining internal representations: the role of the human superior parietal lobe. Nat Neurosci 1:529–533 Longo MR, Azañón E, Haggard P (2010) More than skin deep: body representation beyond primary somatosensory cortex. Neuropsy­ chologia 48:655–668 1 3
https://openalex.org/W4387095106
https://link.springer.com/content/pdf/10.1007/s13235-023-00528-5.pdf
English
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Benefits of Rent Sharing in Dynamic Resource Games
Dynamic games and applications
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cc-by
8,261
Dynamic Games and Applications (2024) 14:20–32 https://doi.org/10.1007/s13235-023-00528-5 Dynamic Games and Applications (2024) 14:20–32 https://doi.org/10.1007/s13235-023-00528-5 Frederick van der Ploeg1,2 Accepted: 18 August 2023 / Published online: 27 September 2023 © The Author(s) 2023 Accepted: 18 August 2023 / Published online: 27 September 2023 © The Author(s) 2023 Keywords Political conflict cycles · Rent sharing · Resource wars · Rapacious depletion · Hold-up problem B Frederick van der Ploeg rick.vanderploeg@economics.ox.ac.uk Benefits of Rent Sharing in Dynamic Resource Games Frederick van der Ploeg1,2 2 CESifo and CEPR, University of Amsterdam, Amsterdam, Netherlands 2 An example where this occurs is Nigeria, a country where there are two different political parties representing each a different political clientele. The two political parties alternate in office depending on election outcomes. Nigeria is an oil-rich country where the incumbent controls what happens with oil rents. When in office these political factions hand out more of the oil rents to their own political clientele than to the political clientele of the rival faction. Resource conflict are a key feature of the natural resource curse (e.g. [22]. 1 Introduction Long [17] was the first to show that the adverse effects of a risk of future expropriation of the profits of an owner of natural resources under anticipated nationalisation leads to more rapacious extraction. 1 The insights are akin to those for over-exploitation of the common. Here we demonstrate how this pans out in a political context with two rival political factions, which alternatively hold political power. We thus depart from the notion of a homogenous government. Rather than analysing the risk of expropriation by a homogenous government as in Long [17], we thus examine the effects of a government that may be removed from office on the dynamics of natural resource depletion. We first show how rent sharing might attenuate the adverse effects of the risk of expropriation or the risk of a future 1-sided regime shift. We then show how the framework can be extended to analyse 2-sided regime switches and never-ending conflict cycles using the theory of differential games. Finally, we indicate how the framework can be used to analyse resource wars with hazard rates that are endogenous and depend on relative fighting efforts also using the theory of differential games.2 We assume iso-elastic demand for natural resources and zero variable extraction costs. With no risk of being removed from office, monopolistic resource extraction is efficient [8, 29]. We thus focus on the inefficiencies that follow from expropriation risk, perennial political cycles, and dynamic resource wars. In addition, (Table 1) we assume that the incumbent incurs upfront costs of exploration investment to make natural resource reserves endogenous (cf., [11]. Our paper is also related to Leonard and Long [16], which analyses the endogenous evolution of a society’s property rights regime and regime switches albeit in an overlapping generation framework.3 Abstract Ngo Van Long’s classic paper on the risk of expropriation of natural resources published in a 1975 issue of the Journal of Economic Theory was an instant classic, which spawned a huge literature. Here I pay tribute to this wonderful brilliant yet modest scholar by briefly reviewing his contribution and then sketching how his insights can be used to analyse dynamic conflict over natural resources both as expropriation game and as a differential game on which Long has published extensively too. We discuss three results. First, if an incumbent faces a threat of a rival faction, extraction is more voracious if the factions do not share rents equally. Second, never-ending political conflict cycles are more inefficient if constitutional cohesiveness or rent sharing is strong and political instability is high. Third, resource wars are more intense if rent sharing is weak, reserves of resources are high, the wage is low, and elections occur less frequently. Keywords Political conflict cycles · Rent sharing · Resource wars · Rapacious depletion · Hold-up problem JEL Classification D81 · H20 · Q31 · Q38 JEL Classification D81 · H20 · Q31 · Q38 1 Other studies that followed are Konrad et al. [13], Bohn and Deacon [6], and Laurent-Luchetti and Santaguni [14]. Stroebel and van Benthem [30] find that expropriation occurs more often with asymmetric information on extraction costs. It also occurs more often if the oil price is higher and checks and balances on the executive are weak. This paper is inspired by pioneering work by Ngo Van Long and is based on earlier results in parts of van der Ploeg [23]. I am grateful to two anonymous referees for their helpful comments on an earlier version. This article is part of the topical collection “Dynamic Games in Economics in Memory of Ngo Van Long” edited by Hassan Benchekroun and Gerhard Sorger. 1 University of Oxford, Manor Road Building, Manor Road, Oxford OX1 3UQ, UK Dynamic Games and Applications (2024) 14:20–32 21 3 This paper shows that enforcement of property rights increases as the stock of capital grows, since then households become richer and prefer commitment to better protection of property rights. 2 Rent Sharing and the Risk of a Future Regime Shift With a poor constitution, 0 < τ A  τ B  τ < 0.5 and oil rents are less equally shared (cf. [3–5]. a fully cohesive constitution the incumbent government must divide up oil rents equally in which case τ A  τ B  τ  0.5. With a poor constitution, 0 < τ A  τ B  τ < 0.5 and oil rents are less equally shared (cf. [3–5]. rents are less equally shared (cf. [3–5]. A special case of our model corresponds to Long [17], which considers an international, typically foreign-owned oil company that owns the stock of natural resources and is subject to the risk of confiscation by the government. This case emerges if we assume that the internationaloilcompanykeepsalloftheoilrents,i.e.τ A  0,andh istheriskofconfiscation. Once the government has confiscated the oil company, it keeps all the rents so that τ B  0. Another possibility is that the government at an unexpected future time increase the profit tax on this company. We will come back to this case when we discuss our results in Sect. 2.3. We study the effect of constitutional cohesiveness (i.e. full rent sharing), but also analyse two departures from the constitution. The first one is when the incumbent adheres to the constitution and shares rents according to τ A  τ, but faces a threat of being removed from office by a rogue rival faction that offers less than what is stipulated by the constitution, i.e. τ B < τ A  τ. The second one is where the incumbent is the rogue administration that faces a threat of being replaced by an administration that shares rents more fairly. This corresponds to τ A < τ B  τ. We show that both types of departures make oil extraction more voracious and depress exploration investment. A special case of our model corresponds to Long [17], which considers an international, typically foreign-owned oil company that owns the stock of natural resources and is subject to the risk of confiscation by the government. This case emerges if we assume that the internationaloilcompanykeepsalloftheoilrents,i.e.τ A  0,andh istheriskofconfiscation. Once the government has confiscated the oil company, it keeps all the rents so that τ B  0. Another possibility is that the government at an unexpected future time increase the profit tax on this company. 2 Rent Sharing and the Risk of a Future Regime Shift We will come back to this case when we discuss our results in Sect. 2.3. We study the effect of constitutional cohesiveness (i.e. full rent sharing), but also analyse two departures from the constitution. The first one is when the incumbent adheres to the constitution and shares rents according to τ A  τ, but faces a threat of being removed from office by a rogue rival faction that offers less than what is stipulated by the constitution, i.e. τ B < τ A  τ. The second one is where the incumbent is the rogue administration that faces a threat of being replaced by an administration that shares rents more fairly. This corresponds to τ A < τ B  τ. We show that both types of departures make oil extraction more voracious and depress exploration investment. We use the Principle of Dynamic Programming and work backwards in time by first analysing what happens after the change in government at some unknown future date T and then analysing what happens in the preceding period when the incumbent A is still in office. 2 Rent Sharing and the Risk of a Future Regime Shift Consider a political system with two rival political parties. There is an incumbent political party A that has a one-off risk of being removed from office by the rival opposition political party B at some unknown future date T . Our model is specified in continuous time. This instantaneous risk is h > 0, so that the expected time that A stays in office is 1/h. To keep mat- ters tractable, we make three simplifying assumptions. First, demand for oil (from now on a shorthand for natural resources) is iso-elastic, and marginal revenue is positive. The price elas- ticity, ε, is thus assumed to be constant and greater than one. Second, oil extraction is costless. Third, political parties are risk neutral. Hence, utility is given by U(R)  R1−1/ε/(1 −1/ε), where R > 0 denotes the rate of oil depletion. Since welfare is the area under the demand curve, the inverse demand function is given by p  U′(R)  R−1/ε, ε > 1. As marginal revenue is always finite and positive, reserves are exhausted asymptotically. Demand for oil, R  p−ϵ, is iso-elastic. As a result of selling oil, the incumbent receives at time t oil rents p(t)R(t) given that extraction costs are zero. We define τ B to be the share of oil rents that B, when in office, gives to A, and, similarly, τ A to be the share of oil rents that A, when in office, gives to B. With 3 This paper shows that enforcement of property rights increases as the stock of capital grows, since then households become richer and prefer commitment to better protection of property rights. Dynamic Games and Applications (2024) 14:20–32 22 Table 1 Sensitivity of speed of oil extraction and fighting efforts V V* V + V* L  R/S f Benchmark: τ  0.25, β  φ  1, H  0.1 16.94 10.40 27.34 0.196 0.082 Double the initial oil stock: S(0)  200 23.96 14.71 38.67 0.196 0.116 Half the wage: W  4 16.94 10.40 27.34 0.196 0.163 Halving the interest rate: r  0.02 20.59 14.23 34.82 0.133 0.079 Table 1 Sensitivity of speed of oil extraction and fighting efforts a fully cohesive constitution the incumbent government must divide up oil rents equally in which case τ A  τ B  τ  0.5. 4 Exploration investment in the narrow sense is concerned with finding oil or gas fields that can be explored (with a probabilistic return), and exploitation investment is concerned with the investments that are needed to pump up the oil or gas once the reserves have been found (e.g. platforms or pipes). These two types have different cost structures. From now on, I will use the term exploration investment to indicate exploration investment in the narrow sense as well as exploitation investment. 2.1 Actions of the New Incumbent Starting at Some Future Date Working backwards in time in accordance with the principle of optimality of dynamic pro- gramming, the political party B once in office maximises its rent net of cohesive payments, Max R,I  ∞ T (1 −τ B)p(t)R(t)e−r(t−T )dt, (1) (1) subject to the inverse demand function and the oil depletion equations, subject to the inverse demand function and the oil depletion equations, ˙S(t)  −R(t), ∀t ≥T ,  ∞ T R(t)dt ≤S(T ), andS(0)  (I), (2) (2) where I denotes investment in initial oil exploration, S denotes the stock of oil reserves, and r is the exogenous and constant market interest rate. Here τ B denotes the government take of where I denotes investment in initial oil exploration, S denotes the stock of oil reserves, and r is the exogenous and constant market interest rate. Here τ B denotes the government take of Dynamic Games and Applications (2024) 14:20–32 23 oil revenues when B is in office given to the opposition in the form of cohesiveness payments (rent sharing). We discuss the setting of optimal oil exploration investment in Sect. 2.2.4 oil revenues when B is in office given to the opposition in the form of cohesiveness payments ( t h i ) W di th tti f ti l il l ti i t t i S t 2 2 4 revenues when B is in office given to the opposition in the form of cohesiveness payments 4 oil revenues when B is in office given to the opposition in the form of cohesiveness payments (rent sharing). We discuss the setting of optimal oil exploration investment in Sect. 2.2.4 g pp p y (rent sharing). We discuss the setting of optimal oil exploration investment in Sect. 2.2.4 The optimal policy requires that marginal oil revenue net of the government take must equal the scarcity rent, λ, which according to the Hotelling rule must rise at a rate equal to the market interest rate, r:  1 −τ B 1 −1 ε  R(t)−1 ε  λ(t), ˙λ(t)/λ(t)  r, t ≥T . (3) (3) It follows from (2) and (3) that under the regime of B the price and oil depletion paths are efficient from time T onwards despite the country being a monopolist on world markets. 2.1 Actions of the New Incumbent Starting at Some Future Date Welfare to go for A from the moment it has been removed from office consists of cohesiveness payments it receives from B and equals V A S(T ), τ B ≡τ B(εr)−1/εS(T )1−1/ε, ∀t ≥T . (7) (7) 2.1 Actions of the New Incumbent Starting at Some Future Date This can be seen from the price of oil following a competitive price path with prices rising at a rate equal to the interest rate according to the Hotelling rule: It follows from (2) and (3) that under the regime of B the price and oil depletion paths are efficient from time T onwards despite the country being a monopolist on world markets. This can be seen from the price of oil following a competitive price path with prices rising at a rate equal to the interest rate according to the Hotelling rule: ˙p(t) p(t)  r > 0, ˙R(t) R(t)  −εr < 0, t ≥T , (4) (4) Hence, oil producers are indifferent between extracting now and in a future point of time. The efficiency of oil extraction from time T onwards stems from the assumptions of costless extraction and iso-elastic demand for oil. Hence, oil producers are indifferent between extracting now and in a future point of time. The efficiency of oil extraction from time T onwards stems from the assumptions of costless extraction and iso-elastic demand for oil. Using (4) in the oil depletion Eqs. (2), we obtain the policy function for oil, R(t)  εr S(t), and thus obtain R(t)  εre−εr(t−T )S(T ), S(t)  e−εr(t−T )S(T ) ≤S(T ), p(t)  er(t−T )(εr S(T ))−1/ε, ∀t > T . (5) (5) The optimal depletion and price paths follow Hotelling paths and are constrained efficient (conditional on S(T )). They do not depend on the government take, τ B, because this take corresponds to a lump-sum tax. Substituting (5) in (1), we get B’s welfare to go at time T : The optimal depletion and price paths follow Hotelling paths and are constrained efficient (conditional on S(T )). They do not depend on the government take, τ B, because this take corresponds to a lump-sum tax. Substituting (5) in (1), we get B’s welfare to go at time T : V B S(T ), τ B ≡ ∞ ∫ T  1 −τ B R(t)1−1/εe−r(t−T )dt   1 −τ B (εr)−1/εS(T )1−1/ε. (6) (6) where V B(.) denotes B’s (undiscounted) value function. Welfare to go for B increases in the stock of oil it inherits and decreases in the interest rate. 2.2 Actions of the Incumbent Before Being Removed from Office Given its future welfare to go (7), A must choose initial exploration investment, I, and the extraction path to maximise expected welfare to go with uncertainty regarding the end of its regime T. With an exponential density function for T, i.e. hexp(−hT ) with h the constant Dynamic Games and Applications (2024) 14:20–32 24 hazard rate, the problem for A from time zero is hazard rate, the problem for A from time zero is hazard rate, the problem for A from time zero is Max R,I E T ∫ 0  1 −τ A p(t)R(t)e−rtdt + e−rT V A S(T ), τ B −qI  ∞ ∫ 0 hexp(−hT ) T ∫ 0  1 −τ A p(t)R(t)e−rsdt + e−rT V A S(T ), τ B  dT  (8) (8) subject to the oil demand function, the oil depletion equations, ˙S(t)  −R(t), ∀0 ≤t ≤T , (0)  S0 > 0,  T 0 R(t)dt ≤S0 −S(T ), (9) (9) and the oil exploration investment schedule, S0  (I)  ω0I ω, ′ > 0, " < 0, ω0 > 0, 0 < ω < 1, (10) S0  (I)  ω0I ω, ′ > 0, " < 0, ω0 > 0, 0 < ω < 1, (10) (10) where q is the exogenous price of oil exploration investment. All exploration invest- ments have to be done up front, so ongoing exploration investments are ruled out for simplicity (cf. [11]). Concavity of (.) ensures decreasing returns to exploration invest- ment. The cumulative probability that the incumbent is removed from office by time t is Pr(T ≤t)  1 −exp(−ht), ∀t ≥0, h ≥0, and the probability that A is still in office by time t is Pr(T > t)  exp(−ht). The conditional probability that A is still in office during a small interval is independent of time. Expected duration and the standard deviation of A’s term of office are 1/h. The Hamilton–Jacobi–Bellman (HJB) equation for the incumbent A is where q is the exogenous price of oil exploration investment. All exploration invest- ments have to be done up front, so ongoing exploration investments are ruled out for simplicity (cf. [11]). Concavity of (.) ensures decreasing returns to exploration invest- ment. 2.2 Actions of the Incumbent Before Being Removed from Office The cumulative probability that the incumbent is removed from office by time t is Pr(T ≤t)  1 −exp(−ht), ∀t ≥0, h ≥0, and the probability that A is still in office by time t is Pr(T > t)  exp(−ht). The conditional probability that A is still in office during a small interval is independent of time. Expected duration and the standard deviation of A’s term of office are 1/h. The Hamilton–Jacobi–Bellman (HJB) equation for the incumbent A is is (11) rV (S, τ A, τ B)  Max R (1 −τ A)U′(R)R −V ′(S, τ A, τ B)R −h V (S, τ A, τ B) −V A(S, τ B) , (11) where p  U′(R) is the inverse oil demand function, V (S, τ A, τ B) the value function excluding exploration investment for A when is still in office, and V A(S, τ B) is the value function for A after it has lost office. The optimality condition for oil extraction sets marginal revenue of oil extraction equals to the marginal value of oil reserves and gives optimal oil extraction, R(t)  V ′ (S(t), τ A, τ B (1 −τ A)(1 −1/ε) −ε , 0 ≤t ≤T . (12) (12) Upon substituting (12) into (11) and using the method of undetermined coefficients, we obtain V (S, τ A, τ B)  K S1−1/ε, where the unique value for K follows from the algebraic equation: Upon substituting (12) into (11) and using the method of undetermined coefficients, we obtain V (S, τ A, τ B)  K S1−1/ε, where the unique value for K follows from the algebraic equation: (1 −τ A)ε ε K 1−ε + hτ B(εr)−1/ε  (r + h)K. (13) (13) The oil price and depletion rate when A is in office are The oil price and depletion rate when A is in office are p(t)  K S(t)−1/ε/(1 −τ A) and R(t)  (1 −τ A)εK −εS(t), 0 ≤t < T . (14) Defining L ≡  (1 −τ A)/K ε and solving for the time paths from (14) and (2), we obtain p(t)  eLt/ε(LS0)−1/ε, R(t)  Le−Lt S0, S(t)  e−Lt S0, 0 ≤t < T . (15) p(t)  K S(t)−1/ε/(1 −τ A) and R(t)  (1 −τ A)εK −εS(t), 0 ≤t < T . 2.3 Effects of a Regime Switch on the Time Paths for Oil Depletion Since oil depletion of the incumbent A is excessively fast, initially the path for the oil depletion rate is above the efficient path and the oil price path is below the Hotelling path. Along a Hotelling path, the market is indifferent between extraction in future and today so that today’s price of oil must equal the present discounted value of tomorrow’s price of oil. Along a Hotelling path, the price of oil thus grows at the market rate of interest, r. However, in a situation with a risk of a regime switch, the price of oil grows at the rate L/ϵ, which exceeds the rate of interest. It thus follows that the rate of oil extraction is faster than that under a Hotelling path too. If the realised date of the change of government is far enough in future, the oil depletion rate before the switch can fall below and the oil price path can rise above the efficient path. Once A is removed, the oil depletion rate jumps down, and the price jumps up by a discrete amount. From then on depletion and reserves follow Hotelling paths, but they are inefficient as they start out from fewer oil reserves than without political risk. Oil prices rise at the interest rate but start from a higher level than without political uncertainty as voracious extraction during A’s incumbency has made oil scarcer when B enters office. Figure 1 reports the results from setting ε  2, r  0.04, S0  100, h  0.1, τ A  0 and τ B  0.4. The expected political take-over thus occurs at time 10 while K  2.46 and L  0.165. The crossing time is 8.5. The reserves to production ratios before and after the switch are 6.1 and 12.5, respectively. Dotted lines indicate the efficient outcomes in case h  0. Solid lines indicate the inefficient outcomes for if the switch occurs after 5 units of time. Since T  5 < 8.5, oil depletion rates are always higher and oil prices are always lower than the efficient ones. Dashed lines correspond to a later change of government with T  15 > 8.5, so the oil depletion and price paths cross the efficient paths before A leaves office. 2.2 Actions of the Incumbent Before Being Removed from Office (14) Defining L ≡  (1 −τ A)/K ε and solving for the time paths from (14) and (2), we obtain p(t)  eLt/ε(LS0)−1/ε, R(t)  Le−Lt S0, S(t)  e−Lt S0, 0 ≤t < T . (15) )  K S(t)−1/ε/(1 −τ A) and R(t)  (1 −τ A)εK −εS(t), 0 ≤t < T . (14) (14) (15) Dynamic Games and Applications (2024) 14:20–32 25 Result 1: After the government switch, the oil price rises at the rate r and the oil depletion rate and reserves decline at the rate εr. Before the switch, the oil depletion rate and reserves decline at the rate L > εr and the oil price rises at the rate L/ε > r. More voracious oil depletion (higher L) and lower welfare to go for A occur with a higher risk of a change in government (higher h), and less rent sharing (smaller τ Aor τ B). Amorecohesiveconstitutionwithmorerentsharing(higherτ A  τ B)giveslessvoracious depletion and fewer inefficiencies. Whenever the incumbent or the rival faction shares rents less fairly oil depletion will be more voracious and A’s welfare will be lower The incumbent Amorecohesiveconstitutionwithmorerentsharing(higherτ A  τ B)giveslessvoracious depletion and fewer inefficiencies. Whenever the incumbent or the rival faction shares rents less fairly, oil depletion will be more voracious, and A’s welfare will be lower. The incumbent faction thus has an incentive to share more equally. Oil extraction of the incumbent will be less voracious if it can count on cohesiveness payments when out of office from the new government B. If both factions share oil rents equally, we have L  εr, so that the rate of extraction is efficient, and there are no political distortions whatsoever. This is also the case if there is no political risk, h  0. Returning to a special case inspired by Long [17], i.e. τ A  τ B  0, Eq. (13) and the definition of L yields L  K −ε  ϵ(r + h) and thus the oil price charged by the international oil company before its oil rents are confiscated rises at a rate larger than the market rate of interest (i.e. L ϵ  r +h > r). Long [17] also studies time-varying probabilities of confiscation and rising trends in the effective profit rate. 2.3 Effects of a Regime Switch on the Time Paths for Oil Depletion The simulations confirm that political uncertainty boosts oil depletion rates and depresses oil prices in the period before the change of government. After that, oil depletion jumps down while oil prices jump up and then continue at their less aggressive Hotelling rates. Dynamic Games and Applications (2024) 14:20–32 26 0 2 4 6 8 10 12 14 16 18 0 5 10 15 20 25 regime switch at T = 15 efficient Oil depleon rate, R regime switch at T = 5 Fig. 1 Time paths of oil extraction before and after a regime switch regime switch at T = 15 25 Fig. 1 Time paths of oil extraction before and after a regime switch 2.4 Exploration Investment and the Hold-up Problem Optimal exploration investment follows from setting its marginal value to its cost: (1 −1/ε)K(I)−1/ε′(I)  q ⇒ I  I(h, τ A, τ B, q), Ih,Iτ A, Iq < 0, Iτ B > 0. (16) (16) Higher political risk and more rent sharing (higher value of h, τ A, and τ B) make it less attractive to undertake exploration investment so that the discovered stock of oil reserves is less. This hold-up problem exacerbates the inefficiencies highlighted in Result 1. 5 Sekeris [27] also studies natural-resource exploration game with the possibility of expropriating natural resources through violent conflict. Its objective is, however, different as it demonstrates that threats to support cooperative outcomes will no longer be subgame perfect and will thus not be credible. This paper also shows within the context of its model that conflict occurs when natural resources are scarce while players deplete resources non-cooperatively without conflict when resources are abundant. 3 Ongoing Conflict Cycles with 2-Sided Regime Switches Consider ongoing switches in government. Assume that both factions share rents equally, τ A  τ B ≡τ, and let the probability of a switch of government, h, be the same whoever is in office. With symmetric, asymptotic, Nash equilibrium outcomes, we do not need to distinguish the value functions for the two factions separately. Denote the in-office value function by V (S) and the out-of-office value function by V ∗(S). The HJB equation for the incumbent is Max R  (1 −τ)U ′(R)R −V ′(S)R  −h  V (S) −V ∗(S)   rV (S). (17) (17) The value of being out of office is the present value of expected cohesiveness payments received when out of office plus the expected net rents upon gaining office, so that the HJB equation is rV ∗(S)  τU ′(R)R −V ∗′(S)R + h  V (S) −V ∗(S)  . (18) (18) Dynamic Games and Applications (2024) 14:20–32 27 From (17) marginal revenue equals the shadow value of oil, so (1 −1/ε)(1 −τ)U ′(R)  V ′(S). Result 2: With ongoing political cycles, exhaustible resource depletion is rapacious and faster than predicted by the competitive Hotelling rule (L > εr), especially with weak consti- tutional cohesiveness (low τ), and frequent changes of government (high h). Political uncertainty and less cohesive constitutions in a situation with ongoing politi- cal conflict (i.e. perennial two-sided regime shift) lead to more voracious extraction of the exhaustible resource. Furthermore, a partisan in-office bias also makes resource extraction more voracious. 4 Resource Wars To study dynamic interactions between exhaustible resource extraction and resource wars, we let the hazard rates of a regime switch depend on fighting by the two factions. By fighting and diverting labour from productive activities, faction try to hold on to office or get into power. We thus study resource wars with repeated switches of government regime.5 If A is in office, A and B fight f A and f B∗units of time, respectively, and have N −f A and N −f B∗units of time left for work with N exogenous labour supply of each faction. If B is in office, A and B fight, respectively, f A∗and f B units of time and work N −f A∗and N −f B units of time. The opportunity cost of fighting is the exogenous wage W. We let the hazard rates of A being replaced by B and of B by A depend on fighting be equal to h A  2H f B∗ f A + f B∗, hB  2H f A∗ f A∗+ f B , H > 0 (19) (19) (cf. [12, 31]. By fighting more, a faction improves its chances of staying in or gaining office A (cf. [12, 31]. By fighting more, a faction improves its chances of staying in or gaining office and gaining control of oil. A rebel faction that does not fight, never gains office, h A  0. If both the incumbent and the rebel faction fight with the same intensity, the hazard of being removed from office is h A  hB  H. If the incumbent does not fend off rebels, its hazard of being removed from office is twice as high, h A  2H, and B once in office will stay in forever, hB  0. H stands for how fast elections take place or for core political instability while 0 < τ ≤0.5 is the cohesiveness of the constitution or share of oil rents the incumbent gives to the rebel faction. A’s HJB equations for the non-cooperative subgame-perfect Nash equilibrium outcome are: Max f A,R A  (1 −τ)U ′(R A)R A −V A S (S)R A + W(N −f A) −h A V A(S) −V A∗(S)   rV A(S), (20) Max f A∗  τU ′(RB)RB −V A∗ S (S)RB + W(N −f A∗) +hB V A(S) −V A∗(S)   rV A∗(S). 4.1 Non-cooperative Outcomes The non-cooperative outcome assumes that, if A is in office, it takes as given rebel fighting efforts f B∗when choosing its optimal fighting efforts f A and oil depletion rate R A. If A is out of office, it takes f B, as given when deciding f A∗. A’s marginal expected gain from, respectively, fighting in and out of office is set to the wage: 2H f B∗  f A + f B∗2  V A(S) −V A∗(S)  2H f B  f A + f B∗2  V A(S) −V A∗(S)  W (22) (22) and similarly for B. This gives two reaction functions for when A is in and out of office indicating that A will fight more if B fights more (both if A is in and out of office). If the intersection with the reaction functions for B exists, we obtain the non-cooperative sym- metric Markov-perfect Nash equilibrium. Since hazard rates are given by symmetric contest functions, this equilibrium is symmetric and gives the fighting intensities: f A  f A∗ f B  f B∗=φH 2W  V (S) −V ∗(S)  . (23) (23) Fighting is thus more intense if the expected gain from office is high relative to the opportunity cost of fighting (W) and if it is easier to remove the incumbent from office (higher H). There is no direct effect of cohesiveness on fighting efforts, only via the value functions. In equilibrium h A  hB  H. The HJB equations for when in and out of office become, respectively: Max R A  (1 - τ)U ′(R)R −V ′(S)R + W N −1.5H  V (S)−V ∗(S)   rV (S), (24) τU ′(R)R −V ∗′(S)R + W N + 0.5H  V (S) −V ∗(S)   rV ∗(S). (25) (24) (25) The incumbent sets marginal oil revenue net of cohesiveness payments to the marginal social cost of oil, (1 −τ)(1 −1/ε)p  V ′(S). This implies that the oil price is low and the rate of oil depletion high if oil is abundant (high S and low V ′(S)), and cohesiveness is weak (low τ). 4 Resource Wars (20) (21) f (21) There are two similar HJB equations for B in V B(S) and V B∗(S). Equation (20) states that the A’s maximum oil rents (net of the share of oil rents given to B and net of the shadow There are two similar HJB equations for B in V B(S) and V B∗(S). Equation (20) states that the A’s maximum oil rents (net of the share of oil rents given to B and net of the shadow Dynamic Games and Applications (2024) 14:20–32 28 cost of oil) plus income from productive activities minus the expected loss in value terms of losing office must equal the return from investing oil proceeds at the market rate of interest. Equation (21) states that the contender’s cohesiveness transfers plus wage income plus the expected gain of entering office must equal the market rate of return. Asymptotically, the effect of which faction started in office withers away and the in- and out-office value functions for the two factions converge. We will thus focus on the asymptotic subgame-perfect Nash equilibrium outcome. 4.1 Non-cooperative Outcomes We conjecture V A(S)  V B(S)  K S1−1/ε + W N/r and V A∗(S)  V B∗(S)  K ∗S1−1/ε + W N/r with K and K* to be found, so we have the optimal oil price and oil depletion rates: p  K β(1 −τ) S−1/ε, R  LS, L ≡[β(1 −τ)]εK −ε. (26) (26) Using (26) in (24) and (25) and equating coefficients on S1−1/ε, we get: Using (26) in (24) and (25) and equating coefficients on S1−1/ε, we get: 1 ε [(1 −τ)]εK 1−ε −1.5H(K −K ∗)  r K, (27) τ[(1 −τ)]ε−1K 1−ε −(1 −1/ε)[(1 −τ)]εK −εK ∗+ 0.5H(K −K ∗)  r K ∗. (28) 1 ε [(1 −τ)]εK 1−ε −1.5H(K −K ∗)  r K, (27) (27) τ[(1 −τ)]ε−1K 1−ε −(1 −1/ε)[(1 −τ)]εK −εK ∗+ 0.5H(K −K ∗)  r K ∗. (28) (28) Dynamic Games and Applications (2024) 14:20–32 29 which can be solved for K and K*. We thus find from (23) the fighting efforts: which can be solved for K and K*. We thus find from (23) the fighting efforts: f  f ∗ H 2W (K −K ∗)S1−1/ε. (29) (29) If all oil rents must be shared equally (τ  0.5), we have K  K ∗ 0.5(εr)−1/ε, L  εr and thus R  εr S and f  0. Hence, a perfectly cohesive constitution ensures efficiency no armed conflict. If factions cannot be removed from office,H  0, there is no fighting while K  β(1 −τ)(εr)−1/ε gives p  (εr S)−1/ε and R  εr S. The outcome is also efficient independent of the degree of rent sharing,τ. Result 3: Resource wars are more intense if reserves are high, and workers are paid poorly. Oil depletion is less rapid if a big part of oil rents is shared (big τ) and government stability is high (low H). 5 Conclusion We have shown that Long’s [17] analysis of expropriation risk can be extended to study the effects of (i) rent sharing and a one-off change in government, (ii) fighting to stay in or gain political power and of rent sharing on the voracity of oil extraction and initial oil exploration investments. We have thus analysed the effects of one-off and ongoing regime shifts with both exogenous and endogenous hazard rates. We have discussed how political uncertainty induces rapacious oil depletion and holds back exploration investment, especially if constitutional cohesiveness and rent sharing is weak. We have also shown why fighting for the control of oil is more intense with large oil reserves and a low wage, especially if there is little rent sharing. It will take too much space to do justice to all the innovative contributions of Ngo Van Long, but it is worthwhile mentioning a few here that are related to the matters that have been discussed. First, Ngo Van Long is together with Hans-Werner Sinn the founding father of the extensive literature on the so-called green paradox, first exposited in Long and Sinn [19] and then much later worked out in more detail in Sinn [28]. As the review in van der Ploeg and Withagen [24] indicates, these crucial insights from almost forty years ago have spawned an extensive and ongoing literature about why a future surprise increase in the price due to an increase in future carbon tax increases resource extraction and accelerates global warming today. The intertemporal mechanisms are not that different from those underlying Long’s [17] theory of expropriation. The green paradox effect is particularly strong if the price elasticity of resource reserves is small. Second, Long [18] shows in his masterful review of dynamic (differential and differ- ence) games in the economics of natural economics how useful this type of analysis is to improve our understanding of this part of economics. The survey discusses both exhaustible and renewable resources, discusses the tragedy of the commons, considers different market structures varying from a monopoly, an oligopoly, a cartel with a competitive fringe, and open access, gives applications to carbon taxation, and pays attention to technology and strategic aspects of investments in R&D. A prominent example of the wide applicability of these tools is Benchekroun, Gaudet and Long [2]. 4.4 Empirical Evidence Countries with high oil exports have more armed conflict especially in sub-Saharan Africa (e.g. [7, 10, 26]. This type of evidence also suggests that natural resources boost conflict, especially in ethnically polarised societies (e.g. [21, 25]). Giant oil discoveries increase armed conflict especially for countries that experienced armed conflict or coups in the previous decade [15]. Within-country evidence also points in this direction (e.g. [1] and finds that a higher price of capital-intensive oil induces guerrilla and paramilitary attacks [9]. 4.2 Some Simulations Figure 2 shows the effects of rent sharing, τ, on oil depletion and fighting and Fig. 2 plots the values to go with baseline parameters set to ε  2,r  0.04, S0  100, H  0.1, N  0.2 and W  8. We see that more rent sharing (higher τ) makes oil depletion less rapacious and curbs armed conflict (cf. Result 3). Rent sharing also raises the value to go for the rebels and the joint value to go, but the incumbent’s value to go first decreases slightly and then increases slightly with the degree of rent sharing. The short-dashed lines indicate that more political instability (H  0.2 > 0.1) leads to more aggressive oil depletion and more fighting, especially if the political system is less cohesive and there is less rent sharing. If the chance of losing office is more imminent, the incumbent fears losing control of the oil stake and the rebels fight more. Political instability depresses value to go for the incumbent and the rebel factions, especially if there is little rent sharing. The model predicts that oil-rich countries with regular, hotly contested elections have more conflict. Finally, it is easy to demonstrate that if the oil stake (proxied by oil reserves, S) is high or the opportunity cost of fighting (proxied by the wage, W), conflict is more intense even though the speed of oil depletion is unaffected. Also, armed conflict is less severe if rebels are more patient as then oil depletion is less rapid and payoffs to both the ruling and the rebel faction are higher. 0.08 0.13 0.18 0.23 0.28 0.33 0.38 0.43 0.48 0.53 0 0.1 0.2 0.3 0.4 0.5 Rate of oil depleon, R/S = L H = 0.2 0 0.005 0.01 0.015 0.02 0.025 0.03 0 0.1 0.2 0.3 0.4 0.5 Fighng efforts, f = f* H = 0.2 Fig. 2 Oil depletion and fighting (f  f .*) versus rent sharing (τ) 0.08 0.13 0.18 0.23 0.28 0.33 0.38 0.43 0.48 0.53 0 0.1 0.2 0.3 0.4 0.5 Rate of oil depleon, R/S = L H = 0.2 0 0.005 0.01 0.015 0.02 0.025 0.03 0 0.1 0.2 0.3 0.4 0.5 Fighng efforts, f = f* H = 0.2 Fig. 2 Oil depletion and fighting (f  f .*) versus rent sharing (τ) Dynamic Games and Applications (2024) 14:20–32 30 4.3 Effects on Oil Exploration Investment and Initial Reserves Since I  I(K(τ, H)), IH < 0, political instability depresses oil exploration investment and initial oil reserves. This hold-up problem depresses welfare further. Rent sharing has a non-monotonic effect on value to go for the incumbent and on oil exploration and initial reserves. 5 Conclusion They analyse why a non-renewable resource cartel that anticipates being forced, at some date in future, to break up into an oligopolistic market Dynamic Games and Applications (2024) 14:20–32 31 will produce more over the same interval of time than it would if there were no threat of dissolution. Furthermore, they show that the rate of extraction decreases with the life of the cartel and that the depletion rate may increase during the cartel phase. will produce more over the same interval of time than it would if there were no threat of dissolution. Furthermore, they show that the rate of extraction decreases with the life of the cartel and that the depletion rate may increase during the cartel phase. Third, Long [20] is an authoritative survey on how to manage, induce, or prevent regime shifts. It deals with regime shifts, thresholds, and tipping points and pays careful attention to uncertainty including the role ambiguity aversion. It discusses applications to political econ- omy, where it distinguishes between gradual and big-push political regime shifts including issues such as repression, redistribution, and gradual democratisation. It also discusses games of resource exploitation and games in industrial organisation with regime shifts (e.g. R&D contests). This survey illustrates the broad width of Long’s scholarship and achievements in the areas of optimal control theory and differential games and his genuine interests to apply to them to important and policy relevant problems in economic theory. The political economy and dynamic resource wars games discussed in this paper owe much to the many insights offered over many decades by my dear friend and colleague Ngo Van Long. He will be sorely missed by young and old alike for his kindness, curiosity, enormous intellect, and unbounded generosity. Author contributions I am the only author, so am responsible for this paper. Conflict of interest I have no competing interests of a financial or personal nature. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References 1. Angrist J, Kugler A (2008) Rural windfall or a new resource curse? Coca, income, and civil conflict in Colombia. Rev Econ Stat 90:191–215 2. Benchoukron H, Gaudet G, Van Long N (2006) Temporary natural resource cartels. J Environ Econ Manag 52:663–674 3. BesleyT,PerssonT(2011)Pillarsofprosperity:thepoliticaleconomicsofdevelopmentclusters.Princeton University Press 4. Besley T, Persson T (2011) The logic of political violence. Quart J Econ 126:1411–1445 5. Besley T, Persson T (2011) Fragile states and development policy. J Eur Econ Assoc 9(3):371–398 6. Bohn H, Deacon R (2000) Ownership risk, investment, and the use of natural resources. 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Stroebel J, van Benthem A (2013) Resource extraction contracts under threat of expropriation: theory and evidence. Rev Econ Stat 95(5):1622–1639 g p y 30. Stroebel J, van Benthem A (2013) Resource extraction contracts under threat of expropriation: theory and evidence. Rev Econ Stat 95(5):1622–1639 31. Tullock G (1967) The welfare costs of tariffs, monopolies, and theft. Western Econ J 5:224–232 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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https://periodicos.sbu.unicamp.br/ojs/index.php/bjos/article/download/8663981/26938
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Comparison of microleakage of an alkasite restorative material, a composite resin and a resin-modified glass ionomer
Brazilian journal of oral sciences/Brazilian Journal of Oral Sciences
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cc-by
5,510
Original Article Volume 20 2021 e213981 Comparison of microleakage of an alkasite restorative material, a composite resin and a resin-modified glass ionomer , Hamid Kermanshah1* Fariba Motevasselian1 2 Ebrahim Rasoulkhani , Mutlu Özcan3 Department of Restorative Dentistry, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran 1 Tehran University of Medical Sciences, School of Dentistry, Tehran, Iran 2 Division of Dental Biomaterials, Clinic for Reconstructive Dentistry, Center for Dental and Oral Medicine, University of Zürich, Switzerland 3 Corresponding author: Hamid Kermanshah Address: Restorative Dentistry Department, School of Dentistry, Tehran University of medical Sciences, North Karegar Street, Tehran, Iran. Postal code: 1439955991 Tel: + 98-21-88015801 E-mail: kermanshahhamid@yahoo. com Received: January 15, 2021 Accepted: April 8, 2021 Editor: Dr Altair A. Del Bel Cury , Aim: To compare the microleakage of Cention N, a subgroup of composite resins with a resin-modified glass ionomer (RMGI) and a composite resin. Methods: Class V cavities were prepared on the buccal and lingual surfaces of 46 extracted human molars. The teeth were randomly assigned to four groups. Group A: Tetric N-Bond etch-and-rinse adhesive and Tetric N-Ceram nanohybrid composite resin, group B: Cention N without adhesive, group C: Cention N with adhesive, and group D: Fuji II LC RMGI. The teeth were thermocycled between 5°-55°C (×10,000). The teeth were coated with two layers of nail vanish except for 1 mm around the restoration margins, and immersed in 2% methylene blue (37°C, 24 h) before buccolingual sectioning to evaluate dye penetration under a stereomicroscope (×20). The data were analyzed by the Kruskal-Wallis and Wilcoxon tests (α=0.05). Results: Type of material and restoration margin had significant effects on the microleakage (p<0.05). Dentin margins showed a higher leakage score in all groups. Cention N and RMGI groups showed significant differences at the enamel margin (p=0.025, p=0.011), and for the latter group the scores were higher. No significant difference was found at the dentin margins between the materials except between Cention N with adhesive and RMGI (p=0.031). Conclusion: Microleakage was evident in all three restorative materials. Cention N groups showed similar microleakage scores to the composite resin and displayed lower microleakage scores compared with RMGI. Keywords: Cention N, Composite resins. Dental leakage. Glass ionomer cements. http://dx.doi.org/10.20396/bjos.v20i00.8663981 1 Motevasselian et al. Introduction Class V cervical carious lesions remain a major oral health problem in the elderly and those at high risk of caries1. A wide variety of restorative materials have been suggested for restoration of these lesions; among which, composite resins and resin-modified glass ionomer (RMGI) cements are most commonly used1. RMGI has been recommended for restoration of class V lesions because it has the combined benefits of chemical adhesion to the tooth substrate, fluoride release potential, and caries-preventive effect2. Achieving adequate marginal integrity at the dentin margins of class V restorations extending beyond the cementoenamel junction (CEJ) remains a challenge1,3. Secondary carious lesions develop in absence of adequate marginal seal of restorations3. Several factors can affect the marginal adaptation of adhesive restorative materials such as polymerization shrinkage and contraction stress4,5, and the difference in the linear coefficient of thermal expansion (LCTE) of restorative material and that of tooth structure6. All these factors can lead to gap formation and marginal microleakage4-6. During the past two decades, researchers have extensively focused on developing dental restorative materials with improved physical and bioactive properties to minimize interfacial gap and secondary caries. Recently, a bulk-fill resin-based powder-liquid composite containing alkaline fillers (alkasite) was introduced to the market by Ivoclar Vivadent (Schaan, Liechtenstein). It is a bioactive restorative material, and the manufacturer claims that it has low polymerization shrinkage. Also, the manufacturer claims that it releases large amounts of fluoride and calcium ions at low pH and deposits minerals in the form of calcium phosphate and calcium fluoride layers. Furthermore, the hydroxide ions released from Cention N have been claimed to have a protective buffering capacity to neutralize cariogenic acids. This material can be used with or without an adhesive7-10. Preclinical screenings and in vitro studies simulating oral conditions are useful for estimation and predication of the performance of restorative materials. Various in vitro methods are used for evaluation of marginal quality of restorations such as penetration test in class II and V cavities and assessment of marginal interface under a light microscope or a scanning electron microscope. Dye penetration test is still the most commonly used method for evaluation of the sealability of restorative materials. Various types of dyes can be used for this purpose. No specific dye tracer has been recommended by the ISO standard for this test. The most commonly used dyes for this test include basic fuchsine, methylene blue, and silver nitrate. Thermocycling (TC) and/or mechanical loading have been recommended for microleakage tests to better simulate the clinical conditions11. The available studies about the microleakage mostly compared the conventional GICs rather than the RMGIs with Cention N12-14. Cention N is a resin-based (UDMA-based) material containing fillers. RMGIs contain resin and their composition is more similar to alkasite restorative materials than conventional GICs7,9,10. In addition, the frequency of thermal cycles in previous studies on this topic was 500 or less while 500 cycles are the minimum cycles recommended by ISO 1140515. 2 Motevasselian et al. The aim of this study was to evaluate the effect of three restorative materials namely an alkasite restorative material (Cention N®; Ivoclar Vivadent, Schaan, Liechtenstein) with and without an adhesive (Tetric® N-bond Vivadent, Schaan, Liechtenstein), a nanohybrid composite resin (Tetric-N® Ceram, Ivoclar Vivadent, Schaan, Liechtenstein), and a RMGI (GC Fuji II LC®, GC Corporation., Tokyo, Japan) on the marginal integrity of class V restorations submitted to 10,000 thermal cycles using the dye penetration test. The null hypotheses were that the location of restoration margin (in the enamel or dentin) or type of restorative material would have no significant effect on the marginal microleakage of restorations. Materials and Methods The commercial materials used in this study and their composition are presented in Table 1. The materials were used according to the manufacturers’ instructions. The study was approved by the Ethics Committee in Research of School of Dentistry of Tehran University of Medical Sciences (IR.TUMS.DENTISTRY. REC.1398.055). Table 1. The materials used and their classification, manufacturer, and composition6,8,29,30 Material (manufacturer) Liquid Powder Batch number UDMA, DCP, Aromatic aliphatic-UDMA PEG-400 DMA Ca-F-Silicate glass, Ba-Al silicate glass, Ca-Ba-Al fluorosilicate glass, YtF3, isofiller (78.4 wt% ) W07418 Polyacrylic acid, HEMA, 2,2,4 TMHEDC, TEGDMA Fluoro-alumino-silicate glass 1704011 Tetric® N-Ceram (Ivoclar Vivadent, Schaan, Liechtenstein) Bis-GMA, Bis-EMA UDMA Ba glass; YbF3; mixed Oxide; prepolymer (80%wt%) W84901 Tetric® N-Bond (Ivoclar Vivadent, Schaan, Liechtenstein) Bis-GMA, UDMA, HEMA, Phosphonic acid acrylate, ethanol, nanofiller, catalysts and stabilizer, nanofiller Cention® N (Ivoclar Vivadent, Schaan, Liechtenstein) GC Fuji II LC® (GC Corp., Tokyo, Japan) W83533 UDMA: Urethane dimethacrylate; DCP: Tricyclodecan-dimethanol dimethacrtylate; An aromatic aliphatic-UDMA: Tetramethyl-xylylen diurethane dimethacrylate; PEG-400 DMA: polyethylene glycol 400 dimethacrylate; Ca-F-Silicate glass: Calcium fluorosilicate glass; Ba-Al silicate glass: Barium aluminum silicate glass; Ca-Ba-Al-F glass: Calcium barium aluminum fluorosilicate glass; YtF3: Ytterbium trifluoride; Isofiller: Copolymer, HEMA: Hydroxyethyl methacrylate; 2,2,4 TMHEDC: Trimethyl hexamethylene dicarbonate; TEGDMA: Triethylene glycol dimethacrylate; Bis-GMA: bisphenol A diglycidylether methacrylate; Bis-EMA: ethoxylated bisphenol-A dimethacrylate; Ba glass: Barium glass. Specimen preparation Forty-six sound, non-carious, unrestored human third molars, extracted for periodontal reasons or as part of orthodontic treatment, were collected after informed consent was obtained from the patients. The minimum sample size for each study group was calculated to be 23, based on a previous study 12 con3 Motevasselian et al. sidering α=0.05, β= 0.2 and pooled standard deviation of 2.1 using SPSS 11 (SPSS Inc., Chicago, IL). The teeth were debrided of residual plaque, calculus and residual soft tissue, and stored in a solution of distilled water and 0.5% chloramine T at 4°C until usage. All teeth had been extracted within the past 3 months. The teeth were visually inspected under a stereomicroscopic at ×10 magnification (Leica, LEICA EZ4D, MEL SOBEL Microscopes, Italy), and sound teeth without fracture lines and cracks were selected for this study. Class V cavities (with 3 mm occlusogingival width, 3 mm mesiodistal width, and 1.5 mm depth) were prepared in the buccal and lingual surfaces of each tooth using a cylindrical diamond bur (≠ 838-012-FG; Hager & Meisinger GmbH, Neuss, Germany) and a high-speed handpiece under copious water irrigation. The occlusal margin of the cavities was located 2 mm coronal to the CEJ, while the gingival margin was located 1 mm apical to the CEJ. The diamond bur was replaced after five preparations. Non-retentive cavities with divergent walls were prepared as such. All the internal line-angles were rounded. The dimensions of all cavities were measured using a periodontal probe. Restorative procedure Before restoration, the prepared cavities were gently cleaned with a slurry of pumice paste and water using a prophy cup, and thoroughly rinsed with tap water. The prepared teeth were randomly divided into four groups (n=23) according to the type of material used, as follows: Group A: (composite resin): 37% phosphoric acid gel (Ivoclar Vivadent, Schaan, Liechtenstein) was applied on the enamel and subsequently on the dentin margins for 15 s. Afterwards, the etchant was thoroughly rinsed off with water spray for 15 s, and the excess water was removed with a small cotton pellet to avoid excessive drying. Tetric-N Bond nanofilled single-component adhesive (Ivoclar Vivadent, Schaan, Liechtenstein) was applied in one thick layer and rubbed on the enamel and dentin surfaces with a micro-applicator brush for 10 s. Excess adhesive in the line angles and the solvent were removed by gentle air stream for 10 s. The adhesive was light-cured for 10 s using a light-emitting diode (LED) curing unit with a light intensity of 1200 mW/cm2 (Bluephase; Ivoclar Vivadent, Schaan, Liechtenstein). Light output was measured using a radiometer (Bluephase Meter II, Ivoclar Vivadent AG, Schaan, Lichtenstein). A2 shade of Tetric-N Ceram nanohybrid composite resin (Ivoclar Vivadent, Schaan, Liechtenstein) was used to restore the cavities in two layers with oblique incremental application technique. The first oblique layer was applied and extended from the gingival floor to the axial wall. The second increment was applied to fill the remainder of the cavity. Each layer was polymerized with LED curing unit (Bluephase, Ivoclar Vivadent, Schaan, Liechtenstein) for 20 s. Group B: (Cention N without adhesive) This product is only available in A2 shade. The prepared cavities were gently dried with air stream. One spoon of powder and one drop of liquid were dispended on a 4 Motevasselian et al. mixing pad according to the manufacturer’s instructions. The powder was gradually added to the liquid and thoroughly mixed for 60 s until a homogenous mass with a slight shine was obtained to wet the tooth substrate. The restorative material was immediately applied and condensed in the cavity with a spatula in one increment. Excess material was carefully removed, and the restoration was cured for 20 s using a LED curing unit. Group C: (Cention N with adhesive) The same steps were followed for adhesive application in this group as in group A. Cention N was then mixed and delivered into the cavity with the same sequence as in group B. Group D: (RMGI) A2 Vita shade of RMGI was chosen. The cavities were conditioned with 10% polyacrylic acid (Dentin Conditioner, GC Corporation, Tokyo, Japan) applied with a micro-applicator brush for 20 s, and were then thoroughly rinsed with water spray for 20 s and blot-dried with cotton pellets to avoid desiccation. One level scoop of powder and two drops of liquid were placed on a mixing pad according to the manufacturer’s instructions. The powder was divided by half and mixed with the liquid within 25 s until a homogenous mass was achieved and applied and packed in bulk into the cavities with a spatula as long as the surface of the mixed cement was shiny. Afterwards, it was polymerized for 20 s using a LED curing unit. All restored cavities were stored in distilled water at 37°C for 24 h, and were then finished and polished with graded series of Sof-Lex discs (3M ESPE, Dental products St Paul, MN, USA). The same operator prepared all the specimens. Microleakage test The specimens were thermocycled for 10,000 cycles between 5˚ C and 55˚C with a dwell time of 30 s and a transfer time of 10 s. Following TC, the teeth were dried, and the root apex of each tooth was sealed with sticky wax. The entire tooth surface including the crown and root structures were covered with two layers of nail varnish, except for a 1 mm band around the restoration margins. All the specimens were then immersed in freshly prepared 2% methylene blue solution for 24 h at 37°C. The teeth were then rinsed with running water. Afterwards, the specimens were mounted in auto-polymerizing acrylic resin (Acropars, Marlic Medical Co., Tehran, Iran) and longitudinally sectioned in half at the center of the restoration in buccolingual direction with a low-speed diamond saw under water coolant. The sectioned teeth were evaluated under a stereomicroscope (Leica, LEICA EZ4D, MEL SOBEL Microscopes, Italy) at ×20 magnification. The extent of dye penetration at the restorative material-tooth interface was scored from zero to three along the occlusal and cervical walls13(Figure. 1). The dye penetration scores were determined by one single operator who was blinded to the type of restorative material used for each group. 5 Motevasselian et al. Occlusal wall Enamel 0 Dentin CEJ Gingival wall Pulpal tissue 2 3 2 3 DEJ Pulpal tissue Occlusal wall 1 Occlusal wall CEJ Gingival wall 0 1 Gingival wall Figure 1. Schematic view of the cavity prepared in the buccal and lingual walls of a molar tooth. The maximum degree of dye penetration was recorded according to the followiing scoring system :0, no dye penetration; 1, dye penetration to 1/3 of the cavity wall; 2, dye penetration up to 2/3 of the cavity wall, 3, dye penetration extending to the axial wall and beyond. CEJ: Cementoenamel junction; DEJ: Dentinoenamel junction Score 0: No dye penetration Score 1: Dye penetration extending to 1/3 of the occlusal or cervical wall Score 2: Dye penetration extending to two-thirds of the occlusal or cervical wall Score 3: Dye penetration extending to the axial wall and beyond Statistical analysis The statistical analysis of microleakage data was performed using SPSS version 25 (SPSS Inc., Chicago, IL, USA). The Kruskal-Wallis test was applied for multiple comparisons followed by the Dunn test. The occlusal and gingival microleakage scores were compared using the Wilcoxon signed rank test. Level of significance was set at 0.05 for the main analysis, and Dunn adjusted p-values were used for multiple comparisons. Results Table 2 presents the microleakage scores (number and percentage) at the enamel and dentin margins of the four groups. The microleakage scores at the enamel margin were significantly lower than the corresponding values at the dentin margins in all groups (p<0.001). 6 Motevasselian et al. Table 2. Microleakage (number and percentage) of the study groups at the enamel and dentin margins and pairwise comparison Enamel Number/percentage Material 0 A Tetric N-Ceram 1 14(61%) 8(34%) Dentin Number/percentage 2 3 1(4%) 0 ab 1(4.34%) 0 1 2 3(13%) 2(8.6%) 3 17(73.9%) cd B Cention N 18(78%) 5(22%) without adhesive 0 0 b C Cention N with adhesive 0 0 b 3(13%) 3(13%) 8(34.8%) 9(39%) c a 0 2(8.6%) 3(13%) 18(78%) d D RMGI* 19(83%) 4(17%) 12(25%) 6(26%) 3(13%) 2(8.6%) P value=0.041 1(4.34%) 1(4.34%) 4(17.39%) 17(73.9%) cd P=0.020 Similar letters show that the distribution of the leakage scores are not significantly different. *RMGI: Resin-modified glass ionomer The Kruskal-Wallis test indicated significant differences between the restorative materials for the occlusal and gingival margin microleakage scores (p=0.041 for the enamel margins, and p=0.020 for dentin margins). Table 2 shows pairwise comparisons of the differences in the microleakage scores of the study groups at 0.05 level of significance. The only significant differences at the enamel margins were found between Cention N with/without adhesive and RMGI groups (p=0.011 and p= 0.025, respectively). However, no significant difference was found between the other groups (p≥0.121). There was no statistically significant difference in microleakage between the groups at dentin margins (p≥0.076), except for Cention N with adhesive and RMGI (p=0.031). Discussion The purpose of this in vitro study was to compare the microleakage of different types of restorative materials for restoration of class V cavities in the cervical region of the teeth using the dye penetration test. Furthermore, microleakage scores of Cention N cavities restored with or without adhesive were compared. The recommended adhesives by the manufacturer of Cention N product are either universal bonding agents such as Tetric N-Bond Universal or etch and rinse adhesive systems such as Tetric N-Bond8. In the current study the latter surface treatment was selected to compare the microleakage scores since the phosphoric acid agent removes the smear layer13. Based on the results of the present study, the score of microleakage was greater at the dentin margin than the enamel margin in all groups regardless of the type of restorative material used. Thus, the first null hypothesis of the study was rejected. With regard to the type of restorative material, the results of the present study showed significant differences between Cention N and RMGI after immersion in 2% methylene blue for 24 h. Therefore, the second null hypothesis regarding insignificant effect of type of restorative material on microleakage score was also rejected. The tooth samples were exposed to 10,000 thermal cycles corresponding to 1-year of clinical service in the oral cavity, as claimed by Gale and Darvell. TC simulates the thermal alterations in the oral cavity that lead to stress build-up at the interface and 7 Motevasselian et al. can adversely affect marginal integrity of the restoration, causing microleakage. TC was performed between 5-55 ˚C according to ISO1140514,15. The results of the present study regarding lower microleakage at the enamel margins were similar to the findings of a previous study16. Bonding of adhesive restorative materials to dental substrate depends on either micromechanical interlocking and hybridization due to the penetration of bonding resin into the microscopic porosities on the surface of enamel and dentin, or chemical interactions with the inorganic content of dental substrate. Both mechanisms depend on the amount of surface free energy of dental substrate, which is directly proportional to the mineral content of the tooth structure and inversely correlated with the percentage of organic content. Enamel has a more homogenous structure than dentin due to higher mineral content and lower water and organic content. Therefore, one primary requisite for better marginal seal is already provided17,18. The comparison between RMGI and Cention N showed that Fuji II LC group revealed significantly higher leakage scores at the enamel margins compared with Cention N groups with/without adhesive. Dentin margins in class V cavities restored with Fuji II LC showed significantly higher leakage scores than Cention N group with adhesive. There are several factors that may explain these findings such as viscosity, polymerization rate, monomer conversion and linear coefficient of thermal expansion (LCTE). There is an inverse relationship between viscosity of a resin-based material and its rate of polymerization19. It has been demonstrated that powder/liquid (P/L) ratio of a resin-based cement affects its viscosity. Higher P/L ratio leads to higher viscosity20. Viscosity also depends on the filler loading, size (and hence the surface area), and shape of fillers as well as the heterogeneity of particle sizes and monomer types in the mixture. It was demonstrated that decreasing the filler size increased the viscosity of experimental composites19. The recommended P/L ratio for Cention N is 4.6:1, which is higher than that of Fuji II LC (3.2:1)20. The average particle size of Cention N is between 0.1 μm and 35 μm8, a wide size distribution; while, that of Fuji II LC is 5.9 μm21. Considering the abovementioned explanation, Cention N appears to have higher viscosity. High viscosity decreases the mobility of free radicals, leading to a reduction in polymerization rate, which has a great impact on shrinkage stress relief and interfacial gap reduction4,5,19. Therefore, the authors assume that lower microleakage scores in Cention N groups compared with the RMGI group might be explained by its probably lower rate of polymerization. Furthermore, it has been stated that lower particle content can cause higher volumetric shrinkage4. Therefore, it might be assumed that Fuji II LC with lower P/L ratio can undergo higher volumetric shrinkage and contraction stress, that can cause interfacial debonding and higher leakage scores. Also, a direct correlation exists between the degree of polymerization and volumetric shrinkage4,5. It also has been demonstrated that monomers with lower molecular weight and viscosity and higher mobility have higher degree of monomer conversion22. UDMA and HEMA are the base monomers of Cention N and Fuji II LC, respectively6,8,23. UDMA has higher molecular weight and viscosity than HEMA22. As a result, higher degree of conversion is expected in Fuji II LC. Panpisut and Toneluck found the same results20. This is another factor which might explain higher leakage scores in Fuji II LC group. 8 Motevasselian et al. LCTE is another parameter influencing the volumetric change of restorative materials in the oral cavity. If the difference between the LCTE of the tooth substrate and that of a restorative material is high, marginal seal at the tooth substrate-restoration interface might be breached6. Pinto-Sinai et al. explained that LCTE of resin-based restorative materials is influenced by the amount of filler. RMGI cements do not contain fillers6. Cention N has 78.4% filler content by weight in the final mass8,23, and a higher P/L ratio than Fiji II LC20, which might decrease its LCTE. It has been shown that Fuji II LC has a high LCTE during heating and cooling cycles (15ºC-50ºC) i.e. 25.4 and 30 ppm, respectively. The LCTE of dentin and enamel is 11 and 17 ppm, respectively in this temperature range6. The LCET of molar teeth in the cervical region has been reported to be around 5 ppm24. In the current study, the difference in expansion and contraction at the tooth and RMGI interface might have caused marginal deterioration and higher leakage score in the RMGI group, compared with CN groups. RMGI and composite resin displayed no difference in leakage score in the current study. The bonding of composite resin to tooth structure is mediated by micromechanical adhesion following etching of dental substrate and penetration of bonding resin17. The bond strength of composite resin to tooth structure is higher than that of RMGI18. an interfacial gap is expected if the adhesion of the restorative material to the tooth structure does not compensate for the shrinkage stress induced during setting and polymerization4,5. Similar leakage scores of these two materials can be explained by several properties of the materials such as their modulus of elasticity, hygroscopic expansion, and the application technique of the material. RMGI has lower modulus of elasticity than composite resin25, which can relieve the induced polymerization shrinkage stress. A material with low modulus of elasticity has higher capacity for plastic flow and stress relaxation during polymerization4,5. Studies have shown that both composite resins and RMGI cements absorb water26,27. In the humid oral environment, polymerization shrinkage may be partly relieved by hygroscopic expansion following water sorption. Therefore, marginal gaps caused by polymerization shrinkage can be decreased by hygroscopic expansion26,27. Water uptake by a restorative material is a diffusion-controlled process through the resin matrix. The diffusion coefficient of water sorption is controlled by hydrophilicity/hydrophobicity of the resin matrix and filler level/resin content ratio27,28. Panpisut and Toneluck showed that the studied nanofilled composite resin had lower water sorption than Fuji II LC, and they attributed this finding to the hydrophilic nature of resin matrix (HEMA) and polyacrylic salt network in the studied RMGI cement20. HEMA accounts for nearly 25%-50% of the liquid content of Fuji II LC6; while, more hydrophobic and rigid monomers such as Bis-GMA and UDMA comprise the polymer network of Tetric N-Ceram29. The filler content of the composite is 80-81% by weight29; while, Fuji II LC has no filler content6. Furthermore, there is a relationship between the cement maturity and water balance. Acid-base reactions in light-cure RMGI are slower than photo-initiated polymerization25. Thus, a longer time for water uptake and maturation is required, and the maturation occurs over a prolonged period of time. However, lower post-curing is expected to occur in composite resin, and the majority of monomer to polymer conversion reactions possibly occur upon the initial light irradiation. This means that composite resin is close to maturity. All these factors probably cause 9 Motevasselian et al. higher water sorption in RMGI than resin composite25. Several studies found that water exposure of RMGI cements and consequent hygroscopic expansion were beneficial for reversing tensile or pulling stresses into compressive stress to minimize gaps in teeth restored with resin-based materials26,27. Furthermore, the composite resin was placed in two oblique increments to avoid contact with the opposing occlusal and gingival margins at the same time. The rational was that this technique decreases the overall polymerization shrinkage and consequently the polymerization stress4,5. Incremental application is also helpful to reduce the probability of bond failure along the gingival margins located in dentin. Dentin provides a weaker bond compared with enamel17, and polymerization shrinkage in one increment may cause debonding at the weaker interface. Composite resin and Cention N groups with/without adhesive also showed comparable leakage scores. This finding could be attributed to degree of conversion (DC), polymerization shrinkage and contraction stress of these materials. Degree of polymerization and polymerization stress are related to interfacial gap formation4,5. DC is influenced by the filler/resin ratio and resin content. The former factor for the composite resin and Cention N is relatively the same (Table 1). However, they do not contain the same resin content (Table 1). Tetric N-Ceram is a Bis-GMA-based composite resin29; while, Cention N is a UDMA-based polymer7,8. Bis-GMA monomer has higher molecular weight and viscosity but lower DC than UDMA monomer22. Panpisut and Toneluck20 found a higher monomer conversion in Cention N compared with a Bis-GMA nanofilled composite resin. Therefore, Cention N is expected to have higher microleakage score. However, there are other factors that might compensate for the higher DC, and consequent polymerization shrinkage and contraction stress. Ilie23 showed that Cention N polymerization initiates instantly following light irradiation in 2 mm material thickness and reaches a plateau in 1 h; whereas, composite resin polymerization continues for more than 24 h. This polymerization behavior might explain the similar leakage scores in these two groups. It was interesting that cavities restored by Cention N with or without adhesive did not reveal significant differences in leakage scores. It shows that marginal adaptation of cavities restored with this material is unrelated to micromechanical retention provided by the adhesive resin. According to the manufacturer and several authors, it is a self-adhering bulk fill restorative material that obviates the need for a separate adhesive7-9. The improved adaptation of the material to tooth margins and smear layer might be related to its hydrophilic character and its ability to wet the tooth surface which is attributed to its resin composition containing a hydrophilic dimethacrylate (polyethylene glycol dimethacrylate)8,10 (Table 1). In summary, Cention N with and without adhesive showed comparable leakage scores to the composite resin, and the values were lower than those in Fuji II LC. Tetric N-Ceram nanohybrid composite resin, which was incrementally applied on the tooth substrate showed similar leakage scores in comparison with Cention N groups and RMGI. Comparison of the abovementioned commercial materials is not simple. There are many factors related to different formulations of resin polymers that complicate a precise comparison such as the initiator type and concentration, and filler dispersion 10 Motevasselian et al. that can affect the physicochemical properties. In addition, thermomechanical loading can better simulate the harsh oral environment and its adverse effect on the longevity of restorations. Therefore, future studies are required on other properties of materials such as their DC, polymerization shrinkage, shrinkage stress, volumetric shrinkage, elastic modulus, and water sorption after thermomechanical cycling of specimens to provide more detailed information and explain the variations in data. Conclusions In vitro microleakage of Cention N was comparable to Tetric N-Ceram at enamel and dentin margins. However, Cention N showed significantly lower leakage scores than Fuji II LC. References: 1. Wierichs R, Kramer E, Meyer-Lückel H. Risk factors for failure of class V restorations of carious cervical lesions in general dental practices. J Dent. 2018;77:87-92. doi: 10.1016/j.jdent.2018.07.013. 2. Nagi SM, Moharam LM, El Hoshy AZ. Fluoride release and recharge of enhanced resin modified glass ionomer at different time intervals. Future Dent J. 2018;4(2):221-4. doi: 10.1016/j.fdj.2018.06.005. 3. Kim J, Cho J, Lee Y, Cho B. The survival of class V composite restorations and analysis of marginal discoloration. Oper Dent. 2017;42(3):E93-101. doi: 10.2341/16-186-C. 4. Soares CJ, Rodrigues MdP, Vilela ABF, Pfeifer CS, Tantbirojn D, Versluis A. Polymerization shrinkage stress of composite resins and resin cements–What do we need to know? Braz Oral Res. 2017;31(Suppl 1):e62. doi: 10.1590/1807-3107BOR-2017.vol31.0062. 5. Kaisarly D, El Gezawi M. Polymerization shrinkage assessment of dental resin composites: a literature review. Odontology. 2016;104(3):257-70. doi: 10.1007/s10266-016-0264-3. 6. Pinto-Sinai G, Brewster J, Roberts H. Linear coefficient of thermal expansion evaluation of glass ionomer and resin-modified glass ionomer restorative materials. Oper Dent. 2018;43(5):E266-72. doi: 10.2341/17-381-L. 7. Van Ende A, De Munck J, Lise DP, Van Meerbeek B. Bulk-fill composites: a review of the current literature. J Adhes Dent. 2017;19(2):95-109. doi: 10.3290/j.jad.a38141. 8. Todd JC. Scientific Documentation: Cention N. Ivoclar-Vivadent. Press: Schaan, Liechtenstein. 2016. p.1-58. 9. Aljunayh MS, Alharbi AG, Aldosari AO, Alghamdi MM, Algahtani NM, Elmarakby AM. One bulk fill composite restorative material: advantages and application technique. EC Dental Science. 2020;19(10):10-7. 10. Roulet J, Hussein H, Abdulhameed N, Shen C. In vitro wear of two bioactive composites and a glass ionomer cement. Dtsch Zahnärztl Z Int. 2019;1(1):24-30. doi: 10.3238/dzz-int.2019.0024-0030. 11. Heintze SD. Clinical relevance of tests on bond strength, microleakage and marginal adaptation. Dent Mater. 2013;29(1):59-84. doi: 10.1016/j.dental.2012.07.158. 12. Scotti N, Comba A, Gambino A, Paolino DS, Alovisi M, Pasqualini D, et al. Microleakage at enamel and dentin margins with a bulk fills flowable resin. Eur J Dent. 2014;8(1):1-8. doi: 10.4103/1305-7456.126230. 13. Khoroushi M, Ehteshami A. Marginal microleakage of cervical composite resin restorations bonded using etch-and-rinse and self-etch adhesives: two dimensional vs. three dimensional methods. Restor Dent Endod. 2016;41(2):83-90. doi: 10.5395/rde.2016.41.2.83. 14. Hepdeniz O, Ermis R. Comparative evaluation of marginal adaptation and microleakage of lowshrinking composites after thermocycling and mechanical loading. Niger J Clin Pract. 2019 May;22(5):633-41. doi: 10.4103/njcp.njcp_567_18. 11 Motevasselian et al. 15. Morresi AL, D’Amario M, Capogreco M, Gatto R, Marzo G, D’Arcangelo C, et al. Thermal cycling for restorative materials: does a standardized protocol exist in laboratory testing? A literature review. J Mech Behav Biomed Mater. 2014;29:295-308. doi: 10.1016/j.jmbbm.2013.09.013. 16. Ebaya MM, Ali AI, Mahmoud SH. Evaluation of marginal adaptation and microleakage of three glass ionomer-based Class V restorations: in vitro study. Eur J Dent. 2019;13(4):599-606. doi: 10.1055/s-0039-3401435. 17. Sofan E, Sofan A, Palaia G, Tenore G, Romeo U, Migliau G. Classification review of dental adhesive systems: from the IV generation to the universal type. Ann Stomatol. (Roma)2017; 3;8(1):1-17. doi: 10.11138/ads/2017.8.1.001. 18. Nicholson JW. Adhesion of glass-ionomer cements to teeth: a review. Int J Adhes Adhes. 2016;69:33-8. doi: 10.1016/j.ijadhadh.2016.03.012. 19. Habib E, Wang R, Zhu X. Correlation of resin viscosity and monomer conversion to filler particle size in dental composites. Dent Mater. 2018;34(10):1501-8. doi: 10.1016/j.dental.2018.06.008. 20. Panpisut P, Toneluck A. Monomer conversion, dimensional stability, biaxial flexural strength, and fluoride release of resin-based restorative material containing alkaline fillers. Dent Mater J. 2020; 39(4):608-15. doi: 10.4012/dmj.2019-020. 21. Bala O, Arisu HD, Yikilgan I, Arslan S, Gullu A. Evaluation of surface roughness and hardness of different glass ionomer cements. Eur J Dent. 2012;6(1):79-86. 22. Pratap B, Gupta RK, Bhardwaj B, Nag M. Resin based restorative dental materials: characteristics and future perspectives. Jpn Den Sci Rev. 2019;55(1):126-38. doi: 10.1016/j.jdsr.2019.09.004. 23. Ilie N. Comparative effect of self-or dual-curing on polymerization kinetics and mechanical properties in a novel, dental-resin-based composite with alkaline filler. Running Title: Resin-composites with alkaline fillers. Materials. 2018;11(1):108. doi: 10.3390/ma11010108. 24. Roberts HW. Temperature-influenced dimensional change of different molar anatomical areas. Arch Oral Biol. 2014;59(12):1312-5. doi: 10.1016/j.archoralbio.2014.08.001. 25. Suiter EA, Tantbirojn D, Watson LE, Yazdi H, Versluis A. Elastic modulus maturation effect on shrinkage stress in a primary molar restored with tooth-colored materials. Pediat Dent. 2018;40(5):370-4. 26. Suiter E, Watson L, Tantbirojn D, Lou J, Versluis A. Effective expansion: balance between shrinkage and hygroscopic expansion. J Dent Res. 2016;95(5):543-9. doi: 10.1177/0022034516633450. 27. Sokolowski K, Szczesio-Wlodarczyk A, Bociong K, Krasowski M, Fronczek-Wojciechowska M, Domarecka M, et al. Contraction and hydroscopic expansion stress of dental ion-releasing polymeric materials. Polymers (Basel). 2018;10(10):1093.doi: 10.3390/polym10101093. 28. Alshali RZ, Salim NA, Satterthwaite JD, Silikas N. Long-term sorption and solubility of bulk-fill and conventional resin-composites in water and artificial saliva. J Dent. 2015;43(12):1511-8. doi: 10.1016/j.jdent.2015.10.001. 29. Yan Y, Chen C, Chen B, Shen J, Zhang H, Xie H. Effects of hydrothermal aging, thermal cycling, and water storage on the mechanical properties of a machinable resin-based composite containing nanozirconia fillers. J Mech Behav Biomed Mater. 2020;102:103522. doi: 10.1016/j.jmbbm.2019.103522. 30. Hass V, Dobrovolski M, Zander-Grande C, Martins GC, Gordillo LAA, Accorinte MdLR, et al. Correlation between degree of conversion, resin–dentin bond strength and nanoleakage of simplified etch-andrinse adhesives. Dent Mater. 2013;29(9):921-8. doi: 10.1016/j.dental.2013.05.001. 12
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Participation of<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mrow><mml:msub><mml:mrow><mml:mtext>GABA</mml:mtext></mml:mrow><mml:mtext>A</mml:mtext></mml:msub></mml:mrow></mml:math>Chloride Channels in the Anxiolytic-Like Effects of a Fatty Acid Mixture
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Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 121794, 7 pages http://dx.doi.org/10.1155/2013/121794 Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 121794, 7 pages http://dx.doi.org/10.1155/2013/121794 Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 121794, 7 pages http://dx.doi.org/10.1155/2013/121794 Academic Editor: Brynn Levy Academic Editor: Brynn Levy Copyright © 2013 Juan Francisco Rodr´ıguez-Landa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Human amniotic fluid and a mixture of eight fatty acids (FAT-M) identified in this maternal fluid (C12:0, lauric acid, 0.9 𝜇g%; C14:0, myristic acid, 6.9 𝜇g%; C16:0, palmitic acid, 35.3 𝜇g%; C16:1, palmitoleic acid, 16.4 𝜇g%; C18:0, stearic acid, 8.5 𝜇g%; C18:1cis, oleic acid, 18.4 𝜇g%; C18:1trans, elaidic acid, 3.5 𝜇g%; C18:2, linoleic acid, 10.1 𝜇g%) produce anxiolytic-like effects that are comparable to diazepam in Wistar rats, suggesting the involvement of 𝛾-aminobutyric acid-A (GABAA) receptors, a possibility not yet explored. Wistar rats were subjected to the defensive burying test, elevated plus maze, and open field test. In different groups, three GABAA receptor antagonists were administered 30 min before FAT-M administration, including the competitive GABA binding antagonist bicuculline (1 mg/kg), GABAA benzodiazepine antagonist flumazenil (5 mg/kg), and noncompetitive GABAA chloride channel antagonist picrotoxin (1 mg/kg). The FAT-M exerted anxiolytic-like effects in the defensive burying test and elevated plus maze, without affecting locomotor activity in the open field test. The GABAA antagonists alone did not produce significant changes in the behavioral tests. Picrotoxin but not bicuculline or flumazenil blocked the anxiolytic-like effect of the FAT-M. Based on the specific blocking action of picrotoxin on the effects of the FAT-M, we conclude that the FAT-M exerted its anxiolytic-like effects through GABAA receptor chloride channels. Correspondence should be addressed to Carlos M. Contreras; ccontreras@uv.mx Correspondence should be addressed to Carlos M. Contreras; ccontreras@uv.mx Received 4 April 2013; Accepted 20 August 2013 1. Introduction of diazepam and some neurosteroids [1, 5, 6]. Their actions are well known. Bicuculline is a competitive antagonist of the GABA binding site and blocks the anxiolytic-like effects of 4󸀠-chlorodiazepam and progesterone [7], among others. Flumazenil is a selective antagonist of the benzodiazepine allosteric binding site and blocks the anxiolytic-like effects of diazepam, alprazolam, and some neurosteroids [4, 8, 9]. Picrotoxin is a noncompetitive antagonist of GABAA receptor chloride channels and blocks the anxiolytic-like effects of diazepam, 4󸀠-chlorodiazepam, progesterone, and allopregnanolone [3, 7, 10, 11], among others. Therefore, these antagonists constitute effective tools in the pharmacological screening of drugs that interact with GABAA receptors [12].l The 𝛾-aminobutyric acid-A (GABAA) receptor is a het- eropentameric structure that consists of several subunits with GABA, benzodiazepine, alcohol, barbiturate, and neu- rosteroid recognition sites [1]. The activation of GABAA receptors increases the intraneuronal concentration of chlo- ride ions, leading to hyperpolarization, an action shared by anxiolytic, hypnotic, sedative, and anticonvulsant drugs [2]. Consequently, GABAA receptors are considered the main target for clinically effective anxiolytic drugs and some neurosteroids with potential anxiolytic properties [3, 4]. Several GABAA receptor antagonists, such as bicuculline, flumazenil, and picrotoxin, are currently used to identify the precise sites of action of drugs on the GABAA receptor. These antagonists are able to block the anxiolytic-like effects g g A p Recently it was demonstrated that human amniotic fluid and a mixture of eight fatty acids (FAT-M) contained therein produce anxiolytic-like effects similar to diazepam in male 2 2 BioMed Research International and female adult Wistar rats [13]. The FAT-M included eight FATs (C12:0, lauric acid; C14:0, myristic acid; C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1cis, oleic acid; C18:1trans, elaidic acid; C18:2, linoleic acid) that were consistently detected in human amniotic fluid, colostrum, and milk. In addition to its anxiolytic effects [13], the FAT- M produced appetitive responses in human newborns [14]. The anxiolytic-like action of the FAT-M may involve the participation of GABAA receptors, given that some FATs modulate the opening of GABAA receptor chloride channels in vitro [15], but this possibility needs to be assayed in vivo.hi rat incidentally touched the electrode, it received an electric shock and began to vigorously displace the sawdust to cover the electrode (burying behavior). 1. Introduction All of the sessions were recorded with a digital videocamera (Sony, DCR-SR85, 25x optical zoom, Carl Zeiss lens) for subsequent analysis by two independent observers to measure burying latency and total cumulative burying time during a 10 min test, starting from the first electric shock. After each test session, the bed of fine sawdust was removed and replaced by clean sawdust bedding. Only observations with more than 95% agreement between observers were included in the data analysis. Therefore, the present study confirmed the anxiolytic- like effect of a FAT-M using two validated experimental animal models currently used to test the effectiveness of clinically effective anxiolytics, the defensive burying test [16] and elevated plus maze [17]. We then explored the GABAA site of action involved in the anxiolytic-like effects of a FAT- M using bicuculline (a competitive antagonist of the GABAA receptor), flumazenil (a blocker of the benzodiazepine recog- nition site), and picrotoxin (a noncompetitive antagonist of chloride channels). 2.3.2. Elevated Plus Maze. The apparatus was constructed of wood and situated in a brightly lit room (40 lux). The apparatus consisted of two opposite open and closed arms set in a perpendicular configuration. The open and closed arms were painted white and black, respectively. The dimensions of the open arms were 50 cm length × 10 cm width and the closed arms were 50 cm length × 10 cm width × 40 cm height. The entire maze was elevated 50 cm from the floor. The rats were placed in the center of the maze, facing an open arm, and the time spent on and number of entries into the open arms was recorded [20]. The total number of entries (open arms + closed arms) and percentage of open arm entries ((open entries)/(total entries) × 100) were calculated. The elevated plus maze was cleaned with a 5% ethanol solution after each session. 2. Material and Methods 2.1. Ethics. All of the experimental procedures in the present study followed the principles of animal care based on the Guide for the Care and Use of Laboratory Animals (National Research Council, [18]). The protocol received authorization from the Biomedical Research Institute Ethical Committee (Universidad Nacional Aut´onoma de M´exico). 2.3.3. Open Field Test. To evaluate the effects of the treat- ments on spontaneous locomotor activity, which could inter- fere with performance in the defensive burying test and elevated plus maze, the rats were subjected to a 5 min test in the open field after the defensive burying and elevated plus maze tests. We used an automated motor activity mon- itor (Acti-Track v2.7.10, PanLab, S.L. Instrument, Barcelona, Spain) in a Perspex box (45 × 45 cm base, 35 cm height). A total of 32 infrared beams, 16 each on perpendicular walls, were mounted 3 cm above the box frame floor and connected to an interface (LE 8811, LSI Letica Scientific Instruments, Barcelona, Spain), and the data were sent to a computer. For data analysis, the floor of the cage was virtually divided into five zones (four peripheral and one central). The total number of entries into the zones (i.e., crossings), time spent active, and time spent resting were recorded. Because of the relatively small cage, we did not compare central versus peripheral exploration.ti 2.2. Animals. Male Wistar rats were obtained from a local strain supplied by Harlan (M´exico City, M´exico). They were housed in local housing facilities at a mean temperature of 25 ± 2∘C with a 12 h/12 h light/dark cycle (lights on at 7:00 AM). All of the rats included in the study were approximately 2 months old, weighed 250–300 g, and five to six rats were housed per cage in acrylic boxes (44 cm width × 33 cm length × 20 cm height) with ad libitum access to food (Teklad Lab Animal Diets; Harlan, Indianapolis, IN, USA) and purified water. All of the experiments were performed during the light period (approximately 12:00 PM). 2.6. Experimental Groups The number of entries into the open arms was also significantly different (𝐹4,35 = 3.66, 𝑃= 0.014). The post hoc test revealed that this variable was higher in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle- 1 + vehicle-2 and picrotoxin + FAT-M groups (Figure 2(b)), but no significant differences between these latter two groups were detected.h 3.2.2. Elevated Plus Maze. The analysis of the time spent in the open arms revealed significant differences between treatments (𝐹4,35 = 8.47, 𝑃= 0.001). The post hoc test revealed that the time spent in the open arms was longer in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2 and picrotoxin + FAT- M groups, which displayed similar values (Figure 2(a)). The number of entries into the open arms was also significantly different (𝐹4,35 = 3.66, 𝑃= 0.014). The post hoc test revealed that this variable was higher in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle- 1 + vehicle-2 and picrotoxin + FAT-M groups (Figure 2(b)), but no significant differences between these latter two groups were detected.h 2.7. Statistical Analysis. All of the data were statistically ana- lyzed using one-way analysis of variance (ANOVA). Values of 𝑃≤0.05 were followed by the Student-Newman-Keuls post hoc. We first analyzed the intrinsic activity of GABAA antagonists on the variables in the behavioral tests. We then evaluated the effects of antagonism of different binding sites of the GABAA receptor on the effects of the FAT-M. The results are expressed as mean ± standard error. The analysis of the percentage of entries into the open arms also revealed significant differences (𝐹4,35 = 4.54, 𝑃 = 0.005). The percentage of entries into the open arms was greater in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2 and picrotoxin + FAT-M groups (Figure 2(d)). No significant differences were found between the vehicle-1 + vehicle-2 and picrotoxin + FAT-M groups. Finally, the total entries into the arms were not significantly different between groups (𝐹4,35 = 2.54, 𝑃= 0.060; Figure 2(c)). 3.2. Interactions between GABAA Antagonists and FAT-M 3.2. Interactions between GABAA Antagonists and FAT-M BioMed Research International BioMed Research International not affect the number of crossings (𝐹3,42 = 2.65, 𝑃= 0.061), activity time (𝐹3,42 = 1.79, 𝑃= 0.164), or resting time (𝐹3,42 = 2.33, 𝑃= 0.088) in the open field test. Likewise, the antagonists did not produce significant changes in the elevated plus maze, including the time spent in the open arms (𝐹3,28 = 0.23, 𝑃= 0.875), number of entries into the open arms (𝐹3,28 = 0.64, 𝑃= 0.592), total number of entries into the arms (𝐹3,28 = 0.84, 𝑃= 0.483), and percentage of entries into the open arms (𝐹3,28 = 0.88, 𝑃= 0.462). Similarly, no significant changes were found in the open field test, including the number of crossings (𝐹3,28 = 1.61, 𝑃= 0.208), activity time (𝐹3,28 = 2.19, 𝑃= 0.111), and resting time (𝐹3,28 = 2.02, 𝑃= 0.134). We used two vehicles, one for the GABAA antagonists (vehicle-1: 1% Tween-80, 14% propylene glycol, and 85% saline) and another for the FAT-M (vehicle-2: 96% propylene glycol and 4% ethanol). All of the chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.5. Treatment. The FAT-M preparation and treatment schedule were based on previous reports [13]. The FAT- M consisted of lauric acid (0.4 mg), myristic acid (3.0 mg), palmitic acid (15.3 mg), palmitoleic acid (7.1 mg), stearic acid (3.7 mg), oleic acid (8.0 mg), elaidic acid (1.5 mg), and linoleic acid (4.4 mg) in 100 mL of vehicle-2 at a temperature <40∘C. The FAT-M (1 mL/rat) or vehicle-2 was subcutaneously injected 60 min before the behavioral tests. Analytical-grade FATs were purchased from Sigma-Aldrich. 2.5. Treatment. The FAT-M preparation and treatment schedule were based on previous reports [13]. The FAT- M consisted of lauric acid (0.4 mg), myristic acid (3.0 mg), palmitic acid (15.3 mg), palmitoleic acid (7.1 mg), stearic acid (3.7 mg), oleic acid (8.0 mg), elaidic acid (1.5 mg), and linoleic acid (4.4 mg) in 100 mL of vehicle-2 at a temperature <40∘C. The FAT-M (1 mL/rat) or vehicle-2 was subcutaneously injected 60 min before the behavioral tests. Analytical-grade FATs were purchased from Sigma-Aldrich. 2.3. Behavioral Tests 2.3.1. Defensive Burying Test. An acrylic box (27 cm width × 17.5 cm length × 30 cm height) with the floor covered by a 5 cm bed of fine sawdust (Teklad Sani-Chips 7090, 2.2 cubic feet; Harlan, Indianapolis, IN, USA) was placed inside a noise- isolated box (65 cm width × 55 cm length × 45 cm height; Coulbourn Instruments, Whitehall, PA, USA). An electrode (7 cm length, 0.5 cm diameter) protruded 2 cm above the saw- dust bed horizontally from one wall of the box (17.5 × 30 cm) [16, 19]. The electrode delivered a constant-intensity current (0.3 mA, direct current) through an electronic stimulator (Grass Instruments S44, Quincy, MA, USA) coupled in series to a stimulus isolation unit (Grass Instruments SIU5) and constant-current unit (Grass Instruments CCUIA). When a After each experimental session, the open field was carefully cleaned and deodorized with a 5% ethanol cleaning solution. Five minutes elapsed between each test to allow the scent of the substances to dissipate. 2.4. Pretreatment. The doses and pretreatment schedules were based on previous reports [4, 7, 21] that effectively antagonized the behavioral effects produced by anxiolytic drugs: bicuculline, 1 mg/kg; flumazenil, 5 mg/kg; picrotoxin, 1 mg/kg. The antagonists were administered intraperitoneally in an equivalent volume of 1 mL/kg 30 min before the FAT-M treatment, including the vehicles. 3 2.6. Experimental Groups 3.2.1. Defensive Burying Test. The analysis of burying latency revealed significant differences between treatments (𝐹4,56 = 3.45, 𝑃= 0.014). The post hoc test showed that burying latency was significantly longer in the FAT-M groups com- pared with the vehicle-1 + vehicle-2 group, but no significant differences were detected in the FAT-M groups pretreated with GABAA antagonists compared with the vehicle-1 + vehicle-2 group (Figure 1(a)).h 2.6.1. Intrinsic Activity of GABAA Antagonists. To identify the intrinsic activity of the GABAA antagonists on anxiety-like behavior and open field activity, four independent groups were evaluated in the defensive burying test and subsequently open field test. The treatment conditions included four combinations: vehicle-1 before vehicle-2 (vehicles; 𝑛= 11), bicuculline before vehicle-2 (bicuculline; 𝑛= 12), flumazenil before vehicle-2 (flumazenil; 𝑛= 12), and picrotoxin before vehicle-2 (picrotoxin; 𝑛 = 11). Other four independent groups (𝑛= 8 rats per group) received a similar treatment schedule but were tested in the elevated plus maze and subsequently open field test. The analysis of cumulative burying time also revealed significant differences between treatments (𝐹4,56 = 10.80, 𝑃= 0.001). The post hoc test revealed that cumulative burying time was significantly shorter in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle- 1 + vehicle-2 group. However, the picrotoxin + FAT-M group was not significantly different from the vehicle-1 + vehicle-2 group (Figure 1(b)). 2.6.2. Interaction between GABAA Antagonists and FAT-M. The defensive burying test included five independent groups that received five different combinations: vehicle-1 before vehicle-2 (vehicles; 𝑛= 11), vehicle-1 before FAT-M (FAT- M; 𝑛= 15), bicuculline before FAT-M (bicuculline + FAT- M; 𝑛= 11), flumazenil before FAT-M (flumazenil + FAT-M; 𝑛= 11), and picrotoxin before FAT-M (picrotoxin + FAT- M; 𝑛= 13). The elevated plus maze test included another five independent groups (𝑛= 8 rats per group) that received similar treatments. The open field test was conducted less than 5 min after the elevated plus maze test. 3.2.2. Elevated Plus Maze. The analysis of the time spent in the open arms revealed significant differences between treatments (𝐹4,35 = 8.47, 𝑃= 0.001). The post hoc test revealed that the time spent in the open arms was longer in the FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2 and picrotoxin + FAT- M groups, which displayed similar values (Figure 2(a)). 4. Discussion The present study explored the participation of the GABAA receptor complex in the anxiolytic-like effects of a mixture of eight FATs in male Wistar rats subjected to the defensive burying test and elevated plus maze. Pretreatment with picrotoxin but not bicuculline or flumazenil blocked the anxiolytic-like effects of FAT-M, without affecting behavior in the open field test. i In the defensive burying test, the time that elapsed between the first shock and first attempt at burying (i.e., burying latency) is inversely related to the rat’s reactivity. The time spent burying (i.e., total cumulative burying) is an indicator of anxiety as discussed by Treit [16]. “Anxious” animals spend more time burying than animals treated with anxiolytic drugs, such as diazepam, which spend less time burying. In the present study, the FAT-M reduced cumulative burying time and increased burying latency, confirming its anxiolytic-like effects, as previously reported [13].h (a) 0 20 40 60 80 100 120 140 Cumulative burying (s) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (b) (a) Cumulative burying (s) f The elevated plus maze is widely used to explore anxiety- like behavior and the anxiogenic- or anxiolytic-like effects of drugs [20]. Anxiety-like behavior in the elevated plus maze is assumed when both the percentage of entries into and time spent on the open arms are reduced. The present results showed that the FAT-M increased both the percentage of entries into and time spent on the open arms, confirming an anxiolytic-like effect in a second test of anxiety. This anxiolytic-like effect was only blocked by picrotoxin and not by the other GABAA receptor antagonists tested in the present study.h (b) Figure 1: Defensive burying test. (a) Burying latency was signif- icantly longer in the FAT-M group pretreated with vehicle-1, an effect attenuated by pretreatment with GABAA antagonists. (b) Cumulative burying time was significantly shorter in the vehicle-1 + FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2, an effect not detected in the picrotoxin + FAT-M group ( ∗𝑃< 0.05, Student-Newman-Keuls post hoc test). FAT-M, fatty acid mixture. The present results suggest that GABAA receptor chloride ion channels, but not benzodiazepine or GABA binding sites, participate in the anxiolytic-like effects of the FAT- M. Certainly, some FATs (i.e., oleic, linoleic, ricinoleic, and arachidonic acids) seemingly participate in the regulation of chloride ion channels [22]. 3. Results 3.1. Intrinsic Activity of GABAA Antagonists. The GABAA antagonists alone did not affect burying latency (𝐹3,42 = 0.46, 𝑃= 0.712) or cumulative burying time (𝐹3,42 = 0.11, 𝑃= 0.958) in the defensive burying test. The antagonists also did BioMed Research International 4 Table 1: Open field test. Table 1: Open field test. Group Crossings Activity time (s) Resting time (s) Vehicle-1 + Vehicle-2 21.8 ± 2.88 75.1 ± 7.05 221.9 ± 7.09 Vehicle-1 + FAT-M 37.5 ± 5.37 109.7 ± 8.82 194.8 ± 10.42 Bicuculline + FAT-M 36.0 ± 6.33 103.9 ± 8.73 201.3 ± 7.00 Flumazenil + FAT-M 38.1 ± 8.70 97.2 ± 14.56 184.0 ± 16.67 Picrotoxin + FAT-M 34.0 ± 7.63 90.3 ± 13.03 213.5 ± 13.87 No significant differences were found in the evaluated variables. The data are expressed as mean ± standard error. FAT-M, fatty acid mixture. 0 100 200 300 400 Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin Burying latency (s) ∗ (a) 0 20 40 60 80 100 120 140 Cumulative burying (s) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (b) Figure 1: Defensive burying test. (a) Burying latency was signif- icantly longer in the FAT-M group pretreated with vehicle-1, an effect attenuated by pretreatment with GABAA antagonists. (b) Cumulative burying time was significantly shorter in the vehicle-1 + FAT-M, bicuculline + FAT-M, and flumazenil + FAT-M groups than in the vehicle-1 + vehicle-2, an effect not detected in the picrotoxin + FAT-M group ( ∗𝑃< 0.05, Student-Newman-Keuls post hoc test). FAT-M, fatty acid mixture. 0 100 200 300 400 Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin Burying latency (s) ∗ (a) 3.2.3. Open Field Test. In the open field test, the analysis of the number of crossings did not reveal significant differences between treatments (𝐹4,35 = 1.06, 𝑃= 0.391). Similarly, no significant differences were detected in activity time (𝐹4,35 = 1.53, 𝑃= 0.213) or resting time (𝐹4,35 = 1.65, 𝑃= 0.183; Table 1). 4. Discussion 0 40 80 120 160 Time into the open arms (s) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin 0 2 4 6 8 Entries into the open arms (n) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin Entries into the open arms (n) (a) 0 5 10 15 20 Total entries into the arms (n) Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (c) (b) 0 10 20 30 40 50 Entries into the open arms (%) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (d) (b) (a) (d) (c) Figure 2: Elevated plus maze. The FAT-M produced anxiolytic-like effects that were blocked by pretreatment with picrotoxin. (a) Time spent in open arms. (b) Total number of entries into the arms. (c) Number of entries into the open arms. (d) Percentage of entries into the open arms. The elevated plus maze test lasted for 5 min. ∗𝑃< 0.05, compared with vehicle-1 + vehicle-2 and picrotoxin + FAT-M groups (Student- Newman-Keuls post hoc test). FAT-M, fatty acid mixture. elevated plus maze. The open field test was conducted after the aforementioned behavioral tests, allowing us to exclude possible nonspecific locomotor effects of antagonists or FAT treatments. In the open field test, we found that none of the GABAA antagonists alone or the FAT-M at the doses tested produced any significant changes on motor activity, similar to previous reports [4, 11, 13, 35]. Additionally, the dose administered of bicuculline and picrotoxin did not pro- duce behaviour linked to seizures (i.e., facial/ear twitching, myoclonic jerks, among others), which are detected with higher doses than those used in the present study. Therefore, the anxiolytic-like effect of the FAT-M and blockade of this effect by GABAA antagonists do not appear to be associated with nonspecific effects of the treatments on spontaneous locomotion. Chloride channels participate in the actions of substances with anxiolytic potency [26–28]. Other compounds that act on chloride channels produce similar anxiolytic-like effects as the FAT-M. Some endogenous steroids, as progesterone and allopregnanolone, are allosteric modulators of GABAA chlo- ride ion channels [29–32]. At physiological (i.e., nanomolar) concentrations, some steroids with a reduced A-ring promote channel opening frequency and increase chloride flux [28, 33, 34]. Majewska [28] demonstrated that steroids interact at the membrane protein and lipid interface, leading to an increased frequency of chloride channel opening. 4. Discussion Interestingly, oleic and linoleic acids are components of the FAT-M tested in the present study. Oleic acid increases the affinity of agonists for the benzodiazepine site of GABAA receptors [23], thus modulat- ing the opening of chloride channels. The modulation of ion channels by other FATs (e.g., myristic and arachidonic acids) occurs through indirect effects that involve their metabolic conversion to active oxygenated metabolites and other direct effects [24] by accumulating FATs in the phospholipid mem- brane bilayer and modifying membrane tension, leading to conformational changes in ion channels and altering ion conductance [25]. 3.2.3. Open Field Test. In the open field test, the analysis of the number of crossings did not reveal significant differences between treatments (𝐹4,35 = 1.06, 𝑃= 0.391). Similarly, no significant differences were detected in activity time (𝐹4,35 = 1.53, 𝑃= 0.213) or resting time (𝐹4,35 = 1.65, 𝑃= 0.183; Table 1). 5 BioMed Research International 0 40 80 120 160 Time into the open arms (s) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (a) 0 2 4 6 8 Entries into the open arms (n) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (b) 0 5 10 15 20 Total entries into the arms (n) Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (c) 0 10 20 30 40 50 Entries into the open arms (%) ∗ ∗ ∗ Vehicle-1 + vehicle-2 Vehicle-1 + FAT-M Bicuculline Flumazenil Picrotoxin (d) Figure 2: Elevated plus maze. The FAT-M produced anxiolytic-like effects that were blocked by pretreatment with picrotoxin. (a) Time spent in open arms. (b) Total number of entries into the arms. (c) Number of entries into the open arms. (d) Percentage of entries into the open arms. The elevated plus maze test lasted for 5 min. ∗𝑃< 0.05, compared with vehicle-1 + vehicle-2 and picrotoxin + FAT-M groups (Student- Newman-Keuls post hoc test). FAT-M, fatty acid mixture. References [17] G. Griebel, D. J. Sanger, and G. Perrault, “The use of the rat elevated plus-maze to discriminate between non-selective and BZ-1 (𝜔1) selective, benzodiazepine receptor ligands,” Psychopharmacology, vol. 124, no. 3, pp. 245–254, 1996. [1] J. Bormann, “The “ABC” of GABA receptors,” Trends in Phar- macological Sciences, vol. 21, no. 1, pp. 16–19, 2000. [18] National Research Council, Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, USA, 1985. [2] S. J. Enna, “The GABA receptors,” in ,The GABA Receptors, S. J. Enna and H. M¨ohler, Eds., pp. 1–22, Humana Press, Totowa, NJ, USA, 2007. [19] J. P. J. Pinel and D. Treit, “Burying as a defensive response in rats,” Journal of Comparative and Physiological Psychology, vol. 92, no. 4, pp. 708–712, 1978. [3] D. Bitran, M. Shiekh, and M. McLeod, “Anxiolytic effect of progesterone is mediated by the neurosteroid allopregnanolone at brain GABAA receptors,” Journal of Neuroendocrinology, vol. 7, no. 3, pp. 171–177, 1995. [20] A. A. Walf and C. A. Frye, “The use of the elevated plus maze as an assay of anxiety-related behavior in rodents,” Nature Protocols, vol. 2, no. 2, pp. 322–328, 2007. [4] A. Fern´andez-Guasti and O. Picazo, “Flumazenil blocks the anxiolytic action of allopregnanolone,” European Journal of Pharmacology, vol. 281, no. 1, pp. 113–115, 1995. [21] K. Hirani, A. N. Sharma, N. S. Jain, R. R. Ugale, and C. T. Chopde, “Evaluation of GABAergic neuroactive steroid 3𝛼- hydroxy-5𝛼- pregnane-20-one as a neurobiological substrate for the anti-anxiety effect of ethanol in rats,” Psychopharmacol- ogy, vol. 180, no. 2, pp. 267–278, 2005. [5] M. Farrant and R. A. Webster, “GABA antagonists, their use and mechanisms of action,” in Drugs as Tools in Neurotransmitter Research. 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Discussion In fact, the actions of neurosteroids resemble the actions of flunitrazepam, musci- mol, and pentobarbital on chloride channel function and are blocked by the noncompetitive GABA antagonist picrotoxin [26], suggesting some similarity between neurosteroids and the FAT-M, given that they share common actions, such as anxiolytic-like effects in experimental models of anxiety that can be blocked by picrotoxin.i A possible limitation of the present study was that the FAT-M was administered in addition to the rats’ normal diet (i.e., purine), possibly providing an additional source of energy that may impact spontaneous behavior. However, we did not observe any change in locomotion associated with the FAT-M treatment, similar to previous reports [13, 36]. Finally, some drugs are able to produce nonspecific changes in spontaneous locomotor activity that may inter- fere with performance in the defensive burying test and 6 BioMed Research International 6 Although we did not directly assess the increased metabolic sources provided by the FATs, this may be considered inconsequential for interpreting the present results.f [9] S. K. Kulkarni and K. Sharma, “Alprazolam modifies animal behaviour on elevated plus-maze,” Indian Journal of Experimen- tal Biology, vol. 31, no. 11, pp. 908–911, 1993. [10] D. Treit, J. P. J. Pinel, and H. C. Fibiger, “The inhibitory effect of diazepam on conditioned defensive burying is reversed by picrotoxin,” Pharmacology Biochemistry and Behavior, vol. 17, no. 2, pp. 359–361, 1982. g In conclusion, the anxiolytic-like effects of the FAT-M involved actions on GABAA receptors, specifically chloride channels, providing additional evidence of the anxiolytic-like effects of FATs and the possible site of action on GABAA receptor of the FAT-M used in the present study. However, the participation of other receptors linked to chloride ion channel (i.e., strychnine-sensitive glycine receptors) could not to be discarded, which needs to be explored in particular studies. [11] D. Bitran, R. J. Hilvers, and C. K. Kellog, “Anxiolytic effects of 3a-hydroxy-5a[𝛽]-pregnan-20-one: endogenous metabolites of progesterone that are active at the GABAA receptor,” Brain Research, vol. 561, no. 1, pp. 157–161, 1991. [12] G. A. R. Johnston, “GABAA antagonists,” Seminars in Neuro- science, vol. 3, no. 3, pp. 205–210, 1991. Acknowledgments [14] C. M. Contreras, A. G. Guti´errez-Garc´ıa, R. Mendoza-L´opez, J. F. Rodr´ıguez-Landa, B. Bernal-Morales, and C. D´ıaz-Marte, “Amniotic fluid elicits appetitive responses in human newborns: fatty acids and appetitive responses,” Developmental Psychobiol- ogy, vol. 55, no. 3, pp. 221–231, 2013. This study was partially supported by a Grant from the Consejo Nacional de Ciencia y Tecnolog´ıa, M´exico (CONA- CyT: CB-2006-1, 61741) and DGAPA-PAPIIT IN211111-3 from Universidad Nacional Aut´onoma de M´exico, which did not participate in the study design, data collection, data analysis, interpretation of the data, writing of the paper, or decision to submit the paper for publication. R.I. Garc´ıa-R´ıos and J. Cueto-Escobedo received fellowships from CONACyT (Reg. 203280 and 197556, resp.). [15] M. Samochocki and J. 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Quercus wutaishanica shrub affects temperate forest community composition and soil properties under different restoration stage
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Peng Kang1,2, Jiming ChengID1, Jinpeng Hu1, Yongshun Jing3, Jing Wang1,2, Hui Yang1, Xiaodong DingID1,2*, Xingfu Yan1,2* 1 College of Biological Sciences and Engineering, North Minzu University, Yinchuan, China, 2 Key Laboratory of Ecological Protection of Agro-Pastoral Ecotones in the Yellow River Basin, National Ethnic Affairs Commission, Yinchuan, China, 3 Forest Tree Breeding Center, Liupanshan Forestry Bureau, Guyuan, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * dingxd@nmu.edu.cn (XD); xxffyan@126.com (XY) * dingxd@nmu.edu.cn (XD); xxffyan@126.com (XY) PLOS ONE RESEARCH ARTICLE Abstract Quercus wutaishanica is the dominant tree species in the natural ecosystem restoration of temperate forests in China, and it plays an active role in maintaining ecological balance. However, little is known about how ecosystem versatility develops during the restoration of forest ecosystems dominated by Q. wutaishanica. In this study, we investigated the species composition of the Q. wutaishanica community, soil nutrients, and their functional traits at various restoration stages, and comprehensively analyzed the correlations among them. At the early stage of restoration (10 years of restoration), there were Spiraea pubescens and Syringa pubescens in Q. wutaishanica community (87% of the total species), while had a larger niche width. In the middle of restoration (30 years of restoration), shannon and even- ness indices were the largest, while soil total carbon, ammonium nitrogen and chlorophyll content of Q. wutaishanica leaves were the highest; among them, soil total carbon was 15.7% higher than that in 10 years of restoration, 32.4% higher than that in 40 years of resto- ration, ammonium nitrogen was 71.7% higher than that in 40 years of restoration, and chlo- rophyll content was 217.9% higher than that in 10 years of restoration, and 51.8% higher than that in 40 years of restoration. At the later stage of restoration (40 years of restoration), Lonicera ferdinandii occupied the dominant ecological niche, and soil available nitrogen, available phosphorus content and leaf thickness were the largest; while AN was 10.9% higher than that of 10 years of restoration, 16.5% higher than that of 30 years of restoration, AP was 60.6% higher than that of 10 years of restoration, 21.6% higher than that of 30 years of restoration, leaf thickness was 22.3% higher than that of 10 years of restoration, 84.9% higher than that of 30 years of restoration. However, the restriction of various soil nutrients was reduced. Our study highlighted the effectiveness of soil resource availability in plant communities during restoration, reduced competition for light among plants, and altered species richness. Furthermore, changes in the interrelationship between plant community composition and leaf functional traits of the dominant species responded positively to com- munity restoration. These results further deepen our understanding of forest management OPEN ACCESS Citation: Kang P, Cheng J, Hu J, Jing Y, Wang J, Yang H, et al. (2023) Quercus wutaishanica shrub affects temperate forest community composition and soil properties under different restoration stage. PLoS ONE 18(11): e0294159. https://doi. org/10.1371/journal.pone.0294159 Editor: Xiao Guo, Qingdao Agricultural University, CHINA Editor: Xiao Guo, Qingdao Agricultural University, CHINA Editor: Xiao Guo, Qingdao Agricultural University, CHINA Copyright: © 2023 Kang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLOS ONE PLOS ONE 1 Introduction The Grain to Green Program (GTGP), Natural Forest Protection Program (NFPP), and Three-North Shelterbelt Program (TNSP) are a series of important plans for the restoration of natural ecosystems in China, which significantly impacts the maintenance of ecosystem func- tions, biological diversity, and carbon balance [1–3]. With the implementation of ecological restoration plans, most evergreen broad-leaved forests are currently at different restoration stages after being restored to secondary forests [4, 5]. Quercus wutaishanica is the dominant tree species in warm and temperate deciduous broad-leaved forests and mixed broadleaf-coni- fer forests in China and plays a positive role in soil and water conservation and ecological bal- ance maintenance [6]. Therefore, improving our understanding of the changes in plant diversity, soil properties, and plant functional traits during ecosystem restoration is essential for coping with abiotic and biotic stresses (climate change, insect pests, drought, etc.) and for the proper management of forests. During forest ecosystem restoration, plant diversity has been studied as an indicator of com- munity species composition and diversity [7]. Long-lived tree species can reduce the fragmenta- tion of the forest landscape at the spatiotemporal scale [8], and tree species with larger canopy gaps are conducive to the replenishment of early stages [9]. With restoration, the number of dominant species and pioneer species has increased [10, 11]. Therefore, the study of plant diver- sity at various stages of restoration can provide a theoretical basis for the restoration and recon- struction of forest ecosystems. However, unlike the traditional use of plant diversity in assessing the restoration stage of forest ecosystems, various aspects of nutrient accumulation in forest eco- systems have been developed in recent studies [12, 13]. An increase in forest carbon sink can improve the diversity and stability of plant community structure [14, 15], while nutrient inputs (e.g., nitrogen or phosphorus) have significant negative effects on plant community richness, thus increasing plant community similarity and decreasing species diversity [16]. An increasing number of studies have shown that in addition to disturbances, environmen- tal conditions (soil properties) also one of the factors affect species diversity during the restora- tion stage [17]. Soil nutrients, as important regulators of vegetation regeneration, exhibit different characteristics at different forest restoration stages, and are closely related to forest restoration speed [18]. The establishment of woody plants is delayed by the coverage of annual plants when nutrients are abundant [19]. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was financially supported by the National Natural Science Foundation of China (31960228, 31660195), the Key Research and Development Project in Ningxia (2018BEG02001) and Natural Science Foundation of Ningxia Province (2020AAC03228, 2022AAC03227). The funders had no role in study design, data collection 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits and restoration of forest communities. In the future, it is necessary to comprehensively con- sider the influence of various factors on forest community restoration. and analysis, decision to publish, or preparation of the manuscript. and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 1 Introduction With the depletion of soil nutrient resources, plants with slow growth rates have more advantages than those with fast growth and high reproduc- tive rates [20]. In addition, the accumulation of soil nutrients depends on the vegetation com- position. During the restoration stages, soil total phosphorus content decreased gradually, whereas total nitrogen content and alkali-hydrolyzable nitrogen in organic matter increased significantly [21]. Other studies have indicated that soil carbon, nitrogen, and phosphorus contents first decreased and then increased with restoration, and were higher during the late restoration periods [22]. More studies have suggested that with an increase in tree biomass, soil nutrients, organic matter, and carbon storage also increase [1]. In conclusion, the differ- ences between the dominant species and the number of species in the forest restoration stage are closely related to soil nutrients [23]. Soil nutrients, including exchangeable cations, total nitrogen, and phosphorus, have strong geographical heterogeneity and affect the composition of plant species at different restoration stages as well as species functional traits [24]. 2 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits In recent years, studies on plant functional traits have greatly improved our understanding of plant functions and characteristics during forest restoration [25, 26]. Previous studies have shown that the ecological strategies of plants switch between resource acquisition and conser- vation during the restoration stage [27]. However, plants must make trade-offs between leaf physiological traits and nutrient storage to better adapt to biotic and abiotic factors during res- toration [28]. From dry to wet conditions, plants use more water to maintain their growth while decreasing photosynthesis to increase protein consumption in leaves with lower nitrogen content [29]. At the early stage of restoration, herbaceous plants dominate, and woody plants improve their photosynthetic capacity by accumulating nitrogen in leaves to maintain domi- nant growth [30]. Overall, biomass allocation among plant organs is driven by environmental conditions, but functional traits may also be potential variables for biomass allocation [31]. Previous studies have suggested that the dominant geographical boundary factors (climate and disturbance intensity) affect the distribution of Q. wutaishanica in China [32], which focused on the relationship between spatial distribution patterns, plant functional traits, nutri- ents, soil carbon, nitrogen, and phosphorus in Q. wutaishanica communities in the Taiyue and Ziwuling Mountains [33–36]. 1 Introduction Therefore, we proposed the following hypotheses: (1) there would be differences in species diversity and richness within communities at different restora- tion stages of Q. wutaishanica communities; (2) changes in species composition during the res- toration stage of Q. wutaishanica communities may also have an effect on soil characteristics; and (3) changes in leaf functional traits of dominant plants in Q. wutaishanica communities would respond differently to the restoration stage. PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits PLOS ONE Table 1. Restoration time, latitude, longitude, and altitude of the three study sites in the Liupanshan Mountains. Study site Restoration time Latitude and longitude Altitude (m) Xilianggou 10 years 35˚230N, 106˚210E 1849.13 Dawan 30 years 35˚230N, 106˚210E 1882.13 Dadaogou 40 years 35˚230N, 106˚210E 1912.98 https://doi.org/10.1371/journal.pone.0294159.t001 Table 1. Restoration time, latitude, longitude, and altitude of the three study sites in the Liupanshan Mountains. The National Nature Reserve of the Liupanshan Mountains is located at the edge of the northern agriculture/pastoral ecotone and a semi-humid to semi-arid transitional zone in North China. Cold and dry air flows were interlaced. It is hot and rainy in the summer and dry and cold in the winter. The mean annual temperature is 5.8˚C, the coldest month (January) is −7˚C, and the hottest month (July) is 17.4˚C. The mean annual precipitation is 676 mm, and the mean annual evaporation is approximately 1426 mm. The frost-free period was approxi- mately 120 days, and the annual total sunshine duration was 2100–2400 h. There are more than 80 rivers in the Liupanshan Mountains, all of which belong to the Yellow River System. The soils in the area are grayish brown. Through visits to the Forest Tree Breeding Center, Liupanshan Forestry Bureau, Guyuan, and combined with information detailed by local villagers, we selected areas that were completely destroyed before restoration, where the shrubs were cut down and the dominant tree species were restored naturally. In July 2019, we identified three shady slope sample stands that gradually extended from east to west, from near the Yejia village in Baimian Town to Liu- panshan National Nature Reserve. The first sample stand was Xilianggou (E106˚210, N35˚230), which was restored late and only gradually with the recent reduction of human activities such as logging and grazing, and the restoration time is about 10 years from now (10 y). And the ele- vation was about 1849.13 m. The second sample stand was Dawan (E106˚210, N35˚230), which was fenced and restored in the 1990s, and the restoration time was about 30 years from now (30 y). The elevation was about 1882.13 m. The third sample stand was Dadaogou (E106˚210, N35˚230), located in the Liupanshan Nature Reserve, which was fenced and restored in the late 1970s, about 40 years ago (40 y). And the elevation was about 1912.98 m (Table 1). PLOS ONE The main vegetation types in the Liupanshan Mountains are temperate coniferous forests, evergreen Bambusoideae shrubs, secondary deciduous broad-leaved forests, deciduous broad- leaved shrubs, and grasslands. The temperate coniferous forest is dominated by Pinus arman- dii, the evergreen bambusoideae shrub by Fargesia nitida, and the secondary deciduous broad- leaved forest by Q. wutaishanica, Betula platyphylla, Betula albosinensis and Populus davidiana [37]. 2.1 Study site This study was conducted in the National Nature Reserve of the Liupanshan Mountains (35˚ 150 to 35˚410 N and 106˚090 to 106˚300 E), which is located in the south of the Liupanshan Mountains in Guyuan City (Longde and Jingyuan counties) of the Ningxia Autonomous Region, with a total area of 67,800 ha, and is one of the largest forest areas in northwest China (Fig 1). And we obtained the approval of Forest Tree Breeding Center, Liupanshan Forestry Bureau, Guyuan Fig 1 from https://apps.nationalmap.gov/viewer/. Fig 1. Distribution of sampling sites in Liupanshan Mountains of Ningxia, China. https://doi.org/10.1371/journal.pone.0294159.g001 Fig 1. Distribution of sampling sites in Liupanshan Mountains of Ningxia, China. https://doi.org/10.1371/journal.pone.0294159.g001 3 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 2.3 Analysis of soil nutrients at various restoration stages Soil samples were collected from three sample stands at various restoration stages with consis- tent environmental and terrain conditions. In each 10 × 10 quadrate, nine sampling points were selected according to the S-shape sampling method, and the soil from each of the three sampling points was mixed into one soil sample, while soil mixtures with a depth of 0–10 cm were collected with a shovel. Twelve soil samples were collected at three different restoration stages, and the collected soil samples were placed in valve bags and brought back to the labora- tory. Each soil sample was divided into two parts. One part was screened a 0.25 mm sieve and then dried naturally. After grinding, the total organic carbon (TC, g/kg), total nitrogen (TN, g/ kg), total phosphorus (TP, g/kg), available phosphorus (AP, mg/kg), and available potassium (AK, mg/kg) content were measured [44]. Another portion of fresh soil from each sample was screened using a 2 mm sieve and stored at 4˚C for the measurements of soil ammonium nitro- gen (NH4 +-N, mg/kg), nitrate nitrogen (NO3 —N, mg/kg), and available nitrogen (AN, mg/kg) contents [45]. Pianka index: (5) In the formula, Bi is the niche width of i species; Pij = nij/ Ni, nij represents the importance value of i species in j resources, Ni is the sum of the importance values of i species in total resources, Pij is the proportion of the importance value of i species in j resources to that species in total resources, r the number of quadrats, and Oik is the overlap index of species i and k. 2.2 Community investigation and species composition index calculation at various restoration stages We investigated the composition of plant communities in three sample stands and determined soil physicochemical properties and plant functional traits. Four transect lines with the same slope direction were set in each stand, and the spacing of each line was greater than 20 m. A 10 ×10 m quadrate was randomly set on each line transect using a simple random method. Each sample stand has 4 quadrats, for a total of 12 quadrats. All woody plants with a base diameter 1 cm in each quadrate were counted, and their species names, plant height, plant number, and coverage were recorded. The plant species were identified with the method of Cheng et al. (2020) and combined with data from http://ppbc.iplant.cn/ [38]. Species diversity indices, including shannon index [39], evenness index, and richness index [40, 41], were calculated according to the following equations: 4 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits shannon index: (1) evenness index: (2) richness index: (3) where Pi = ni/N, ni represents the number of individuals of a species, N is the total number of species, and S is the number of species. Niche width was calculated using the shannon formula [42] and niche overlap was calcu- lated using the Pianka index [43] (Fig 3). shannon index: (4) PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 3.1 Species composition of Q. wutaishanica community among various restoration stages By surveying all species in three different restoration stages, we found 10 species of arbors, 31 species of shrubs, and the most herbaceous species with 42. Among the three different restora- tion stages, 30 years of restoration contained the most trees with nine species, 10 years of resto- ration was the second with eight species, and 40 years of restoration had the fewest tree species with four. The number of shrubs after 10 years of restoration was the largest, with 27 species, accounting for 87% of the total; 30 years of restoration was the next most abundant with 16 species, accounting for 51% of the total; and 40 years of restoration was the least abundant with 15 species, accounting for 48% of the total (Fig 2A). At the three restoration stages, shannon and evenness indices were the largest at 30 years of restoration, which were significantly different from those at 40 years of restoration (p < 0.05). The richness index was the largest at 10 years of restoration and significantly higher than that at 40 years (p < 0.05) (Fig 2B). NMDS analysis showed that plant communities were similar after 10, 30, and 40 years of restoration (Fig 2C). At 10 years of restoration, Q. wutaishanica had a higher niche overlap with Spiraea Pubes- cens, Populus Davidiana and Syringa Pubescens (Fig 3A). At 30 years of restoration stage, Q. wutaishanica had a higher niche overlap with Corylus mandshurica, Rosa davurica and Spiraea pubescens (Fig 3B). At 40 years of restoration stage, Q. wutaishanica had a higher niche overlap with Populus davidiana, L. ferdinandii and Berberis brachypoda (Fig 3C). 2.5 Statistical analysis Soil nutrients and plant functional traits were analyzed by one-way analysis of variance (ANOVA) using SPSS (25.0), and multiple comparisons were performed for all data using the least significant difference method (LSD). All data are presented as mean ± SE (n = 12). The "Vegan" package was used in R software for non-metric multidimensional scaling (NMDS) analysis [49]. “Spaa” package in R software (Version 4.1.0) was used to calculate niche overlap based on levins method, and the corrplot package was used for visualization. The functional richness index (FRic), functional evenness index (FEve), functional divergence index (FDiv), functional dispersion index (FDis) were calculated by “FD” package. The correlations between soil physicochemical properties and plant functional traits and between plant functional traits were calculated using the "psych" package. Redundancy analysis (RDA) was used to establish relationships among species composition, soil physicochemical properties, and plant func- tional traits at various restoration stages [50]. 2.4 Analysis of plant functional traits of Q. wutaishanica We collected the upper canopy and sunny, pest-free, and fully unfolded leaves of Q. wutaisha- nica plants from each stand in September 2019. All leaves were placed between two pieces of wet filter paper, placed in a valve bag, and brought to the laboratory for measurement [46]. The leaves were divided into two parts on average. One part was used to measure plant func- tional traits: leaf length-width ratio (LWR), leaf thickness (LT, mm), leaf dry matter content (LDMC, g), specific leaf area (SLA, cm2/g), and leaf chlorophyll content (LCC, mg/g). Leaf length was measured as the distance from the petiole-blade junction to the blade tip, and leaf width was measured at the widest part of the blade. A digimatic micrometer was used to mea- sure three positions (front, middle, and back) of the blade close to the main veins, and LT was the average value obtained from the three measurements. Furthermore, leaves were soaked in a petri dish filled with water under 5˚C dark conditions for 12 h, the water on the surface was quickly wiped off with filter paper, and the saturated fresh weight of the leaves was weighed immediately. The leaves were then dried in an oven at 75˚C to a constant weight and their dry weights were measured. LDMC was calculated as leaf dry weight divided by leaf saturated fresh weight. The leaf area was measured using a por. leaf area meter (LI-COR 3000C Area PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 5 / 19 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits Meter, LI-COR, Lincoln, USA), and SLA was calculated as leaf area divided by leaf dry weight [47]. Chlorophyll in fresh leaves was extracted using acetone, and the chlorophyll content was measured using spectrophotometry [48]. The remaining leaf samples were placed in an oven at 80˚C for 30 min and then at 60˚C for 72 h. After complete drying, the samples were crushed and sieved through a 0.25 mm. These leaf samples were used to measure the total carbon (LTC, g/kg), total nitrogen (LTN, g/kg), and total phosphorus (LTP, g/kg), according to previ- ous studies [44]. PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 3.3 Plant functional traits of Q. wutaishanica at various restoration stages Our study indicated that the LTC content of Q. wutaishanica leaves showed little difference between the three restoration stages. LTN content after 30 and 40 years of restoration was higher than that after 10 years, and LTP content decreased with restoration time. The LT index was the highest at 40 years of restoration and was lower at 30 years; LCC was the highest at 30 years of restoration and significantly higher than that at 10 and 40 years of restoration (p < 0.05) (Fig 5A). RDA showed that leaf functional traits and soil physicochemical properties had strong directivity to restoration years, while RDA1 and RDA2 explained 50.9% and 21.37% of the variables, respectively. Among them, LWR, LN:P, LTC, and LC:P were corre- lated with AP, AN, and NO3 —N after 30 years of restoration, whereas LTP, SLA, LCC, and LTN were significantly correlated with soil TC, TP, and AK after 40 years of restoration (Fig 5B). 3.2 Characteristics of soil nutrients at various restoration stages The soil TC content was highest at 30 years of restoration and significantly higher than that at 40 years of restoration (p < 0.05). The NH4 +-N content after 30 years of restoration was higher 6 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits Fig 2. Species composition characteristics (A), the indices of species diversity (B) and non-metric multidimensional scaling analysis (C) of Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. https://doi.org/10.1371/journal.pone.0294159.g002 Fig 2. Species composition characteristics (A), the indices of species diversity (B) and non-metric multidimensional scaling analysis (C) of Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. https://doi.org/10.1371/journal.pone.0294159.g002 https://doi.org/10.1371/journal.pone.0294159.g002 than that after 40 years of restoration. However, the AN and AP contents in soil after 40 years of restoration were significantly higher than those after 10 and 30 years of restoration (p < 0.05) (Fig 4A). Furthermore, the correlation analysis between the community structure characteristic index and physicochemical properties showed that the richness index of the plant community was significantly negatively correlated with AN, while shannon was posi- tively correlated with TC, but negatively correlated with AN. In addition, FDis and RaoQ scores were negatively correlated with AP (p < 0.05) (Fig 4B). 3.4 Relationship between community diversity index and plant functional traits of Q. wutaishanica community Further analysis showed that LTC and LC:P had a negative effect on FDis and RaoQ after 10 years of restoration, while LCC had a positive correlation with evenness (p < 0.05). With the 7 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits Fig 3. Niche overlap of shrubs in Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. A: restoration for 10 years; B: restoration for 30 years; C: restoration for 40 years. The plants in the figure are the top 10 ecological niches. https://doi.org/10.1371/journal.pone.0294159.g003 Fig 3. Niche overlap of shrubs in Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. A: restoration for 10 years; B: restoration for 30 years; C: restoration for 40 years. The plants in the figure are the top 10 ecological niches. https://doi.org/10.1371/journal.pone.0294159.g003 https://doi.org/10.1371/journal.pone.0294159.g003 8 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits Fig 4. Relationship between soil physicochemical properties (A) and community diversity index (B) at various restoration stages in Liupanshan Mountains. TC: total carbon; TN: total nitrogen; TP: total phosphorus; NH4 +-N: ammoniacal nitrogen; NO3 —N: nitrate nitrogen; AN: available nitrogen; AP: available phosphorus; AK: available potassium; FRic: functional richness index; FEve: functional evenness index; FDiv: functional divergence index; FDis: functional dispersion index. (*p < 0.05; **p < 0.01). Fig 4. Relationship between soil physicochemical properties (A) and community diversity index (B) at various restoration stages in Liupanshan Mountains. TC: total carbon; TN: total nitrogen; TP: total phosphorus; NH4 +-N: ammoniacal nitrogen; NO3 —N: nitrate nitrogen; AN: available nitrogen; AP: available phosphorus; AK: available potassium; FRic: functional richness index; FEve: functional evenness index; FDiv: functional divergence index; FDis: functional dispersion index. (*p < 0.05; **p < 0.01). https://doi.org/10.1371/journal.pone.0294159.g004 https://doi.org/10.1371/journal.pone.0294159.g004 restoration of plant communities, LTN, SLA, and LCC were positively correlated with the plant community indices (p < 0.05). At the 40 years of restoration, LTC and LC:N were posi- tively correlated with richness, while LTN and LN:P were negatively correlated with richness (p < 0.05) (Fig 6). PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 4.1 At the later restoration stages, the composition of the species community changes The spearman correlation with plant functional traits and community diversity index of Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. A: restoration for 10 years; B: restoration for 30 years; C: restoration for 40 years. LTC: leaf total carbon; LTN: leaf total nitrogen; LTP: leaf total phosphorus; LC:N: leaf carbon-nitrogen ratio; LC:P: leaf carbon-phosphorus ratio; LN:P: leaf nitrogen-phosphorus ratio; SLA: specific leaf area; LDMC: leaf dry matter content; LT: leaf thickness; LWR: leaf length-width ratio; LCC: leaf chlorophyll content; FRic: functional richness index; FEve: functional evenness index; FDiv: functional divergence index; FDis: functional dispersion index. (*p < 0.05; **p < 0.01). https://doi org/10 1371/journal pone 0294159 g006 https://doi.org/10.1371/journal.pone.0294159.g006 communities changed with prolonged restoration time, which was consistent with the findings of Craven et al. [52]. In general, small-seeded species predominate in the early restoration of young (4–12 years) and middle (16–20 years) forests. However, in old-growth forests, there are some plants related to late restoration (such as large-seeded species and trees) [53]. After 10 years of restoration, the Q. wutaishanica community had more total species, which was similar to the result that plant species accumulated rapidly during the initial stage of restoration (Fig 2). Although environmental factors (sunlight, evaporation, and soil nutrients) have a significant impact on the distribution of plant species [54], ephemeral plants often dominate the commu- nity in the early restoration stage [55]. Abbas et al. also found that in the restoration of tropical forest plant communities, the accumulation of plant species mainly occurs in the early stage of restoration (15–20 years) [56]. With restoration, the number of shrubs in the middle of the res- toration stage (30 y of restoration) decreased. This is similar to previous results showing that with the prolonging of the restoration period, the competition among species in the community intensified, leading to the decline of species [57, 58]. It is more likely that the community vegeta- tion was dominated by sunny species in the early stage of restoration, and the species composi- tion was differentiated in the middle stage due to random environmental filtering and dispersal restrictions of species colonization and recruitment [56]. Especially in forest community resto- ration, pioneer species dominate the secondary forest community structure [59]. communities changed with prolonged restoration time, which was consistent with the findings of Craven et al. [52]. 4.1 At the later restoration stages, the composition of the species community changes Q. wutaishanica plays an important role in forest restoration in the Loess region of northern China [51]. Our study highlighted that the species diversity dominated by Q. wutaishanica Fig 5. Plant functional traits (A) and RDA analysis (B) of Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. LTC: leaf total carbon; LTN: leaf total nitrogen; LTP: leaf total phosphorus; LC:N: leaf carbon-nitrogen ratio; LC:P: leaf carbon-phosphorus ratio; LN:P: leaf nitrogen- phosphorus ratio; SLA: specific leaf area; LDMC: leaf dry matter content; LT: leaf thickness; LWR: leaf length-width ratio; LCC: leaf chlorophyll content. https://doi.org/10.1371/journal.pone.0294159.g005 Fig 5. Plant functional traits (A) and RDA analysis (B) of Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. LTC: leaf total carbon; LTN: leaf total nitrogen; LTP: leaf total phosphorus; LC:N: leaf carbon-nitrogen ratio; LC:P: leaf carbon-phosphorus ratio; LN:P: leaf nitrogen- phosphorus ratio; SLA: specific leaf area; LDMC: leaf dry matter content; LT: leaf thickness; LWR: leaf length-width ratio; LCC: leaf chlorophyll content. https://doi.org/10.1371/journal.pone.0294159.g005 https://doi.org/10.1371/journal.pone.0294159.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 9 / 19 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits Fig 6. The spearman correlation with plant functional traits and community diversity index of Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. A: restoration for 10 years; B: restoration for 30 years; C: restoration for 40 years. LTC: leaf total carbon; LTN: leaf total nitrogen; LTP: leaf total phosphorus; LC:N: leaf carbon-nitrogen ratio; LC:P: leaf carbon-phosphorus ratio; LN:P: leaf nitrogen-phosphorus ratio; SLA: specific leaf area; LDMC: leaf dry matter content; LT: leaf thickness; LWR: leaf length-width ratio; LCC: leaf chlorophyll content; FRic: functional richness index; FEve: functional evenness index; FDiv: functional divergence index; FDis: functional dispersion index. (*p < 0.05; **p < 0.01). https://doi.org/10.1371/journal.pone.0294159.g006 Fig 6. The spearman correlation with plant functional traits and community diversity index of Quercus wutaishanica shrub at various restoration stages in Liupanshan Mountains. A: restoration for 10 years; B: restoration for 30 years; C: restoration for 40 years. LTC: leaf total carbon; LTN: leaf total nitrogen; LTP: leaf total phosphorus; LC:N: leaf carbon-nitrogen ratio; LC:P: leaf carbon-phosphorus ratio; LN:P: leaf nitrogen-phosphorus ratio; SLA: specific leaf area; LDMC: leaf dry matter content; LT: leaf thickness; LWR: leaf length-width ratio; LCC: leaf chlorophyll content; FRic: functional richness index; FEve: functional evenness index; FDiv: functional divergence index; FDis: functional dispersion index. (*p < 0.05; **p < 0.01). https://doi.org/10.1371/journal.pone.0294159.g006 Fig 6. PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 4.1 At the later restoration stages, the composition of the species community changes In general, small-seeded species predominate in the early restoration of young (4–12 years) and middle (16–20 years) forests. However, in old-growth forests, there are some plants related to late restoration (such as large-seeded species and trees) [53]. After 10 years of restoration, the Q. wutaishanica community had more total species, which was similar to the result that plant species accumulated rapidly during the initial stage of restoration (Fig 2). Although environmental factors (sunlight, evaporation, and soil nutrients) have a significant impact on the distribution of plant species [54], ephemeral plants often dominate the commu- nity in the early restoration stage [55]. Abbas et al. also found that in the restoration of tropical forest plant communities, the accumulation of plant species mainly occurs in the early stage of restoration (15–20 years) [56]. With restoration, the number of shrubs in the middle of the res- toration stage (30 y of restoration) decreased. This is similar to previous results showing that with the prolonging of the restoration period, the competition among species in the community intensified, leading to the decline of species [57, 58]. It is more likely that the community vegeta- tion was dominated by sunny species in the early stage of restoration, and the species composi- tion was differentiated in the middle stage due to random environmental filtering and dispersal restrictions of species colonization and recruitment [56]. Especially in forest community resto- ration, pioneer species dominate the secondary forest community structure [59]. PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 10 / 19 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits An important question in forest restoration is whether the diversity of plant species has improved [58]. The species diversity of the Q. wutaishanica community increased significantly after 30 years of restoration compared to that after 10 years of restoration. It is well known that the species richness of immature secondary forests is higher than that of young secondary for- est [60, 61]. Species heterogeneity and diversity changed, probably because of the stronger response of new community species to soil nutrients and environmental changes at the early stages of restoration, as well as the limitation of seed dispersal [57, 62]. Lasky et al. found that species diversity in tropical forests decreased with increasing restoration period (from 10 to 40 years) [63]. 4.1 At the later restoration stages, the composition of the species community changes Similarly, it was found that after a long period of forest restoration (50 years), spe- cies diversity decreased significantly [64, 65]. Our study found that the species diversity of the Q. wutaishanica community decreased after 40 years of restoration compared to that after 30 years of restoration. This may be because of the existence of pioneer species in the early resto- ration of the Q. wutaishanica community, which recruited a large number of species, and the lack of abiotic or biotic filtration, resulting in an increase in species diversity. With restoration, the species colonization rate was higher and the mortality rate was lower in the understory, while some dominant species and herbs eventually dominated in the understory, thus chang- ing species diversity [66]. In the present study, plant species diversity first increased and then decreased (Fig 2). This unimodal curve was probably due to communities constructed by ran- dom dispersal and species colonization during early restoration. In view of the closer relation- ship between species, the environment was suitable for their expansion, which maximized species diversity in the middle stage of restoration. However, at the later stages of restoration, related species were gradually eliminated due to environmental filtration, which reduced the species diversity in the community [56, 67]. Our observations indicated that the plant species richness of the Q. wutaishanica commu- nity decreased gradually from 10 to 40 years after restoration (Fig 2B). Studies have shown that species richness gradually stabilizes with community restoration. The former was gradually shaded by the crown canopy, whereas the latter gradually occupied the crown canopy with res- toration [61], thus playing the role of filtering restrictions and reducing species richness [68]. In addition, a higher soil resource supply and light availability at the initial restoration stage of the plant community increased plant richness. However, as plant community restoration pro- ceeds, the availability of soil resources decreases, reducing consumer competition for light and altering species richness [69]. Furthermore, competition among plant consumers, which increases intraspecific community height, also decreases interspecific SLA [69, 70]. Plant com- munity responses to restoration drivers depend on the underlying characteristics of resident species that influence community diversity during restoration [69]. In the restoration of plant communities, the larger the niche width of the species, the higher the resource utilization efficiency of the species, and the stronger the adaptive capac- ity to the environment [71]. PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 4.2 At the later restoration stages, the limitation of soil nutrients decreased Forest ecosystem restoration is determined by the soil environment, especially soil nutrient availability. However, plant growth rate and biomass accumulation respond rapidly to soil nutrients [73]. It is believed that species in the early stage of restoration compete with other species by increasing the utilization efficiency of light resources through nutrient accumula- tion [74]. The reason why TC and NH4 +-N in our study were the largest after 30 years of resto- ration was also confirmed, while the competitive growth of species increased the utilization of NH4 +-N by plants and carbon input in soil (such as litter) [75]. With the restoration of the Q. wutaishanica community, the dominant species had a niche advantage and the demand for AN content further increased (Fig 4). The competing species died successively, which changed the carbon input to the soil [76]. This may explain the strong correlation between TC and community diversity observed in this study. The change in soil AN content was opposite to that of TC and TP contents in this study and was the lowest after 30 years of restoration. On one hand, species diversity increased the AN utilization by plants, resulting in nitrogen limitation of soil nutrients [77, 78]; on the other hand, leguminous plants (Medicago lupulina and Lespedeza bicolor) appeared in 10 and 40-year restoration communities, which increased the fixation of available nitrogen in soil [79]. Another study pointed out that soil ammonium and nitrate nitrogen play key roles in community β-diversity composition [80]. In our study, the carbon nitrogen ratio and NH4 + were higher in the community after 30 years of restoration than those after 40 years of restora- tion, indicating that the ability of soil nutrient mineralization and decomposition gradually decreased during community restoration, and similar results were recorded by Bauhus and Pare [81]. 4.1 At the later restoration stages, the composition of the species community changes Our results indicated that the niche width of Q. wutaishanica was the largest among the three restoration stages, indicating that Q. wutaishanica was dominant in each stage. The niche width of community restoration dominated by Q. wutaishanica was controlled by environmental selection and random processes; however, random processes may play a large role in the early stage of restoration, while environmen- tal selection plays a dominant role in the middle and late stage [67, 72]. In addition, niche overlap reflects the similarity in resource utilization among populations. We found that spe- cies niche overlap significantly decreased after 40 years of restoration, which was similar to the results of previous studies that symbiotic relationships among plants gradually weak- ened over time, and competition among species decreased during community restoration at a regional scale [71, 72]. 11 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits 4.3 Species composition and leaf functional traits of Q. wutaishanica communities differ in different restoration stages Plant functional traits are indicators of plant resource utilization strategies and are closely related to various restoration stages of the community. The analysis of plant functional traits can deepen our understanding of community restoration processes and community structure [82–85]. It was observed that the strategy of resource utilization in plants was changed from acquisitive type to conservative type during restoration [86]. Here, we found that the SLA and LCC indices initially increased and then decreased with restoration. In plant restoration, owing to differences in soil nutrient allocation, the strategy was changed to a conservative strategy, which was consistent with the findings of Pinho et al. [73]. The SLA index increased with the accrue of crown canopy and nutrient utilization, and the SLA index of dominant spe- cies decreased with the complexity of the community structure during restoration [87]. Our research also confirmed that the SLA of Q. wutaishanica showed similar changes in commu- nity diversity and soil NH4 +-N. Moreover, SLA is affected by both LT and LDMC indices [88]. In our study, the LT and LDMC indices decreased at the beginning and then increased with restoration time, and the change in LT was greater. As the LT index was positively correlated with the utilization efficiency of light resources [89], Q. wutaishanica gradually occupied the canopy position as the dominant species in community restoration, which improved the utili- zation efficiency of light resources by the leaves [90]. The changes in plant functional traits of the dominant species are driven by carbon and other nutrient cycles [91]. The higher the leaf nitrogen and phosphorus contents, the greater the photosynthetic rate, while the growth rate and resource competitiveness increased accord- ingly [92]. We found that leaf TN content gradually increased and tended to be stable with the prolonging of the restoration period, which further supports the conclusion that TN content PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 12 / 19 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits increases with increasing stand age [93]. The leaf TP content decreased gradually with restora- tion, which was caused by various nutrient utilization strategies of different plant types and explained the functional redundancy of soil AP at different restoration stages [94]. Plant-soil interactions are an important determinant of vegetation restoration [95, 96]. 4.3 Species composition and leaf functional traits of Q. wutaishanica communities differ in different restoration stages We found a strong correlation between soil TC, FDis, and RaoQ after 10 years of restoration, and the limiting effect gradually decreased, indicating that the resource utilization strategy of Q. wutaishanica changed during community restoration. It has been reported that the reduction in the utilization efficiency of light resources by plants leads to the weakening of the nutrient cycle restriction [73]. They also showed that stand age is an important driver of soil carbon input [28]. At the early stage of restoration, plants have a strong demand for nutrients, but less input of leaf litter makes it difficult to achieve effective carbon sequestration [91]. On the other hand, with community restoration, soil carbon input increased, which increased soil func- tional redundancy and improved geobiochemical circulation [97]. In addition, the effect of higher species richness in the initial restoration stages of plant communities on plant seed quality was also influenced by the supply of soil resources. As restoration proceeds, competi- tion among plant consumers increases intraspecific community height and decreases interspe- cific SLA [69, 70]. Our study further confirmed that plant leaf nitrogen content had an important effect on species richness from the late stage of community restoration, which was consistent with previous findings that also reported that changes in plant functional traits affected species richness [98]. 5 Conclusion Q. wutaishanica is the dominant tree species in natural ecosystem restoration of temperate for- ests in China, which plays an active role in maintaining ecological balance. However, little is known about how ecosystem versatility develops during the restoration of forest ecosystems dominated by Q. wutaishanica. In this study, we investigated the species composition of the Q. wutaishanica community, soil nutrients, and their functional traits at various restoration stages, and comprehensively analyzed the correlations among them. At the early stage of resto- ration (10 years of restoration), there were more shrubs in Q. wutaishanica community, while had a larger niche width. In the middle of restoration (30 years of restoration), shannon and richness indexes were the largest, while soil total carbon, total phosphorus, ammonium nitro- gen and leaf shape index, chlorophyll content of Q. wutaishanica leaves were the highest. Moreover, the correlation between leaf and soil nutrients was the strongest. At the later stage of restoration (40 years of restoration), soil available nitrogen, available phosphorus content and leaf thickness were the largest. However, the restriction of various soil nutrients was reduced. Our study highlighted the effectiveness of soil resource availability in plant communi- ties during restoration, reduced competition for light among plants, and altered species rich- ness. Furthermore, changes in the interrelationship between plant community composition and leaf functional traits of the dominant species responded positively to community restora- tion. These results further deepen our understanding of forest management and restoration of forest communities. In the future, it is necessary to comprehensively consider the influence of various factors on forest community restoration. Supporting information S1 Data. (XLSX) 13 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0294159 November 17, 2023 PLOS ONE Ccommunity restoration, Quercus wutaishanica, species composition, soil nutrients, plant functional traits Conceptualization: Peng Kang. Conceptualization: Peng Kang. Data curation: Peng Kang, Jinpeng Hu, Jing Wang. Formal analysis: Jiming Cheng, Jinpeng Hu, Yongshun Jing. Investigation: Jiming Cheng, Hui Yang. Methodology: Peng Kang, Jing Wang. Project administration: Xingfu Yan. Writing – original draft: Peng Kang. Writing – review & editing: Xiaodong Ding. Conceptualization: Peng Kang. Data curation: Peng Kang, Jinpeng Hu, Jing Wang. Formal analysis: Jiming Cheng, Jinpeng Hu, Yongshun Jing. Investigation: Jiming Cheng, Hui Yang. Methodology: Peng Kang, Jing Wang. Project administration: Xingfu Yan. Writing – original draft: Peng Kang. Writing – review & editing: Xiaodong Ding. Project administration: Xingfu Yan. Writing – original draft: Peng Kang. Writing – review & editing: Xiaodong Ding. Acknowledgments We thank Prof. Jinlin Zhang for its linguistic assistance during the preparation of this manuscript. 12. Hipp A.L.; Larkin D.J.; Barak R.S.; Bowles M.L.; Cadotte M.W.; Jacobi S.K.; et al. Phylogeny in the ser- vice of ecological restoration. 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Does functional trait diversity predict above-ground biomass and productivity of tropical forests? Testing three alternative hypotheses. J Ecol. 2014, 103, 191–201. https://doi.org/10.1111/1365-2745. 12346 References Does functional trait diversity predict above-ground biomass and productivity of tropical forests? Testing three alternative hypotheses. J Ecol. 2014, 103, 191–201. https://doi.org/10.1111/1365-2745. 12346 19 / 19
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Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study
BMC geriatrics
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Download date: 24. Oct. 2024 Citation for pulished version (APA): Elkjær, M., Wolff, D. L., Primdahl, J., Backer Mogensen, C., Brabrand, M., & Gram, B. (2021). Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study. BMC Geriatrics, 21, Article 696. https://doi.org/10.1186/s12877-021-02644-6 Open Access Abstract Background:  Older adults admitted to an emergency department (ED) who are dependent on homecare may be especially challenged with respect to readmission and mortality. This study aimed to assess whether receiving homecare prior admission was associated with readmission or mortality within 30 days of a short ED admission and to explore whether the amount of homecare received was associated with an increased risk of readmission or mortality. Methods:  This nationwide register-based cohort study included patients aged 65 or above who were admitted to an ED at any Danish hospital from 1 December 2016 to 30 November 2017 and discharged within 48 h. Data were extracted from national registers through Statistics Denmark. Homecare was categorized into groups; patients without homecare and three groups according to the amount of homecare received per week. Logistic regression analyses were used to explore the association between the four homecare groups and outcomes, readmissions and mortality. Results:  In total, 80,517 patients (51% female, median age 75 years) were included in the study. Overall, 64,886 patients without homecare, 15,631 (19%) patients received homecare (64% female, median age 83 years), of which 4938 patients received homecare ≤30 min, 4033 received > 30 min to ≤120 min and 6660 received > 120 min per week. The risk of readmission and mortality increased concurrently with the minutes of homecare received: Patients receiving homecare > 120 min per week had the highest odds ratios (ORs) for readmission within 30 days (OR 1.8 95% CI: 1.7–1.9) and mortality within 30 days (OR 4.5 95% CI: 4.1–4.9) compared with patients without homecare. Conclusion:  Receiving homecare was associated with an increased risk of readmission and death following a short ED admission. Collaboration between the ED and primary health care sector in relation to rehabilitation and end- of-life care is essential to improve quality of care for older adults who receive homecare, particularly those receiving homecare > 2 h a week, because of their increased risk of readmission and mortality. Keywords:  Primary health care, Older adults, Death, Acute illness, Acute hospitalisation, Multimorbidity, Comorbidities, Emergency medicine Mette Elkjær1,2,3*  , Donna Lykke Wolff2,4, Jette Primdahl2,5,6, Christian Backer Mogensen1,2, Mikkel Brabrand2,7,8 and Bibi Gram2,3 Mette Elkjær1,2,3*  , Donna Lykke Wolff2,4, Jette Primdahl2,5,6, Christian Backer Mogensen1,2, Mikkel Brabrand2,7,8 and Bibi Gram2,3 Mette Elkjær1,2,3*  , Donna Lykke Wolff2,4, Jette Primdahl2,5,6, Christian Backer Mogensen1,2, Mikkel Brabrand2,7,8 and Bibi Gram2,3 Mette Elkjær1,2,3*  , Donna Lykke Wolff2,4, Jette Primdahl2,5,6, Christian Backer Mogensen1,2, Mikkel Brabrand2,7,8 and Bibi Gram2,3 Background *Correspondence: mette.elkjaer@rsyd.dk 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark Full list of author information is available at the end of the article *Correspondence: mette.elkjaer@rsyd.dk 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark Full list of author information is available at the end of the article *Correspondence: mette.elkjaer@rsyd.dk 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark Full list of author information is available at the end of the article Admission at emergency department (ED) is especially challenging for older adults aged 65 or above owing to a high prevalence of multimorbidity [1–3]. One-fifth of University of Southern Denmark Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study Elkjær, Mette; Wolff, Donna Lykke; Primdahl, Jette; Backer Mogensen, Christian; Brabrand, Mikkel; Gram, Bibi Citation for pulished version (APA): Elkjær, M., Wolff, D. L., Primdahl, J., Backer Mogensen, C., Brabrand, M., & Gram, B. (2021). Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study. BMC Geriatrics, 21, Article 696. https://doi.org/10.1186/s12877-021-02644-6 Citation for pulished version (APA): Elkjær, M., Wolff, D. L., Primdahl, J., Backer Mogensen, C., Brabrand, M., & Gram, B. (2021). Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study. BMC Geriatrics, 21, Article 696. https://doi.org/10.1186/s12877-021-02644-6 Citation for pulished version (APA): Elkjær, M., Wolff, D. L., Primdahl, J., Backer Mogensen, C., Brabrand, M., & Gram, B. (2021). Older adults who receive homecare are at increased risk of readmission and mortality following a short ED admission: a nationally register-based cohort study. BMC Geriatrics, 21, Article 696. https://doi.org/10.1186/s12877-021-02644-6 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: g y y y Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only • You may download this work for personal use only. Y t f th di t ib t th t i l it f y y p g • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk Elkjær et al. BMC Geriatrics (2021) 21:696 https://doi.org/10.1186/s12877-021-02644-6 © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 2 of 9 Page 2 of 9 all acutely admitted older adults are readmitted within 30 days, leading to a significant financial burden for society [4]. Moreover, an acute admission is associated with a critical increase in risk of mortality [5]. Among acutely admitted older adults aged 65 or above who are discharged, 20% will die within 1 year of discharge [5]. Although considerable research has been conducted on older adults who are acutely admitted, there is a lack of agreement on how to identify patients at risk of readmis- sion and mortality. receive practical help, the majority of patients receiving > 30 to ≤120 min per week receive some assistance with personal care in addition to practical help and the major- ity of the patients receiving > 120 min of homecare are dependent on daily assistance for ADL. Thus, we hypoth- esise that the amount of help received reflects the degree of dependency on external help. Examination of whether older adults receive homecare at the time of admission to the ED may be a simple way to identify patients at high risk of readmission or mortality. Thus, this study aimed to assess whether receiving home- care was associated with readmission or mortality within 30 days in older adults, after a short ED admission, and to explore whether the amount of homecare received was related to an increased risk of readmission or mortality. A 2015 study from Belgium reported that older adult patients 75 years or above who were discharged from an ED had a higher readmission rate compared with other hospitalised patients [3]. ED care involves high activ- ity, and the accelerated treatment processes and rapid discharges might affect the readmission rate. A Danish study concluded that 49% of readmissions were prevent- able among older adults 75 years or above and these were more likely to occur soon after hospital discharge [6]. In addition, a study from the Netherlands found that (adults median aged 71) the most common reason for prevent- able readmission was a lack of coordination within the health care system [7]. Identifying patients at risk for readmission would allow health professionals to apply target interventions to prevent readmissions among the patients with the greatest needs. Study design and setting A retrospective register-based cohort study was con- ducted. National data on acutely admitted patients were extracted from Danish registers to construct a database on readmission rates, mortality and associated variables. The Danish health care system is tax funded for all 5.8 million residents, and Danish patients do not bear any additional financial costs for hospital care and treatment or for homecare.h The Danish EDs are organised with hospital beds for patients in need of a short admission and where the discharge is planned directly from the ED. A short ED admission is nationally defined as an admission less than 48 h. Acutely admitted older adults are often extraordinar- ily fragile with multiple comorbidities, functional limita- tions and cognitive impairments [1, 2, 8]. Municipalities offer cost-free homecare to residents in Denmark for those who find activities of daily living (ADL) chal- lenging because of functional limitations or cognitive impairments [9]. The probability of receiving homecare increases with age, and two-thirds of those who receive homecare are aged 65 or above [10]. In 2015, around 12% of older adults aged 65 or above received homecare in Denmark [9]. Homecare to support ADL consists of “personal care”, which includes assistance with personal hygiene, dress- ing, morning routines and mealtimes, and “practical help”, which includes assistance with cleaning, laundry and shopping. The type and amount of homecare allo- cated to a resident is based on an individual assess- ment, with the specific aim of restoring, maintaining and improving mental and physical functionality [9]. Indi- vidual needs are continually adjusted and are planned by a specialist. Data on the type (personal/practical) and amount of homecare are shared electronically between the municipality of residence and local and regional hos- pitals at admission and discharge. Around 35% of medical patients in a Danish ED aged 65 or above, receive homecare [11]. A population-based cohort study from Denmark included 3775 medical patients from a single municipality and reported that receiving homecare before an acute admission was a strong predictor of subsequent readmission and mortal- ity [11]. However, it is not known whether these results are transferable to an ED population and whether the risk of readmission and mortality is associated with the amount of homecare received. Exposure variable Th d l The study population was categorised into one of the fol- lowing four groups according to the proportion of home- care received: 1) patients without homecare, 2) patients receiving homecare ≤30 min per week, 3) patients receiv- ing homecare between > 30 to ≤120 min per week and 4) patients receiving homecare > 120 min per week.i The Danish Registration of Elderly contains informa- tion about services supplied by the municipalities to resi- dents. Information about health care use and homecare services among acutely admitted older adult patients was extracted [15].h Homecare is defined as personal and practical help received and recorded in minutes per week. To assess the total time of homecare, we merged time allocated to practical help and time allocated to personal help. The homecare variable was assessed a month before the index admission and was calculated as a mean (minutes) of homecare received per week throughout the entire month. Patients receiving homecare for less than 1 min per week were categorised as patients without homecare.h The Danish National Prescription Registry (NPR) con- tains individual-level data on prescriptions dispensed from pharmacies [16]. Information about the prescrip- tion of medication for mental health Anatomical Thera- peutic Chemical (ATC) codes (N05–N07) was extracted and used to describe mental comorbidity.h The Income Statistics Register provides data on the income composition of the entire Danish population. The patients’ disposable income was extracted from the Income Statistics Register [17]. The Danish Registration of Elderly receives monthly data from the municipalities. Unfortunately, data from some municipalities are inadequate and there may be variations over the year that are not recorded. If the municipalities do not approve the data as valid, Statis- tics Denmark uses the data delivered the month previ- ously. Statistics Denmark provides information on those months for which data might not be valid. Using this information, we performed a sensitivity analysis (see Appendix B). Data sources Data were extracted from the following registries in the Statistics Denmark [12].h Data were extracted from the following registries in the Statistics Denmark [12].h We hypothesise that the risk of readmission and mor- tality among older adults receiving homecare is associ- ated with their ADL difficulties, which is reflected in the amount (in time) of homecare they receive per week. We categorise homecare based on the assumption that the majority of patients receiving ≤30 min per week only The Danish Civil Registration System (CRS) contains individual-level information on all persons residing in Denmark [13]. The unique 10-digit identification num- ber assigned to all Danish citizens in the CRS at birth or immigration enables linking data from different data Elkjær et al. BMC Geriatrics (2021) 21:696 Page 3 of 9 sources. We extracted information about marital status, age, sex and the date of death among the study popula- tion from this database. as the primary outcome as we do not consider them to be outpatient appointments. Secondary outcomes were readmission within 7 days and mortality within 30, 180 and 360 days. Primary and secondary outcome variables were assessed from the day of discharge from the index admission. The Danish National Patient Registry (DNPR) pos- sesses longitudinal data on inpatient and outpatient con- tacts, as well as discharge diagnosis codes according to the International Classification of Diseases, Revision 10 (ICD10) [14]. The DNPR was used to define the study population and obtain information about readmission and comorbidity. Study population From 1 December 2016 to 30 November 2017, patients aged 65 years or above with an admission to any Dan- ish ED were included in the study population. The study population was extracted from the DNPR. In the DNPR, patients are registered as inpatients or outpatients (patient type) and as admission type, acute or planned. As a proxy for ED admissions, we included all acute inpa- tients (admissions) with a length of stay below 48 h in the study population. Covariables Explanatory variables were selected based on their pos- sible influence on readmission and mortality. We used information about the patients’ age, sex and marital sta- tus (unmarried/married) recorded on the date of their index admission. Disposable income was included as a socio-economic factor. Disposable income is defined as the amount of money a person has available to spend and save after accounting for income tax. We calculated the median yearly disposable income over the past 5 years up to the index admission. The updated version of the Charlson Comorbidity Index (CCI) [18] was used to calculate and describe the number of patients’ chronic diseases using ICD-10 diagnoses and included both A (primary) and B (secondary) diagnoses. All registered diagnoses from 5 years before the index admission were included. Since the CCI contains little to no information about mental disorders, we used information on medi- cal prescriptions for mental disorders at any time within Since each medical specialty, department or hospital contact is coded as an individual contact in the DNPR, contacts of less than 3 h apart were merged into a single contact. An additional file shows this in more detail (see Additional file 1). We excluded patients registered as an acute outpatient visit only, patients living in residential care and patients without Danish residency. The patient’s first contact at the hospital in the study period was regis- tered as the index admission. The follow-up periods for the study population were 30 days regarding readmission and 360 days regarding mortality. Statistical methods Descriptive statistics were used to report demographic and clinical characteristics for the following categories: all patients, patients without homecare and patients with homecare. Categorical data were reported as frequen- cies and continuous variables as median and interquartile range (IQR). The distribution of data was assessed with Q-Q plots. Differences between groups were tested with the Wilcoxon rank sum test or Pearson’s chi-square test, depending on the distribution of the data. The associations between received homecare and out- comes (readmission rate on day 7 and 30 and mortal- ity on day 30, 180 and 360) were tested using logistic regression analyses and presented as crude ORs with 95% confidence intervals (CIs). Furthermore, the regres- sion analyses were preformed adjusting for age, Charlson Comorbidity Index and income (as continuous variables) and sex, marital status and treatment for mental disor- der (as categorical variables) and presented as adjusted ORs with 95% CIs. Finally, a margins plot calculating the probability for mortality and readmission within the homecare categories from the adjusted logistic regres- sion analysis was created. Patients who died before read- mission were excluded from the analyses calculating the risk of readmission. Further, we repeated all the analy- ses excluding the months with inadequate data from the municipalities in sensitivity analyses. All analyses were performed using Stata version 16.1 (StataCorp, College Station, TX, USA). P-values of < 0.05 were considered statistically significant. Fig. 1  Flowchart of study inclusions and exclusions 1 [0–2] vs. 0 [0–1]) compared with patients who did not receive homecare (Table  1). Further, patients receiv- ing homecare experienced a significant increased rate of readmission and mortality after an acute admission (Table 1). Outcome variablesh The primary outcome was readmission, which was defined as an acute contact, occurring within 30 days of the day of previous discharge. We included acute outpa- tient contacts above 6 h equal to inpatients (admissions) Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 4 of 9 the year before the index admission as a proxy for men- tal disorder (yes/no). Different types of medication for mental disorders included antipsychotics, anxiolytics, hypnotics and sedatives, antidepressants, and medica- tion for dementia. Psychostimulants were rarely used (< 0.03%) and were therefore not included. Type of medi- cation was used to describe the cohort and to compare patients receiving homecare with patients not receiving homecare. Type of medication was not included in the analyses. the year before the index admission as a proxy for men- tal disorder (yes/no). Different types of medication for mental disorders included antipsychotics, anxiolytics, hypnotics and sedatives, antidepressants, and medica- tion for dementia. Psychostimulants were rarely used (< 0.03%) and were therefore not included. Type of medi- cation was used to describe the cohort and to compare patients receiving homecare with patients not receiving homecare. Type of medication was not included in the analyses. Fig. 1  Flowchart of study inclusions and exclusions Outcomesh The distribution of the amount of homecare was as fol- lows: 4938 patients (32%) received ≤30 min (~ practical help), 4033 patients (26%) received ≥31 and ≤ 120 min (~ personal care in addition to practical help) and 6660 patients (42%) received ≥121 min per week (~ dependent on daily assistance for ADL). Participants In total, 80,517 patients were included as the study popu- lation (Fig. 1). Among the study population, 5371 patients (7%) were readmitted within 7 days and 10,895 patients (14%) were readmitted within 30 days. Table 2 shows the association between amount of homecare received and risk of read- mission. The risk of readmission within 30 days increased with the amount of time for which patients received homecare; patients receiving homecare for > 120 min Among the study population, 15,631 patients (19%) received homecare. Patients receiving homecare before the index admission were older (median [IQR]: age 83 [76–88] vs. 74 [70–80]) years, fewer were married (24% vs. 62%), the majority were female (64% vs. 48%), a larger proportion received treatment for mental disorders (53% vs. 30%), and they had a higher CCI score (median [IQR]: Elkjær et al. BMC Geriatrics (2021) 21:696 Page 5 of 9 Table 1  Sociodemographic characteristics of the study population Table 1  Sociodemographic characteristics of the study population Note: IQR is interquartile range and n is numbers of patients. The difference between groups was tested with the Wilcoxon rank sum test (unequal distribution) or Pearson’s chi-squared test. *Patients can appear in more than one category Variables All patients N = 80,517 Patients with no homecare n = 64,886 Patients receiving homecare n = 15,631 P-value Homecare vs. Participants no homecare Aged (year) Median (IQR) 75 (70–82) 74 (70–80) 83 (76–88) <  0.001 Marital status (married) 43,498 (54%) 39,725 (62%) 3773 (24%) <  0.001 Sex (female) 40,882 (51%) 30,918 (48%) 9964 (64%) <  0.001 In treatment for mental disorders 27,517 (34%) 19,243 (30%) 8274 (53%) <  0.001 In treatment with *   Antipsychotics 1696 (2%) 1078 (2%) 618 (4%) <  0.001   Anxiolytics 4311 (5%) 3125 (5%) 1186 (8%) <  0.001   Hypnotics & sedative 9069 (11%) 6740 (10%) 2329 (15%) <  0.001   Antidepressants 6706 (8%) 4558 (7%) 2148 (14%) <  0.001   Medicine for dementia 1272 (2%) 628 (1%) 644 (4%) <  0.001 Income after taxes (Euro)   median (IQR) 22,162 (17,756–29,300) 22,323 (17,513–30,435) 21,801 (18,710–25,796) <  0.001 Charlson Comorbidity Index   median (IQR) 0 (0–2) 0 (0–1) 1 (0–2) <  0.001   0 48,548 (60%) 41,358 (64%) 7190 (46%)   1 10,066 (12%) 7414 (11%) 2652 (17%)   2 13,562 (17%) 10,418 (16%) 3144 (20%)    > 3 8341 (11%) 5696 (9%) 2645 (17%) Mortality    < 30 days 3010 (4%) 1790 (3%) 1220 (8%) <  0.001    < 180 days 7022 (9%) 4320 (7%) 2702 (17%) <  0.001    < 360 days 10,191 (13%) 6.325 (10%) 3866 (25%) <  0.001 Readmission    < 7 days 5371 (7%) 4094 (6%) 1277 (8%) <  0.001    < 30 days 10,895 (14%) 8127 (13%) 2768 (18%) <  0.001 Table 2  Association between homecare categories and readmission Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with** Variables Patients with no homecare Patients receiving homecare ≤ 30 min/week Patients receiving homecare >  30 to ≤120 min/week Patients receiving homecare >  120 min/week Readmission < 7 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.26 (1.13–1.40)** 1.15 (1.02–1.29)* 1.26 (1.12–1.42)** 1.13 (1.00–1.29) 1.53 (1.40–1.68) ** 1.30 (1.17–1.43) ** Readmission < 30 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.41 (1.30–1.52) ** 1.22 (1.12–1.31) ** 1.46 (1.34–1.59) ** 1.24 (1.13–1.35) ** 1.82 (1.70–1.94) ** 1.43 (1.33–1.54) ** CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity l disorder. Participants Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with** mental disorders, patients receiving homecare > 120 min a week still had significant increased odds for readmis- sion – OR 1.43 (95% CI: 1.33–‍1.54) – compared with patients without homecare (Table 2). The increased risk a week had the highest odds for readmission – OR 1.82 (95% CI: 1.70–1.94) – compared with patients without homecare in the crude analysis (Table 2). When adjusting for age, sex, marital status, income, CCI and treatment for Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 6 of 9 The sensitivity analyses excluding the months with inadequate data from the municipalities revealed no sig- nificance for the results regarding readmission and mor- tality. An additional file shows this results in detail (see Additional file 2). of readmission within 30 days among patients receiving homecare for ≤30 min and between > 30 and ≤ 120 min was similar to that found in the crude analysis. In the study population, 3010 (4%) patients died within 30 days, 7022 (9%) within 180 days and 10,191 (13%) within 360 days. Table 3 shows the association between amount of homecare received and mortality. The crude risk for 30-days mortality increased related to the amount of time for which patients received homecare (Table 3). The patients receiving homecare for > 120 min per week had the highest OR for 30-days mortality – OR 4.51 (95% CI: 4.12–4.93) – when comparing the three homecare categories (Table 3). Discussion Overall, our study showed that older adults aged 65 or above receiving homecare within the month prior to an ED admission were at a significantly higher risk of read- mission and a significantly higher risk of dying compared with older adults not receiving homecare. Although we adjusted for explanatory variables such as comorbidity, sex and age, receiving homecare was a strong predictor of readmission and early death. Older adults who were dependent on daily assistance for ADL (receiving home- care for > 2 h) were at the highest risk among the home- care categories. When adjusting for age, sex, marital status, income, CCI and treatment for mental disorders, patients receiving homecare for > 120 min per week still had an increased OR of 2.25 (95% CI: 2.01–2.50) for 30-days mortality compared with patients without homecare (Table 3). Among our study population, 19% received homecare, which was fewer than found in other studies [1, 11]; how- ever, our study population differs because it consists of both medical and surgical complaints and solely com- prises short ED admissions of < 48 h. Receiving homecare was associated with an increased probability of readmission and mortality and patient’s receiving homecare > 120 min per week had the high- est probability within 30 days from the day of discharge (readmission: 21% (95% CI: 20.1–22.1) and mortality: 11% (95% CI: 10.6–12.1)). For comparison, older adults without homecare had a probability of readmission 13% (95% CI: 12.5–13.1) and of mortality 3% (95% CI: 2.6–2.9) within 30 days. Mortality and homecare We found that receiving homecare was a significant pre- dictor of the risk of dying within 30 days of discharge. Moreover, older adults receiving homecare for more than 2 h a week had a nearly fourfold risk (OR 4.5) and prob- ability (11% vs 3%), compared with patients who did not receive homecare. Similarly, Tanderup et al. reported that Danish medi- cal patients receiving homecare had an increased risk of mortality within 30 days of an acute admission, with a HR of 2.8, compared with patients not receiving homecare [11]. In addition, we found different risks between the homecare groups. Older adults receiving homecare for > 120 min a week had a significantly higher risk of mortal- ity compared with adults receiving homecare for ≤30 min a week (OR 4.5 vs. OR 1.7). Thus, our study contributes Readmission and homecare BMC Geriatrics (2021) 21:696 Page 7 of 9 Fig. 2  Probability for readmission and mortality within 30 days for the categories of homecare. Note: Results show probability (%) with 95% CI for readmission and mortality within 30 days calculated from the adjusted logistic regression analyses Fig. 2  Probability for readmission and mortality within 30 days for the categories of homecare. Note: Re readmission and mortality within 30 days calculated from the adjusted logistic regression analyses Fig. 2  Probability for readmission and mortality within 30 days for the categories of homecare. Note: Results show probability (%) with 95% CI for readmission and mortality within 30 days calculated from the adjusted logistic regression analyses important results to the existing knowledge since older adults dependent on daily assistance for ADL (i.e. receiv- ing homecare for > 2 h) have a markedly increased risk of dying after an ED admission compared to patients with- out homecare. (≥ 75 years) reported no significant association between readmission and homecare [3]. However, the study from Belgium was relatively small, with only 97 patients [3]. Our study clearly demonstrates that older adult Dan- ish residents receiving homecare are at increased risk of readmission following an ED admission. We adjusted for age, comorbidities, income, sex, marital status and treat- ment for mental disorders. However, there might be some overlooked confounding variables not included in the analyses affecting to which extent homecare is an inde- pendent predictor of an increased risk of readmission. A Spanish cohort study identified male sex, comor- bidity, hospital admission and degree of pressure ulcers as significant predictors of mortality among older adults receiving homecare [19]. Male sex, age and comorbid- ity were also identified as independent predictors of one-year mortality in the Danish population of medical patients admitted ≥65 years [11]. We adjusted for age, comorbidities, income, sex, marital status and treatment for mental disorders, and when comparing the crude and the adjusted analyses, the results indicated that some of the selected explanatory variables influenced the results. However, further research is required to elu- cidate the influence of the covariables (e.g. age, sex and comorbidity). Readmission and homecare We found that receiving homecare was a significant predictor of readmission within 30 days of discharge. Older adults receiving homecare > 2 h per week had an increased risk (OR 1.8) corresponding to nearly doubled probability, compared to older adults without homecare (21% vs 13%). When adjusting for age, sex, marital status, income, CCI and treatment for mental disorders, patients receiv- ing homecare for > 120 min per week had a probability for readmission within 30 days of 17% (95% CI: 16.3–18.2) and 5% (95% CI: 4.3–5.3) for mortality compared to a probability of readmission of 13% (95% CI: 12.5–13) and 2% (95% CI: 2.1–2.3) of mortality within 30 days among older adults without homecare (Fig. 2). Tanderup et al. also reported that receiving homecare was a strong predictor of readmission among Danish medical patients (≥ 65 years) compared with medical patients without homecare (HR for competing risk 1.8) [11]. In contrast, a study from Belgium of ED patients Table 3  Association between homecare categories and mortality Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with** Variables Patients with no homecare Patients receiving homecare ≤ 30 min/week Patients receiving homecare >  30 to ≤120 min/week Patients receiving homecare >  120 min/week Mortality < 30 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.72 (1.50–1.99)** 1.08 (0.92–1.26) 2.18 (1.90–2.50)** 1.26 (1.07–1.47)* 4.51 (4.12–4.93)** 2.25 (2.01–2.50)** Mortality < 180 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.71 (1.56–1.88) ** 1.08 (0.97–1.20) 2.23 (2.03–2.45)** 1.31 (1.18–1.46)** 4.48 (4.20–4.77)** 2.28 (2.11–2.46) ** Mortality < 360 days Crude OR (95% CI) Adjusted OR (95% CI) 1 (ref.) 1.81 (1.67–1.96)** 1.14 (1.05–1.25) * 2.34 (2.16–2.54)** 1.39 (1.27–1.52) ** 4.71 (4.45–4.99)** 2.45 (2.28–2.62)** Table 3  Association between homecare categories and mortality Notes: OR is the odds ratio and CI is confidence intervals. The logistic regression analyses were adjusted for age, sex, marital status, income, Charlson Comorbidity Index and treatment for mental disorder. Significance levels with a p-value of < 0.05 are marked with * and significance levels with a p-value of < 0.001 are marked with** Elkjær et al. Acknowledgments g The authors thank Andreas Kristian Pedersen, Hospital Sønderjylland, Univer- sity Hospital of Southern Denmark, for statistical support, and Caroline Mar- garet Moss, Hospital Sønderjylland, University Hospital of Southern Denmark, for providing language help. Further, we thank OPEN (Odense Patient data Explorative Network), Region of Southern Denmark, for assisting with prepar- ing the application for the data and with data management. Our study had some limitations. As described, there is no specific registration of ED patients and we thus had to develop a proxy for ED admissions. This cod- ing may have imposed a risk of misclassification bias. However, this data management method is well known and used in other register studies [22, 23]. We included both primary and secondary diagnoses calculating patients CCI but did not differentiate between acute and chronic conditions. Furthermore, lifestyle factors Abbreviations d ED: Emergency department; ADL: Activities of daily living; DNPR: Danish National Patient Registry; NPR: National Prescription Registry; CRS: Danish Civil Registration System; CCI: Charlsons Comorbidity Index; IQR: Interquartile range; CI: Confidence interval; OR: Odds ratio; HR: Hazard ratio. Clinical implications Knowledge about the risk profile of patients receiving homecare can be used to organise and coordinate ED care and preventative treatment. To be able to identify patients at high risk of readmission and death will allow health professionals to implement supportive interven- tions to ease transfer from the ED to primary health care and prevent unnecessary readmission and early death. Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Page 8 of 9 Early follow-up visits at home after hospital discharge can reduce preventable readmissions among geriatric patients [6]. Combining several rehabilitative interven- tions before discharge (individual planning, geriatric assessment and patient education) with follow-up after discharge may further reduce readmissions [20]. In Denmark, information about whether patients receive homecare or not is easy to access from the electronic communication between the municipality and local hos- pitals, and it is relatively reliable and requires no addi- tional training for use. such as smoking and overweight can be confounding factors, but was not possible to included using registry data. Further, we used data on marital status (married/ unmarried); thus, we could not tell whether the older adults lived with adult children or cohabitants. How- ever, the study population was aged 65 or above, so we assumed that relatively few persons lived together with- out being married according Danish culture. Finally, the Danish municipalities had months where data transfer was inadequate due to change of registrations system; however, we completed sensitivity analyses excluding these months, which confirmed the robustness of our results. A study from Canada found that older adults receiving homecare experienced at least one care transition (moves between care settings such as a home nursing, home and hospital) during the last 30 days before death, and one- fifth experienced two or more hospitalisations [21]. If this knowledge is combined with our results demonstrating a nearly fourfold increased risk of dying within 30 days of discharge among older adults receiving homecare, the ED admission may be considered a time-limited window of opportunity to offer help to fragile patients. Strengths and limitations Our study has important strengths. This is the first study to investigate readmission rates after a short ED admission comparing older adult patients related to the amount of homecare received, in a large national cohort. We used the DNPR to identify our study population and linked the patients between the registries using the unique 10-digit identification number. Registry data have high completeness and validity [12–17], and to supple- ment the CCI, we added data from the Danish NPR to describe the patients’ mental health to obtain a complete picture of the patients’ total comorbidities. Conclusion h Receiving homecare prior admission was associated with an increased risk of readmission and death follow- ing admission to an ED for older adults aged 65 or older. Older adults who received homecare for more than 2 h a week were at the highest risk compared with those who received less or no homecare. Collaboration between the ED and primary health care facility in relation to reha- bilitation and end-of-life care is essential to improve the quality of care for older adults who receive homecare, particularly those receiving homecare for more than 2 h a week, because of their increased risk of readmission and mortality. f We suggest increased attention given to older adults dependent on daily assistance for ADL (receiving home- care for > 2 h). Care interventions could include evalu- ation of their needs and preferences for rehabilitative interventions or end-of-life care. Whether older adults receive homecare or not and the amount of time allo- cated to homecare can also be used to develop a plan for future treatment and care and ensure that collaboration between the ED and primary health care sector focuses on preventative interventions. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12877-​021-​02644-6. Additional file 1. Additional file 2. Funding visits for geriatric patients discharged from hospital. Eur Geriatr Med. 2018;9(5):613–21. g The Novo Nordisk Foundation and Hospital Sønderjylland, University Hospital of Southern Denmark, supported the study financially but were not involved in any aspect of study design, data collection, data analysis or interpretation of the data. 7. Fluitman KS, van Galen LS, Merten H, Rombach SM, Brabrand M, Cooksley T, et al. Exploring the preventable causes of unplanned readmissions using root cause analysis: coordination of care is the weakest link. Eur J Intern Med. 2016;30:18–24. Consent for publication Not applicable. 13. Schmidt M, Pedersen L, Sorensen HT. The Danish civil registration system as a tool in epidemiology. Eur J Epidemiol. 2014;29(8):541–9. 14. Schmidt M, Schmidt SA, Sandegaard JL, Ehrenstein V, Pedersen L, Sorensen HT. The Danish National Patient Registry: a review of content, data quality, and research potential. Clin Epidemiol. 2015;7:449–90. Declarations 10. Wilson DM, Truman CD, Hewitt JA, Els C. Are chronically ill patients high users of homecare services in Canada? Am J Manag Care. 2015;21(10):e552–9. Availability of data and materialsi 8. Gray LC, Peel NM, Costa AP, Burkett E, Dey AB, Jonsson PV, et al. Profiles of older patients in the emergency department: findings from the interRAI multinational emergency department study. Ann Emerg Med. 2013;62(5):467–74. The data that support the findings of this study are available from Statistics Denmark. However, data were used under licence for the current study and are not publicly available. 9. Ministry of Health. Healthcare in Denmark: an overview. Copenhagen: Ministry of Health; 2017. ISBN: 978-87-7601-365-3. Competing interests The authors declare that they have no competing interests. y 15. Danish Registration of Elderly [Web Page]. Statistics Denmark 2020 [cited 2020 2108]. Available from: https://​www.​dst.​dk/​da/​Stati​stik/​dokum​entat​ ion/​Times/​aeldr​edoku​menta​tion. Accessed 29 Mar 2021. References 22. Fløjstrup M, Bogh SB, Henriksen DP, Bech M, Johnsen SP, Brabrand M. Increasing emergency hospital activity in Denmark, 2005–2016: a nation- wide descriptive study. BMJ Open. 2020;10(2):e031409. 1. Buurman BM, Frenkel WJ, Abu-Hanna A, Parlevliet JL, de Rooij SE. Acute and chronic diseases as part of multimorbidity in acutely hospitalized older patients. Eur J Intern Med. 2016;27:68–75. 1. Buurman BM, Frenkel WJ, Abu-Hanna A, Parlevliet JL, de Rooij SE. Acute and chronic diseases as part of multimorbidity in acutely hospitalized older patients. Eur J Intern Med. 2016;27:68–75. y 23. The Danish database for acute and emergency hospital contacts. Clin Epidemiol. 2016;8:469–74. 2. Kennelly SP, Drumm B, Coughlan T, Collins R, O’Neill D, Romero-Ortuno R. Characteristics and outcomes of older persons attending the emergency department: a retrospective cohort study. QJM. 2014;107(12):977–87. Author details 1 1 Department of Emergency Medicine, University hospital of Southern Denmark, Kresten Philipsens Vej 15, 6200 Aabenraa, Denmark. 2 Department of Regional Health Research, University of Southern Denmark, Odense, Denmark. 3 Research Unit of Health Sciences, Hospital of South West Jut- land, Esbjerg, University hospital of Southern Denmark, Esbjerg, Denmark. 4 Department of Internal Medicine, University hospital of Southern Denmark, Aabenraa, Denmark. 5 Sygehus Sønderjylland, University hospital of Southern Denmark, Aabenraa, Denmark. 6 Danish Hospital for Rheumatic Diseases, University hospital of Southern Denmark, Sønderborg, Denmark. 7 Department of Emergency Medicine, University hospital of Southern Denmark, Esbjerg, Denmark. 8 Department of Emergency Medicine, Odense University Hospital, Odense, Denmark. 16. Pottegard A, Schmidt SAJ, Wallach-Kildemoes H, Sorensen HT, Hallas J, Schmidt M. Data resource profile: The Danish National Prescription Regis- try. Int J Epidemiol. 2017;46(3):798–f. y p 17. Baadsgaard M, Quitzau J. Danish registers on personal income and trans- fer payments. Scand J Public Health. 2011;39(7 Suppl):103–5. 18. Quan H, Li B, Couris CM, Fushimi K, Graham P, Hider P, et al. Updating and validating the Charlson comorbidity index and score for risk adjustment in hospital discharge abstracts using data from 6 countries. Am J Epide- miol. 2011;173(6):676–82. 19. Badia JG, Santos AB, Segura JCC, Terén CA, González LC, Ramírez EL, et al. Predictors of mortality among elderly dependent home care patients. BMC Health Serv Res. 2013;13(1):316. Received: 14 April 2021 Accepted: 16 November 2021 Received: 14 April 2021 Accepted: 16 November 2021 20. Åhsberg E. Discharge from hospital – a national survey of transition to out-patient care. Scand J Caring Sci. 2019;33(2):329–35. 21. Abraham S, Menec V. Transitions between care settings at the end of life among older homecare recipients: a population-based study. Gerontol- ogy & geriatric medicine. 2016;2:2333721416684400. Authors’ contributions BG, MB, JP and ME designed the study. BG, MB and JP supervised the research project. DLW and ME performed the preparation of data for analysis. ME ana- lysed the data. BG, MB, JP, CBM, DLW and ME contributed to data interpreta- tion and the manuscript. All authors provided substantial contribution revising the manuscript critically for important intellectual content and approved the version to be published. Page 9 of 9 Elkjær et al. BMC Geriatrics (2021) 21:696 Elkjær et al. BMC Geriatrics (2021) 21:696 Ethics approval and consent to participate The study was reported to the Danish Data Protection Agency (file no. 2008-58-0035). In accordance with Danish law, register studies are exempted from gaining informed consent from participants. The need for formal ethi- cal approval was waived by the Regional Scientific Ethical Committee for the Region of Southern Denmark (file no. 20182000–68) because the study did not involve biomedical interventions. Reporting followed the STROBE guidelines. 11. Tanderup A, Ryg J, Rosholm JU, Lassen AT. Association between the level of municipality healthcare services and outcome among acutely older patients in the emergency department: a Danish population-based cohort study. BMJ Open. 2019;9(4):e026881. 12. Thygesen LC, Daasnes C, Thaulow I, Bronnum-Hansen H. Introduction to Danish (nationwide) registers on health and social issues: structure, access, legislation, and archiving. Scand J Public Health. 2011;39(7 Suppl):12–6. Publisher’s Note 3. Deschodt M, Devriendt E, Sabbe M, Knockaert D, Deboutte P, Boonen S, et al. Characteristics of older adults admitted to the emergency depart- ment (ED) and their risk factors for ED readmission based on compre- hensive geriatric assessment: a prospective cohort study. BMC Geriatr. 2015;15:54. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 4. Klinge M, Aasbrenn M, Öztürk B, Christiansen CF, Suetta C, Pressel E, et al. Readmission of older acutely admitted medical patients after short- term admissions in Denmark: a nationwide cohort study. BMC Geriatr. 2020;20(1):203. 4. Klinge M, Aasbrenn M, Öztürk B, Christiansen CF, Suetta C, Pressel E, et al. Readmission of older acutely admitted medical patients after short- term admissions in Denmark: a nationwide cohort study. BMC Geriatr. 2020;20(1):203. 5. Fløjstrup M, Henriksen DP, Brabrand M. An acute hospital admission greatly increases one year mortality – getting sick and ending up in hos- pital is bad for you: a multicentre retrospective cohort study. Eur J Intern Med. 2017;45:5–7. 5. Fløjstrup M, Henriksen DP, Brabrand M. An acute hospital admission greatly increases one year mortality – getting sick and ending up in hos- pital is bad for you: a multicentre retrospective cohort study. Eur J Intern Med. 2017;45:5–7. 6. Pedersen LH, Brynningsen P, Foss CH, Gregersen M, Barat I, Damsgaard EM. Avoidable readmissions seem to be reduced by early follow-up 6. Pedersen LH, Brynningsen P, Foss CH, Gregersen M, Barat I, Damsgaard EM. Avoidable readmissions seem to be reduced by early follow-up
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Plasma and Cerebrospinal Proteomes From Children With Cerebral Malaria Differ From Those of Children With Other Encephalopathies
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Plasma and Cerebrospinal Proteomes From Children With Cerebral Malaria Differ From Those of Children With Other Encephalopathies Evelyn N. Gitau,1,5 Gilbert O. Kokwaro,2,3,4 Henry Karanja,1 Charles R. J. C. Newton,1,6 and Stephen A. Ward5 1Centre for Geographic Medicine–Coast, KEMRI-Wellcome Trust Research Programme, Kilifi, and 2African Centre for Clinical Trials, 3Consortium for National Health Research, 4Department of Pharmaceutics and Pharmacy Practice, School of Pharmacy, University of Nairobi, Kenyatta National Hospital, Nairobi, Kenya; and 5Liverpool School of Tropical Medicine, and 6Department of Psychiatry, University of Oxford, United Kingdom at Liverpool University Library on Novembe http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ Downloaded from Clinical signs and symptoms of cerebral malaria in children are nonspecific and are seen in other common en- cephalopathies in malaria-endemic areas. This makes accurate diagnosis difficult in resource-poor settings. Novel malaria-specific diagnostic and prognostic methods are needed. We have used 2 proteomic strategies to identify differentially expressed proteins in plasma and cerebrospinal fluid from children with a diagnosis of cerebral malaria, compared with those with a diagnosis of malaria-slide-negative acute bacterial meningitis and other nonspecific encephalopathies. Here we report the presence of differentially expressed proteins in ce- rebral malaria in both plasma and cerebrospinal fluid that could be used to better understand pathogenesis and help develop more-specific diagnostic methods. In particular, we report the expression of 2 spectrin pro- teins that have known Plasmodium falciparum–binding partners involved in the stability of the infected red blood cell, suppressing further invasion and possibly enhancing the red blood cell’s ability to sequester in micro- vasculature. ds. proteomics; P. falciparum; cerebral malaria; spectrin; platelet activation. Keywords. proteomics; P. falciparum; cerebral malaria; spectrin; platelet activation. significant proportion who fulfilled the World Health Organization definition of cerebral malaria had viruses detected in the cerebrospinal fluid (CSF) [3]. Whereas bacterial meningitis can be excluded by the examina- tion and culture of CSF [4], exclusion of other encepha- lopathies remains a significant challenge. This seriously confounds studies on the pathophysiology of cerebral malaria and also delays critical decisions on appropri- ate clinical management. Therefore, we need better ways of identifying children who have or may develop cerebral malaria to facilitate early clinical decisions on management. Identifying characteristics that distinguish different encephalopathies occurring in children in malaria- endemic regions can be difficult, especially in resource- poor settings. Plasmodium falciparum is often assumed to be the main cause, but there are many other causes of encephalopathy, including bacterial meningitis or viral meningitis [1]. M A J O R A R T I C L E M A J O R A R T I C L E Plasma and Cerebrospinal Proteomes From Children With Cerebral Malaria Differ From Those of Children With Other Encephalopathies Frequently, no evidence of an in- fectious agent is found [2], and between 2007 and 2011, 51% of comatose children admitted to KilifiDistrict Hospital on the Kenyan coast had coma with no cause identified. In addition, an earlier study of Kenyan children with acute encephalopathy found that a We have previously demonstrated the measurable presence of differentially expressed proteins in plasma from a mouse model of cerebral malaria, compared with noninfected mice [5]. To determine whether differentially expressed proteins could be identified in body fluids from patients with cerebral malaria, we undertook a similar proteomic study, using CSF and plasma from children with cerebral malaria, and compared the protein profiles in these biological matrices with those in samples from children with confirmed acute bacterial meningitis and other nonspecific encephalopathies. Correspondence: Evelyn N. Gitau, PhD, Centre for Geographic Medicine Research, KEMRI/Wellcome Trust Research Programme, PO Box 230, 80108, Kilifi Kenya (egitau@kemri-wellcome.org). Received 15 January 2013; accepted 9 April 2013; electronically published 25 July 2013. Received 15 January 2013; accepted 9 April 2013; electronically published 25 July 2013. Correspondence: Evelyn N. Gitau, PhD, Centre for Geographic Medicine Research, KEMRI/Wellcome Trust Research Programme, PO Box 230, 80108, Kilifi Kenya (egitau@kemri-wellcome.org). The Journal of Infectious Diseases 2013;208:1494–503 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/infdis/jit334 Subjects The study used archived plasma and CSF samples collected from clinically well-characterized children attending KilifiDis- trict Hospital between 2001 and 2002. The Kenya Medical Re- search Institute Ethics Review Committee approved all studies. The children came from a geographic region described in detail elsewhere [6]. Children were grouped according to the results of a malaria slide, CSF leukocyte count, and microbiological findings [6]. Cerebral malaria (n = 12) was defined according to World Health Organization criteria: (1) a Blantyre coma score of <3 in the presence of peripheral asexual malarial parasites on the blood film, and (2) negative results of CSF or blood cultures and a CSF leukocyte count of ≤10 cells/μL. In addition, we se- lected patients with a parasitemia of >2500 parasites/µL, since this cutoff has the most specificity in this area [6]. Acute bacte- rial meningitis (n = 12) was defined as the absence of asexual malaria parasites in 3 slides of blood specimens obtained over 24 hours; the presence of a CSF leukocyte count of >10 cells/μL or a positive CSF culture result, a positive blood culture result, or detection of bacterial antigen in CSF [1]. Nonspecific en- cephalopathy (n = 12) was defined as impaired consciousness, no detection of asexual-stage parasites in 3 slides of blood spec- imens obtained over 24 hours, and no growth on CSF or blood cultures. Matched CSF and plasma samples and complete medical records were available from each patient. at Liverpool University Library on Novemb http://jid.oxfordjournals.org/ Downloaded from The Journal of Infectious Diseases 2013;208:1494–503 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/infdis/jit334 1494 • JID 2013:208 (1 November) • Gitau et al MATERIALS AND METHODS samples, Coomassie-stained gels were not prepared, and spots were cut directly from silver-stained gels and digested using a modified method previously described by Terry et al [7]. Mass spectra of tryptic digests were obtained using a MALDI-ToF mass spectrometer (MS; Shimadzu CFR Plus, Manchester, United Kingdom) as previously described [5]. When definitive protein identification could not be formally made by MALDI- ToF, the tryptic digests of the spots of interest were further separated by reverse-phase–high-performance liquid chroma- tography performed on an UltiMate 3000 LC system (Dionex, United Kingdom). A total of 1 µL of the concentrated sample was diluted with 4 µL of 2.5% v/v acetonitrile in water contain- ing 0.1% formic acid and injected onto a monolithic capillary column (200-µm internal diameter × 5 cm; Dionex). Peptides were eluted at a flow rate of 1.5 µL/minute, using a solvent gra- dient of solvent A (2.5% v/v acetonitrile in water with 0.1% formic acid) and solvent B (90% v/v acetonitrile in water with 0.1% formic acid), starting at 5% solvent B, linearly ramped to 40% solvent B over 12 minutes, and then to 90% solvent B for a further 2 minutes. Solvent B was then decreased to 5%, and this was maintained to the end of the run at 27 minutes. Resulting ions were eluted into a LCQ Deca XP Plus ion trap MS (Ther- moFinnigan, United States) equipped with a nanospray source connected to a PicoTip column. Further details of the methods used can be found in the Supplementary Materials. Sample Preparation The protein separation on gels was subsequently replaced by a 2-dimensional LC-MS/MS–based approach. An equivalent of 100 µg of CSF protein or 200 µg of plasma protein was injected onto a ProSwift RP-1S monolith column (4.6 × 50 mm, Dionex). The high-performance liquid chromatography was performed on an UltiMate 3000 LC system (Dionex UK). Samples were eluted with Solvent A, 2.5% acetonitrile in water with 0.1% trifluoroacetic acid and solvent B, and 90% acetoni- trile in water with 0.1% trifluoroacetic acid. The flow rate was maintained at 200 µL/minute, and the separated proteins were eluted into a 96-well plate. Forty-eight fractions per sample were collected. The fractions were then dried down overnight in an oven set at 50°C. A total of 25 µL of 100 mM ammonium bicarbonate was added to the sample, followed by 5 µL of a 20-µg/mL solution of trypsin in 25 mM ammonium bicarbon- ate. Samples were thoroughly mixed and incubated overnight at 37°C to achieve complete digestion. The resulting digest was subjected to reverse-phase–high-performance liquid chroma- tography MS as described above. Archived CSF samples that had been stored at −80°C for 5–6 years were thawed at 4°C and desalted using Micorocon YM-3 centrifugal units (Millipore, United States). Archived plasma samples stored at −80°C were also thawed at 4°C. Protein con- centrations for the plasma (1:100 v/v dilution with water) and desalted CSF sample were determined using the Bradford assay as previously described [5]. Before storage, CSF samples were centrifuged at 450 ×g. All but the bottom 0.5 mL of the supernatant was transferred to new container and stored at −80°C. For plasma, heparinized blood was centrifuged at 450 ×g and the plasma was removed, aliquoted, and stored at −80°C. 2-Dimensional Gel Electrophoresis Proteins were separated using 2-dimensional gel electrophore- sis and analyzed as previously described [5]. Master gels were prepared by analyzing duplicate gels of samples from 12 indi- vidual patients. Spots matched in >75% of the gels were includ- ed in the master gel, using PDQuest 2-dimensional software, with semiquantitative analysis performed using Progenesis 200 software. For plasma samples, protein spots of interest were excised from Coomassie-stained gels and digested as previously described [5]. Because of the low protein content in CSF P. falciparum Histidine-Rich Protein 2 (HRP2) Double-Site Antigen-Capture Enzyme-Linked Immunosorbent Assay (ELISA) ELISA was used to determine the presence of pHRP2 in frozen plasma and CSF samples. Plates were coated with 100 µL/well P. falciparum Histidine-Rich Protein 2 (HRP2) Double-Site Antigen-Capture Enzyme-Linked Immunosorbent Assay (ELISA) ELISA was used to determine the presence of pHRP2 in frozen plasma and CSF samples. Plates were coated with 100 µL/well Plasma and CSF Proteomes in Cerebral Malaria • JID 2013:208 (1 November) • 1495 of 1.0 µg/mL immunoglobulin M monoclonal anti-HRP2 anti- body (MPFM-55A, Immunology Consultants Laboratories, Newberg, OR) diluted in phosphate-buffered saline (PBS) and incubated overnight at 4°C. Plates were saturated for 2 hours at room temperature with 200 µL of 3% skimmed milk (Marvel; catalog no. UKFF 005M EC) in PBS. Plates were then washed 3 times in PBS/Tween (Sigma; catalog no. P1379; 500 mL; 0.05%) washing solution. A total of 100 µL of diluted plasma samples (1:64) or 100 µL of CSF samples was added to the plates, which were sealed and incubated at room tempera- ture in a humid chamber for 2 hours and then washed 5 times. A total of 100 µL of secondary antibody conjugated with horseradish peroxidase (MPFG-55P, Immunology Con- sultants Laboratories; 0.2 μg/mL diluted in 2% bovine serum albumin, 1% Tween 20, and PBS) was added to the wells and incubated for 1 hour at room temperature. After incubation, substrate (Sigmafast OPD; catalog no. P9187-50SET) was added and incubated for 30 minutes at room temperature, and the reaction was stopped by adding 50 µL of 2N sulfuric acid. Plates were read at an optical density of 490 nm. Standards were made by serially diluting plasma samples of known para- sitemia, with a high parasitemia of 0.2% and a low of 0.003125% (this gave a 0.1 absorbance value above baseline). A cutoff of 0.025 in plasma and 0.004 in CSF had been deter- mined to separate children with cerebral malaria from all other children with impaired consciousness in a separate group of children. Spectra obtained from the MALDI-ToF were used to search through the NCBInr database, using the Mascot Peptide Mass Fingerprinting software [8]. Protein scores were considered sig- nificant in accordance with cutoff scores recommended by Mascot for Homo sapiens 65 P < .05 or for P. falciparum 55 P < .05. MS/MS spectra were evaluated using the TurboSEQUEST al- gorithm in BioWorks v 3.1 software provided by ThermoFinni- gan and were searched against the human and P. falciparum subsets of the NCBInr database and the P. falciparum database (PlasmoDB, version 4.4) downloaded from the Sanger Institute. P. falciparum Histidine-Rich Protein 2 (HRP2) Double-Site Antigen-Capture Enzyme-Linked Immunosorbent Assay (ELISA) ELISA was used to determine the presence of pHRP2 in frozen plasma and CSF samples. Plates were coated with 100 µL/well All searches were performed according to search parameters described in the Supplementary Materials. Proteins identified were stored in Stata, version 11.2, and a heat map showing the presence or absence of a protein in a patient was generated using Stata, version 11.2. Proteins were included in the pheno- type database if they were identified in samples from at least 6 of the 12 patients in the group. at Liverpool University Library on Novemb http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ Downloaded from Functional cataloging of proteins was performed as de- scribed elsewhere [5]. Further protein and pathway analysis was undertaken using tools available at the Universal Protein Re- source [9]. Patient Characteristics Samples from 36 children were used in this study. Table 1 reports clinical features of the children. As expected, children with acute bacterial meningitis had significantly higher levels of white blood cell counts in the CSF and lower levels of glucose in the CSF. Children with cerebral malaria had significantly fewer platelet counts. Data are median (interquartile range). Abbreviations: CSF, cerebrospinal fluid; HRP2, histidine-rich protein 2; iRBC, infected red blood cell; WBC, white blood cell. a By the Kruskal-Wallis test. Data are median (interquartile range). Abbreviations: CSF, cerebrospinal fluid; HRP2, histidine-rich protein 2; iRBC, infected red blood cell; WBC, white blood cell. a By the Kruskal-Wallis test. Data are median (interquartile range). Abbreviations: CSF, cerebrospinal fluid; HRP2, histidine-rich protein 2; iRBC, infected red blood cell; WBC, white blood cell. Protein Separation by 2-Dimensional LC-MS/MS Analysis of plasma samples using 2-dimensional LC-MS/MS revealed a total of 339 host proteins and 573 falciparum pro- teins. In CSF, we identified 113 host proteins and 254 falcipa- rum proteins. Heat maps of all proteins identified in all 36 patients are shown in Figure 2. We selected 259 host proteins of interest (Figure 3 and Sup- plementary Table 1). Selection was based on whether the protein was of known function and unique to the cerebral malaria phenotype in plasma (n = 1) or CSF (n = 7) or whether it was identified in the other 2 phenotypes but not in cerebral malaria. Seventy-six proteins were found in CSF but not in plasma, and 29 were found in both plasma and CSF. Of at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on Nove http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ loaded from Figure 1. Composite gel showing the differences between gels in (A) plasma collected from children with a diagnosis of cerebral malaria (CM) and acute bacterial meningitis (ABM; A), plasma collected from children with a diagnosis of CM and nonspecific encephalopathy (B), cerebrospinal fluid (CSF) collected from children with a diagnosis of CM and ABM (C), and CSF collected from children with a diagnosis of CM and ABM (D). Composite gel maps were created using the comparison tool in PDQuest. The gel maps were created by analyzing duplicate gels for 12 patients’ biological replicates (2 gels per patient sample). The extent of the correlation of protein spots between the individual gels was >0.77 (a coefficient of 1.00 indicates that the replicate gels are perfectly similar). Red spots are spots found in the CM gel map, blue spots are unique to the comparator, and green spots depict spots found in both. Figure 1. Composite gel showing the differences between gels in (A) plasma collected from children with a diagnosis of cerebral malaria (CM) and acute bacterial meningitis (ABM; A), plasma collected from children with a diagnosis of CM and nonspecific encephalopathy (B), cerebrospinal fluid (CSF) collected from children with a diagnosis of CM and ABM (C), and CSF collected from children with a diagnosis of CM and ABM (D). Composite gel maps were created using the comparison tool in PDQuest. Data Management and Statistical Analysis Differences in clinical characteristics of the 3 disease pheno- types were evaluated using Stata, version 11.2. Medians were calculated, and Kruskal–Wallis P values reported. Table 1. Clinical Characteristics of Patients Characteristic Acute Bacterial Meningitis Cerebral Malaria Nonspecific Encephalopathy Pa Age, months 32.75 (6.07–86.03) 31.83 (19.98–39.47) 25.55 (13.47–35.50) .7730 Parasite density, iRBCs ×103/μL 0 (0–0) 566 000 (22 434–1 289 500) 0 (0–56.5) .0001 Hemoglobin level, g/dL 9.7 (9.05–11.5) 6.3 (5.3–7.7) 9.6 (8–10.15) .0002 Platelet level, platelets/μL 494 (320.5–655.5) 93 (76–184) 419 (273–605) .0021 CSF WBC count, cells/μL 228 (34–1000) 1 (0–2) 2 (0–2) .0003 CSF protein level, mg/dL 1.865 (0.875–2.645) 0.24 (0.205–0.375) 0.195 (0.165–0.24) .0001 Blood glucose, mg/dL 5.9 (3.8–7.1) 4.6 (3.75–5.05) 4.5 (3.85–6.65) .4281 CSF glucose, mg/dL 0.95 (0.6–2.15) 3.4 (3–3.7) 3.3 (2.85–3.9) .0004 Ratio of CSF to blood glucose 0.19 (0.12–0.41) 0.72 (0.65–0.96) 0.70 (0.61–0.77) .0011 Plasma HRP2 level 0.0025 (0–0.006) 6.1915 (0.4845–13.3285) 0 (0–0.2235) .0001 CSF HRP2 level 0.002 (0–0.0035) 0.0265 (0.0085–0.108) 0.003 (0.001–0.01) .0002 Hospitalization duration, d 9.5 (8.5–14) 3 (2.5–3.5) 4 (2.5–6) .0003 Data are median (interquartile range). Abbreviations: CSF, cerebrospinal fluid; HRP2, histidine-rich protein 2; iRBC, infected red blood cell; WBC, white blood cell. a By the Kruskal-Wallis test. Table 1. Clinical Characteristics of Patients 1496 • JID 2013:208 (1 November) • Gitau et al binding. CSF proteins of interest were mainly involved in apo- ptosis and proteolysis. Protein Separation by 2-Dimensional Gel Electrophoresis Reference gels for each phenotype were created using PDQuest. For plasma, averages of 200, 194, and 71 spots were included in the cerebral malaria, nonspecific encephalopathies, and acute bacterial meningitis gels, respectively. For CSF, aver- ages of 150 spots were included in the cerebral malaria and nonspecific encephalopathies reference gels, and 80 spots were included in the acute bacterial meningitis gel. The 2-dimen- sional gel electrophoresis patterns of both plasma and CSF samples showed significant differences between the 3 clinical phenotypes studied (Figure 1). Proteins of interest that were definitively identified using mass spectrometry are listed in Table 2 (plasma) and Table 3 (CSF). Plasma proteins identi- fied were mainly involved in platelet activation and aggrega- tion, protein transport, endocytosis and cell communication, lipid metabolism, and binding and protein/antigen/nucleotide Protein Separation by 2-Dimensional LC-MS/MS The gel maps were created by analyzing duplicate gels for 12 patients’ biological replicates (2 gels per patient sample). The extent of the correlation of protein spots between the individual gels was >0.77 (a coefficient of 1.00 indicates that the replicate gels are perfectly similar). Red spots are spots found in the CM gel map, blue spots are unique to the comparator, and green spots depict spots found in both. Plasma and CSF Proteomes in Cerebral Malaria • JID 2013:208 (1 November) • 1497 Table 2. Host Proteins Identified as Differentially Expressed When Gels of Plasma From Individuals With Cerebral Malaria Were Compared to Other Phenotypes Biological Process, Accession No. Description Spot Difference Acute Bacterial Meningitis Nonspecific Encephalopathy Platelet activation/aggregation P01009 Alpha-1-antitrypsin Unique Unique P02647 Apolipoprotein A . . . Down P02787 Serotransferrin precursor Unique Missing P02787 C chain of human serum transferrin . . . Missing P02679 F chain of fragment D of fibrinogen . . . Missing P52735 Guanine nucleotide exchange factor VAV2 Unique Unique D3DP16 Fibrinogen γ chain, isoform CRA_a . . . Missing Protein transport/endocytosis/cell communication O60493 Sorting nexin-3 (protein SDP3) Down Down P19652 Alpha-1-acid glycoprotein, type 2 Up Up P02763 Alpha-1-acid glycoprotein, type 1 Up Up P02768 Albumin, isoform CRA_g . . . Unique Lipid metabolism and binding P02647 A chain of human apolipoprotein A-I . . . Down Protein/antigen/nucleotide binding Q96Q89 M-phase phosphoprotein 1 Up Unique P16871 Interleukin 7 receptor, isoform CRA_b . . . Missing particular interest were 3 brain-specific proteins found in plasma namely brain-specific angiogenesis inhibitor 2, calcium/calmodulin-dependent protein kinase IV in acute bac- terial meningitis, and spectrin nonerythroid β chain 3 in cere- bral malaria. In addition, we also identified 14 other proteins that have been implicated in cerebral malaria or other brain injury (Table 4). Table 2. Host Proteins Identified as Differentially Expressed When Gels of Plasma From Individuals With Cerebral Malaria Were Compared to Other Phenotypes Table 2. Host Proteins Identified as Differentially Expressed When Gels of Plasma From Individuals With Cerebral Malaria Were Compared to Other Phenotypes In total, we compiled a list of 107 nonhypothetical P. falcipa- rum proteins of interest (Figure 3 and Supplementary Table 2) that were found in plasma and CSF. Protein Separation by 2-Dimensional LC-MS/MS This list included proteins involved in host cell modification, such as heat shock protein 40; in antigenic variation and host cell interaction, such as 17 variants of erythrocyte membrane protein 1; and 7 rifins. We wondered whether the presence of parasite proteins in slide-negative children could be due to a recent infection, and we therefore measured levels of pHRP2, a parasite protein that has been shown to have a half-life of about 2 weeks after infec- tion [10]. All children with cerebral malaria had pHRP2 levels in plasma and CSF that were above set cutoff levels. One child with acute bacterial meningitis and 3 children with nonspecific encephalopathy had high levels in plasma. The 3 children with nonspecific encephalopathy also had levels of pHRP2 in CSF that were equal to or above the set cutoff level. Interestingly, 3 children with acute bacterial meningitis had levels of pHRP2 in CSF equal to or above the set cutoff level but did not have any pHRP2 detected in the plasma. at Liverpool University Library on Novemb http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on Nov http://jid.oxfordjournals.org/ Downloaded from 1498 • JID 2013:208 (1 November) • Gitau et al DISCUSSION Pathogenic states in children with impaired consciousness in malaria-endemic areas could be reflected by changes in protein biomarkers in both plasma and CSF. Proteomic approaches allow for the analysis of large numbers of proteins at the same time, and this may help elucidate pathways that can be targeted for therapy. Additionally, proteomic platforms allow for the measurement of low-abundance proteins in complex materials Table 3. Host Proteins Identified as Differentially Expressed When Gels of Cerebrospinal Fluid From Individuals With Cerebral Malaria Were Compared to Other Phenotypes UniProt KB Accession No. Description Spot Difference Gene Ontology Acute Bacterial Meningitis Nonspecific Encephalopathy P02766 Transthyretin precursor . . . Unique Protein transport/hormone activity and binding P62745 ρ-related GTP-binding protein Down . . . Angiogenesis/apoptosis/cell differentiations P50213 Isocitrate dehydrogenase [NAD] subunit α, mitochondrial precursor Unique . . . Carbohydrate metabolic processing Q9NRR2 Tryptase γ preproprotein Down . . . Proteolysis O60423 Probable phospholipid-transporting ATPase IK Unique . . . Cation transport A4D1V4 39S ribosomal protein L32 Unique . . . Translation Table 3. Host Proteins Identified as Differentially Expressed When Gels of Cerebrospinal Fluid From Individuals With Cerebral Malaria Were Compared to Other Phenotypes 1498 • JID 2013:208 (1 November) • Gitau et al Figure 2. Heat maps showing distribution of proteins expressed using a 2-dimensional liquid chromatography tandem mass spectrometry strategy. A, Host proteins in plasma and cerebrospinal fluid (CSF). B, Plasmodium falciparum proteins in plasma and CSF. C, Graph showing breakdown of the gene on- tology categorization of host proteins identified in plasma. at Liverpool University Library on Novemb http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ Downloaded from Figure 2. Heat maps showing distribution of proteins expressed using a 2-dimensional liquid chromatography tandem mass spectrometry strategy. A, Host proteins in plasma and cerebrospinal fluid (CSF). B, Plasmodium falciparum proteins in plasma and CSF. C, Graph showing breakdown of the gene on- tology categorization of host proteins identified in plasma. community controls. The presence of pathogen proteins in CSF could be due to a leaky blood brain barrier. However, not all proteins found in the CSF were found in plasma, and addition- ally we did not always find a correlation with plasma pHRP2 levels and CSF pHRP2 levels. We therefore cannot rule out that proteins in the CSF are there as a result of sequestration in the brain and/or leukocyte migration into the brain. such as plasma. Plasma and CSF Proteomes in Cerebral Malaria • JID 2013:208 (1 November) • 1499 DISCUSSION Venn diagram showing distribution of proteins of interest expressed using a 2-dimensional liquid chromatography tandem mass spectrometry strategy. The red circles represent cerebral malaria, the green circles represent acute bacterial meningitis, and the blue circles represent nonspecific encephalopathy. Abbreviation: CSF, cerebrospinal fluid. cell-adhesion molecules at initial segments and nodes of Ranvier [15]. Additionally, spectrin proteins have also been identified as binding partners for various P. falciparum pro- teins. In particular, spectrins have been shown to associate with heat shock protein 40 [16], and in ring-stage infected red blood cells (RBCs), ring-infected erythrocyte surface antigen associ- ates with spectrin and stabilizes the membrane skeleton. In mature-stage parasitized RBCs, knob-associated His-rich protein molecules self-associate to form conical structures that interact with spectrin. Pf332 and mature-parasite-infected erythrocyte surface antigen bind to the junction complex, while P. falciparum erythrocyte membrane protein 3, identified in this study in plasma of children with cerebral malaria and acute bacterial meningitis, binds to spectrin, further compromising RBC membrane deformability [17]. These interactions stabilize spectrin tetramers in the infected RBC, increasing resistance to further parasite invasion of the cell by increasing infected RBC rigidity, and could facilitate sequestration and inhibit splenic clearance [18]. We hypothesize that the difference in spectrin expression in our study could be linked to these interactions with the infected RBC. Differentially expressed proteins identified in this study, such as α 1 acid glycoprotein type 1 and type 2 (orosomucoid 2), re- ticulon 4, and retinol binding protein 2 could be markers of an uncontrolled and harmful inflammatory response. An increase in orosomucoid 2, differentially expressed in both plasma and CSF of patients with cerebral malaria, together with an in- creased production of ceruloplasmin and glutathione, would enhance antioxidant defenses and limit the stimulatory effects of oxidant molecules on cytokine production. This acute-phase protein has previously been studied in connection with malaria [13], and it has been suggested that its production may reflect the severity of the acute phase response [14]. Spectrin β chain brain 3, a protein that belongs to a family of spectrin proteins, was differentially expressed in both plasma and CSF of children with cerebral malaria. In our previous mouse study [5], this protein was differentially expressed in plasma of mice infected with Plasmodium berghei, compared with expression in noninfected mice. DISCUSSION This article describes the use of proteomic plat- forms to elucidate differences in plasma and CSF proteomes collected from children in a malaria-endemic area presenting with impaired consciousness. In this analysis, by use of 2 differ- ent proteomic strategies, 259 host proteins and 107 parasite (ie, P. falciparum) proteins were identified as differentially ex- pressed in both plasma and CSF, and these could help elaborate mechanistic differences in the different encephalopathies de- scribe here. Importantly, some of the proteins could help point to the cause of disease in the group of patients with nondefined encephalopathy. We found 14 spots differentially regulated based on gels from the cerebral malaria group, compared with gels from the other 2 groups. Seven of these were identified as proteins that play a role in platelet activation and aggregation (Table 2). In addition, using the 2-dimensional LC-MS/MS strategy, we identified 13 proteins that play a role in coagulation and that were missing in plasma from the cerebral malaria group (Supplementary Table 1) or were found in CSF from acute bacterial meningitis group and not in the other groups. This is not surprising because thrombocytopenia is associated with cerebral malaria [11, 12], and in fact children in this study had lower levels of platelets. Parasite platelet complexes could be a cause of reduced platelet counts, and a proteomic study of platelet microparticles could help clarify mechanisms leading to thrombocytopenia. The first striking observation was the presence of P. falcipa- rum proteins in both plasma and CSF of slide-negative children with acute bacterial meningitis. This result raises 2 possibilities: (1) the children have had a recent episode of malaria, which could predispose them to acute bacterial meningitis; and (2) children in malaria-endemic areas have persistently low levels of parasite proteins in their sera, although one wonders why fewer proteins were identified in the slide-negative children without acute bacterial meningitis. To confirm the second point, we would need to analyze samples from healthy Figure 3. Venn diagram showing distribution of proteins of interest expressed using a 2-dimensional liquid chromatography tandem mass spectrometry strategy. The red circles represent cerebral malaria, the green circles represent acute bacterial meningitis, and the blue circles represent nonspecific encephalopathy. Abbreviation: CSF, cerebrospinal fluid. at Liverpool University Library on Novembe http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ Downloaded from Figure 3. 1500 • JID 2013:208 (1 November) • Gitau et al DISCUSSION In the brain, this protein is enriched in myelinated neurons, where it colocalizes with ankyrin at axon initial segments and nodes of Ranvier and par- ticipates in the clustering of voltage-gated Na+ channels and Table 4. Host Proteins of Interest Implicated in Brain Injury Identified Using a 2-Dimensional Liquid Chromatography Tandem Mass Spectrometry Strategy Protein UniProt KB Accession No. CSF Plasma Reference(s) CM; NE; ABM CM; NE; ABM Cytoplasmic protein NCK2 O43639 Yes; No; Yes No; No; Yes [29] Brain-specific angiogenesis inhibitor 2 O60241 No; No; No No; No; Yes [30] Spectrin beta chain, erythrocyte P11277 No; No; No No; No; Yes [31, 32] Secretogranin-2 precursor P13521 Yes; No; No No; No; Yes [33] Sodium/glucose cotransporter 1 P13866 Yes; No; No No; No; No [34, 35] Vinculin P18206 No; Yes; No No; No; No [36] Neurofibromin P21359 No; No; Yes No; No; No [37, 38] Alanine glyoxylate aminotransferase P21549 Yes; No; No No; No; No [39] Retinol-binding protein 2 P50120 No; No; No No; Yes; yes [5, 40] Retinal guanylyl cyclase 2 precursor P51841 Yes; No; No Yes; No; Yes [19, 41] Neurexophilin-1 precursor P58417 No; No; No No; No; Yes [42] Calcium/calmodulin-dependent protein kinase IV; brain Ca Q16566 No; No; No No; No; Yes [41, 43] phospholipase C, β 2 Q59F77 Yes; No; No No; No; No [44] Spectrin β chain, brain 3 Q9H254 Yes; No; No Yes; No; No [15] Reticulon 4 Q9NQC3 No; No; No No; No; Yes [45, 46] Bromodomain adjacent to zinc finger domain protein 2B Q9UIF8 Yes; No; Yes No; No; Yes [47] NDRG2 protein Q9UN36 No; Yes; No Yes; No; No [48, 49] Endothelial lipase precursor Q9Y5X9 Yes; No; No No; No; No [50] Abbreviations: ABM, acute bacterial meningitis; CM, cerebral malaria; CSF, cerebrospinal fluid; NE, nonspecific encephalopathies. Table 4. Host Proteins of Interest Implicated in Brain Injury Identified Using a 2-Dimensional Liquid Chromatography Tandem Mass Spectrometry Strategy Table 4. Host Proteins of Interest Implicated in Brain Injury Identified Using a 2-Dimensional Liquid Chromatography Tandem Mass Spectrometry Strategy at Liverpool University Library on Novembe http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ Downloaded from collection to remove all cellular components, although protease inhibitors, which can minimize degradation, were not added. DISCUSSION These inhibitors can interfere with peptide and amino acid MS signals [26], and some studies suggest that there are no differ- ences in CSF and plasma proteomes determined in the pres- ence or absence of protease inhibitors [27, 28]. Second, a “normal” control could not be established for this study because CSF can only be ethically obtained from children with impaired consciousness. However, future studies could include a nonfebrile group of children with impaired consciousness, such as those with epileptic seizures or poisoning or those who have a lumbar puncture to rule out meningitis. Third, the samples were obtained at admission, and therefore the effects of antimalarial treatment and other effects of disease progres- sion, such as the inflammatory responses that may influence the protein levels, could not be fully assessed. Fourth, cytokines and chemokines that have previously been identified as playing a role in cerebral malaria were not differentially expressed in our study. Two possible reasons for this finding are that (1) semiquantitative 2-dimensional gel analysis may not be sensi- tive enough to detect such low abundant proteins and that (2) the 2-dimensional LC-MS/MS method described in this study was not quantitative and therefore only reported the pres- ence or absence of proteins in a patient group. Most cytokines and chemokines implicated in malaria were present in all 3 groups. Excitotoxic cell death in CNS disorders is partly due to dys- function of the sodium/potassium pump, resulting in an in- creased uptake of water, which could as a consequence lead to an increased influx of calcium. Retinal guanylyl cyclase 2 pre- cursor is a gene that displays calcium-dependent regulation [19]. In addition, sodium-dependent glucose transport could also be affected, and this could result in the differential expres- sion of sodium/glucose cotransporter 1 (SGLT1) seen in the CSF of patients with cerebral malaria. SGLT1 has been shown to be expressed in neurons and is upregulated during metabolic stress when there is a decrease in D-glucose content [20]. The sorting nexin family of proteins, which contain a Phox homolo- gy domain, play crucial roles in regulating the intracellular membrane trafficking of the endocytic pathway [21]. SNX3, which was differentially expressed in this study, is associated with the early endosome through the PX domain, which is capable of interaction with phosphatidylinositol-3-phosphate. Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Plasma and CSF Proteomes in Cerebral Malaria • JID 2013:208 (1 November) • 1501 DISCUSSION It is important to consider potential limitations of our study design and analysis strategy. First, proteomic studies require careful sample collection and storage. Prolonged contact of CSF and plasma with cellular components has been shown to affect protein and peptide quality because of the presence of proteo- lytic enzymes in plasma [22, 23] and CSF [24, 25]. Samples ana- lyzed in this study were centrifuged within an hour of Plasma and CSF Proteomes in Cerebral Malaria • JID 2013:208 (1 November) • 1501 Despite the limitations mentioned above, the results from this study show that novel disease specific biomarkers can be identified using proteomic strategies. In addition to providing insight into underlying pathophysiological mechanisms, they have the potential after thorough validation to be used as bio- marker panels, which may be of value in the early diagnosis of disease and in monitoring responses to therapies. In particular, this study has shown that there are proteins uniquely expressed in cerebral malaria. The next phase of this work will be to rigor- ously assess and validate the diagnostic value of these differen- tially expressed proteins before transfer to a suitable platform that can generate an affordable point-of-care diagnostic. 7. Terry DE, Umstot E, Desiderio DM. Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. J Am Soc Mass Spectrom 2004; 15:784–94. 8. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spec- trometry data. Electrophoresis 1999; 20:3551–67. 9. Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res 2012; 40:D71–5. 10. Ochola LB, Vounatsou P, Smith T, Mabaso ML, Newton CR. The reliabil- ity of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard. Lancet Infect Dis 2006; 6:582–8. in the absence of a gold standard. Lancet Infect Dis 2006; 6:58 11. Cserti-Gazdewich CM, Dhabangi A, Musoke C, et al. Cytoadherence in paediatric malaria: ABO blood group, CD36, and ICAM1 expression and severe Plasmodium falciparum infection. Br J Haematol 2012; 159:223–36. 12. Wassmer SC, Taylor T, Maclennan CA, et al. Platelet-induced clumping of Plasmodium falciparum-infected erythrocytes from Malawian pa- tients with cerebral malaria-possible modulation in vivo by thrombocy- topenia. J Infect Dis 2008; 197:72–8. References 23. Rai AJ, Gelfand CA, Haywood BC, et al. HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples. Proteomics 2005; 5:3262–77. 1. Berkley JA, Mwangi I, Mellington F, Mwarumba S, Marsh K. Cerebral malaria versus bacterial meningitis in children with impaired con- sciousness. QJM 1999; 92:151–7. 2. Gwer S, Thuo N, Idro R, et al. Changing trends in incidence and aetiol- ogy of childhood acute non-traumatic coma over a period of changing malaria transmission in rural coastal Kenya: a retrospective analysis. BMJ Open 2012; 2:e000475. 2. Gwer S, Thuo N, Idro R, et al. Changing trends in incidence and aetiol- ogy of childhood acute non-traumatic coma over a period of changing malaria transmission in rural coastal Kenya: a retrospective analysis. BMJ Open 2012; 2:e000475. 24. Schoonenboom NS, Mulder C, Vanderstichele H, et al. Effects of pro- cessing and storage conditions on amyloid beta (1–42) and tau concen- trations in cerebrospinal fluid: implications for use in clinical practice. Clin Chem 2005; 51:189–95. 3. WHO. Severe falciparum malaria. World Health Organization, Com- municable Diseases Cluster. Trans R Soc Trop Med Hyg 2000; 94 (Suppl 1):S1–90. 25. Rosenling T, Stoop MP, Smolinska A, et al. The impact of delayed storage on the measured proteome and metabolome of human cerebro- spinal fluid. Clin Chem 2011; 57:1703–11. 3. WHO. Severe falciparum malaria. World Health Organization, Com- municable Diseases Cluster. Trans R Soc Trop Med Hyg 2000; 94 (Suppl 1):S1–90. 4. Berkley JA, Mwangi I, Ngetsa CJ, et al. Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet 2001; 357:1753–7. 26. Schuchard MD, Mehigh RJ, Cockrill SL, et al. Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor, monitored by 2-dimensional electrophoresis and mass spectrometry. Biotechniques 2005; 39:239–47. 4. Berkley JA, Mwangi I, Ngetsa CJ, et al. Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet 2001; 357:1753–7. 5. Gitau EN, Kokwaro GO, Newton CR, Ward SA. Global proteomic anal- ysis of plasma from mice infected with Plasmodium berghei ANKA using two dimensional gel electrophoresis and matrix assisted laser de- sorption ionization-time of flight mass spectrometry. Malar J 2011; 10:205. 5. Gitau EN, Kokwaro GO, Newton CR, Ward SA. at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ aded from 15. Berghs S, Aggujaro D, Dirkx R Jr., et al. betaIV spectrin, a new spectrin localized at axon initial segments and nodes of ranvier in the central and peripheral nervous system. J Cell Biol 2000; 151:985–1002. Notes 16. Acharya P, Chaubey S, Grover M, Tatu U. An exported heat shock protein 40 associates with pathogenesis-related knobs in Plasmodium falciparum infected erythrocytes. PLoS One 2012; 7:e44605. Acknowledgments. We thank the director of the Kenya Medical Research Institute, for permission to publish this work, and Prof Kevin Marsh and Dr Britta Urban, for critical discussion and review of the manuscript. 17. Maier AG, Cooke BM, Cowman AF, Tilley L. Malaria parasite proteins that remodel the host erythrocyte. Nat Rev Microbiol 2009; 7:341–54. E. G. performed the proteomic experiments and drafted the manuscript. C. J. R. C. N., S. A. W., and G. K. participated in the design of the study and helped draft the manuscript. All authors read and approved the final manuscript. 18. Oh SS, Voigt S, Fisher D, et al. Plasmodium falciparum erythrocyte membrane protein 1 is anchored to the actin-spectrin junction and knob-associated histidine-rich protein in the erythrocyte skeleton. Mol Biochem Parasitol 2000; 108:237–47. Financial support. This work was supported by the World Health Organization (TDR/MIM grant 980074 to G. O. K.), the KEMRI /Wellcome Trust Research Programme Kenya (to E. N. G.), and the Wellcome Trust (senior fellowship 050533 to C. R. J. C. N.). 19. Burgoyne RD. Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling. Nat Rev Neurosci 2007; 8:182–93. 20. Poppe R, Karbach U, Gambaryan S, et al. Expression of the Na+-D- glucose cotransporter SGLT1 in neurons. J Neurochem 1997; 69:84–94. p Potential conflicts of interest. All authors: No reported conflicts. Potential conflicts of interest. All authors: No reported conflicts. 21. Xu Y, Hortsman H, Seet L, Wong SH, Hong W. SNX3 regulates endo- somal function through its PX-domain-mediated interaction with PtdIns(3)P. Nat Cell Biol 2001; 3:658–66. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. 22. Hulmes DJ, Marsden ME, Strachan RK, Harvey RE, McInnes N, Gardner DL. Intra-articular hyaluronate in experimental rabbit osteo- arthritis can prevent changes in cartilage proteoglycan content. Osteo- arthritis Cartilage 2004; 12:232–8. Supplementary Data 13. Beesley R, Filteau S, Tomkins A, et al. Impact of acute malaria on plasma concentrations of transferrin receptors. Transactions of the Royal Society of Tropical Medicine and Hygiene 2000; 94:295. at Liverpool University Library on Novembe http://jid.oxfordjournals.org/ Downloaded from at Liverpool University Library on November 18, 2014 http://jid.oxfordjournals.org/ Downloaded from Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. 14. Filteau SM, Morris SS, Abbott RA, et al. Influence of morbidity on serum retinol of children in a community-based study in northern Ghana. Am J Clin Nutr 1993; 58:192–7. References Global proteomic anal- ysis of plasma from mice infected with Plasmodium berghei ANKA using two dimensional gel electrophoresis and matrix assisted laser de- sorption ionization-time of flight mass spectrometry. Malar J 2011; 10:205. 27. Berven FS, Kroksveen AC, Berle M, et al. Pre-analytical influence on the low molecular weight cerebrospinal fluid proteome. Proteomics Clin Appl 2007; 1:699–711. 28. Ayache S, Panelli M, Marincola FM, Stroncek DF. Effects of storage time and exogenous protease inhibitors on plasma protein levels. Am J Clin Pathol 2006; 126:174–84. 6. Bejon P, Berkley JA, Mwangi T, et al. Defining childhood severe falcipa- rum malaria for intervention studies. PLoS Med 2007; 4:e251. 6. Bejon P, Berkley JA, Mwangi T, et al. Defining childhood severe falcipa- rum malaria for intervention studies. PLoS Med 2007; 4:e251. 1502 • JID 2013:208 (1 November) • Gitau et al 29. Round JE, Sun H. The adaptor protein Nck2 mediates Slit1-induced changes in cortical neuron morphology. Mol Cell Neurosci 2011; 47:265–73. 40. Tabone MD, Muanza K, Lyagoubi M, et al. The role of interleukin-6 in vitamin A deficiency during Plasmodium falciparum malaria and possi- ble consequences for vitamin A supplementation. Immunology 1992; 75:553–4. 30. Zencir S, Ovee M, Dobson MJ, Banerjee M, Topcu Z, Mohanty S. Iden- tification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein. Biochem Biophys Res Commun 2011; 411:792–7. 41. Lang B, Newbold CI, Williams G, Peshu N, Marsh K, Newton CR. An- tibodies to voltage-gated calcium channels in children with falciparum malaria. J Infect Dis 2005; 191:117–21. 31. Pei X, Guo X, Coppel R, et al. The ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum stabilizes spectrin tetramers and suppresses further invasion. Blood 2007; 110:1036–42. 42. Petrenko AG, Ullrich B, Missler M, Krasnoperov V, Rosahl TW, Sudhof TC. Structure and evolution of neurexophilin. J Neurosci 1996; 16:4360–9. 43. Hanissian SH, Frangakis M, Bland MM, Jawahar S, Chatila TA. Expres- sion of a Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, in human T lymphocytes. Regulation of kinase activity by T cell recep- tor signaling. J Biol Chem 1993; 268:20055–63. 32. Lauterbach SB, Coetzer TL. The M18 aspartyl aminopeptidase of Plas- modium falciparum binds to human erythrocyte spectrin in vitro. Malar J 2008; 7:161. 33. Shyu WC, Lin SZ, Chiang MF, et al. Secretoneurin promotes neuropro- tection and neuronal plasticity via the Jak2/Stat3 pathway in murine models of stroke. J Clin Invest 2008; 118:133–48. 44. Plasma and CSF Proteomes in Cerebral Malaria • JID 2013:208 (1 November) • 1503 References Bertagnolo V, Marchisio M, Pierpaoli S, et al. Selective up-regulation of phospholipase C-beta2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors. J Leukoc Biol 2002; 71:957–65. 34. Vemula S, Roder KE, Yang T, Bhat GJ, Thekkumkara TJ, Abbruscato TJ. A functional role for sodium-dependent glucose transport across the blood-brain barrier during oxygen glucose deprivation. J Pharmacol Exp Ther 2009; 328:487–95. 45. Lackner P, Beer R, Broessner G, et al. Nogo-A expression in the brain of mice with cerebral malaria. 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Europol’s international cooperation between ‘past present’ and ‘present future’: reshaping the external dimension of EU police cooperation.* * Assistant Professor in European Law, Erasmus University Rotterdam, The Netherlands. The author would like to thank the anonymous reviewers for their valuable comments. All websites accessed 31 December 2017.
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Peer Review: This article has been peer reviewed through the journal’s standard double blind peer-review, where both the reviewers and authors are anonymised during review. Copyright: © 2018, Florin Coman-Kund • This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY) 4.0 https://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited • DOI: https://doi.org/10.14324/111.444.ewlj.2018.01. Europol’s international cooperation between ‘past present’ and ‘present future’: reshaping the external dimension of EU police cooperation Europol’s international cooperation between ‘past present’ and ‘present future’: reshaping the external dimension of EU police cooperation Dr Florin Coman-Kund1,* How to cite: Coman-Kund, F., ‘Europol’s international cooperation between ‘past present’ and ‘present future’: reshaping the external dimension of EU police cooperation.’ Europe and the World, 2018, 2 (1): 1, pp. 1–37. DOI: https://doi.org/10.14324/111.444.ewlj.2018.01 Published: 20 April 2018 Europe and the World: A law review Europe and the World: A law review *Correspondence: comankund@law.eur.nl 1 Erasmus School of Law, Erasmus University Rotterdam, Netherlands Open Access: Europe and the World: A law review is a peer-reviewed open access journal. *Correspondence: comankund@law.eur.nl 1 Erasmus School of Law, Erasmus University Rotterdam, Netherlands *Correspondence: comankund@law.eur.nl © 2018, Dr Florin Coman-Kund • This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY) 4.0 https://creativecommons.org/licenses/by/4.0/, which permits ­unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited • DOI: https://doi.org/10.14324/111.444.ewlj.2018.01. Europol’s international cooperation between ‘past present’ and ‘present future’: reshaping the external dimension of EU police cooperation Dr Florin Coman-Kund* *  Assistant Professor in European Law, Erasmus University Rotterdam, The Netherlands. The author would like to thank the anonymous reviewers for their valuable comments. All websites accessed 31 December 2017. 1  This article is based on the author’s PhD dissertation: Florin Coman-Kund, European Union Agencies as Global Actors: A Legal Study of the European Aviation Safety Agency, Frontex and Europol (PhD dissertation, Universitaire Pers Maastricht 2015). A revised version of this study will be published as a monograph as follows: Florin Coman-Kund ‘European Union Agencies as Global Actors: A Legal Study of the European Aviation Safety Agency, Frontex and Europol’ (Routledge 2018, forthcoming). For the purpose of this paper, the category of EU agencies covers what the Common Approach on EU agencies designates as ‘decentralized agencies’ encompassing both the ‘regulatory’ or former ‘Community’ agencies and the former ‘third pillar’ agencies – see Joint Statement of the European Parliament, the Council of the EU and the European Commission on decentralized agencies of 19 July 2012 (referred to hereafter as the ‘Common Approach on EU agencies’) ˂<http:// europa.eu/about-eu/agencies/overhaul/index_en.htm>. 2  Andrea Ott, ‘EU Regulatory Agencies in EU External Relations: Trapped in a Legal Minefield between European and International Law’ (2008) 13 EFAR 515, 518. 3  See Florin Coman-Kund, ‘Assessing the Role of EU Agencies in the Enlargement Process: the Case of the European Aviation Safety Agency’ (2012) 8 CYELP 335, 339–40. _ 2  Andrea Ott, ‘EU Regulatory Agencies in EU External Relations: Trapped in a Legal Minefield between European and International Law’ (2008) 13 EFAR 515, 518. 3  See Florin Coman-Kund, ‘Assessing the Role of EU Agencies in the Enlargement Process: the Case of the European Aviation Safety Agency’ (2012) 8 CYELP 335, 339–40. © 2018, Dr Florin Coman-Kund • This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY) 4.0 https://creativecommons.org/licenses/by/4.0/, which permits ­unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited • DOI: https://doi.org/10.14324/111.444.ewlj.2018.01. 8  Ott (n 2); Andrea Ott, Ellen Vos and Florin Coman-Kund, ‘EU Agencies on the Global Scene: EU and International Law Perspectives’ in Michelle Everson, Cosimo Monda and Ellen Vos (eds), European Agencies in-between Institutions and Member States (Kluwer Law International 2014) 87–122. p y y   5  Martijn Groenleer, ‘Linking up Levels of Governance: Agencies of the European Union and their Interaction with International Organisations’ in Oriol Costa and Knud Erik Jørgensen (eds), The Influence of International Institutions on the European Union (Palgrave Macmillan 2012) 144. IOLR 1 (2004) 163. 10  See, for instance, Conny Rijken, ‘Legal and Technical Aspects of Co-operation between Europol, Third States, and Interpol’ in Vincent Kronenberger (ed), The European Union and the International Legal Order: Discord or Harmony? (Asser Press 2001) 577–603; Dick Heimans, ‘The External Relations of Europol – Political, Legal and Operational Considerations’ in Berndt Martenczuk and Servaas van Thiel (eds), Justice, Liberty and Security: New Challenges for EU External Relations (VUBPRESS 2008) 367–89; Laura Surano, ‘L’Action Extérieure d’Eurojust’ in Marianne Dony (ed), La Dimension Externe de l’Espace de Liberté, Sécurité et de Justice au Lendemain de Lisbonne et de Stockholm: un Bilan à mi-Parcours (Éditions de l’Université de Bruxelles 2012) 211–19; Melanie Fink, ‘Frontex Working Arrangements: Legitimacy and Human Rights Concerns regarding Technical Relationships’ (2012) 28 Utrecht J Int’l & Eur L 20; Coman-Kund (n 3) on the European Aviation Safety Agency (EASA); Groenleer and Gabbi (n 4) on the European Food Safety Authority (EFSA); Juan Santos Vara, ‘The External Activities of AFSJ Agencies: the Weakness of Democratic and Judicial Controls’ (2015) 20 EFAR 115. 9  Gregor Schusterschitz, ‘European Agencies as Subjects of International Law’ (2004) 1 IOLR 1 (2004) 163. 4  See also Martijn Groenleer and Simone Gabbi, ‘Regulatory Agencies of the European Union as International Actors: Legal Framework, Development over Time and Strategic Motives in the Case of the European Food Safety Authority’ (2013) 4 EJRR 479, 481–2. 4  See also Martijn Groenleer and Simone Gabbi, ‘Regulatory Agencies of the European Union as International Actors: Legal Framework, Development over Time and Strategic Motives in the Case of the European Food Safety Authority’ (2013) 4 EJRR 479, 481–2.   5  Martijn Groenleer, ‘Linking up Levels of Governance: Agencies of the European Union and their Interaction with International Organisations’ in Oriol Costa and Knud Erik Jørgensen (eds), The Influence of International Institutions on the European Union (Palgrave Macmillan 2012) 144.   6  Groenleer and Gabbi (n 4) 482–3.   7  Groenleer (n 5) 135–54.   8  Ott (n 2); Andrea Ott, Ellen Vos and Florin Coman-Kund, ‘EU Agencies on the Global Scene: EU and International Law Perspectives’ in Michelle Everson, Cosimo Monda and Ellen Vos (eds), European Agencies in-between Institutions and Member States (Kluwer Law International 2014) 87–122.   9  Gregor Schusterschitz, ‘European Agencies as Subjects of International Law’ (2004) 1 IOLR 1 (2004) 163. 10  See, for instance, Conny Rijken, ‘Legal and Technical Aspects of Co-operation between Europol, Third States, and Interpol’ in Vincent Kronenberger (ed), The European Union and the International Legal Order: Discord or Harmony? (Asser Press 2001) 577–603; Dick Heimans, ‘The External Relations of Europol – Political, Legal and Operational Considerations’ in Berndt Martenczuk and Servaas van Thiel (eds), Justice, Liberty and Security: New Challenges for EU External Relations (VUBPRESS 2008) 367–89; Laura Surano, ‘L’Action Extérieure d’Eurojust’ in Marianne Dony (ed), La Dimension Externe de l’Espace de Liberté, Sécurité et de Justice au Lendemain de Lisbonne et de Stockholm: un Bilan à mi-Parcours (Éditions de l’Université de Bruxelles 2012) 211–19; Melanie Fink, ‘Frontex Working Arrangements: Legitimacy and Human Rights Concerns regarding Technical Relationships’ (2012) 28 Utrecht J Int’l & Eur L 20; Coman-Kund (n 3) on the European Aviation Safety Agency (EASA); Groenleer and Gabbi (n 4) on the European Food Safety Authority (EFSA); Juan Santos Vara, ‘The External Activities of AFSJ Agencies: the Weakness of Democratic and Judicial Controls’ (2015) 20 EFAR 115. 6  Groenleer and Gabbi (n 4) 482–3. 7  Groenleer (n 5) 135–54. 1.  Introduction EU agencies1 are interacting in various ways with actors outside the EU and they are becoming arguably more and more visible on the interna- tional arena. Increasingly reflected in their own legal framework and practice,2 the international dimension of EU agencies entails, among other things,3 providing assistance and support to the Commission, the Council and the Member States with regard to aspects of various EU external actions and initiatives, as well as establishing more or less auton- omously formal and informal relations with international organizations reshaping the external dimension of EU police cooperation 1 and third countries. The development of this dimension of EU agencies’ profiles can be explained by the progressive extension of the globaliza­ tion phenomenon leading to an amplification of trans-boundary issues and triggering a focus on external aspects in various EU policy fields.4 In these circumstances, engaging in international cooperation activities has arguably become a ‘must’ for the EU agencies in order to enhance their effectiveness.5 The need for EU agencies to boost their autonomy and legitimacy has also been advanced as a reason for the proliferation of international cooperation activities by these bodies.6 Recently, scholars have begun to pay more attention to various aspects of EU agencies’ international dimension. Existing studies range from exploring the forms and consequences of interactions between EU agencies and international institutions,7 to more horizontal studies mapping out the external relations of these bodies and pointing to legal problems and tensions this may cause,8 or addressing the legal status of EU agencies on the international plane,9 to studies covering various facets of the international dimension of specific EU agencies.10 From a legal perspective, EU agencies’ evolving international dimension poses complex problems regarding the legal nature of their EUROPE AND THE WORLD 2 activities, their status as global actors and the scope and limits of their international cooperation. First, the international dimension of the European agencies raises important legal questions in terms of insti­ tutional balance and the Meroni doctrine of delegation of powers, as well as with regard to the distribution of competences and tasks in the field of EU external relations. In this respect it is important to clarify how far and under which conditions EU agencies are allowed to pursue international cooperation under the EU constitutional framework, and how the international dimension of EU agencies links to the roles of the main EU external action actors. 11  Coman-Kund (n 1) 85–176; see also Florin Coman-Kund, ‘The International Dimension of the EU Agencies: Framing a Growing Legal–Institutional Phenomenon’ (2018) 23 EFAR 97. 12  Usually, EU agencies’ legal framework and practice features international cooperation instruments labelled ‘arrangements’ or ‘memoranda of understanding’. 13  Police cooperation for the purpose of preventing and combatting crime is a particularly sensitive operational field in which Member States displayed reluctance over time towards more ‘communitarization’, and in which cooperation with national police and enforcement authorities is essential. 1.  Introduction Second, the external actions of EU agencies also trigger effects from an international law perspective. This requires examining European agencies through the lens of international law in order to clarify the legal nature and implications of their actions, in particular their treaty-like cooperation instruments concluded with third countries and international organizations. Addressing these issues is of utmost importance as this may lead to a reappraisal of the position of agencies within the EU constitutional framework, revealing clashes between EU and international law, and enhancing scrutiny and account­ ability of EU agencies’ international activities. Whilst legal scholarship has dealt with these aspects in a sparing and fragmented manner, recently a more comprehensive legal–analytical framework featuring EU and international law parameters has been proposed to tackle them.11 This article builds upon this legal–analytical framework in examining the above-mentioned legal aspects with regard to Europol’s international dimension. Europol is the embodiment of an agency with a strong interna­ tional profile, being one of the few EU agencies whose international cooperation record consists of formal ‘agreements’ concluded with third countries and international organizations.12 As such, Europol could be seen as an ‘exemplary’ case for the outer limits of EU agencies’ international cooperation powers. At the same time, Europol is a rather peculiar EU agency, acting within a specific legal–policy environment13 and having the ‘pedigree’ of an intergovernmental body established reshaping the external dimension of EU police cooperation 3 by the Member States via a convention.14 This ‘intergovernmental pedigree’ had also been reflected in the legal framework based on Council Decision 2009/371/JHA (Europol Council Decision)15 as regards the agency’s international cooperation. The new Europol Regulation,16 applicable as of 1 May 2017, redesigns the agency’s international cooperation framework arguably in order to fully align it with the post-Lisbon constitutional framework and the Common Approach on EU agencies. One important change concerns Europol’s international cooperation instruments, in that the agency will conclude in the future working and administrative arrangements instead of cooperation agreements as provided by the former Europol Council Decision. However, the new Europol Regulation ensures partly the ‘survival’ of the agency’s legal framework based on the Europol Council Decision17 and, importantly, it also preserves the validity of Europol’s cooperation agreements concluded before 1 May 2017.18 Against this background the aim of this article is two-fold. 14  Convention based on Article K.3 of the Treaty on the European Union of 27 November 1995, on the establishment of a European Police Office (Europol Convention) [1995] OJ C 316/1. 15  Council Decision 2009/371/JHA of 6 April 2009 establishing the European Police Office (Europol) [2009] OJ L 131/37. 16  Regulation (EU) 2016/794 of the European Parliament and of the Council of 11 May 2016 on the European Union Agency for Law Enforcement Cooperation (Europol) and replacing and repealing Council Decisions 2009/371/JHA, 2009/934/JHA, 2009/935/ JHA, 2009/936/JHA and 2009/968/JHA [2016] OJ L 135/53. 15  Council Decision 2009/371/JHA of 6 April 2009 establishing the European Police Office (Europol) [2009] OJ L 131/37. 16  Regulation (EU) 2016/794 of the European Parliament and of the Council of 11 May 2016 on the European Union Agency for Law Enforcement Cooperation (Europol) and replacing and repealing Council Decisions 2009/371/JHA, 2009/934/JHA, 2009/935/ JHA, 2009/936/JHA and 2009/968/JHA [2016] OJ L 135/53. 17  According to Article 76 of Regulation (EU) 2016/794, ‘rules and measures adopted by the Management Board on the basis of Decision 2009/371/JHA shall remain in force after 1 May 2017, unless otherwise decided by the Management Board in the application of this Regulation’. 18  Article 71(2) of Regulation (EU) 2016/794. 19  Some aspects of the analysis of Europol’s international cooperation practice draw on two semi-structured interviews conducted with Europol officials involved in international cooperation aspects of the agency. 14  Convention based on Article K.3 of the Treaty on the European Union of 27 November 1995, on the establishment of a European Police Office (Europol Convention) [1995] OJ C 316/1. 17  According to Article 76 of Regulation (EU) 2016/794, ‘rules and measures adopted by the Management Board on the basis of Decision 2009/371/JHA shall remain in force after 1 May 2017, unless otherwise decided by the Management Board in the application of this Regulation’. 19  Some aspects of the analysis of Europol’s international cooperation practice draw on two semi-structured interviews conducted with Europol officials involved in international cooperation aspects of the agency. 18  Article 71(2) of Regulation (EU) 2016/794. 1.  Introduction First, to provide an assessment of Europol’s international dimension based on its former legal framework and its international cooperation practice.19 This is motivated not only by the fact that Europol’s inter­ national cooperation instruments under the former legal framework remain valid bases for cooperation, but also by the fact that the new Europol Regulation amends the agency’s international dimension in response to perceived legal problems and shortcomings rooted in the Europol Council Decision. Second, it gives a fresh appraisal of Europol’s redesigned international dimension based on the new Europol Regulation with a view to mapping prospectively its legal effects and potential problems regarding Europol’s operation as a global actor, and 18  Article 71(2) of Regulation (EU) 2016/794. 19  Some aspects of the analysis of Europol’s international cooperation practice draw on two semi-structured interviews conducted with Europol officials involved in international cooperation aspects of the agency. EUROPE AND THE WORLD to assess the way in which it addresses crucial aspects of the agency’s international cooperation compared to the ‘moribund’ Europol legal framework. In this respect, the article addresses the more general question of the room left under the Founding Treaties for EU agencies like Europol to carry out international cooperation. The legal–analytical blueprint employed for this purpose uses the principle of institutional balance, the Meroni doctrine and the concept of (binding) ‘international agreement’ as parameters for determining the legal nature and impli­ cations of Europol’s international cooperation instruments, as well as the compatibility with the EU constitutional framework of the agency’s international dimension under the former Europol Council Decision and the new Europol Regulation. In order to address these legal questions in a dynamic and comparative fashion, the analysis focuses on essential formal legal design aspects of Europol’s old and new international dimensions. The paper investigates specifically the way in which the international cooperation mandate of Europol is defined, the agency’s international cooperation tasks and powers, substantive and procedural aspects regarding the agency’s formal international cooperation instruments, as well as limitations, supervision and controls over the agency’s inter­ national cooperation activities. 20  The Common Approach on EU agencies (para 25) foresees the conclusion by EU agencies of arrangements with third countries and international organizations excluding the possibility to commit the EU to international obligations. 1.  Introduction Special attention is given to Europol’s cooperation agreements as they represent arguably one of the most problematic aspects as to the compatibility of the agency’s interna­ tional dimension with the EU constitutional framework concerning external relations.20 Europol’s international cooperation practice, which is relevant for gaining a deeper understanding into how the agency’s international dimension actually works and for determining the legal nature of Europol’s concrete international cooperation instruments, is examined only with regard to the former legal framework as at the moment of writing there was no practice under the new Regulation yet. The possibility to apply mutatis mutandis findings concerning a special agency like Europol to the wider population of EU agencies should be regarded with caution. Yet the study of Europol on the basis of the above legal–analytical framework mentioned previously may offer valuable insights with regard to broader issues, such as the nature, scope and limits of the external powers that may be conferred on EU agencies, the conditions for the exercise of such powers, the application reshaping the external dimension of EU police cooperation 5 of the principle of institutional balance, and the assessment of EU agencies’ international cooperation practices and instruments. Also the transformation of Europol’s international dimension through the new Europol Regulation might indicate a broader trend towards stream­ lining EU agencies’ international cooperation, entailing that some of the elements introduced by the agency’s new legal framework might be transplanted to other EU agencies in the future. The article begins with a brief overview of the evolution and design of the agency’s general mandate (section 2). Thereafter, Europol’s international dimension according to the Europol Council Decision is scrutinized, by addressing both the formal legal framework for the agency’s international cooperation and its international practice. Besides examining the international cooperation mandate and tasks of the agency, the terminology and types, content and structure, effects and practical aspects concerning the procedure for the negotiation and conclusion of Europol’s cooperation agreements are addressed (section  3). This is followed by a mapping out of the design, the legal implications and potential problems of Europol’s international dimension according to the new Europol Regulation (section 4). Next, the article advances a legal–analytical blueprint providing the benchmarks for assessing Europol’s international dimension both under the old and the new legal frameworks (section 5). 1.  Introduction Section 6 offers an assessment of Europol’s international dimension with regard to the legal nature and effects of its international cooperation instruments, and as to the compatibility of its international cooperation with the European Union’s legal–institutional framework. 21  The Europol Convention can be best described as an intergovernmental agreement concluded by the Member States within the framework of the EU; see Rijken (n 10) 582–3. 22  Heimans (n 10) 369. 25  Steve Peers, EU Justice and Home Affairs Law (OUP 2011) 932. 26  Alexandra de Moor and Gert Vermeulen, ‘The Europol Council Decision: Transforming Europol into an Agency of the European Union’ (2010) 47 CML Rev 1089, 1097–1104 and 1120–1. 27  Council Decision 2009/934/JHA of 30 November 2009 adopting the implementing rules governing Europol’s relations with partners, including the exchange of personal data and classified information [2009] OJ L325/6, and Council Decision 2009/935/JHA of 30 November 2009 determining the list of third states and organizations with which Europol shall conclude agreements, [2009] OJ L325/12. 23  Rijken (n 10) 583. 24  Heimans (n 10) 369–70. 2.  Europol’s legal design and incremental evolution: from a Member States’ agency to an EU agency Europol was established on the basis of Article K.1 TEU by the 1995 Europol Convention21 and became operational on 1 July 1999. This prompted the conclusion that Europol was not really an EU agency but a ‘fully fledged international organization’ equipped with ‘full legal personality, including the capacity to enter into binding agreements under international law’22 or at least a ‘European organization sui EUROPE AND THE WORLD 6 generis of the EU’.23 The centre of gravity of Europol’s mandate was on information processing and exchange tasks.24 After ten years of operation as an intergovernmental body, Europol’s legal framework has been replaced by Council Decision 2009/371/JHA (Europol Council Decision). This was an important milestone in the evolution of Europol with implications for its legal  status  and operation. Most notably, although it kept its legal personality, Europol was transformed into an EU agency, meaning that it was financed from the EU budget and subject to staff regulations and all relevant EU rules.25 However, with regard to the agency’s main tasks, its governance and control, the Europol Council Decision implemented overall cosmetic changes.26 The same holds for the inter­ national dimension of Europol. The Europol Council Decision, together with the relevant implementing measures,27 basically maintained the status quo of the Europol Convention, including the agency’s capacity to conclude agreements with third countries and international organizations.28 With the entry into force of the Lisbon Treaty, the fields from the former ‘third pillar’ were brought together with the other elements of the Area of Freedom, Security and Justice (AFSJ) within Title V of the Treaty on the Functioning of the European Union (TFEU). Accordingly, police cooperation became a shared competence,29 triggering the application of the standard EU instruments and decision-making procedures. In this vein, Article 88 TFEU (Europol’s treaty legal basis) ensures that the legal framework of the agency would be established by regulations adopted according to the ordinary legislative procedure. Whereas Protocol No. 36 on Transitional Provisions still allowed Europol to operate under the Europol Council Decision,30 the Lisbon Treaty has further consolidated 28  Moor and Vermeulen (n 26) 1121. 29  Article 4(2)(j) TFEU. j 30  According to Article 9 of Protocol No. 36, the legal effects of acts adopted on the basis of the EU Treaty before the entry into force of the Treaty of Lisbon are preserved ‘until those acts are repealed, annulled or amended in implementation of the Treaties’. 30  According to Article 9 of Protocol No. 36, the legal effects of acts adopted on the basis of the EU Treaty before the entry into force of the Treaty of Lisbon are preserved ‘until those acts are repealed, annulled or amended in implementation of the Treaties’. 27  Council Decision 2009/934/JHA of 30 November 2009 adopting the implementing rules governing Europol’s relations with partners, including the exchange of personal data and classified information [2009] OJ L325/6, and Council Decision 2009/935/JHA of 30 November 2009 determining the list of third states and organizations with which Europol shall conclude agreements, [2009] OJ L325/12. 25  Steve Peers, EU Justice and Home Affairs Law (OUP 2011) 932. 26  Alexandra de Moor and Gert Vermeulen, ‘The Europol Council Decision: Transforming Europol into an Agency of the European Union’ (2010) 47 CML Rev 1089, 1097–1104 and 1120–1. 27  Council Decision 2009/934/JHA of 30 November 2009 adopting the implementing rules governing Europol’s relations with partners, including the exchange of personal data and classified information [2009] OJ L325/6, and Council Decision 2009/935/JHA of 30 November 2009 determining the list of third states and organizations with which Europol shall conclude agreements, [2009] OJ L325/12. 28  Moor and Vermeulen (n 26) 1121. 29  Article 4(2)(j) TFEU. 30  According to Article 9 of Protocol No. 36, the legal effects of acts adopted on the basis of the EU Treaty before the entry into force of the Treaty of Lisbon are preserved ‘until those acts are repealed, annulled or amended in implementation of the Treaties’. 23  Rijken (n 10) 583. 24  Heimans (n 10) 369–70. 29  Article 4(2)(j) TFEU. 31  Article 47 TEU. 33  See Jean-Claude Piris, The Lisbon Treaty: a Legal and Political Analysis (CUP 2010) 259–60. 2.  Europol’s legal design and incremental evolution: from a Member States’ agency to an EU agency reshaping the external dimension of EU police cooperation 7 7 the international legal status of the EU, by providing explicitly for the European Union’s legal personality,31 by codifying its competence to conclude international agreements32 and, arguably, by establishing a ‘single treaty-making procedure’ under Article 218 TFEU.33 Both the Europol Council Decision34 and Article 88(1) TFEU emphasize the role of Europol as a supportive structure for the enforcement authorities of the Member States, and not as a ‘European FBI’ with fully fledged investigative and enforcement powers. Above all, Europol’s information-related tasks defined the ‘core business’ of the agency.35 The new Europol Regulation maintains the core principles upon which the role and functioning of Europol were premised, but also brings some noteworthy novelties. First, it expands Europol’s ratione materiae competence by supplementing the list of serious crimes,36 and by adding ‘forms of crime which affect a common interest covered by a Union policy’.37 Second, it further consolidates Europol’s core informa­ tion-related tasks by establishing an obligation for the Member States to provide information to the agency.38 Third, it aims to strengthen through the involvement of the European Data Protection Supervisor (EDPS) the supervision over Europol’s processing of personal data,39 and to increase parliamentary supervision over Europol’s activities in line with Article 88(2) TFEU.40 Fourth, the Commission is given a more influential role with regard to the agency.41 Overall, the new Regulation can be seen as an important step towards transforming Europol from a ‘special’ agency into an ‘ordinary’ EU agency in line with the Common Approach on EU agencies. 35  Moor and Vermeulen (n 26) 1099. 36  For example, as compared to the Europol Council Decision, Annex I of Regulation 2016/794 adds the category ‘genocide, crimes against humanity and war crimes’ to Europol’s mandate. p 37  Article 3(1) of Regulation 2016/794; an example is the category of ‘crimes against the financial interests of the Union’ listed in Annex I of the Regulation. 38  Recital (13) and Article 7(6) of Regulation 2016/794. 38  Recital (13) and Article 7(6) of Regulation 2016/794. 39  Recitals (50)–(54) and Article 43 of Regulation 2016/794. 39  Recitals (50)–(54) and Article 43 of Regulation 2016/794. 40  Recital (58) and Articles 51–52 of Regulation 2016/794. 40  Recital (58) and Articles 51–52 of Regulation 2016/794. 41  See, for instance, Articles 12 and 68 of Regulation 2016/794. 41  See, for instance, Articles 12 and 68 of Regulation 2016/794. 32  Articles 3(2) and 216–17 TFEU. 32  Articles 3(2) and 216–17 TFEU. 33  See Jean-Claude Piris, The Lisbon Treaty: a Legal and Political Analysis (CUP 2010) 259–60. 34  Article 3. 35  Moor and Vermeulen (n 26) 1099. 36  For example, as compared to the Europol Council Decision, Annex I of Regulation 2016/794 adds the category ‘genocide, crimes against humanity and war crimes’ to Europol’s mandate. 37  Article 3(1) of Regulation 2016/794; an example is the category of ‘crimes against the financial interests of the Union’ listed in Annex I of the Regulation. 38  Recital (13) and Article 7(6) of Regulation 2016/794. 39  Recitals (50)–(54) and Article 43 of Regulation 2016/794. 40  Recital (58) and Articles 51–52 of Regulation 2016/794. 41  See, for instance, Articles 12 and 68 of Regulation 2016/794. 31  Article 47 TEU. 2.  Europol’s legal design and incremental evolution: from a Member States’ agency to an EU agency EUROPE AND THE WORLD 8 42  See concisely on these issues also Coman-Kund (n 11) 108, 110–12 and 114–15. 43  Article 23(3)–(9) of the Europol Council Decision. 42  See concisely on these issues also Coman-Kund (n 11) 108, 110–12 and 114–15. 43  Article 23(3)–(9) of the Europol Council Decision. 44  See Title III, Articles 8–20 of Council Decision 2009/934/JHA. 45  Article 1(g) and (h) of Council Decision 2009/ 934/JHA. 46  The ‘list’ took the form of Council Decision 2009/935/JHA and initially included twenty-five third countries and three organizations. The Council’s ‘list’ was amended in May 2014, when four third countries were added (Brazil, Georgia, Mexico, United Arab Emirates) – see Article 1 of Council Implementing Decision of 6 May 2014 amending Decision 2009/935/JHA as regards the list of third States and organizations with which Europol shall conclude agreements, [2014] OJ L138/104. 46  The ‘list’ took the form of Council Decision 2009/935/JHA and initially included twenty-five third countries and three organizations. The Council’s ‘list’ was amended in May 2014, when four third countries were added (Brazil, Georgia, Mexico, United Arab Emirates) – see Article 1 of Council Implementing Decision of 6 May 2014 amending Decision 2009/935/JHA as regards the list of third States and organizations with which Europol shall conclude agreements, [2014] OJ L138/104. 45  Article 1(g) and (h) of Council Decision 2009/ 934/JHA. 43  Article 23(3)–(9) of the Europol Council Decision. 3.  Europol’s international cooperation legal framework and practice under Council Decision 2009/371/JHA42 3.1  Legal framework The legal framework for Europol’s international cooperation activities was provided primarily by Article 23 of the Europol Council Decision complemented by Council Decision 2009/934/JHA. Article 23(1) of the Europol Council Decision stipulated clearly that Europol’s inter­ national cooperation activities were instrumental to the core mandate and tasks of the agency. This provision also made clear that the quasi exclusive way for the agency to formalize its relationship with partners outside the EU was by concluding cooperation agreements. What is more, it exhibited that exchange of information related to crimes within Europol’s mandate was the main concern of such instruments.43 Council Decision 2009/934/JHA also touched on the issue of information exchange between Europol and its external partners,44 and introduced a distinction between operational and strategic agreements concluded by Europol with its partners, depending on the information which could be exchanged under each type of instrument.45 Yet importantly, the Europol Council Decision provided two important limitations on Europol’s international cooperation. According to Article 23(2) in combination with Article 26(1) of Council Decision 2009/371/JHA the agency was bound to conclude agreements only with third countries and international organizations put on a ‘list’ drawn up by the Council, on the proposal of Europol’s Management Board.46 Furthermore, Article 23(2) subjected each Europol’s agreement to prior approval by the Council. reshaping the external dimension of EU police cooperation 47  Article 6(1) and (2) of Council Decision 2009/934/JHA. 48  Article 6(3) of Council Decision 2009/934/JHA. 49  Article 6(4) of Council Decision 2009/934/JHA. 50  Article 6(4) of Council Decision 2009/934/JHA. Post Lisbon, the approval took the shape of a Council implementing decision. 51  Emma Disley et al, ‘Evaluation of the Implementation of the Europol Council Decision and of Europol’s Activities’ (technical report), RAND Europe (2012) 113. 52  Article 5(4) of Council Decision 2009/934/JHA. 53  Established as an independent body outside the structure of the agency, the JSB was Europol’s data protection control authority; see Article 34 of the Europol Council Decision and Heimans (n 10) 371. 54  Article 6(1) of Council Decision 2009/934/JHA. 47  Article 6(1) and (2) of Council Decision 2009/934/JHA. 48  Article 6(3) of Council Decision 2009/934/JHA. 55  Article 6(3) of Council Decision 2009/934/JHA. 56  Article 37(9)(a) of the Europol Council Decision. 57  For example, Europol Strategy 2016–2020, (2015) EDOC# 796794v19B, available at <https://www.europol.europa.eu/content/europol-strategy-2016-2020>. 58  For example, Europol External Strategy 2010–2014, (2010) EDOC # 434663; Europol External Strategy 2017–2020 (2016) EDOC#865852v3. 59  According to a Europol official, ‘the setting of Europol’s international strategy is a mix of (politically) predetermined elements and of operationally driven considerations’, Interview 5, The Hague, 29 January 2013. 60  Interview 13 with a Europol official, The Hague, 1 July 2013. 61  This is in line with the Common Approach on EU agencies (para 25) providing for Commission’s role to oversee EU agencies’ international cooperation. 62  E.g. the Group on External JHA issues (JAIEX), Coordinating Committee in the area of police and judicial cooperation in criminal matters (CATS), Law Enforcement Working Party (LEWP). 63  See Madalina Busuioc, Deirdre Curtin and Martijn Groenleer, ‘Agency Growth between Autonomy and Accountability: the European Police Office as a “Living Institution”‘ (2011) J Eur Publ Pol 862. reshaping the external dimension of EU police cooperation 9 3.1.2  The procedure for Europol’s cooperation agreements According to Articles 5–6 of Council Decision 2009/934/JHA, the detailed procedure for negotiating and concluding Europol’s interna­ tional agreements included the following milestones: • the negotiations with a third country or international organization included on the Council’s ‘list’ were carried out by the Executive Director, supported by the relevant internal units of Europol;47 • after the conclusion of negotiations, the Executive Director had to submit the draft agreement to the Management Board for endorsement;48 • the draft agreement endorsed by the Management Board was then submitted to the Council for approval (which made a decision after having consulted the Management Board);49 • the agreement was concluded (signed by the Executive Director) after the Council’s approval.50 Whereas for strategic agreements the steps mentioned previously were sufficient, for operational agreements there were additional procedural requirements.51 First, before initiating negotiations, Europol had to carry out ‘an assessment of the existence of an adequate level of data protection ensured by the third party’.52 This assessment was forwarded to the Management Board, which sent it to the Joint Supervisory Body (JSB) for an opinion.53 After receiving the opinion of the JSB, the Management Board decided whether to authorize the Executive Director to start negotiations on an operational agreement.54 Second, after the negotiations, the draft agreement was submitted to the Management Board, which asked again for the opinion of the JSB, this time on the draft agreement.55 4  Article 6(1) of Council Decision 2009/934/JHA. 4  Article 6(1) of Council Decision 2009/934/JHA. 55  Article 6(3) of Council Decision 2009/934/JHA. EUROPE AND THE WORLD 10 3.2  Europol’s international dimension in practice 3.2  Europol’s international dimension in practice 3.2.1  Setting the international cooperation priorities and actions 3.2.1  Setting the international cooperation priorities and actions Europol’s overall priorities, including in the area of international cooperation, are framed in multiannual documents adopted by the Management Board56 in the shape of four-year general strategies57 and specific strategies focusing on external relations issues.58 The overall planning of Europol’s priorities concerning international cooperation could be depicted as mainly being operationally driven, but also subject to policy-political influences.59 First, although driven mostly by operational needs, setting the agency’s priorities was tightly restricted and controlled by the Council and the Member States via the Management Board. Second, the consistency between Europol’s inter­ national cooperation priorities and the European Union’s operational and political needs and priorities, including taking into account of the sudden changes in the EU external action,60 is guaranteed mainly by the Commission61 and through relevant Council committees and working parties.62 This suggests a rather limited discretion for Europol in conducting international cooperation because of the restrictions set by the agency’s legal framework and the constraints imposed by its operational and political environment.63 56  Article 37(9)(a) of the Europol Council Decision. 63  See Madalina Busuioc, Deirdre Curtin and Martijn Groenleer, ‘Agency Growth between Autonomy and Accountability: the European Police Office as a “Living Institution”‘ (2011) J Eur Publ Pol 862. 3.2.2  Europol’s cooperation agreements 3.2.2  Europol’s cooperation agreements According to Council Decision 2009/934/JHA, the scope of Europol’s strategic agreements was more limited in that they allow for the exchange of information, except for personal data, while operational agreements included the issues covered by strategic agreements plus 63  See Madalina Busuioc, Deirdre Curtin and Martijn Groenleer, ‘Agency Growth between Autonomy and Accountability: the European Police Office as a “Living Institution”‘ (2011) J Eur Publ Pol 862. reshaping the external dimension of EU police cooperation 11 personal data exchanges. Put simply, the two types of agreements provided the ‘toolbox’ allowing Europol to use one instrument or another depending on the level of trust in its partners: strategic agreements to establish formal cooperation with partners whose data protection system or human rights record might be problematic64 and operational agreements with close partners. Europol currently has twenty-six agreements: eighteen operational agreements (seventeen with third countries65 and one with Interpol) and eight strategic agreements (six with third countries66 and the remaining two with United Nations Office on Drugs and Crime (UNODC) and World Customs Organization (WCO)).67 The wording of both strategic and operational agreements features prescriptive ‘shall/will’ clauses. Regarding the structure, both strategic and operational agreements include a preamble, a main body and usually one or more annexes. The provisions of all the cooperation agreements are structured in articles. The core part of all Europol’s agreements deals mostly with the exchange of operational, strategic and technical information relating to the areas of crime within Europol’s mandate. Rules on the detailed procedure for information exchange (including classified information), specific limitations and requirements, as well as confidentiality duties are included. Other aspects normally included in the core part of the agreements deal with issues such as the exchange of expertise and training. Importantly, the agreements provide for the possibility to assign liaison officers. The final provisions of the strategic agreements usually cover dispute settlement, amendment procedures, entry into force, termination of the agreement and the relationship with other international cooperation instruments. The entry into force of the agreements is usually linked to the date on which the third country notifies Europol about the completion of its own internal procedures. The agreements stipulate that Europol may consent to amendments only after approval by the Council, thereby re-confirming that the agency does not act fully independently in its relationship with its partners. 64  Heimans (n 10) 382. 65  Albania, Australia, Bosnia and Herzegovina, Canada, Colombia, Former Yugoslav Republic of Macedonia (FYROM), Georgia, Iceland, Liechtenstein, Moldova, Monaco, Montenegro, Norway, Serbia, Switzerland, Ukraine, USA (2002). 66  Brazil, China, Russian Federation, Turkey, United Arab Emirates, USA (2001). 67  A list of cooperation agreements is available at <https://www.europol.europa.eu/ partners-agreements>. 68  Operational agreements with Canada and USA. 69  Article 3(3). 70  Articles 11 and 13. 71  Elspeth Guild et al, ‘Implementation of the EU Charter of Fundamental Rights and its Impact on EU Home Affairs Agencies: Frontex, Europol and the European Asylum Support Office’, Study for the European Parliament, Committee on Civil Liberties, Justice and Home Affairs (LIBE) (PE 453.196/2011) 74; see also Heimans (n 10) 385–7. 72  Interview 5 (n 59). 73  Interview 13 (n 60). 3.2.2  Europol’s cooperation agreements According to the standard procedure, the termination of the agreements requires a written notification and a three-month notification period. EUROPE AND THE WORLD 12 12 Additionally, the operational agreements include detailed provisions on the procedures and obligations of the parties concerning the exchange, processing and handling of personal data, including conditions and restrictions for the onward transmission of the data exchanged with third parties. They also lay down a right for the individuals and private entities to have access to personal data concerning them that is processed under the agreement, and usually comprise elaborated provisions on the liability of the contracting parties for damages caused to individuals as a result of errors in the exchange and processing of personal data. It should be also noted that few of Europol’s operational agreements depart from the standard model described above.68 The 2002 Supplemental Agreement with the United States deserves special attention for its emphasis on the fact that it does not give rise to a right on behalf of any private party to obtain, suppress, exclude any evidence or to impede the execution of a request.69 Next, quite surprisingly, the agreement does not mention anything about the adequate level protection of personal data and says little about data protection safeguards. What is more, this agreement does not contain clear provisions on liability, and it does not include elaborated provisions on dispute settlement.70 These issues raised legal concerns as it was suspected that this agreement did not meet essential legal and data protection safeguards provided by the Europol legal framework, thereby potentially infringing fundamental rights.71 Regarding the procedure for the negotiation and conclusion of the cooperation agreements, in practice, the initiation of contacts with potential partners started after receiving a request, which was normally channelled through the Management Board.72 An important practical aspect is that both JSB’s (formally non-binding) opinions on the data protection report and on the draft agreement to be signed between the parties seemed to be quite influential.73 Also, throughout the whole process (initiation, negotiations, conclusion) there were informal and formal interactions between the agency and the Council, reshaping the external dimension of EU police cooperation 74  Interviews 5 (n 59) and 13 (n 60). 75  Case C-363/14 Parliament v Council EU:C:2015:579. In that case, the EP argued essentially that the contested Council implementing decision had been adopted based on an invalid secondary law legal basis (ie, Article 26 of the Europol Council Decision), and that, moreover, EP’s prerogative to be consulted before the adoption of that decision had been breached. 76  See for previous cases on this matter, Joined Cases C-317/13 and C-679/13 Parliament v Council EU:C:2015:223, and Case C-540/13 Parliament v Council EU:C:2015:224. 77  Case C-363/14 Parliament v. Council, paras 70–72. 78  ibid, paras 82–85. 79  Council doc. 12035/15, Judgment of the Court of 10 September 2015 in Case C-363/14, ‘European Parliament v Council for the European Union – legality of a Council implementing Decision based on the 2009 Europol Decision concerning the list of third states with which Europol can conclude cooperation agreements’, 7 October 2015, 6. 80  For a brief analysis, see also Coman-Kund (n 11). reshaping the external dimension of EU police cooperation 13 the Commission as well as the Member States.74 This suggests dynamics going beyond the formal legal procedures, entailing more involvement of the main EU external action actors in Europol’s most important inter­ national cooperation agreements than on paper. A recent development regarding the procedure for the conclusion of Europol’s agreements flowed from Court of Justice of the European Union (CJEU)’s judgment in Case C-363/1475 concerning the action for annulment of Council Implementing Decision 2014/269/EU extending the Council’s ‘list’. The Court confirmed that Article 26(1)(a) of the Europol Council Decision represented a valid secondary legal basis76 for adopting Council implementing acts such as the one under consideration,77 and established a duty for the Council to consult the European Parliament (EP) based on former Article 39(1) TEU when adopting such measures.78 The Council gave an extensive interpretation to this consultation duty by extending it to all implementing measures adopted under the old ‘third pillar’ framework.79 Accordingly, after September 2015, the Council imple­ menting decisions approving Europol’s agreements with third countries were also subject to prior EP consultation. In short, this resulted in strengthening EP’s oversight by adding an extra condition to the procedure for the negotiation and conclusion of the agency’s cooperation agreements. A recent development regarding the procedure for the conclusion of Europol’s agreements flowed from Court of Justice of the European Union (CJEU)’s judgment in Case C-363/1475 concerning the action for annulment of Council Implementing Decision 2014/269/EU extending the Council’s ‘list’. The Court confirmed that Article 26(1)(a) of the Europol Council Decision represented a valid secondary legal basis76 for adopting Council implementing acts such as the one under consideration,77 and established a duty for the Council to consult the European Parliament (EP) based on former Article 39(1) TEU when adopting such measures.78 The Council gave an extensive interpretation to this consultation duty by extending it to all implementing measures adopted under the old ‘third pillar’ framework.79 Accordingly, after September 2015, the Council imple­ menting decisions approving Europol’s agreements with third countries were also subject to prior EP consultation. In short, this resulted in strengthening EP’s oversight by adding an extra condition to the procedure for the negotiation and conclusion of the agency’s cooperation agreements. , p 79  Council doc. 12035/15, Judgment of the Court of 10 September 2015 in Case C-363/14, ‘European Parliament v Council for the European Union – legality of a Council implementing Decision based on the 2009 Europol Decision concerning the list of third states with which Europol can conclude cooperation agreements’, 7 October 2015, 6. 74  Interviews 5 (n 59) and 13 (n 60). 78  ibid, paras 82–85. 80  For a brief analysis, see also Coman-Kund (n 11). 81  ‘Impact Assessment on Adapting the European Police Office’s Legal Framework with the Lisbon Treaty’ SWD (2013) 98 final, 3–4. 82  Commission, ‘Proposal for a Council Decision establishing the European Police Office (Europol)’ COM (2006) 817 final, 11; Commission, ‘Impact Assessment on Adapting the European Police Office’s Legal Framework with the Lisbon Treaty’ SWD (2013) 98 final, 3. See also Heimans (n 10) 389; Moor and Vermeulen (n 26) 1107–8. 83  See House of Commons, European Scrutiny Committee, Reforming Europol 3rd Report of Session 2013–2014 (TSO 2013), HC 83-iii (Session 2013/14), 13. 84  Busuioc, Curtin and Groenleer (n 63) 857–8. On the legal concerns raised by this agreement, see Guild et al (n 71) 74. 85  EP legislative resolution of 17 January 2008 on the proposal for a Council decision establishing the European Police Office (EUROPOL), P6_TA (2008)0015; Committee on Civil Liberties, Justice and Home Affairs, Meeting 4-5 November 2009, Joint debate on Europol related files, LIBE(2009)1104_1; see also Moor and Vermeulen (n 26) 1096–1097. 86  Commission, ‘Proposal for a Regulation of the European Parliament and of the Council on the European Union Agency for Law Enforcement Cooperation and Training (Europol) and repealing Decisions 2009/371/JHA and 2005/681/JHA’ COM (2013) 173 final, 4. 87  See in particular para 25 of the Common Approach (n 1). 4.  The new Europol Regulation: redesigning Europol’s international dimension The new Europol Regulation substantively redesigns Europol’s inter­ national dimension.80 Among the factors that arguably triggered the overhaul of the agency’s international cooperation were the demand for aligning Europol’s international relations with the post-Lisbon EUROPE AND THE WORLD 14 14 EU external action constitutional framework, the concerns regarding the insufficient parliamentary/democratic scrutiny over the agency’s international activities, and the need to streamline its international dimension in line with the Common Approach on EU agencies.81 The Commission in particular had been sensitive over time as regards Europol’s power to conclude its own international cooperation agreements as this could affect the coherence of the Union’s external relations and collide with the institutional balance established by the Founding Treaties regarding EU external representation and treaty-making.82 Post Lisbon, Articles 216 and 218 TFEU offered arguably more support for an exclusive EU treaty-making procedure which rendered allegedly the possibility for Europol to conclude binding international agreements highly questionable.83 Also the EP’s frustration regarding its longstanding non-involvement in Europol’s design and activities (including highly controversial aspects of the agency’s international cooperation, such as the 2002 Supplemental Agreement with USA)84 had been arguably one of the drivers behind the reform of Europol’s international dimension. As such, the EP had been eager to seize the opportunity offered by Article 88 TFEU in order to gain the possibility to decide on an equal footing with the Council upon Europol’s legal design, operation and scrutiny (including inter­ national exchanges of data).85 As stated in the Commission’s proposal for the Europol Regulation,86 the Common Approach on EU agencies, which foresees an enhanced relationship between the Commission and EU agencies for consistency reasons and for ensuring that these bodies do not commit the Union to international obligations, also played a role in the reshaping of Europol’s international dimension.87 reshaping the external dimension of EU police cooperation 15 15 First, Europol is brought in line with other EU agencies, in that the agency will formally establish cooperative relations with the competent authorities of third countries (instead of third countries as provided by the former Europol legal framework).88 Second, in response to the concerns regarding institutional balance in EU external relations and the unity of the Union’s external repre­ sentation, there will no longer be Europol cooperation agreements. 88  Article 23(1) of Regulation 2016/794. 89  According to Article 25(4) of Regulation 2016/794, the Commission will review all Europol’s cooperation agreements by 14 June 2021, in particular with regard to data protection. 90  Europol External Strategy 2017–2020 (2016) EDOC#865852v3, 4. 91  Article 25(1) and (4) of Regulation 2016/794. 92  Article 218 (6)(a) TFEU juncto Article 88(2) TFEU. 93  Article 218(4) TFEU. 88  Article 23(1) of Regulation 2016/794. 89  According to Article 25(4) of Regulation 2016/794, the Commission will review all Europol’s cooperation agreements by 14 June 2021, in particular with regard to data protection. p 90  Europol External Strategy 2017–2020 (2016) EDOC#865852v3, 4. 4.  The new Europol Regulation: redesigning Europol’s international dimension Whilst Europol’s agreements concluded by 1 May 2017 are preserved89 and will remain for the time being the basis for cooperation with third countries and international organizations,90 future international agreements allowing the agency to transfer personal data will be concluded according to Article 218 TFEU.91 However, the Europol Regulation does not clarify whether and how the agency will be involved in the negotiation of the relevant agreements foreseen under Article 218 TFEU. Article 11(2) of the Europol Regulation only provides for the possibility of the Management Board to make a suggestion to the Council to draw the attention of the Commission to the need for concluding an ‘Article 218 TFEU’ inter­ national agreement. From this provision it can be inferred that the initialling of an international agreement allowing Europol to exchange personal data will not so much depend on the agency anymore, but on the Council and the Commission. Furthermore, the application of the procedure for ‘Article 218 TFEU’ agreements will entail Council decisions concerning the opening of negotiations, adopting negotiating directives, authorizing the signing of and concluding the agreement, negotiations by the negotiator designated by the Council, and EP’s consent.92 Within this procedure, Europol representatives might eventually be involved either in the negotiating team or in the special consultation committee which may be designated by the Council in order to support the negotiator.93 In any case, this represents an important shift in Europol’s role in international cooperation, as compared to the Europol Council Decision, where the agency was in the lead as regards the initiation, negotiation and conclusion of the cooperation agreements. This change largely reflects Commission’s EUROPE AND THE WORLD 16 view regarding the revision of Europol’s international cooperation with a view to fully align it with the Lisbon Treaty and to increase coherence in the Union’s external relations.94 With regard to the latter aspect, the negotiation and conclusion of international agreements concerning Europol by the main EU external action actors are expected to ensure better that broader strategic aspects of the Union’s external relations are observed in the agency’s international cooperation activities. Furthermore, it remains to be seen how effective the procedure laid down in Article 218 TFEU will be. Thus, political and non-­ specialized actors might not be sufficiently familiar with the specific and highly technical issues covered by the current Europol agreements. 95  Article 23(4) of Regulation 2016/794. 94  See Moor and Vermeulen (n 26) 1107–8. 94  See Moor and Vermeulen (n 26) 1107–8. 95  Article 23(4) of Regulation 2016/794. 4.  The new Europol Regulation: redesigning Europol’s international dimension What is more, there might be a risk for wider political preferences in the Union’s external action area being privileged to the detriment of Europol’s operational priorities. Also the negotiation and conclusion of agreements under Article 218 TFEU might take longer than in the case of Europol’s cooperation agreements. Besides, it is not very clear what kind of agreements will be concluded under Article 218 TFEU. One option would be to conclude special agreements concerning Europol’s cooperation with a third country or international organization, in particular with regard to exchanges of personal data. Such instruments would be very much like the current Europol’s cooperation agreements. Another option could be the conclusion of broader framework agreements between the EU and the respective international partner covering various cooperation aspects, and including Europol. Under the latter scenario, the negotiation process might meet with specific difficulties depending on the breadth and complexity of the issues covered by such an agreement. Third, Europol is still allowed to conclude specific international cooperation instruments. In this respect, the Europol Regulation distin­ guishes between working arrangements and administrative arrange­ ments in a way that evokes the demarcation between strategic and operational agreements under the old legal framework. The working arrangements are designed to cover cooperative relations, except for exchanges of personal data, and are explicitly characterized as not being binding for the EU and the Member States.95 Europol’s adminis­ trative arrangements are intended to enable personal data exchanges by implementing ‘Article 218 TFEU’ agreements, cooperation agreements concluded by Europol before 1 May 2017, or the Commission’s reshaping the external dimension of EU police cooperation 17 ‘adequacy decisions’ regarding the transfer of personal data, but, unlike the working arrangements, nothing is mentioned about their legal nature.96 While on the face of it, the provisions of the new Regulation suggest that such instruments would likely qualify as soft law measures97 (as shown in section 5), determining their legal nature requires a case-by-case analysis of each particular instrument in light of international law criteria. 96  Article 25(1) of Regulation 2016/794.   97  For a recent analysis of the concept of ‘soft law’ and of soft law instruments at EU level, see Fabien Terpan, ‘Soft Law in the European Union: the Changing Nature of EU Law’ (2015) 21 Eur LJ (2015) 68.   98  Article 11(1)(r) of Regulation 2016/794.   99  Based on Directive (EU) 2016/680 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data by competent authorities for the purposes of the prevention, investigation, detection or prosecution of criminal offences or the execution of criminal penalties, and on the free movement of such data, and repealing Council Framework Decision 2008/977/ JHA, [2016] OJ L 119/89. 100  Article 25(1) of Regulation 2016/794. 101  Article 25(2) of Regulation 2016/794. 102  See in particular paras 29 and 60 of the Common Approach on EU agencies (n 1). 100  Article 25(1) of Regulation 2016/794. 96  Article 25(1) of Regulation 2016/794. 102  See in particular paras 29 and 60 of the Common Approach on EU agencies (n 1). 101  Article 25(2) of Regulation 2016/794. 97  For a recent analysis of the concept of ‘soft law’ and of soft law instruments at EU level, see Fabien Terpan, ‘Soft Law in the European Union: the Changing Nature of EU Law’ (2015) 21 Eur LJ (2015) 68. 102  See in particular paras 29 and 60 of the Common Approach on EU 4.  The new Europol Regulation: redesigning Europol’s international dimension It should be also noted that, unlike the Europol Council Decision, the new Regulation does not provide details regarding the procedure for the negotiation and conclusion of Europol’s working and admin­ istrative arrangements, except that they need to be approved by the Management Board.98 As compared to Europol’s cooperation agreements, the new international cooperation framework does not foresee Council’s approval or EP’s compulsory consultation for the agency’s new international cooperation tools. Fourth, the new Regulation enhances the Commission’s role with regard to Europol’s international dimension. Most obviously, this is exhibited by formalizing the Commission’s ‘adequacy decisions’99 as one of the legal bases, allowing the agency to exchange personal data. As already mentioned, the implementation of such decisions may require the conclusion of specific administrative arrangements between the agency and competent authorities of third countries and international organizations100 moreover, Europol’s Executive Director has a duty to report to the Management Board on the implementa­ tion of the Commission’s ‘adequacy decisions’.101 Additionally, Article 25(4) of the new Regulation further bolsters the Commission’s position by granting it the power to review Europol’s ‘surviving’ cooperation agreements with a view to maintain or replace them with ‘Article 218’ agreements. In line with the Common Approach on EU Agencies,102 EUROPE AND THE WORLD 18 the new Regulation also introduced more general tools, which enable presumably the Commission to influence the agency’s international cooperation. This entails giving the Commission a say with regard to Europol’s multiannual planning  and annual work programmes,103 as well as presumably the role assigned to the Commission as regards the periodic evaluation of the agency.104 Fifth, the new Regulation formally enhances parliamentary scrutiny of Europol’s international cooperation as compared to the former Europol Council Decision. 103  Article 12(1) of Regulation 2016/794. 104  Article 68 of Regulation 2016/794. 105  Article 51(1) of Regulation 2016/794. 106  Article 51(2) of Regulation 2016/794. 107  Article 51(3)–(4) of Regulation 2016/794. 108  Article 51(5) of Regulation 2016/794. 109  Article 52 of Regulation 2016/794. 110  See Articles 51(4) and 52(1)–(2) juncto Article 67 of Regulation 2016/794. 111  See Article 43 of Regulation 2016/794. 4.  The new Europol Regulation: redesigning Europol’s international dimension A Joint Parliamentary Scrutiny Group (JPSG), which brings together members of the EP and of national parliaments, is established with a view to monitor Europol’s activities.105 For this purpose, the Europol Regulation imposes a duty on the chair of the Management Board, the Executive Director and other Europol officials to report before the JPSG on the agency’s activities.106 Moreover, an obligation is placed on Europol to transmit to JPSG various documents, including the administrative arrangements concluded with authorities of third countries and international organizations, for the purpose of enabling exchanges of personal data.107 The outcome of the monitoring carried out by the JPSG materializes in ‘conclusions’, which are submitted to the EP and the national parliaments.108 Additionally, the EP can access non-classified and, under certain conditions, also classified information processed by or through Europol.109 Nevertheless, two caveats should be mentioned both as regards JPSG monitoring and EP’s access to Europol information. To begin with, access to information will be subject to the agency’s own rules regarding protection of non-classified and classified information, which could at least in theory enable Europol to restrict to a certain extent parliamentary scrutiny of its activities.110 Next, the Europol Regulation does not provide anything concrete regarding the legal consequences of the scrutiny of Europol’s activities. Sixth, the supervision of Europol’s processing of personal data is reinforced by replacing the JSB with the European Data Protection Supervisor (EDPS) with far-reaching tasks and powers.111 In this respect, the EDPS is tasked to hear complaints, conduct inquiries, 103  Article 12(1) of Regulation 2016/794. 104  Article 68 of Regulation 2016/794. 105  Article 51(1) of Regulation 2016/794. 106  Article 51(2) of Regulation 2016/794. 107  Article 51(3)–(4) of Regulation 2016/794. 108  Article 51(5) of Regulation 2016/794. 109  Article 52 of Regulation 2016/794. 110  See Articles 51(4) and 52(1)–(2) juncto Article 67 of Regulation 2016/794. 111  See Article 43 of Regulation 2016/794. reshaping the external dimension of EU police cooperation Management Board, EDPS, Commission (formal role), EP/JPSG (more intense ex post formal reporting and information duties) 113  Article 43(3) of Regulation 2016/794. 112  Article 43(2) of Regulation 2016/794. 112  Article 43(2) of Regulation 2016/794. 113  Article 43(3) of Regulation 2016/794. reshaping the external dimension of EU police cooperation 19 monitor and ensure the application of the relevant EU legal framework in order to protect natural persons with regard to processing of personal data by Europol, including in the context of international exchanges of data.112 The EDPS may, inter alia, warn or admonish Europol, compel the agency to rectify, restrict, erase or destroy incorrectly processed personal data, ban Europol’s processing operations, or refer a matter to the EP, the Council, the Commission or the CJEU.113 Table 1. Comparative overview of Europol’s international dimension under the Europol Council Decision and the new Europol Regulation Europol’s International Dimension Europol Council Decision New Europol Regulation Mandate definition Instrumental – ancillary to core tasks Instrumental – ancillary to core tasks Tasks Information exchange (strategic and personal data) Information exchange (strategic and personal data) Formal cooperation instruments Cooperation agreements (strategic and operational) Working arrangements (non- personal data) and (imple­ menting) administrative arrangements (personal data) Procedures Elaborate: Council’s ‘list’, Management Board authorization to start negotiations, JSB opinions (for operational agreements), Management Board endorsement of draft agreement, Council approval Concise: Management Board to decide upon the conclusion of working and administrative arrangements Supervision and controls Council, Management Board, JSB, Commission (informally), EP (ex post information duties) Management Board, EDPS, Commission (formal role), EP/JPSG (more intense ex post formal reporting and information duties) Table 1. Comparative overview of Europol’s international dimension under the Europol Council Decision and the new Europol Regulation 113  Article 43(3) of Regulation 2016/794. EUROPE AND THE WORLD 20 115  Some of the landmark cases affirming the principle of institutional balance are Case 9/56 Meroni & Co, Industrie Metallurgiche, SpA v High Authority of the European Coal and Steel Community EU:C:1958:7 and Case 10/56 Meroni & Co., Industrie Metallurgiche, società in accomandita semplice v High Authority of the European Coal and Steel Community EU:C:1958:8 (the Meroni cases); Case 25/70 Einfuhr- und Vorratsstelle für Getreide und Futtermittel v Köster and Berodt & Co EU:C:1970:115; Case C-70/88 Parliament v Council: Radioactive contamination of foodstuffs (Chernobyl) EU:C:1991:373. f f ff y 116  Sacha Prechal, ‘Institutional Balance: a Fragile Principle with Uncertain Contents’ in Ton Heukel, Niels Blokker and Marcel Brus (eds) The European Union after Amsterdam: a Legal Analysis (Kluwer Law International 1998) 280; Jean-Paul Jacqué, ‘The Principle of Institutional Balance’ (2004) 41 CML Rev 383, 384. 114  Based on the legal–analytical framework for assessing the international dimension of EU agencies developed in Coman-Kund (n 1), and further refined in Coman-Kund (n 11) 100–106. 5.  A legal–analytical framework for assessing Europol’s international dimension114 From an EU law perspective, the principle of institutional balance and the so-called Meroni doctrine, as essential parameters for determining the ‘constitutionality’ of the overall design and powers of EU agencies like Europol, bear particular importance also for assessing Europol’s international cooperation mandate, powers and actions. As for the international law perspective, the concept of ‘international agreement’ enables the clarification of the legal nature of Europol’s international cooperation instruments. 118  The list of ‘institutional balance’ requirements has been advanced initially by Lenaerts and Van Nuffel listing, however, requirements (2) and (3) in a reversed order; see Koen Lenaerts and Piet Van Nuffel, Constitutional Law of the European Union (Sweet & Maxwell 1999) 414. This list has been taken over and adapted by Majone, and adopted in this study as in the author’s view the current sequence of requirements reflects more logically the 5.1  The principle of institutional balance and the doctrine of delegation of powers 5.1  The principle of institutional balance and the doctrine of delegation of powers Developed gradually in CJEU’s case law115 and enshrined in Article 13(2) TEU, the principle of institutional balance has been portrayed by scholars as a tool performing a similar function at EU level to the principle of separation of powers in state constitutional systems.116 Thus, institutional balance is aimed at a system of checks and balances in which the prerogatives of the EU institutions are guaranteed.117 Based on this understanding, institutional balance requires ‘that each institution: (1) has the necessary independence in exercising its powers; (2) must respect the powers of the other institutions; and (3) may not unconditionally assign its powers to other institutions and bodies’.118 In Developed gradually in CJEU’s case law115 and enshrined in Article 13(2) TEU, the principle of institutional balance has been portrayed by scholars as a tool performing a similar function at EU level to the principle of separation of powers in state constitutional systems.116 118  The list of ‘institutional balance’ requirements has been advanced initially by Lenaerts and Van Nuffel listing, however, requirements (2) and (3) in a reversed order; see Koen Lenaerts and Piet Van Nuffel, Constitutional Law of the European Union (Sweet & Maxwell 1999) 414. This list has been taken over and adapted by Majone, and adopted in this study as in the author’s view the current sequence of requirements reflects more logically the reshaping the external dimension of EU police cooperation 21 particular the second and the third observations illustrate the relevance of the principle of institutional balance concerning the delegation of powers to EU agencies like Europol.119 More specifically, when setting up and entrusting powers to an EU agency like Europol, it must be ensured that its legal design neither affects the powers of the EU institu­ tions established by the Treaties nor entails an unconditional surrender of powers by the institutions creating it. The main tool ensuring that EU agencies like Europol do not affect the institutional balance is the delegation of powers doctrine or the so-called Meroni doctrine.120 The delegation of powers doctrine determines the kind of powers that may be entrusted to EU agencies, as well as the conditions and requirements attached to such powers. The delegation of powers doctrine is dynamic, depending on the evolution of the EU constitutional framework and on the interpretation given by the CJEU. essentials of the principle of institutional balance; see Giandomenico Majone, ‘Delegation of Regulatory Powers in a Mixed Polity’ (2002) 8 ELJ 319, 327. 123  ESMA, paras 44–45. See for a detailed analysis of the ESMA judgment, Merijn Chamon, ‘The Empowerment of Agencies under the Meroni Doctrine and Article 114 TFEU: Comment on United Kingdom v Parliament and Council (Short-selling) and the Proposed Single Resolution Mechanism’ (2014) 39 EL Rev 381. 120  Koen Lenaerts, ‘Regulating the Regulatory Process: “Delegation of Powers” in the European Community’ (1993) 18 EL Rev 23, 27 and 40–49. 122  Case C-270/2012 United Kingdom v Parliament and Council (ESMA) EU:C:2014:1, para 41. p y , 121  Case 9/56, Meroni, 152 and Case 10/56, Meroni, 173. essentials of the principle of institutional balance; see Giandomenico Majone, ‘Delegation of Regulatory Powers in a Mixed Polity’ (2002) 8 ELJ 319, 327. 119 Coman-Kund (n 1) 350 124  There is no general consensus on the detailed list of requirements that make up the Meroni doctrine, the only common aspect so far being that ‘discretionary powers cannot be delegated’, see Chamon (n 123) 382; for an overview of the various Meroni conditions devised in literature, see Merijn Chamon, EU Agencies: Legal and Political Limits to the Transformation of the EU Administration (OUP 2016) 191. g y y , 119  Coman-Kund (n 1) 350. essentials of the principle of institutional balance; see Giandomenico Majone, ‘Delegation of Regulatory Powers in a Mixed Polity’ (2002) 8 ELJ 319, 327. 119  Coman-Kund (n 1) 350. 120  Koen Lenaerts, ‘Regulating the Regulatory Process: “Delegation of Powers” in the European Community’ (1993) 18 EL Rev 23, 27 and 40–49. 121  Case 9/56, Meroni, 152 and Case 10/56, Meroni, 173. 122  Case C-270/2012 United Kingdom v Parliament and Council (ESMA) EU:C:2014:1, para 41. 123  ESMA, paras 44–45. See for a detailed analysis of the ESMA judgment, Merijn Chamon, ‘The Empowerment of Agencies under the Meroni Doctrine and Article 114 TFEU: Comment on United Kingdom v Parliament and Council (Short-selling) and the Proposed Single Resolution Mechanism’ (2014) 39 EL Rev 381. 124  There is no general consensus on the detailed list of requirements that make up the Meroni doctrine, the only common aspect so far being that ‘discretionary powers cannot be delegated’, see Chamon (n 123) 382; for an overview of the various Meroni conditions devised in literature, see Merijn Chamon, EU Agencies: Legal and Political Limits to the Transformation of the EU Administration (OUP 2016) 191. 125  Regarding the relevance of institutional balance as a standard for assessing the lawfulness of EU agencies’ powers, see Stefan Griller and Andreas Orator, ‘Everything Under Control? The “Way Forward” for European Agencies in the Footsteps of the Meroni Doctrine’ (2010) 35 EL Rev 3, 31. Whereas, conceptually, the principle of institutional balance, being concerned with preserving the prerogatives of the EU institutions, is different 5.1  The principle of institutional balance and the doctrine of delegation of powers In the Meroni cases, the Court established that EU institu­ tions may delegate to external bodies ‘executive’ powers that they themselves possess according to the Treaties, but only if such powers are ‘clearly defined’ and subject to their supervision.121 In the ESMA judgment, the Court confirmed the application of the Meroni doctrine to EU agencies,122 but at the same it opened the path for entrusting wide-ranging powers to these bodies.123 According to the Meroni logic as reinterpreted by the CJEU in ESMA,124 and in light of the principle of institutional balance,125 EU agencies’ powers seem to be acceptable as long as: 122  Case C-270/2012 United Kingdom v Parliament and Council (ESMA) EU:C:2014:1, para 41. 125  Regarding the relevance of institutional balance as a standard for assessing the lawfulness of EU agencies’ powers, see Stefan Griller and Andreas Orator, ‘Everything Under Control? The “Way Forward” for European Agencies in the Footsteps of the Meroni Doctrine’ (2010) 35 EL Rev 3, 31. Whereas, conceptually, the principle of institutional balance, being concerned with preserving the prerogatives of the EU institutions, is different EUROPE AND THE WORLD 22 (1) they are of an executive nature;126 (2) they are clearly defined, which, however, has been construed broadly by the Court as meaning that they stay within the boundaries of the agency’s broad regulatory framework;127 (3) they there are criteria and conditions limiting the discretion of the agency, which calls for mechanisms ensuring sufficient oversight by the main EU institutions;128 (4) they do not encroach upon the powers conferred by the Treaties on the EU institutions.129 (4) they do not encroach upon the powers conferred by the Treaties on the EU institutions.129 In the aftermath of ESMA, EU agencies like Europol may arguably be entrusted with wide-ranging powers provided that the revamped Meroni conditions are met. Moreover, alternative routes for the delegation of executive powers by the legislator to agencies, outside the Commission’s powers under the Treaties, do not necessarily breach the principle of institutional balance. What seems to matter in terms of institutional balance in light of the revived Meroni doctrine is the system of controlling mechanisms set up under the Treaty framework with regard to the exercise of EU agencies’ powers in order to preserve the system of checks and balances. from the Meroni doctrine, it is maintained that in practice these two parameters are tightly connected. Accordingly, for the purpose of the present analytical framework, conditions regarding delegation of powers to EU agencies stricto sensu (1)–(3) have been integrated with requirements dictated by the principle of institutional balance (4). To be sure, there is no consensus in the literature as regards the precise relationship between institutional balance and Meroni; see for a summary of the different views, Chamon (n 123) 188–9 and 200–1. 5.1  The principle of institutional balance and the doctrine of delegation of powers The principle of institutional balance and the Meroni require­ ments are relevant legal standards for assessing the overall design and delegated powers of EU agencies like Europol. It is therefore maintained that they are also applicable to their international cooperation tasks and activities.130 The specific features of the international dimension of EU agencies like Europol entail in particular that these bodies must not encroach upon the powers conferred by the Treaties on the EU institu­ tions in the Union’s external action area. from the Meroni doctrine, it is maintained that in practice these two parameters are tightly connected. Accordingly, for the purpose of the present analytical framework, conditions regarding delegation of powers to EU agencies stricto sensu (1)–(3) have been integrated with requirements dictated by the principle of institutional balance (4). To be sure, there is no consensus in the literature as regards the precise relationship between institutional balance and Meroni; see for a summary of the different views, Chamon (n 123) 188–9 and 200–1. 126  ESMA, para 67. Here the Court states that the condition that ‘only clearly defined executive powers may be delegated’ is still a valid standard for assessing ESMA’s powers. 127  ibid, para 44. 128  ibid, paras 45–54. 129  ibid, paras 84–86. 130  Coman-Kund (n 1) 88–9. 126  ESMA, para 67. Here the Court states that the condition that ‘only clearly defined executive powers may be delegated’ is still a valid standard for assessing ESMA’s powers. 129  ibid, paras 84–86. reshaping the external dimension of EU police cooperation from the Meroni doctrine, it is maintained that in practice these two parameters are tightly connected. Accordingly, for the purpose of the present analytical framework, conditions regarding delegation of powers to EU agencies stricto sensu (1)–(3) have been integrated with requirements dictated by the principle of institutional balance (4). To be sure, there is no consensus in the literature as regards the precise relationship between institutional balance and Meroni; see for a summary of the different views, Chamon (n 123) 188–9 and 200–1. 126  ESMA, para 67. Here the Court states that the condition that ‘only clearly defined executive powers may be delegated’ is still a valid standard for assessing ESMA’s powers. 127  ibid, para 44. 128  ibid, paras 45–54. 129  ibid, paras 84–86. 130  Coman-Kund (n 1) 88–9. 131  Ott (n 2) 521. See also Commission, Vademecum on the External Action of the European Union, SEC (2011) 881/3, 18–19. 132  Case C-660/13 Council v Commission EU:C:2016:616. In casu, the Council asked for the annulment of a Commission decision on the signature of an addendum to the 2006 Memorandum of Understanding on a Swiss contribution to the new Member States concluded between the EU and Switzerland. 133  ibid, paras 33–34. 134  Case C-327/91 France v Commission EU:C:1994:305. 133  ibid, paras 33–34. 134  Case C-327/91 France v Commission EU:C:1994:305. 132  Case C-660/13 Council v Commission EU:C:2016:616. In casu, the Council asked for the annulment of a Commission decision on the signature of an addendum to the 2006 Memorandum of Understanding on a Swiss contribution to the new Member States concluded between the EU and Switzerland. reshaping the external dimension of EU police cooperation 23 5.1.1  Institutional balance and delegation of powers in the EU external action area 5.1.1  Institutional balance and delegation of powers in the EU external action area Whereas various actors are involved in different ways in the Union’s external action, from Articles 17(1) and 21(3) TEU, as well as Article 220 TFEU it can be inferred that the Commission is mainly in charge of the Union’s external representation and of the daily management of its external relations.131 Recently in Case C-660/13,132 the Court has further clarified the scope and limits of the Commission’s external powers in light of the principle of institutional balance. In its judgment, the Court explained that the EU external action is based on an insti­ tutional system whereby the European Council defines the strategic interests and objectives of the Union, the Council is in charge of policy- making, and more specifically it further elaborates the Union’s external action and ensures its consistency, whereas the Commission exercises executive and management functions and ensures the Union’s external representation.133 Next, the institutional balance regarding EU treaty-making is relevant for the limits of EU agencies’ international cooperation as it triggers the inquiry into whether such actors are allowed to conclude international binding agreements. This directly questions the consti­ tutionality of Europol’s cooperation agreements, and also the agency’s international cooperation instruments according to the new Europol Regulation, provided that these qualify as binding agreements in light of the relevant international law criteria. Thus, Article 218 TFEU features a procedure according to which the Council formally decides on the opening of negotiations, the signing and conclusion of the international agreements, while the Commission, in principle, can only propose and negotiate the Union’s binding international agreements. On this division of tasks, the Court took a strict stance, relying on the principle of institu­ tional balance. The issue arose most famously in Case C-327/91 France v Commission134 around the legal question of whether the Commission could conclude certain international agreements autonomously. 135  ibid, para 36. 136  ibid, para 41. See also James Kingston, ‘External Relations of the European Community: External Capacity versus Internal Competence’ (1995) 44 ICLQ 659, 668. 137  Piris (n 33) 87. 138  See on the delegation of treaty-making powers to the Commission and other EU institutions and bodies, Robert Schütze, Foreign Affairs and the EU Constitution: Selected Essays (CUP 2014) 392–9. 139  Articles 58 and 184–9 of Regulation (EU, Euratom) 966/2012 of the European Parliament and of the Council of 25 October 2012 on the financial rules applicable to the general budget of the Union and repealing Council Regulation (EC, Euratom) No. 1605/2002 [2012] OJ L 298/1; see also Schütze (n 138) 395–9. 140  Besides the former Europol Council Decision, the label ‘agreement’ is explicitly mentioned in the case of Eurojust (Article 26a of 2002/187/JHA Council Decision of 28 February 2002 setting up Eurojust with a view to reinforcing the fight against serious crime, [2002] OJ L 63/1). 141 See for details and examples Coman Kund (n 1) 62 80 140  Besides the former Europol Council Decision, the label ‘agreement’ is explicitly mentioned in the case of Eurojust (Article 26a of 2002/187/JHA Council Decision of 28 February 2002 setting up Eurojust with a view to reinforcing the fight against serious crime, [2002] OJ L 63/1). 141  See, for details and examples, Coman-Kund (n 1) 62–80. 139  Articles 58 and 184–9 of Regulation (EU, Euratom) 966/2012 of the European Parliament and of the Council of 25 October 2012 on the financial rules applicable to the general budget of the Union and repealing Council Regulation (EC, Euratom) No. 1605/2002 [2012] OJ L 298/1; see also Schütze (n 138) 395–9. reshaping the external dimension of EU police cooperation The Court disagreed, essentially arguing that there was a particular institu­ tional balance set by the Treaty with regard to the Union’s international EUROPE AND THE WORLD 24 agreements entailing the conclusion of such agreements by the Council, not the Commission.135 Furthermore, the Court indicated that there was no automatic parallelism between the internal and external powers of the Commission as far as the Union’s treaty-making was concerned.136 The strict stance taken by the Court seems to be backed up by Article 218(1) TFEU, suggesting a single procedure for the negotiation and conclusion of the Union’s agreements with third countries and international organizations.137 However, the delegation of specific treaty-making powers to the Commission and other actors like EU agencies should not be ruled out, provided that the prerogatives of the institutions involved in ‘Article 218 TFEU’ procedure are not affected.138 A more flexible view of the institutional balance in external relations has also been embraced by the EU legislator, by delegating certain external relations tasks to the Commission. The most obvious examples are the financing agreements concluded by the Commission with third countries and international organizations on the basis of the EU Financial Regulation.139 Similarly, the EU legislator has entrusted limited external relations tasks to EU agencies, though the Founding Treaties do not formally assign them with a role in the Union’s external action. The founding acts of some EU agencies provide explicitly for the possibility to conclude agreements140 or arrangements with third countries and international organizations.141 A common thread in all these cases is that the enabled international cooperation is ancillary and instrumental to the implementation of the relevant secondary legislative instruments. If such secondary law provisions are interpreted as allowing legally binding agreements to be concluded by the Commission and the EU agencies, one may argue that the EU legislator distorted the institutional balance under Article 218 TFEU. 141  See, for details and examples, Coman-Kund (n 1) 62–80. reshaping the external dimension of EU police cooperation 25 Yet, in our view, a delineation142 could be made between agreements concluded according to the ordinary procedure laid down in Article 218 TFEU and international agreements concluded by the Commission and other EU bodies. 147  ESMA, para 85. 142  For a similar view, see Schütze (n 138) 392–6. 143  For a parallel between the Union’s international law-making and the making of its ‘internal’ legislation, see Piet Eeckhout, EU External Relations Law (OUP 2011) 193–4. 144  For a more detailed analysis of the concept of ‘international administrative agreement’ at EU level, see Coman-Kund (n 1) 155–65. 145  ESMA, para 79. 146  Coman-Kund (n 1) 132. reshaping the external dimension of EU police cooperation The first category embodies the most important legal–political commitments taken by the Union on the international plane corresponding to a sort of ‘external legislation- making’, and requiring the participation of the EU institutions that are also involved in the legislative process.143 The second category features agreements of a technical–administrative nature regarding the imple­ mentation of Article 218 TFEU agreements and EU legislation, and the daily management of EU external policies or the external dimension of Union’s internal policies.144 These instruments must comply with the international agreements concluded in accordance with Article 218 TFEU and with the Union’s internal legislation, and they must not affect the powers bestowed on other actors in EU external relations. 5.1.2  EU agencies and institutional balance in external relations Whereas the Commission and the EU agencies resemble each other in that they both pursue, as part of the EU administration, international cooperation on a technical–administrative level, the Founding Treaties are silent as regards the role of agencies in the EU external action area. However, in ESMA, the Court admitted generally that ‘while the treaties do not contain any provision to the effect that powers may be conferred on’ EU agencies, such a possibility exists nevertheless.145 Since there is no distinction between the delegation of powers exercised by EU agencies internally, (within the Union), and powers concerning external relations, one may assume that the possibility to delegate holds in principle for both categories.146 Moreover, the Court clarifies that post Lisbon, the Commission’s prerogatives under the Treaties should not be undermined per se if executive powers delegated to EU agencies are part of the regulatory framework in a policy area that requires specific technical expertise, and they are necessary to attain the objectives of that policy area.147 Similarly, limited international cooperation tasks and actions performed by the EU agencies within 5.1.2  EU agencies and institutional balance in external relations 5.1.2  EU agencies and institutional balance in external relations Whereas the Commission and the EU agencies resemble each other in that they both pursue, as part of the EU administration, international cooperation on a technical–administrative level, the Founding Treaties are silent as regards the role of agencies in the EU external action area. 42  For a similar view, see Schütze (n 138) 392–6. reshaping the external dimension of EU police cooperation However, in ESMA, the Court admitted generally that ‘while the treaties do not contain any provision to the effect that powers may be conferred on’ EU agencies, such a possibility exists nevertheless.145 Since there is no distinction between the delegation of powers exercised by EU agencies internally, (within the Union), and powers concerning external relations, one may assume that the possibility to delegate holds in principle for both categories.146 Moreover, the Court clarifies 5.1.2  EU agencies and institutional balance in external relations Whereas the Commission and the EU agencies resemble each other in that they both pursue, as part of the EU administration, international cooperation on a technical–administrative level, the Founding Treaties are silent as regards the role of agencies in the EU external action area. However, in ESMA, the Court admitted generally that ‘while the treaties do not contain any provision to the effect that powers may be conferred on’ EU agencies, such a possibility exists nevertheless.145 Since there is no distinction between the delegation of powers exercised by EU agencies internally, (within the Union), and powers concerning external relations, one may assume that the possibility to delegate holds in principle for both categories.146 Moreover, the Court clarifies that post Lisbon, the Commission’s prerogatives under the Treaties should not be undermined per se if executive powers delegated to EU agencies are part of the regulatory framework in a policy area that requires specific technical expertise, and they are necessary to attain the objectives of that policy area.147 Similarly, limited international cooperation tasks and actions performed by the EU agencies within EUROPE AND THE WORLD 26 their mandate, and with a view to attain the objectives established by the EU legislator, should not be in principle irreconcilable with the powers of the Commission in EU external relations. The possibility for EU agencies to pursue international cooperation has been acknowledged in the Common Approach on EU agencies148 through an all-embracing formula covering instances where the mandate or work programme of these bodies ‘foresees cooperation with third countries and/or international organizations’. 148  Common Approach on EU agencies (n 1), para 25. 149  The Court examined the overall setting of Europol’s international relations, including the agency’s cooperation agreements, with a view to determine whether the Council decision amending the ‘list’ of Europol’s partners pertained to an essential element of the matter regulated by the Europol Council Decision, which would have required using the legislative procedure established by primary law instead of the more flexible procedure provided for in Article 26(1)(a) of the Europol Council Decision. 150  Case C-363/14 Parliament v Council, paras 49–50. 151  See Schütze (n 138) 397–9. 152  See Ott, Vos and Coman-Kund (n 8) 91–93 and 115–16. 151  See Schütze (n 138) 397–9. 152  See Ott, Vos and Coman-Kund (n 8) 91–93 and 115–16. 150  Case C-363/14 Parliament v Council, paras 49–50. 156  Duncan B. Hollis, ‘Defining Treaties’ in Duncan B. Hollis (ed.), The Oxford Guide to Treaties (OUP 2012) 12 and Philippe Gautier, ‘Article 1’ and ‘Article 2’ in Olivier Corten and Pierre Klein (eds), The Vienna Conventions of the Law of Treaties: a Commentary, v I (OUP 2011) 45 and 60. 153  See, for a comprehensive overview, Jan Klabbers, The Concept of Treaty in International Law (Kluwer 1996). 154  1969 Vienna Convention on the Law of Treaties between States (the 1969 Vienna Convention) and 1986 Vienna Convention on the Law of Treaties between States and International Organizations or between International Organizations (the 1986 Vienna Convention). 155  Article 2(1)(a) of the 1969 Vienna Convention and Article 2(1)(a) of the 1986 Vienna Convention. 156  Duncan B. Hollis, ‘Defining Treaties’ in Duncan B. Hollis (ed.), The Oxford Guide to Treaties (OUP 2012) 12 and Philippe Gautier, ‘Article 1’ and ‘Article 2’ in Olivier Corten and Pierre Klein (eds), The Vienna Conventions of the Law of Treaties: a Commentary, v I (OUP 2011) 45 and 60. 157  Land and Maritime Boundary between Cameroon and Nigeria (Cameroun v Nigeria: Equatorial Guinea Intervening), Judgment, 2002 ICJ 303, para 263. 158  Hollis (n 156) 26. See also Maritime Delimitation and Territorial Questions between Qatar and Bahrain, Jurisdiction and admissibility, Judgment, 1994 ICJ 112, paras 23–25. 55  Article 2(1)(a) of the 1969 Vienna Convention and Article 2(1)(a) of the 1986 Vienna Convention. reshaping the external dimension of EU police cooperation Moreover, the CJEU has for the first time examined the international dimension of an agency with regard specifically to Europol in Case C-363/14, Parliament v Council149 without questioning the ‘constitutionality’ of the agency’s international dimension as long as it was ancillary and necessary for the performance of its core tasks, and provided that it took place within the framework defined by the EU legislature.150 Unlike the Commission, which has a general vocation in EU external relations, EU agencies may only become involved in interna­ tional cooperation as sectoral actors, with a role that is restricted to what is necessary to fulfil their core mandate entrusted by the legislator in a certain policy area. Accordingly, entrusting certain international cooperation tasks to EU agencies is acceptable, but the Meroni require­ ments listed previously and the institutional balance in the EU external action area must be observed. It is also maintained that EU agencies can be delegated limited powers to conclude binding international agreements inherent to the fulfilment of their mandate by an act of secondary legislation.151 Similarly  to the Commission’s administrative agreements, such agreements concluded by the EU agencies are a special form of EU external administrative action which does not automatically disturb the institutional balance laid down in Article 218 TFEU.152 In order for such instruments to be validated in light of the Meroni requirements and the principle of institutional balance, it is essential that they: (1) remain within the core mandate of the agency, and are consistent with reshaping the external dimension of EU police cooperation 27 27 ‘Article 218 TFEU’ agreements and EU legislation; and (2) the agency is subject to sufficient supervision and control, ensuring that the powers of the main actors in the EU external action area are not affected. In particular, the Council and the Commission should be involved in view of their roles under Articles 17(1), 16(6) and 21(3) TEU, as well as Articles 218 and 220 TFEU. 5.2  The concept of a (binding) ‘international agreement’ 157  Land and Maritime Boundary between Cameroon and Nigeria (Cameroun v Nigeria: Equatorial Guinea Intervening), Judgment, 2002 ICJ 303, para 263. 153  See, for a comprehensive overview, Jan Klabbers, The Concept of Treaty in International Law (Kluwer 1996). 54  1969 Vienna Convention on the Law of Treaties between States (the 1969 Vienna Convention) and 1986 Vienna Convention on the Law of Treaties between States and nternational Organizations or between International Organizations (the 1986 Vienna Convention). q g g p 158  Hollis (n 156) 26. See also Maritime Delimitation and Territorial Questions between Qatar and Bahrain, Jurisdiction and admissibility, Judgment, 1994 ICJ 112, paras 23–25. 159  See, for more elaborated discussions, Henry G. Schermers and Niels M. Blokker, International Institutional Law: Unity within Diversity (5th revised edn, Martinus Nijhoff Publishers 2011) 1121–3, and Anthony Aust, Modern Treaty Law and Practice (CUP 2013) 17–18. 160  Hollis (n 156) 26–8. 5.2  The concept of a (binding) ‘international agreement’ Moving away from the controversies surrounding the concept of ‘treaty’ or binding ‘international agreement’,153 the most authoritative definition is to be found in the two Vienna Conventions on the Law of Treaties.154 These instruments define a treaty as ‘an international agreement’ concluded in a written form and governed by interna­ tional law, regardless of whether it is embodied in one, two or more instruments and whatever its particular designation.155 This definition is widely accepted by the international law scholarship156 and it is also supported by the International Court of Justice (ICJ)157 as reflecting customary international law. Based on this definition, it follows that under international law an essential criterion for assessing the legally binding character of international instruments is the genuine intention of the parties to create binding effects governed by international law, regardless of the name or the form of the instrument.158 Accordingly, one may identify two essential requirements for an instrument (agreement) to become binding under international law: 157  Land and Maritime Boundary between Cameroon and Nigeria (Cameroun v Nigeria: Equatorial Guinea Intervening), Judgment, 2002 ICJ 303, para 263. 158  Hollis (n 156) 26. See also Maritime Delimitation and Territorial Questions between Qatar and Bahrain, Jurisdiction and admissibility, Judgment, 1994 ICJ 112, paras 23–25. EUROPE AND THE WORLD 28 (1) there must be an intention to create legally binding effects; (2) such effects must be governed by international law.159 161  Maritime Delimitation and Territorial Questions between Qatar and Bahrain, paras 23–25. See also Kirsten Schmalenbach, ‘Article 2. Use of Terms’, in Oliver Dörr and Kirsten Schmalenbach (eds), Vienna Convention on the Law of Treaties: A Commentary (Springer 2012), 40–1. 159  See, for more elaborated discussions, Henry G. Schermers and Niels M. Blokker, International Institutional Law: Unity within Diversity (5th revised edn, Martinus Nijhoff Publishers 2011) 1121–3, and Anthony Aust, Modern Treaty Law and Practice (CUP 2013) 17–18. 160  Hollis (n 156) 26–8. 161  Maritime Delimitation and Territorial Questions between Qatar and Bahrain, paras 23–25. See also Kirsten Schmalenbach, ‘Article 2. Use of Terms’, in Oliver Dörr and Kirsten Schmalenbach (eds), Vienna Convention on the Law of Treaties: A Commentary (Springer 2012), 40–1. 162  Anthony Aust, ‘Alternatives to Treaty-making: MOUs as Political Commitments’, in Hollis (ed), The Oxford Guide to Treaties (n 156) 48–9. 163  Maritime Delimitation and Territorial Questions between Qatar and Bahrain, para 27. 164  Hollis (n 156) 27. 165  ibid. 162  Anthony Aust, ‘Alternatives to Treaty-making: MOUs as Political Commitments’, in Hollis (ed), The Oxford Guide to Treaties (n 156) 48–9. 163  Maritime Delimitation and Territorial Questions between Qatar and Bahrain, para 27. 164  Hollis (n 156) 27. 165  ibid. (1) there must be an intention to create legally binding effects; (2) such effects must be governed by international law.159 (1) (2) (2) However, it is more difficult to assess whether particular instruments (agreements) fulfil these requirements.160 Accordingly, one has to use various evidentiary factors in order to assess the legal nature of each particular international instrument. The factors most commonly accepted include the wording and substance, as well as the particular circumstances (context) surrounding the negotiation and conclusion of the instrument.161 Thus, an instrument with an elaborated structure comprising articles and covering issues such as the purpose of the instrument, rights and obligations of the parties, entry into force, amendment, suspension and termination, liability and dispute settlement is likely to constitute a treaty. Moreover, binding international agreements normally use a certain language featuring ‘shall’ and ‘will’ clauses and a certain terminology such as ‘parties’, ‘agree’, ‘agreement’, ‘enter into force’.162 Additionally, the wider legal framework within which the instrument has been enacted, the procedure used, as well as decisions taken after the conclusion of the instrument regarding the application of its provisions are relevant circumstances for determining its legal nature.163 The factors mentioned above are mere indicators of intent, as ‘there are no magic words to create a treaty or deny an agreement that status’.164 Therefore, the determination of the legal nature of interna­ tional cooperation instruments requires a careful analysis combining the above-mentioned factors, by giving consideration to the form, terminology and content of the instrument, as well as to the surrounding circumstances.165 Things are no different within the European Union. The CJEU indicated that the binding character of an agreement should be reshaping the external dimension of EU police cooperation 29 established, in principle, based on the intention of the parties as results from the wording of the instrument and the history of negotiations.166 Moreover, the Commission’s Vademecum on the External Action of the European Union stresses the decisive character of the content of an instrument for the purpose of determining its legal nature, and warns about careful drafting in order to avoid an instrument being considered as legally binding.167 Therefore, in order to determine the legal nature of Europol’s international cooperation instruments, a complex analysis is required. This entails an assessment of Europol’s legal framework pertaining to international cooperation, including the procedure for negotiating and concluding international cooperation instruments, together with a more detailed analysis of the international cooperation instruments. 166  Case C-327/1991 France v Commission, para 15. , p 167  Vademecum on the External Action of the European Union (n 131) 52. 169  See generally on this issue, Hollis (n 156) 14. 168  See also Vara (n 10) 127 and 135. 166  Case C-327/1991 France v Commission, para 15. 167  Vademecum on the External Action of the European Union (n 131) 52. 168 See also Vara (n 10) 127 and 135. 167  Vademecum on the External Action of 168 See also Vara (n 10) 127 and 135 167  Vademecum on the External Action of the E 6.  Europol’s international dimension: an assessment 6.1  The legal framework and international cooperation practice under Council Decision 2009/371/JHA 6.1.1  The legal nature of Europol’s cooperation agreements 6.1.1  The legal nature of Europol s cooperation agreements The analysis of the relevant legal framework, the procedure for the negotiation and conclusion, as well as the content and effects support the conclusion that Europol’s cooperation agreements can be qualified as binding international agreements.168 This is most obvious with the operational agreements, which include clear obligations for the contracting parties in terms of data protection, information exchange and processing, rights for third parties affected, and liability provisions. The strategic agreements are lighter in terms of the content of the cooperation, but still the procedure used, and the wording and content of their provisions, suggest their binding nature. The qualification of Europol’s cooperation agreements as binding international agreements triggers the application of the principle pacta sunt servanda, and international responsibility in case of breach of the duties under the agreement.169 EUROPE AND THE WORLD 30 Europol’s cooperation agreements are to be included in the category of international technical–administrative agreements. They have a very limited scope, in line with the mandate of the agency, and lay down a specific framework for technical and operational cooperation. As such, these agreements enable concrete cooperation between administrations without touching presumably the political sphere of external relations. 6.1.2  The principle of institutional balance and the Meroni doctrine 6.1.2  The principle of institutional balance and the Meroni doctrine In the field of police cooperation, Europol is a technical body playing a much more modest role than suggested by its name. Europol’s discretion to set its international cooperation priorities seems limited because of the constraints imposed on the agency in practice by its operational and political environment. The restricted role of Europol as a global actor was reflected in the agency’s legal framework for inter­ national cooperation,170 and in its cooperation agreements. Europol’s agreements primarily cover information exchange and processing, the competence of the agency is delineated,171 and essential requirements in line with Europol’s legal framework regarding data protection, confi­ dentiality and liaison officers are included.172 Overall, Europol’s legal provisions on international cooperation, as well as its agreements and related activities were instrumental to attaining the main objectives set by the relevant legal framework, including Article 88(2) TFEU, and their scope remained within the core mandate of the agency. 170  See Article 23 of the Europol Council Decision. 171  Still one may argue that Europol’s mandate is broadly defined because of the lack of an EU-wide definition of the crimes for which the agency is competent. 172  Note, however, the controversial 2002 Supplemental Agreement between Europol and the USA. 173  Ott, Vos and Coman-Kund (n 8) 108. 170  See Article 23 of the Europol Council Decision. 6.  Europol’s international dimension: an assessment Particularly after Lisbon, the conclusion of binding interna­ tional agreements by Europol arguably posed problems for the insti­ tutional balance in the EU external action area.173 Thus, it could be maintained that ‘Article 218 TFEU’ agreements should have been used instead or, in the alternative, that the legal framework concerning Europol’s cooperation agreements did not account sufficiently for the Commission’s role to ensure the Union’s external representation under Articles 17(1) TEU and 220 TFEU, and for EP’s prerogatives under Article 218 TFEU. reshaping the external dimension of EU police cooperation 31 Based on the analysis of Europol’s agreements, we take the view that these instruments, as technical–administrative agreements remaining outside the scope of Article 218 TFEU, do not per se affect institutional balance in external relations. In our opinion, the CJEU judgment in Case C-363/2014 provides further support for this view by validating Europol’s international dimension provided that it was ancillary to the core activities of the agency, and that it took place within the framework defined by the EU legislator. What is more, the Court highlighted the possibility for Europol to conclude cooperation agreements as an essential element of this framework, and then indicated that the procedure for the negotiation and conclusion of these instruments provided sufficient safeguards preventing the transfer of personal data to third countries from interfering with fundamental rights.174 From this it can be inferred that Europol’s cooperation agreements, as forms of the agency’s ancillary international action, and taking place within the legal framework defined by the legislator, were not precluded by Article 218 TFEU. The legal provisions dealing with international cooperation established important formal conditions and controls concerning Europol’s cooperation agreements. These included the Council’s ‘list’, the role of the Management Board to authorize the negotiation of cooperation agreements and to endorse them, the approval of each cooperation agreement by the Council, as well as the authoritative opinions of the JSB on operational agreements. Insights from practice into the procedure for the negotiation and conclusion of Europol’s agreements revealed additional informal supervisory mechanisms carried out by the Commission. Such formal and informal conditions and mechanisms appeared to be capable of ensuring consistency with the Union’s priorities in external relations, and to preserve the Commission’s role under Articles 17 TEU and 220 TFEU. 174  Case C-363/14 Parliament v Council, paras 53–55. This general finding based on the formal legal design of Europol’s procedure for the negotiation and conclusion of cooperation agreements could be relativized to some extent when looking at the agency’s international cooperation practice, a prime example being the 2002 Supplemental Agreement with the USA. 175  Article 48 of the Europol Council Decision. 175  Article 48 of the Europol Council Decision. 6.  Europol’s international dimension: an assessment Furthermore, the obligation undertaken recently by the Council to consult the EP prior to approving Europol’s cooperation agreements, together with the duty placed on the chair of the Management Board and of the Director to inform the EP on request,175 were presumably sufficient to pass the Meroni conditions and the institutional balance test. EUROPE AND THE WORLD 32 Accordingly, Europol’s cooperation agreements are in line overall with Meroni and do not seem to disturb the institutional balance in EU external relations. Being concluded by an EU body acting on the global level, Europol’s agreements are considered as being carried out ultimately on behalf of the European Union, which is in contrast with the Common Approach on EU agencies, stipulating that EU agencies cannot commit the Union to international obligations.176 Whilst Europol’s international agreements are binding, the agency did not have international legal personality because of the strict controls set on its international cooperation precluding it from enjoying a sufficient degree of autonomy on the international plane.177 Accordingly, since Europol is an element of the Union’s institutional structure and the Union has international legal personality, it follows that the obligations arising from Europol’s cooperation agreements are incumbent in the end upon the EU. 176  Para 25. This statement seems to envisage two possible scenarios: (1) that EU agencies cannot adopt legally binding international cooperation instruments or (2) that EU agencies could adopt legally binding international cooperation instruments but without engaging the Union internationally; the latter scenario would entail that EU agencies have their own international legal personality allowing them to take up in their own right obligations on the international plane. 177  On the international legal personality of EU agencies and, in particular, of Europol, see also Coman-Kund (n 11) 105–6 and 117. 178  Article 23(1) of Regulation 2016/794. 6.2  The new Europol Regulation The legal framework established by the new Regulation arguably marks a weakening of Europol’s role in international cooperation, in parallel with a partial enhancement of controlling mechanisms over the agency’s international dimension. While emphasizing almost identically with the old Europol Council Decision the instrumental– ancillary nature of Europol’s international cooperation,178 the new Regulation also subdues the agency’s core international activities – i.e. exchange of personal data – to ‘Article 218 TFEU’ agreements and the Commission’s ‘adequacy decisions’. The agency is merely left with the possibility to conclude administrative arrangements with a view to implementing the instruments providing the legal basis for interna­ tional cooperation, or to conclude, on the matters covered previously by strategic agreements, working arrangements which are, however, qualified as non-binding for the EU  and the Member States. At the same time, the new Regulation features increased scrutiny from the reshaping the external dimension of EU police cooperation 33 33 Commission and the EP over Europol’s international cooperation. As discussed, for the Commission this entails the possibility to assess Europol’s ‘surviving’ cooperation agreements, getting involved in the agency’s annual and multiannual planning, and undertaking the periodic evaluation of the agency. EP’s increased role vis-à-vis the agency materializes most importantly in the monitoring of Europol’s activities through the JPSG and the possibility to obtain access to Europol information. The previous points suggest that Europol’s redesigned international dimension is likely to respect institutional balance in external relations and the Meroni doctrine. However, the new legal framework also raises some legal and practical problems. First, the application of the criteria for binding ­international agreements discussed under section 5.2 to Europol’s working and administrative arrangements which will be concluded under the new Regulation might conduce to the conclusion that some of them are legally binding instruments.179 In spite of being labelled ‘arrangements’ and acting essentially as implementing tools, if the wording and content of these instruments as well as the particular circumstances surrounding their negotiation and conclusion reveal the intention of the parties to create legally binding effects under international law they will qualify  as binding interna­ tional agreements. Just like under Europol’s former legal framework, this would entail that such specific technical–administrative agreements would ultimately bind  the EU. Hence, replacing Europol’s previous strategic and operational agreements with working and administra­ tive arrangements does not necessarily result in a complete abolition of the agency’s ‘problematic’ power to enact binding international cooperation instruments. 179  Presumably, this could be the case most obviously for the administrative arrangements concluded by Europol for enabling exchanges of personal data. 180  Coman-Kund (n 11) 116–17. 181  In our view more effective oversight would have been ensured by including in the Europol regulation a duty to inform the JPSG before an administrative agreement is concluded (ex ante) as this could prevent potential breaches of the relevant legal framework from taking place in the first place. 6.2  The new Europol Regulation What is more, Europol’s new international cooperation instruments can be seen as problematic because there is more uncertainty as to their legal nature, which might impede legal review and individual protection. Second, the fact that the new Regulation is elliptical as regards the involvement of the Council, the Commission and the EP in the procedure for the negotiation and conclusion of Europol’s working and administrative arrangements raises questions about the sufficiency of controlling mechanisms by the main EU institutions in light of the  Meroni  doctrine.180 From this perspective, the new Regulation EUROPE AND THE WORLD 34 marks a step back as compared to the strict system of controls regarding Europol’s cooperation agreements under the previous legal framework, featuring Council’s approval, the influential opinions of the JSB, and, lately, also the duty to consult the EP. While Europol’s international cooperation instruments will be concluded within the framework of existing international agreements or the Commission’s ‘adequacy decisions’, the lack of express provisions on controlling mechanisms ensuring that the agency’s arrangements remain within its mandate and do not affect the powers of the institutions in the EU external action area is a matter of concern. Arguably this concern is not sufficiently addressed by the Commission’s involvement in the agency’s annual and multiannual planning nor by the duties to provide access to information to the EP or to transmit ex post to the JPSG the administrative agreements concluded with third countries and inter­ national organizations.181 According to Articles 17(1) TEU and 220 TFEU, and in line with the Common Approach on EU agencies, at least involvement and oversight by the Commission should be ensured in line with the role of the latter to ensure the Union’s external repre­ sentation and consistency of EU policy. Moreover, similarly to the JSB under the Europol Council Decision, the EDPS could be granted an advisory role in the procedure for the negotiation and conclusion of Europol’s administrative arrangements with third country authorities and international organizations. 7.  Conclusion The analysis of Europol’s international cooperation framework and practice reveals an agency with an international dimension ancillary to its core tasks, and which was subject to various formal and informal conditions and controls, in particular by the Council, the Commission and the Member States. As such, Europol’s international dimension represents a specific form of EU administrative external action which respects in principle the institutional balance in external relations and with the present-day Meroni requirements. This holds also for Europol’s cooperation agreements having a limited scope which revolves around reshaping the external dimension of EU police cooperation 35 35 the agency’s information-related tasks, and laying down a very specific framework for technical and operational cooperation. As a specific category of international agreements of a technical–administrative nature, these instruments appear to remain outside the scope of Article 218 TFEU, and they comply overall with the relevant EU legal framework. Contrary to the Common Approach on EU agencies, Europol’s cooperation agreements ultimately bind the Union under international law. Regulation 2016/794 substantially redesigns Europol’s inter­ national dimension while ensuring that the agency’s cooperation agreements remain valid bases for international cooperation under the new legal framework. The new Europol Regulation brings Europol’s international dimension in line with that of other EU agencies, and attempts to improve the agency’s accountability for its international activities through strengthening the Commission’s position towards the agency, increasing parliamentary oversight, and establishing supervision by the EDPS. Quite significantly, the new Regulation removes the agency’s power to conclude cooperation agreements by replacing them with ‘Article 218 TFEU’ agreements. While in our view such a change was not imperative in order to ensure the compatibility of Europol’s international dimension with the current Treaty framework, it arguably downplays the role of Europol on the international plane. This suggests a more modest role for Europol in international cooperation, which will likely respect institutional balance in external relations and the Meroni conditions. The application of the new Regulation also raises interesting legal and practical problems, such as: finding the most appropriate type of international agreement under Article 218 TFEU in order to address Europol’s operational needs; determining the legal nature and implications of Europol’s working and administrative arrangements; or the issue of supervision and controls over Europol’s international cooperation powers and instruments. 182  See, on the concept and application of Union’s international actorness, Martijn Groenleer and Louise van Schaik, ‘United We Stand? The European Union’s International Actorness in the Cases of the International Criminal Court and the Kyoto Protocol’ (2007) 45 JCMS 969. 7.  Conclusion Future practice in the application of the new Europol Regulation will offer more insights into the actual shaping of the agency’s international dimension and the way in which these issues are addressed. In brief, this article has shown that Europol’s international dimension under the former legal framework was compatible overall with the EU constitutional framework and, therefore, a reform of this aspect of the agency’s operations was not indispensable from a legal point of view. It also argues that the new Europol Regulation does not seem to address ideally some of the important aspects of the agency’s international cooperation. From a broader perspective, however, the EUROPE AND THE WORLD 36 reform of Europol’s international dimension may signal a more general phenomenon of enhancing the Union’s international actorness182 by streamlining the rather fragmented EU external action institutional landscape, and by instating coordination and control over various EU bodies pursuing international cooperation. 182  See, on the concept and application of Union’s international actorness, Martijn Groenleer and Louise van Schaik, ‘United We Stand? The European Union’s International Actorness in the Cases of the International Criminal Court and the Kyoto Protocol’ (2007) 45 JCMS 969. reshaping the external dimension of EU police cooperation reshaping the external dimension of EU police cooperation 37
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Sensitivity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation
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To cite this version: Stefan Jongen, Joy Perrier, Eric F Vuurman, Johannes G Ramaekers, Annemiek Vermeeren. Sensitiv- ity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation. PLoS ONE, 2015, 10 (2), pp.e0117045. ￿10.1371/journal.pone.0117045￿. ￿inserm-03152637￿ Sensitivity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation Stefan Jongen, Joy Perrier, Eric F Vuurman, Johannes G Ramaekers, Annemiek Vermeeren tefan Jongen, Joy Perrier, Eric F Vuurman, Johannes G Ramaekers, Annemiek Vermeeren HAL Id: inserm-03152637 https://inserm.hal.science/inserm-03152637v1 Submitted on 25 Feb 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/272192656 Sensitivity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation Article  in  PLoS ONE · February 2015 DOI: 10.1371/journal.pone.0117045 · Source: PubMed CITATIONS 28 READS 736 5 authors, including: Some of the authors of this publication are also working on these related projects: Role of histamine in cognition View project CANMEM View project Stefan Jongen Maastricht University 22 PUBLICATIONS   227 CITATIONS    SEE PROFILE Joy Perrier Unité Inserm U1077 32 PUBLICATIONS   164 CITATIONS    SEE PROFILE Eric F P M Vuurman Maastricht University 74 PUBLICATIONS   2,506 CITATIONS    SEE PROFILE Johannes G Ramaekers Maastricht University 290 PUBLICATIONS   7,553 CITATIONS    SEE PROFILE All content following this page was uploaded by Stefan Jongen on 19 February 2015. The user has requested enhancement of the downloaded file. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/272192656 Sensitivity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation Article  in  PLoS ONE · February 2015 DOI: 10.1371/journal.pone.0117045 · Source: PubMed CITATIONS 28 READS 736 5 authors, including: Some of the authors of this publication are also working on these related projects: Role of histamine in cognition View project CANMEM View project Stefan Jongen Maastricht University 22 PUBLICATIONS   227 CITATIONS    SEE PROFILE Joy Perrier Unité Inserm U1077 32 PUBLICATIONS   164 CITATIONS    SEE PROFILE Eric F P M Vuurman Maastricht University 74 PUBLICATIONS   2,506 CITATIONS    SEE PROFILE Johannes G Ramaekers Maastricht University 290 PUBLICATIONS   7,553 CITATIONS    SEE PROFILE See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/272192656 The user has requested enhancement of the downloaded file. All content following this page was uploaded by Stefan Jongen on 19 February 2015. The user has requested enhancement of the downloaded file. All content following this page was uploaded by Stefan Jongen on 19 February 2015. Sensitivity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation Stefan Jongen1*, Joy Perrier1,2, Eric F. Vuurman1, Johannes G. Ramaekers1, Annemiek Vermeeren1 1 Department of Neuropsychology and Psychopharmacology, Faculty of Psychology and Neuroscience, Maastricht University, Maastricht, The Netherlands, 2 U 1075 COMETE, INSERM, Caen, France * stefan.jongen@maastrichtuniversity.nl * stefan.jongen@maastrichtuniversity.nl a1111 Sensitivity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation Article  in  PLoS ONE · February 2015 DOI: 10.1371/journal.pone.0117045 · Source: PubMed Some of the authors of this publication are also working on these related projects: Some of the authors of this publication are also working on these related projects: Role of histamine in cognition View project The user has requested enhancement of the downloaded file. RESEARCH ARTICLE OPEN ACCESS Citation: Jongen S, Perrier J, Vuurman EF, Ramaekers JG, Vermeeren A (2015) Sensitivity and Validity of Psychometric Tests for Assessing Driving Impairment: Effects of Sleep Deprivation. PLoS ONE 10(2): e0117045. doi:10.1371/journal.pone.0117045 Results Copyright: © 2015 Jongen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. On-the-road driving performance was significantly impaired after sleep deprivation, as mea- sured by an increase in Standard Deviation of Lateral Position (SDLP) of 3.1 cm compared to performance after a normal night of sleep. At 5 am, performance in most psychometric tests showed significant impairment. As expected, largest effect sizes were found on perfor- mance in the Psychomotor Vigilance Test (PVT). Large effects sizes were also found in the Divided Attention Test (DAT), the Attention Network Test (ANT), and the test for Useful Field of View (UFOV) at 5 and 11 am during sleep deprivation. Effects of sleep deprivation on SDLP correlated significantly with performance changes in the PVT and the DAT, but not with performance changes in the UFOV. Data Availability Statement: The authors confirm that all data underlying the findings are fully available without restriction. Data can be obtained via the Dutch Dataverse Network: https://dataverse.nl/dvn/ dv/NP. The corresponding Accession number is hdl:10411/20325. Data will also be made available to all interested researchers upon request. Contact person: A. Blokland, a.blokland@maastrichtuniversity.nl. Funding: The authors have no support or funding to Methods Twenty four healthy volunteers participated in a 2-period crossover study in which the high- way driving test was conducted twice: once after normal sleep and once after one night of sleep deprivation. The psychometric tests were conducted on 4 occasions: once after nor- mal sleep (at 11 am) and three times during a single night of sleep deprivation (at 1 am, 5 am, and 11 am). Academic Editor: Linda Chao, University of California, San Francisco, UNITED STATES Academic Editor: Linda Chao, University of California, San Francisco, UNITED STATES Academic Editor: Linda Chao, University of California, San Francisco, UNITED STATES Received: June 30, 2014 Accepted: December 18, 2014 Published: February 10, 2015 Copyright: © 2015 Jongen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Objective To assess drug induced driving impairment, initial screening is needed. However, no con- sensus has been reached about which initial screening tools have to be used. The present study aims to determine the ability of a battery of psychometric tests to detect performance impairing effects of clinically relevant levels of drowsiness as induced by one night of sleep deprivation. Introduction Medicinal and illicit drugs can have detrimental side effects, such as sedation and reduced alertness, which can cause driving impairment possibly leading to traffic accidents (e.g. [1], [2], [3], [4], [5]). Performance testing should be applied to provide meaningful precautions for users and prescribers regarding the impact of particular drugs on driving, either as part of the drug registration process (e.g. [6]) or for already marketed medicinal or illicit drugs (e.g.[7]). Methodological guidelines for experimental studies assessing the effects of drugs on driving in- dicate that relatively simple laboratory tests can be used as a first step in screening a drug’s im- pairing potential as they often provide the earliest evidence of impairment on driving performance [8], and that more sophisticated procedures (e.g. driving simulators, on-the-road testing) should be included in a later stage [9], [10], [11], [12]. The advantage of simple labora- tory tests is that these tests are generally easy to administer, are cost-effective, and have a rela- tive short duration [9], [11], [12], [13]. Many tests are being used to indicate whether drugs impair driving performance and to judge fitness to drive when drugs are being used. However, no consensus has been reached about which specific initial screening tools are best to be used [10], as the link between test outcomes and clinical relevance is often unclear. Therefore, it is needed to establish a link between tests and effects of clinical relevance in order to compare results over separate studies. To provide this information, a requisite of a test is to be sufficiently sensitive to detect clinically relevant impairment [5], [10]. Drugs with known impairing effects can be used to induce clinically relevant levels of impairment, such as alcohol. Epidemiological studies have indicated an increase of traffic accidents with alcohol reaching blood alcohol concentrations of 0.5 g/L [14], [15]. However, when a psychometric test is not sensitive to the impairing effects of clinically relevant levels of alcohol, it does not neces- sarily mean it is not sensitive to more specific drug effects. Another way to establish the clinically relevant performance impairment of a test is to assess its sensitivity to clinically relevant levels in drowsiness induced by one night of sleep depriva- tion [16], [17]. After alcohol, sleepiness is the most frequent cause of motor vehicle accidents [18], [19]. Sensitivity and Validity of Tests for Assessing Driving Impairment impairment. Further studies are needed to assess the sensitivity and validity of these psy- chometric tests after benchmark sedative drug use. impairment. Further studies are needed to assess the sensitivity and validity of these psy- chometric tests after benchmark sedative drug use. Competing Interests: Dr. E.F. Vuurman is a full time employee of Maastricht University and has received grants/research support grants from GlaxoSmithKline, Transcept, and Merck companies. Prof. Dr. J.G. Ramaekers is a full time employee of Maastricht University and has received grants/ research support from NWO, ZonMW, EU, Dutch Ministry of Infrastructure and Environment and Lundbeck. Dr. A. Vermeeren is a full time employee of Maastricht University. As employee of Maastricht University, she participated—as investigator—in studies sponsored by for-profit businesses (Transcept Pharmaceuticals Inc, Purdue Pharma LP, Merck Sharp and Dohme) and non-profit organizations (Dutch Ministry of Infrastructure and Environment) She acted as a consultant for IntecPharma. The other authors have nothing to disclose. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Conclusion From the psychometric tests used in this study, the PVT and DAT seem most promising for initial evaluation of drug impairment based on sensitivity and correlations with driving Funding: The authors have no support or funding to report. Funding: The authors have no support or funding to report. 1 / 22 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Sensitivity and Validity of Tests for Assessing Driving Impairment effects of clinically relevant levels of alcohol reaching a Blood Alcohol Concentration (BAC) of 0.5 g/L [40]. In the field of ageing and dementia, The Concept Shifting Test (CST) or the equivalent Trail Making Test [41], [42] and a test of Useful Field of View (UFOV) [43] are used to assess driv- ing impairment. The Attention Network Test (ANT) is used in the field of neuropsychology [44] as it measures the efficiency of multiple attention networks. After initial screening, measures of driving with higher ecological validity are often used to assess drug induced impairment. The standardized highway driving test used in the Nether- lands [1], [2], [13] is a sensitive and reliable measure to assess drug induced impairment. Standard Deviation of Lateral Position (SDLP) is the primary outcome of this test and has been proven to be sensitive to the effects of many sedative drugs, such as alcohol [45], [46], an- tidepressants [8], antihistamines [47], [48], the residual effects of hypnotics [49], [50], [51], and was sensitive to the effects of sleep deprivation [52]. Therefore, the highway driving test is used as a reference to indicate the magnitude of the effect of clinically relevant levels of impairment after one night of sleep deprivation. Laboratory tests showing comparable effect sizes could be useful as initial screening tools to assess drug induced driving impairment. In addition, laboratory tests can be useful when they are able to predict actual driving im- pairment. Two reviews correlated drug-induced changes driving performance (SDLP) and per- formance in a number of psychometric tests [8], [53]. Ramaekers [8] calculated intrasubject correlations (n = 32) between changes in driving and psychomotor task performance across treatment conditions. Correlations were assessed in two separate studies showing mild and strong drug effects of antidepressant drugs (i.e. doxepin 75 mg and amitriptyline 75 mg, respec- tively). Tests of critical tracking, divided attention, choice reaction time, critical flicker fusion, memory, finger tapping, and vigilance were included. Results showed several significant corre- lations, but these were relatively modest. The highest correlations (r = 0.45) were found be- tween SDLP and tracking performance as measured by the CTT and the DAT for strong drug effects. The strength of the associations depended on the severity of the drug effects; correla- tions diminished with milder drug effects. PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Introduction Being sleep deprived while driving is a serious problem and is a direct or contribut- ing factor in road related accidents [20], [21], [22]. Many experimental studies have confirmed that driving related skills, such as vigilance and divided attention, deteriorates under conditions of sleep deprivation [23], [24], [25], [26], [27], [28], [29]. The present study includes sleep dep- rivation to induce clinically relevant levels of sedation to assess relevant impairment. A number of psychometric tests are often used to assess possible driving impairment, but the choice of tests differs depending on the area of research or practice. The Psychomotor Vigilance Test (PVT) is often used in sleep research for assessing drowsiness resulting from disturbed or insufficient sleep [30], [31], [32]. The Critical Tracking Test (CTT), Divided Attention Test (DAT), the Digit Symbol Substitution Test (DSST) and the Determination Test (DT) as part of the Vienna Test System [33] are often used in psychopharmacological studies to assess drug in- duced effects in healthy volunteers or patients [8], [11], [34], [35], [36], [37], [38]. A Postural Balance Test (PBT) has been indicated as a feasible test to assess drowsiness at the roadside [39]. Furthermore, several of these tests (i.e. PVT, DAT, and PBT) are sensitive to the impairing 2 / 22 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Participants Twenty-four healthy volunteers (12 males, 12 females) aged between 23–45 years were recruited through advertisements at Maastricht University. Initial screening was based on a medical histo- ry questionnaire. Eligible participants were invited for a physical examination, which included urinalysis, tests for drugs of abuse (amphetamines, benzodiazepines, cannabis, cocaine, 3,4- methylenedioxymethamphetamine, and opiates), and a 12-lead electrocardiogram. For partici- pation, the following inclusion criteria had to be met: possession of a valid driving license for three years or more, driving experience of at least 5000 km per year on average over the last three years and a body mass index (BMI) between 19 and 29 kg mˉ². Exclusion criteria included: shift work; history of a sleep disorder; extreme morning or evening type; any history of psychiat- ric or medical illness; history or current drug or alcohol abuse; current use of psycho-active medication; excessive caffeine use, defined as drinking six or more cups of coffee per day. The mean (± SD) age of the participants was 26.9 (± 3.4) years. The study was conducted in accordance with the code of ethics on human experimentation established by the declaration of Helsinki (1964) and amended in Seoul (2008). All participants were informed of the study’s goal, procedures, and potential hazards in writing, and they indicated their informed consent in writing. The Medical Ethics Committees of Maastricht University approved the study. Par- ticipants received a financial compensation for their participation in the study. Twenty-four healthy volunteers (12 males, 12 females) aged between 23–45 years were recruited through advertisements at Maastricht University. Initial screening was based on a medical histo- ry questionnaire. Eligible participants were invited for a physical examination, which included urinalysis, tests for drugs of abuse (amphetamines, benzodiazepines, cannabis, cocaine, 3,4- methylenedioxymethamphetamine, and opiates), and a 12-lead electrocardiogram. For partici- pation, the following inclusion criteria had to be met: possession of a valid driving license for three years or more, driving experience of at least 5000 km per year on average over the last three years and a body mass index (BMI) between 19 and 29 kg mˉ². Exclusion criteria included: shift work; history of a sleep disorder; extreme morning or evening type; any history of psychiat- ric or medical illness; history or current drug or alcohol abuse; current use of psycho-active medication; excessive caffeine use, defined as drinking six or more cups of coffee per day. Design The study was conducted according to a 2-period cross-over design to compare performance after one night of sleep deprivation with performance after a normal night of sleep. The high- way driving test was conducted twice: one after normal sleep and once after one night of sleep deprivation (i.e. 24h of wakefulness) both starting at 9:00 am. The psychometric tests were con- ducted on 4 occasions: once after normal sleep (at 11:00 am) and three times during a single night of sleep deprivation (at 1:00 am, 5:00 am, and 11:00 am, i.e. after 16h, 20h, and 26h of wakefulness). Both conditions were separated by an interval of at least one week, and the order was balanced over participants. To reduce order effects between the tests, the test battery was divided in two parts and these parts were balanced over participants. Participants The mean (± SD) age of the participants was 26.9 (± 3.4) years. The study was conducted in accordance with the code of ethics on human experimentation established by the declaration of Helsinki (1964) and amended in Seoul (2008). All participants were informed of the study’s goal, procedures, and potential hazards in writing, and they indicated their informed consent in writing. The Medical Ethics Committees of Maastricht University approved the study. Par- ticipants received a financial compensation for their participation in the study. Verster and Roth [53] analysed data from three studies (n = 96) showing varying effects of hypnotics, antihistamines, analgesics, and alcohol on actual driving and performance in tests of tracking, divided attention, memory and digit symbol substitution. Similar to the findings of Ramaekers [8], highest correlations with changes in SDLP were found for tracking in a contin- uous tracking test and a divided attention test (overall r = 0.47). Nevertheless, regression analy- sis showed that the combination of all performance parameters explained only 33% of the variance found in driving. Notably, the strength of the associations depended again on the se- verity of the impairing effects. The main objective of the present study was to determine the ability of nine psychometric tests to detect clinically relevant impairing effects of drowsiness as induced by one night of sleep deprivation. More specifically, the sensitivity of these laboratory tests was assessed during a single night of sleep deprivation at 1:00 am, 5:00 am, and 11:00 am, i.e. after 16, 20 and 26 hours of wakefulness and compared with performance after a night of normal sleep. A second- ary objective was to determine and compare the magnitude of the sedative effects on these tests during and after a night of sleep deprivation for future reference in clinical trials. We hypothe- sized that all these tests would be sensitive to the impairing effects of one night of sleep depriva- tion, but that the magnitude of the impairing effect would differ between tests. The magnitude of the effects in the laboratory tests was compared with the magnitude of effect on SDLP in the highway driving test. We expected a large effect of sleep deprivation on SDLP in the highway driving test. A third objective was to determine the correlations between performance changes 3 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment in these tests and those in driving performance to evaluate the validity of each test for predict- ing driving impairment (i.e. SDLP changes). PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Procedure Participants were individually trained to perform the behavioral tests prior to the first test day in two separate sessions. Participants agreed not to use any drugs of abuse or oral medication (except oral contraceptives and paracetamol) during the study. During participation in the study, alcohol intake was not allowed from 24 hours prior to each test day until discharge. On testing days, caffeine intake and smoking was not allowed until discharge. In both conditions, participants wore wrist actimeters and filled in sleep diaries to evaluate the rest/activity rhythm one week before testing. In the sleep deprivation condition, participants were called at 9:00 am to wake them up and to verify a night of good sleep assessed by the Groningen Sleep Quality Scale (GSQS, [54]). Par- ticipants arrived at the site at 9:00 pm in order to accompany them in complying with the pro- cedures. They yielded urine and breathe samples to confirm their compliance with prohibitions against use of drugs and to verify a BAC of 0.0 g/L. At 1:00 am and 5:00 am, the y y p p prohibitions against use of drugs and to verify a BAC of 0.0 g/L. At 1:00 am and 5:00 am, the 4 / 22 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Sensitivity and Validity of Tests for Assessing Driving Impairment participants performed the first and second session of the laboratory tests, comprising the Critical Tracking Test, Divided Attention Test, Psychomotor Vigilance Test, Digit Symbol Sub- stitution Test, Attention Network Test, Concept Shifting Test, Postural Balance Test, Determi- nation Test, and the test of Useful Field of View. At 8:00 am, a standardized breakfast was served. Participants were transported to the highway at 8:30 am. The highway driving test was conducted from 9:00 am to 10:00 am. Upon completion of the driving test the participants re- turned to the testing facilities for a third test session, starting at 11:00 am. See Fig. 1 for a time- line of testing during a single night of sleep deprivation. After a night of normal sleep, participants arrived at the site at 07:45 am. The GSQS was ad- ministered and participants yielded urine and breathe samples to confirm their compliance with the protocol. The highway driving test was conducted from 9:00 am to 10:00 am. The par- ticipants returned to the testing facilities for one test session comprising the laboratory tests described above. Procedure Both testing days ended at 13:00 pm. After normal sleep, participants were dismissed; after sleep deprivation, participants were driven home. Assessment The highest and lowest scores are removed, as extreme high or low scores could cause high skewness of the mean performance. The final score is the average of the three remaining scores. Divided Attention Task. The Divided Attention Task (DAT) measures the ability to divide attention between two simultaneously performed tasks [36]. In the primary task, the partici- pant performs the same tracking task described above, yet at a constant level of difficulty set at 50% of his or her maximum capacity, as measured by the individual’s best mean lambda score in the CTT at the end of training. In the secondary task, the participant monitors 24 peripheral displays in which single digits change asynchronously at 5-s intervals. Participants are in- structed to remove their foot from a pedal as rapidly as possible whenever the digit “2” appears. This signal occurs twice at every location, in random order, at intervals of 5–25 seconds. The primary dependent measures in each subtask are tracking error (in mm) and average reaction time to targets (in ms). Secondary control measures are control losses in the tracking task and number of hits in the target detection task. Digit Symbol Substitution Test. The Digit Symbol Substitution Test (DSST) measures many different psychomotor and cognitive functions at the same time [58]. A computerized version [59] of the original paper-and-pencil test taken from the Wechsler Adult Intelligence Scale is used (e.g. [60]). The participant is required to match each digit with a symbol from the encoding list as rapidly as possible by using a touch screen. The number of digits encoded cor- rectly within 3 min is the performance measure. Attention Network Test. The Attention Network Test (ANT) provides measures of three functions of attention within a single task [61]. Participants are instructed to keep their eyes fixed on a fixation cross throughout the test. Then, at some variable interval (ranging from 400 to 1600 ms) a cue is presented for 100 ms. After the offset of the cue, a target display appears, and remains on until response (i.e., a key-press indicating the direction of the target arrow), or for 1700 ms if no response is made. Dependent variables are total reaction time, alerting (i.e. Assessment Highway Driving Test. In the standardized highway driving test [2, 13] the participant oper- ates a specially instrumented vehicle over a 100-km (61-mile) primary highway circuit, accom- panied by a licensed driving instructor having access to dual controls. The task of the participant is to maintain a constant speed of 95 km/h (58 m/h) and a steady lateral position between the delineated boundaries of the right traffic lane. The vehicle speed and lateral posi- tion are recorded continuously. These signals are digitized at a rate of 4 Hz and edited off-line to remove data recorded during overtaking manoeuvres or disturbances caused by roadway or traffic situations. The remaining data are then used to calculate mean values and standard devi- ation of lateral position and speed. The primary outcome variable is Standard Deviation of Lat- eral Position (SDLP, in cm) which is a measure of road tracking error, or ‘weaving’. The secondary outcome variable is the Standard Deviation of Speed (SDSP), which is an index of the ability to maintain a constant speed. Psychomotor Vigilance Task. The Psychomotor Vigilance Task (PVT) is based on a simple visual reaction time test [55]. Mean reaction time (RT) in ms, inverse reaction time (1/RT), and lapses (i.e. RT > 500 ms) were calculated. 1/RT emphasizes slowing in the optimum and intermediate response domain and it substantially decreases the contribution of long lapses. For calculation mean 1/RT each RT (ms) was divided by 1,000 and then reciprocally trans- formed [56]. Fig 1. Timeline of the sleep deprivation condition. Abbreviation: GSQS = Groningen Sleep Quality Scale. doi:10.1371/journal.pone.0117045.g001 Fig 1. Timeline of the sleep deprivation condition. Abbreviation: GSQS = Groningen Sleep Quality Scale. doi:10.1371/journal.pone.0117045.g001 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 5 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment Critical Tracking Task. The Critical Tracking Task (CTT) measures the ability to control an unstable error signal in a first-order compensatory tracking task [57]. This test is designed to measure psychomotor coordination. Participants are instructed to keep an unstable cursor in the middle of a horizontal plane by counteracting or reverse its movements with the aid of a joystick. The frequency of cursor deviations at which the participant loses control is the critical frequency or lambda (λc), in rad sˉ1. The CTT includes five trials to obtain a reliable mean per- formance score. PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Assessment difference between reaction time in no cue condition and double cue condition), orienting (dif- ference between reaction time in center cue condition and spatial cue condition) and conflict (difference between reaction time with incongruent flankers and congruent flankers). The test duration is approximately 20 minutes. For a full description of the task, see the article of Fan and colleagues [61]. Concept Shifting Task. The computerized Concept Shifting Task (CST) is used to measure processing speed and cognitive flexibility [62]. It consists of three subtasks (A, B, and C). On each display 16 small circles are grouped into one larger circle. In the smaller circles the test items (numbers [A], letters [B] or both [C]) appear in a fixed random order. In part A, partici- pants are asked to cross out numbers (1–16) in the right order as quickly and accurately as pos- sible, using a touch screen. In part B, the circles contain letters (A–P) that have to be crossed out in alphabetical order. In part C, the both numbers and letters are displayed, and the participant is requested to alternate between numbers and letters. The time needed to complete each part is scored (CST-A, CST-B, CST-C in s, respectively). An interference score (CSTi) was obtained by the following formula: (CST-C −½  (CST-A + CST-B)) / (½  (CST-A + CST-B))  100. Postural Balance Test. The Postural Balance Test (PBT) is measured by using the AMTI AccuSway System for Balance and Postural Sway Measurement (Advanced Mechanical Postural Balance Test. The Postural Balance Test (PBT) is measured by using the AMTI AccuSway System for Balance and Postural Sway Measurement (Advanced Mechanical PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 6 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment Technology, Inc., Watertown, MA) force platform [63], [64]. Postural sway is assessed by mea- suring the length of the path of the centre-of-pressure (COP), and the area of the 95% confi- dence ellipse enclosing the COP (A95), which is the primary outcome measure in cm2. The test is conducted in two trials of both 60 seconds: one trial with the participants’ eyes open and one trial with eyes closed, both with feet apart at hip’s width. Determination Test. The Determination Test (DT) [33] is used to measure resilience of at- tention and reaction speed under conditions of sensory stress. Assessment The task of the participant is to identify various stimuli and to react to them by pressing the respective corresponding response buttons, using the response panel of the Vienna Test System. The test is administered as a com- puterized adaptive test whereby the presentation time of the stimuli adjusts itself to the reaction speed of the participant. However, unlike classic computerized adaptive tests, this test form presents the stimuli a little faster than would be optimal given the participants’ reaction speed, thus resulting in a condition of sensory stress. Median reaction time and correct responses were used to assess the performance of the participants. Total duration of the test is approxi- mately 4 min. Useful Field of View test. The test of Useful Field of View (UFOV) is a computer-based test measuring detection time for three subtests (visual processing speed, divided attention, and se- lective attention) which involve attentional tasks of increasing difficulty [65]. A total detection time was computed by summing the threshold scores for the 3 subtests. Total duration of the test is approximately 7 min. For a full description of the task, see the article of Edwards and colleagues [65]. Subjective rating scales. The driving instructors rated each participant’s driving quality and sedation at the conclusion of the highway driving test, using two 100-mm visual analogue scales. Participants rated their subjective feeling of sleepiness and driving quality prior to, half- way, and at the conclusion of the Highway Driving Test using the Karolinska Sleepiness Scale (KSS, [66]) and a modified version of the Driving Quality Scale (DQS, [67]). The KSS is a nine- point rating scale ranging from extremely alert (1) to very sleepy, great effort to keep alert, fighting sleep (9). The modified DQS is a 10 point rating scale ranging from extremely poor (1) to extremely good (10). PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Missing data One participant withdrew from further testing at 5:00 am due to excessive sleepiness. His data were excluded for statistical analyses. Due to technical problems, no data were collected for one participant during the DT and for another during the DAT on a single occasion. Only partici- pants with complete data sets were entered in the analysis of the respective performance pa- rameters. Outliers, defined as a difference by more than two times the standard deviation from the mean, were removed. Sensitivity and Validity of Tests for Assessing Driving Impairment effect sizes in repeated measure designs. Effect sizes between 0 and 0.19 are considered small, between 0.20–0.69 are considered moderate and 0.70 or higher are considered large [71]. effect sizes in repeated measure designs. Effect sizes between 0 and 0.19 are considered small, between 0.20–0.69 are considered moderate and 0.70 or higher are considered large [71]. To determine the correlations between changes in psychometric test performance and driv- ing, Pearson’s correlations were calculated between changes scores of SDLP and psychometric performance immediately before and after driving, i.e. at 5 am and at 11 am. Only significant correlations are reported. All statistical analyses were done by using the Statistical Package for the Social Sciences for Windows (version 21; SPSS Inc, Chicago, IL, USA). doi:10.1371/journal.pone.0117045.t001 Statistical analyses Sample size calculation was based on detecting a minimally relevant difference with an effect size of 0.25 between performance after a normal night of sleep and at 5:00 am, i.e. after 20 hours of wakefulness during a night of sleep deprivation. Given a test-retest reliability of pa- rameters at the Psychomotor Vigilance Test of at least r = 0.80 [68], a group of 24 participants should permit detection of a mean change in reaction time, with a power of at least 95% and an α of .05. For the highway driving test, each variable was analyzed using General Linear Model (GLM) repeated measures with wakefulness (two levels) as within subject factor. For the labo- ratory tests, each variable was analyzed using GLM repeated measures with wakefulness (four levels) as within subject factor. Three separate sleep deprivation-normal sleep contrasts were conducted when an overall effect of sleep deprivation was found. Change scores for each of the dependent variables were transformed to z-scores, which were calculated across the pooled changes in the single night of sleep deprivation on three occasions relative to performance on a separate day after one night of normal sleep. This allows for easy comparison across each of the various performance tests [69]. To determine the magnitude of the simple effects at various times during the night of sleep deprivation, Dunlap et al.’s [70] ef- fect size (ES) statistics (i.e. tc[2(1-r)/n]1/2) were calculated. This statistic is used to calculate PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 7 / 22 Effects of sleep deprivation on highway driving test Table 1 summarizes the mean (SE) scores obtained in the highway driving test. Seven driving tests were terminated before scheduled completion, all in the sleep deprivation condition. One test was terminated by the driving instructor because he judged the participant to be too Table 1. Mean (SE) and effects of one night of sleep deprivation in the on-the-road highway driving test. Time of day Overall effect 9:00 am 9:00 am (+24 h) F Dunlap’s ES SDLP 15.11 (0.6) 18.23 (0.7) 30.29*** 0.97 SDSP 2.09 (0.08) 2.50 (0.14) 8.56** 0.73 Mean lateral position 95.70 (2.6) 97.15 (2.4) 0.76 0.12 Mean speed 93.37 (0.4) 92.69 (0.6) 2.34 0.27 Subjective sleepiness (KSS 1–9) Before driving 2.83 (0.2) 5.78 (0.4) 49.72*** 2.17 At turning point 2.65 (0.3) 6.74 (0.3) 108.60*** 2.89 After driving 3.28 (0.3) 6.94 (0.4) 82.28*** 1.95 Subjective driving quality (1–10) First part 7.48 (0.2) 5.61 (0.4) 19.52*** 1.32 Second part 7.44 (0.2) 5.72 (0.4) 13.83** 1.19 Instructor rating (VAS mm) Sedation 13.65 (2.1) 56.35 (6.0) 59.65*** 1.79 Driving quality 25.96 (2.5) 41.00 (3.3)a 12.15** 1.06 Abbreviations: ES = Effect Size; SDLP = Standard Deviation of Lateral Position, SDSP = Standard Deviation of Speed, KSS = Karolinska Sleepiness Scale, VAS = Visual Analogue Scale. *p < 05 Table 1. Mean (SE) and effects of one night of sleep deprivation in the on-the-road highway driving test. Table 1. Mean (SE) and effects of one night of sleep deprivation in the on-the-road highway driving test. Time of day Ov Abbreviations: ES = Effect Size; SDLP = Standard Deviation of Lateral Position, SDSP = Standard Deviation of Speed, KSS = Karolinska Sleepiness Scale, VAS = Visual Analogue Scale. **p <. 01, ***p <. 001 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 8 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment drowsy to continue safely. Six tests were terminated by participants because they felt too drowsy to continue safely. All premature terminations occurred between 30 and 60 minutes of the driving test. Of these driving tests, SDLP scores were calculated from the data collected until termination of the test. Analysis showed that mean SDLP was significantly increased by 3.1 cm after sleep depriva- tion compared to driving after a night of normal sleep (F1, 22 = 30.29, p <. 001). This increase corresponds to an effect size of 0.97. Effects of sleep deprivation on highway driving test SDSP was significantly increased after one night of sleep deprivation as compared with driving after a normal night of sleep (F1, 22 = 8.56, p <. 01). This increase corresponds to an effect size of 0.73. Subjective rating scales. In the sleep deprivation condition, participants felt significantly more sleepy than normal, as measured with the KSS before (F1, 22 = 49.72, p <. 001) halfway (F1, 22 = 108.60, p <. 001), and at the end (F1, 22 = 82.28, p <. 001) of the driving test. At the same time, participants judged their driving quality to be worse than normal during the first and second half of the driving test (F1, 22 = 19.52, p <. 001; F1, 22 = 13.83, p <. 001, respec- tively). In line with this, instructors judged subjects to appear more sedated (F1, 22 = 59.65, p <. 001) and drive worse (F1, 22 = 12.15, p <. 01) after one night of sleep deprivation com- pared with driving after a normal night of sleep. PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Laboratory tests Table 2 presents a summary of the means and standard errors of the means (SE) of all perfor- mance scores, the results of the analyses of variance, and the simple contrasts. Psychomotor Vigilance Test. Mean reaction time (F3, 18 = 9.20, p <. 01) and lapses (F3, 18 = 6.37, p <. 01) in the PVT were significantly different between the conditions of sleep dep- rivation. Contrast analysis indicated an increase of mean reaction time at 5:00 am (F1, 20 = 10.95, p <. 01) and 11:00 am (F1, 20 = 30.30, p <. 001) during sleep deprivation compared with mean re- action time after a night of normal sleep. Lapses increased at 5:00 am (F1, 22 = 6.95, p <. 05) and 11:00 am (F1, 22 = 20.66, p <. 001) during sleep deprivation compared with lapses after a normal night of sleep. To decrease the contribution of long lapses, inverse reaction times (1/RT) l l d ff f l d f d ( ) Psychomotor Vigilance Test. Mean reaction time (F3, 18 = 9.20, p <. 01) and lapses (F3, 18 = 6.37, p <. 01) in the PVT were significantly different between the conditions of sleep dep- rivation. Contrast analysis indicated an increase of mean reaction time at 5:00 am (F1, 20 = 10.95, p <. 01) and 11:00 am (F1, 20 = 30.30, p <. 001) during sleep deprivation compared with mean re- action time after a night of normal sleep. Lapses increased at 5:00 am (F1, 22 = 6.95, p <. 05) and 11:00 am (F1, 22 = 20.66, p <. 001) during sleep deprivation compared with lapses after a normal night of sleep. To decrease the contribution of long lapses, inverse reaction times (1/RT) were calculated. A main effect of sleep deprivation was found at 1/RT (F3, 20 = 22.19, p <. 001). Contrast analysis indicated a decrease of 1/RT at 5:00 am (F1, 22 = 24.22, p <. 001) and 11:00 am (F1, 22 = 44.26, p <. 001) during sleep deprivation compared with 1/RT after a normal night of sleep (see Fig. 2). g p g p were calculated. A main effect of sleep deprivation was found at 1/RT (F3, 20 = 22.19, p <. 001). Contrast analysis indicated a decrease of 1/RT at 5:00 am (F1, 22 = 24.22, p <. 001) and 11:00 am (F1, 22 = 44.26, p <. Sensitivity and Validity of Tests for Assessing Driving Impairment Table 2. Mean (SE), Overall Effects of Sleep Deprivation and Contrast Analyses of Laboratory Tests. Laboratory tests 001) during sleep deprivation compared with 1/RT after a normal night of sleep (see Fig. 2). Critical Tracking Test. A trend towards a main effect of sleep deprivation was found on mean tracking performance in the CTT (F3, 20 = 2.98, p = .06). Contrast analysis revealed a trend towards an increase of mean tracking performance at 5:00 am (F1, 22 = 3.29, p = .08) and a trend towards a decrease of tracking performance at 11:00 am (F1, 22 = 4.00, p = .06) during sleep deprivation compared with performance after a normal night of sleep (see Fig. 2). Divided Attention Test. Two participants were not able to perform the DAT (i.e. more than 100 control losses) at 11:00 am during sleep deprivation; therefore these participants were not considered for the analysis. The secondary control measures, control losses (F3, 18 = 4.85, p = .01) and misses (F3, 18 = 4.81, p = .01), were significant different between conditions of sleep deprivation. These variables are therefore taken into account for measuring primary and secondary task performance. The distributions of control losses and misses were highly skewed. Therefore, transforma- tions were applied to their logarithmic scores (log 10) before transformation to z-scores. Log 10 was applied to deal with zero values by using the formula NEWX = LG10 (X + 1). ANOVA of tracking performance (i.e. the sum scores of the z-scores of the average tracking error and log10 of the total number of control losses) revealed a significant main effect (F3, 17 = 18.54, PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 9 / 22 Table 2. Mean (SE), Overall Effects of Sleep Deprivation and Contrast Analyses of Laboratory Tests. Normal night of sleep Sleep deprivation Overall effect Simple contrasts 11:00 am 01:00 am 05:00 am 11:00 am 11:00 am versus 01:00 am 11:00 am versus 05:00 am 11:00 am vers 11:00 am Test Mean (SE) F p p p p Psychomotor Vigilance Test Inverse reaction time 3.96 (0.09) 4.02 (0.07) 3.57 (0.14) 3.44 (0.10) 22.19 <0.001 0.22 <0.001 <0.001 Mean reaction time (ms) 258 (4.9) 265 (6.6) 308 (17.2) 328.5 (13.5) 9.20 <0.01 0.09 <0.01 <0.001 Lapses 0.83 (0.30) 1.13 (0.28) 6.30 (2.06) 8.39 (1.68) 6.37 <0.01 0.44 0.02 <0.001 Critical Tracking Test Mean lambda (rad/s) 3.73 (0.12) 3.82 (0.11) 3.65 (0.15) 3.52 (0.16) 2.98 0.06 0.08 0.41 0.06 Divided Attention Test z-AE+z-lg10(cl) -0.65 (0.29) -0.95 (0.29) 0.31 (0.41) 1.27 (0.38) 18.54 <0.001 0.22 <0.01 <0.001 z-RT+ z-lg10(mi) -0.48 (0.36) -0.46 (0.33) 0.20 (0.50) 0.75 (0.44) 6.47 <0.01 0.92 0.05 <0.01 Attention Network Test Overall reaction time (ms) 492 (12) 492 (13) 552 (22) 586 (20) 11.73 <0.001 0.99 <0.01 <0.001 Alerting effect (ms) 53 (6) 55 (5) 59 (10) 69 (7) 1.52 0.24 0.69 0.48 0.06 Orienting effect (ms) 49 (4) 51 (4) 57(5) 32 (5) 2.30 0.11 0.22 0.72 0.27 Conflict effect (ms) 103 (6) 96 (6) 115 (9) 130 (10) 6.23 <0.01 0.10 0.17 0.03 Digit Symbol Substitution Test Correct responses 105.6 (3.0) 101.4 (3.1) 96.4 (3.3) 98.4 (3.6) 3.63 0.03 0.03 0.021 <0.01 Concept Shifting Test Reaction time CST-A (s) 18.6 (0.9) 19.1 (0.9) 19.0 (0.6) 19.0 (0.6) 0.65 0.59 0.59 0.20 0.42 Reaction time CST-B (s) 21.1 (0.9) 21.8 (0.9) 22.1 (0.7) 21.6 (0.7) 1.43 0.27 0.27 0.10 0.06 Reaction time CST-C (s) 24.9 (1.2) 25.7 (1.4) 28.3 (1.2) 24.9 (1.0) 3.22 0.05 0.05 0.36 <0.01 Interference (CSTi) 25.7 (3.2) 24.8 (3.6) 38.2 (5.1) 22.8 (2.8) 2.04 0.14 0.79 0.04 0.53 Determination Test Correct responses 296.4 (9.0) 291.1 (9.1) 278.0 (12.3) 279.0 (12.8) 2.36 0.10 0.28 0.06 0.02 Median reaction time (ms) 646 (14) 665(14) 681 (18) 681 (20) 3.89 0.03 <0.01 <0.01 0.02 Useful Field of View Test Total detection time (ms) 80.6 (6.4) 86.3 (8.2) 140.0 (19.8) 131.4 (17.4) 4.26 0.02 0.38 <0.01 <0.01 Postural Balance Test Eyes open—ln-area 95 (cm2) 0.42 (0.16) 0.43 (0.16) 0.84 (0.13) 0.61 (0.14) 7.34 <0.01 0.91 <0.01 0.15 Eyes closed—ln-area 95 (cm2) 0.61 (0.1) 0.60 (0.1) 1.0 (0.2) 0.90 (0.2) 3.82 0.03 0.97 <0.01 0.04 Abbreviations: z-AE = z-score of average tracking error; z-lg10(cl) = z-score of log transformed total number of control losses; z-RT = z-score of reactio time; z-log10(mi) = z-score of log transformed total number of misses; ln = natural log. Abbreviations: z-AE = z-score of average tracking error; z-lg10(cl) = z-score of log transformed total number of control losses; z-RT = z-score of reaction time; z-log10(mi) = z-score of log transformed total number of misses; ln = natural log. doi:10.1371/journal.pone.0117045.t002 Test a sum of the z-scores of reac- tion time and log10 of total number of misses) was found (F3, 17 = 6.47, p <. 01). Contrast analyses indicated that target detection performance was significant decreased at 5:00 am (F1, 19 = 4.48, p <. 05) and 11:00 am (F1, 19 = 14.50, p = .001) during sleep deprivation com- pared with target detection performance after a normal night of sleep (see Fig. 2). Attention Network Test. A main effect of sleep deprivation was found on total reaction time in the ANT (F3, 20 = 11.73, p <. 001). Total reaction time increased at 5:00 am (F1, 22 = 13.79, p = .001) and 11:00 am (F1, 22 = 27.22, p <. 001) during sleep deprivation compared with total reaction time after a normal night of sleep (see Fig. 3). The conflict effect was different be- tween conditions of sleep deprivation (F3, 20 = 6.23, p <. 01). The conflict effect increased at 11:00 am (F1, 22 = 5.08, p <. 05) during sleep deprivation compared with the conflict effect after a night of normal sleep. No main effects of sleep deprivation were found for the alerting and orienting effect (see Fig. 2). Digit Symbol Substitution Test. The amount of correct responses in the DSST was signifi- cantly different between the conditions of sleep deprivation (F3, 20 = 3.63, p <.05). Participants’ correct responses decreased significantly at 1:00 am (F1, 22 = 6.17, p <. 05), 5:00 am (F1, 22 = 11.13, p <. 01), and 11:00 am (F1, 22 = 9.32, p <. 01) during a night of sleep deprivation com- pared with correct responses after a normal night of sleep (see Fig. 3). Concept Shifting Test. A main effect of sleep deprivation was found on subtest C in the CST (F3, 20 = 3.22, p <. 05). Reaction time of part C increased at 5:00 am (F1, 22 = 8.95, p <. 01) during sleep deprivation compared with reaction time of part C after a normal night of sleep (see Fig. 3). No main effects of sleep deprivation were found at the subtests A and B and at the interference score (CSTi). Determination Test. Median reaction time at the DT was significantly different between the conditions of sleep deprivation (F3, 19 = 3.89, p <. 05). Test PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 10 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment Fig 2. Mean baseline normalized performance at 1:00am (16 hours awake), 5:00am (20 hours awake) and 11:00 am (26 hours awake) compared with performance after a normal night of sleep (at 11:00 am) across dependent variables of the Psychomotor Vigilance Test, Critical Tracking Test, Divided Attention Test, and Attention Network Test. *p < 0.05, **p < 0.01, ***p< 0.001. Error bars indicate the standard error of the mean. doi:10.1371/journal.pone.0117045.g002 Sensitivity and Validity of Tests for Assessing Driving Impairment Fig 2. Mean baseline normalized performance at 1:00am (16 hours awake), 5:00am (20 hours awake) and 11:00 am (26 hours awake) compared with performance after a normal night of sleep (at 11:00 am) across dependent variables of the Psychomotor Vigilance Test, Critical Tracking Test, Divided Attention Test, and Attention Network Test. *p < 0.05, **p < 0.01, ***p< 0.001. Error bars indicate the standard error of the mean. doi:10.1371/journal.pone.0117045.g002 doi:10.1371/journal.pone.0117045.g002 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 11 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment p <. 001) of sleep deprivation. Contrast analyses indicated that tracking performance was sig- nificantly decreased at 5:00 am (F1, 19 = 8.76, p <. 01) and 11:00 am (F1, 19 = 53.29, p <. 001) during sleep deprivation compared with tracking after a normal night of sleep (see Fig. 2). A significant main effect in target detection performance (i.e. a sum of the z-scores of reac- tion time and log10 of total number of misses) was found (F3, 17 = 6.47, p <. 01). Contrast analyses indicated that target detection performance was significant decreased at 5:00 am (F1, 19 = 4.48, p <. 05) and 11:00 am (F1, 19 = 14.50, p = .001) during sleep deprivation com- pared with target detection performance after a normal night of sleep (see Fig. 2). p <. 001) of sleep deprivation. Contrast analyses indicated that tracking performance was sig- nificantly decreased at 5:00 am (F1, 19 = 8.76, p <. 01) and 11:00 am (F1, 19 = 53.29, p <. 001) during sleep deprivation compared with tracking after a normal night of sleep (see Fig. 2). A significant main effect in target detection performance (i.e. PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Test Contrast analysis revealed that medi- an reaction time increased at 1:00 am (F1, 21 = 9.59, p <. 01), 5:00 am (F1, 21 = 8.30, p <. 01), and 11:00 am (F1, 19 = 7.03, p <. 05) during sleep deprivation compared with median reaction time after a night of normal sleep (see Fig. 3). No main effects were found on the other variables (i.e. correct responses, misses, and mistakes). Useful Field of View test. A main effect of sleep deprivation was found on the third subtest (i.e. selective attention) of the UFOV (F3, 18 = 5.05, p = .01). Detection time at the selective at- tention test increased at 5:00 am (F1, 20 = 12.47, p <. 01) and 11:00 am (F1, 20 = 11.93, p <. 01) during sleep deprivation compared with detection time at the selective attention test after a night of normal sleep. No main effects were found on subtest one (i.e. visual processing speed) and two (i.e. divided attention) of the UFOV, although a trend was found toward a main effect of sleep deprivation on the divided attention subtest (F3, 17 = 3.14, p = .052). A main effect of sleep deprivation was found on the total score of the UFOV (F3, 18 = 4.26, p <. 05). Contrast analysis revealed that total detection time increased at 5:00 am (F1, 20 = 11.62, p <. 01), and 11:00 am (F1, 20 = 12.13, p <. 01) during sleep deprivation compared with total detection time after a night of normal sleep (see Fig. 3). Postural Balance Test. As data of the area of the 95% confidence ellipse enclosing the COP (A95) in the PBT was not normally distributed, data was log transformed (e.g. [72]). Main ef- fects of sleep deprivation were found on A95 with both eyes open (F3, 19 = 7.34, p <. 01) and eyes closed (F3, 19 = 3.82, p <. 05). Contrast analysis revealed this effect was due to performance at 5:00 am (F1, 21 = 11.67, p <. 01) compared with A95 with eyes open after a night of normal sleep (see Fig. 3). With eyes closed, contrast analysis revealed this effect was due to 12 / 22 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Sensitivity and Validity of Tests for Assessing Driving Impairment Fig 3. Comparison of performance measures A summary of the mean difference with 95% confidence intervals, mean baseline-normalized z-scores, and Dunlap’s effect sizes (ES) is shown in Table 3. Effect sizes and z-scores indicate that tasks and parameters differ in sensitivity to the effects of one night of sleep deprivation. At 11:00 am after a night of sleep deprivation, largest effect sizes were found in the DAT on prima- ry task performance and false alarms in secondary task performance (ES = 1.26 and 0.86, re- spectively), inverse reaction time, mean reaction time and lapses in the PVT (ES = 1.13, 0.98, and 1.23, respectively), overall reaction time in the ANT (ES = 1.13) and total detection time in the UFOV (ES = 0.70). At 5:00 am during the night of sleep deprivation, all these tests showed smaller effect sizes (0.60  ES  0.88) compared with 11:00 after a night of sleep deprivation. At 5:00 am, effect sizes were moderate (0.56  ES  0.62) on the test parameters of the CST and PBT, but smaller (0.00  ES  0.41) at 11:00 am during sleep deprivation. At 1:00 am during the night of sleep deprivation, two test parameters, correct responses in the DSST and median reaction time in the DT, showed moderate effect sizes (both ES = 0.28). These parameters showed moderate effects at 5:00 am and 11:00 am during and after a night of sleep deprivation: 0.60 and 0.43 on correct responses in the DSST and 0.45 and 0.39 on median reaction time in the DT, respectively. Smallest effect sizes, i.e. 0.11 and 0.29, were found on lambda in the CTT at 5:00 am and 11:00 am, respectively, during sleep deprivation, respectively. Test Mean baseline normalized performance at 1:00 am (16 hours awake), 5:00 am (20 hours awake) and 11:00 am (26 hours awake) compare with performance after a normal night of sleep (at 11:00 am) across dependent variables of the Digit Symbol Substitution Test, Concept Shifti Test, Determination Test, Useful Field of View Test, and Postural Balance Test. *p < 0.05, **p < 0.01, ***p <0.001. Error bars indicate the standa error of the mean. doi:10.1371/journal.pone.0117045.g003 Fig 3. Mean baseline normalized performance at 1:00 am (16 hours awake), 5:00 am (20 hours awake) and 11:00 am (26 hours awake) compared with performance after a normal night of sleep (at 11:00 am) across dependent variables of the Digit Symbol Substitution Test, Concept Shifting Test, Determination Test, Useful Field of View Test, and Postural Balance Test. *p < 0.05, **p < 0.01, ***p <0.001. Error bars indicate the standard error of the mean. doi:10.1371/journal.pone.0117045.g003 doi:10.1371/journal.pone.0117045.g003 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 13 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment performance at 5:00 am (F1, 21 = 8.53, p <. 01) and 11:00 am (F1, 21 = 4.64, p <. 05) during sleep deprivation (see Fig. 3). Correlations between laboratory tasks and highway driving test Table 4 summarizes the significant correlations between changes in laboratory tasks and changes in the on-the-road highway driving test. Changes of mean reaction time in the PVT between 5:00 am and performance after a normal night of sleep correlated with changes in SDLP (r = 0.68, p <. 01) (see Fig. 4). SDLP changes significantly correlated with changes in lapses (r = 0.66, p <. 01) and inverse reaction time (r = -0.52, p <. 05) in the PVT between 5:00 am and performance after a normal night of sleep. Moderate correlations were found be- tween changes of SDLP and changes of total mean reaction time in the ANT (r = 0.48, p <. 05), median reaction time in the DT (r = 0.47, p <. 05), and secondary task performance in the DAT (r = 0.47, p <. 05) between 5:00 am during sleep deprivation and performance after a nor- mal night of sleep. Moderate correlations were found between changes in SDSP and changes of correct re- sponses (r = -0.56, p <. 01) and median reaction time (r = 0.55, p <. 05) in the DT between 5:00 am and performance after a normal night of sleep. Changes in SDSP correlated moderately with changes of correct responses in the DSST (r = -0.42, p <. 05) between 11:00 am during sleep deprivation and performance after a normal night of sleep. Sensitivity and Validity of Tests for Assessing Driving Impairment th 95% confidence intervals with performance after normal night of sleep, mean baseline*-normalized z- of the performance tests. Table 3. Mean difference scores with 95% confidence intervals with performance after normal night of sleep, mean baseline*-normalized z- scores, and effect sizes (Dunlap’s) of the performance tests. Table 3. Mean difference scores with 95% confidence intervals with performance after normal night of sleep, mean baseline*-normalized z- scores, and effect sizes (Dunlap’s) of the performance tests. Discussion The main objective of the present study was to determine the ability of nine psychometric tests to detect performance impairing effects of clinically relevant effects of drowsiness as induced by one night of sleep deprivation. After one night of sleep deprivation, performance at all psy- chometric tests except the CTT, was decreased. Specifically, performance in the DT and DSST was decreased at 1:00 am. At 5:00 am during a night of sleep deprivation, performance was im- paired in the PVT, DAT, ANT, UFOV, DT, PBT, CST and DSST. At 11:00 am after a night of sleep deprivation, performance was impaired in all these tests, except the CST. The magnitude PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 14 / 22 g Abbreviations: RT = Reaction Time; z-AE = z-score of average tracking error; z-lg10(cl) = z-score of log transformed total number of control losses; z-RT = z-score of reaction time; z-log10(mi) = z-score of log transformed total number of misses; ln = natural log. doi:10.1371/journal.pone.0117045.t003 * baseline is performance after a night of normal sleep on a separate day. 01:00 am (16h awake) 05:00 am (20h awake) 11:00 am (26h awake) 1:00 am 5:00 am 11:00 am 11:00 am versus 01:00 am 11:00 am versus 05:00 am 11:00 am versu 11:00 am Test 95% CI 95% CI 95% CI z-scores ES ES ES Psychomotor Vigilance Test Inverse reaction time +0.06 -0.04 to 0.17 -0.39 -0.55 to- 0.22 -0.51 -0.67 to- 0.35 -0.21 0.59 1.09 -0.15 0.60+ 1.13++ Mean reaction time (ms) +6 -1 to 15 +50 18 to 81 +70 44 to 97 0.23 0.47 0.69 0.21+ 0.62+ 0.98++ Lapses +0.30 -0.49 to 1.10 +5.48 1.17 to 9.79 +7.57 4.11 to 11.02 0.22 0.68 1.26 0.22+ 0.78++ 1.23++ Critical Tracking Test Mean Lambda (rad/s) +0.09 -0.01 to 0.19 -0.08 -0.27 to 0.12 -0.21 -0.42 to 0.01 -0.16 0.11 0.27 -0.16 0.11 0.29+ Divided Attention Test z-AE+z-lg10(cl) -0.30 -0.80 to 0.20 +0.96 0.28 to 1.63 +1.92 1.37 to 2.47 -0.23 0.53 1.13 -0.22 0.62+ 1.26++ z-RT+ z-lg10(mi) +0.02 -0.45 to 0.49 +0.68 0.01 to 1.36 +1.23 0.55 to 1.90 0.01 0.31 0.62 0.01 0.32+ 0.67+ Attention Network Test Overall reaction time (ms) 0 -16 to 17 +60 27 to 94 +94 57 to 132 0.00 0.58 0.98 0.00 0.63+ 1.13++ Alerting Effect (ms) +2 -7 to 10 +6 -12 to 24 +16 -1 to 33 0.07 0.14 0.48 0.06 0.16 0.51+ Orienting Effect (ms) +2 -7 to 10 +8 -3 to 18 -2 -16 to 12 0.08 0.33 -0.07 0.08 0.34+ -0.08 Conflict Effect (ms) -8 -17 to 2 +12 -5 to 29 +26 2 to 50 -0.29 0.28 0.53 -0.26 0.32+ 0.63+ Digit Symbol Substitution Test Correct responses -4.2 -7.7 to- 0.69 -9.2 -14.9 to- 3.5 -7.2 -12.0 to- 2.3 0.28 0.58 0.41 0.28+ 0.60+ 0.43+ Concept Shifting Test Reaction time CST-A (s) +0.5 -0.3 to 1.4 +0.5 -0.7 to 1.6 +0.4 -0.9 to 1.8 0.13 0.16 0.14 0.13 0.12 0.11 Reaction time CST-B (s) +0.7 -0.1 to 1.6 +1.0 -0.1 to 2.1 +0.5 -0.8 to 1.7 0.17 0.29 0.13 0.16 0.23+ 0.11 Reaction time CST-C (s) +0.7 -0.9 to 2.4 +3.4 1.0 to 5.7 0.0 -1.8 to 1.8 0.11 0.61 0.01 0.11 0.59+ 0.00 Interference (CSTi) -0.9 -8.0 to 6.2 +12.5 0.6 to 24.4 -2.9 -12.3 to 6.5 -0.05 0.53 -0.21 -0.06 0.62+ -0.20+ Determination Test Correct responses +18 6 to 30 +35 10 to 60 +34 7 to 61 0.28 0.42 0.37 0.13 0.35+ 0.29+ Median reaction time (ms) -5.3 -15.2 to 4.6 -18.5 -37.8 to 0.9 -17.5 -32.1 to- 2.8 0.12 0.32 0.29 0.28+ 0.45+ 0.39+ Useful Field of View Test Total detection time (ms) +5.7 -7.7 to 19.1 +59.4 23 to 96 +50.8 20.4 to 81.3 0.15 0.65 0.64 0.17 0.75++ 0.70++ Postural Balance Test Eyes open—ln-area 95 (cm2) +0.01 -0.24 to 0.27 +0.42 0.17 to 0.68 +0.19 -0.07 to 0.45 0.16 0.38 0.06 0.02 0.62+ 0.27+ Eyes closed—ln-area 95 (cm2) -0.00 -0.26 to 0.25 +0.39 0.11 to 0.67 +0.30 0.01 to 0.58 -0.01 0.50 0.37 0.01 0.56+ 0.41+ i di t d t ff t i Test ++ indicates large effect sizes. Abbreviations: RT = Reaction Time; z-AE = z-score of average tracking error; z-lg10(cl) = z-score of log transformed total number of control losses; z-RT = z-score of reaction time; z-log10(mi) = z-score of log transformed total number of misses; ln = natural log. PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 15 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment Table 4. Significant correlations between changes in laboratory test parameters and changes in on-the-road highway driving parameters between time points during a night of sleep deprivation and testing after a normal night of sleep. Δ SDLP Test Variable Time of day during sleep deprivation na Pearson’s r Psychomotor Vigilance Test Mean reaction time 5:00 am 21 0.68** Lapses 5:00 am 21 0.66** Inverse reaction time 5:00 am 21 -0.52* Attention Network Test Total mean reaction time 5:00 am 23 0.48* Determination Test Median reaction time 5:00 am 20 0.47* Divided Attention Test Mean reaction time and misses 5:00 am 20 0.47* Δ SDSP Determination Test Correct responses 5:00 am 20 -0.56** Median reaction time 5:00 am 20 0.55* Digit Symbol Substitution Test Correct responses 11:00 am 23 -0.42* a outliers, i.e. values larger than ± 2 SD from the mean, were removed. *p <. 05, **p <. 01. Abbreviations: SDLP = Standard Deviation of Lateral Position, SDSP = Standard Deviation of Speed. Table 4. Significant correlations between changes in laboratory test parameters and changes in on-the-road highway driving parameters between time points during a night of sleep deprivation and testing after a normal night of sleep. PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Test p <. 01. Abbreviations: SDLP = Standard Deviation of Lateral Position, SDSP = Standard Deviation of Speed. doi:10.1371/journal.pone.0117045.t004 doi:10.1371/journal.pone.0117045.t004 of effect was different among the tests at 5:00 am and 11:00 am during and after a night of sleep deprivation; largest effect sizes (i.e.  0.70) were found at the PVT, DAT, UFOV, and ANT. of effect was different among the tests at 5:00 am and 11:00 am during and after a night of sleep deprivation; largest effect sizes (i.e.  0.70) were found at the PVT, DAT, UFOV, and ANT. In the highway driving test, a large impairing effect was found on SDLP after one night of sleep deprivation. SDLP increased with 3.1 cm compared with SDLP after a normal night of sleep, which is comparable to impairment at a BAC between 0.5 and 0.8 g/L [45] indicating the clinically relevant impairment after one night of sleep deprivation. p g In the highway driving test, a large impairing effect was found on SDLP after one night of sleep deprivation. SDLP increased with 3.1 cm compared with SDLP after a normal night of sleep, which is comparable to impairment at a BAC between 0.5 and 0.8 g/L [45] indicating the clinically relevant impairment after one night of sleep deprivation. p g In the highway driving test, a large impairing effect was found on SDLP after one night of sleep deprivation. SDLP increased with 3.1 cm compared with SDLP after a normal night of sleep, which is comparable to impairment at a BAC between 0.5 and 0.8 g/L [45] indicating the clinically relevant impairment after one night of sleep deprivation. Fig 4. Correlations between changes (performance at 5:00 am—performance after normal night of sleep) in mean reaction time in the Psychomotor Vigilance Test and changes in SDLP. doi:10.1371/journal.pone.0117045.g004 Fig 4. Correlations between changes (performance at 5:00 am—performance after normal night of sleep) in mean reaction time in the Psychomotor Vigilance Test and changes in SDLP. Fig 4. Correlations between changes (performance at 5:00 am—performance after normal night of sleep) in mean reaction time in the Psychomotor Vigilance Test and changes in SDLP. Test doi:10.1371/journal.pone.0117045.g004 PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 16 / 22 Sensitivity and Validity of Tests for Assessing Driving Impairment As expected, lapses and inverse reaction time in the PVT were among the most sensitive pa- rameters to the impairing effects of being awake for 20 and 26 hours, which is in line with pre- vious studies (e.g. [26], [30], [31], [32], [55]). The PVT was previously found to be sensitive to alcohol doses reaching clinically relevant BAC of 0.5 g/L [40] and lower [73]. The PVT is a promising screening tool to detect impairment, although it has not yet been shown to be sensi- tive to the impairing effect of medicinal drugs, such as zopiclone 7.5 mg [74]. Previous studies have consistently shown that the residual effects of zopiclone 7.5 mg impair driving perfor- mance, as indicated by an increase of SDLP comparable to impairment with a BAC of 0.5 g/L [49], [51]. Further research should examine the sensitivity of the PVT to benchmark sedative drugs to indicate its ability to detect specific drug effects. The effects of sleep deprivation at the DAT are in line with previous studies [24], [27]. The DAT has previously been shown to be sensitive to the effects of many sedative drugs such as doses of alcohol, antidepressants, antihistamines, and the residual effects of hypnotics [37], [40], [46], [48], [60], [74]. The present results support the use of the DAT as screening tool for assessing clinically relevant driving impairment. In the present study, performance at an UFOV test was impaired after sleep deprivation, which is in line with a previous study assessing these effects on performance in the UFOV while driving in a simulator [75]. The UFOV test seems a promising screening tool to assess drug induced impairment, although no significant effects were found in a previous study with alcohol reaching a clinically relevant blood alcohol level of 0.48 g/L [69]. Further research should therefore assess the sensitivity of the UFOV test to the specific effects of sedative drugs. The ANT was also one of the most sensitive tests after sleep deprivation in the current study. The sensitivity of the ANT after sleep deprivation is in line with previous studies [76] [77]. Although total reaction time and the conflict effect were affected, the orienting and alert- ing effects were not increased after sleep deprivation. PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 Sensitivity and Validity of Tests for Assessing Driving Impairment should therefore be conducted to determine the ability of the PVT and other tasks to detect clinically relevant drug induced impairment. To summarize, largest effects of sleep deprivation were found on performance in the PVT, DAT, ANT and UFOV. These effects were comparable to or larger than the effect on SDLP in the highway driving test. Furthermore, these tests were minimally influenced by circadian ef- fects, as performance impairment was larger at 11:00 am compared with 5:00 am during sleep deprivation. In addition, performance changes in the PVT, DAT and ANT correlated signifi- cantly with changes in SDLP. Performance changes in the UFOV did not correlate with changes in driving and the duration of the ANT is considerably long compared with the other tests. The PVT and DAT seem therefore more preferable than the UFOV and the ANT. According to the present study, effects of clinically relevant levels of drowsiness as induced by one night of sleep deprivation can be used as minimally relevant effects of impairment. One limitation of the implications of the present study is, however, that sleep deprivation, alcohol, and sedative drugs have qualitatively differences [81], [82]. Sedative drugs could have more specific effects on simple laboratory tests. Further studies with a double-blind, placebo-con- trolled, cross-over design examining the effects of benchmark sedative drugs are helpful to as- sess the sensitivity of laboratory tests to the potentially impairing effects and whether specific tests can predict drug induced driving impairment. In conclusion, from the psychometric tests used in this study, the PVT and DAT seem most promising for initial evaluation of drug induced impairment based on sensitivity, correlations with driving impairment, and short duration. The effects of one night of sleep deprivation on these tests are similar to or larger than clinically relevant levels of alcohol [40]. Such decreases in arousal after one night of sleep deprivation are clinically relevant, as an increased crash risk has been indicated at night or in the early morning hours [20], [21], [22]. The suggested initial screening tools can be used as a first step to provide meaningful precautions for users and pre- scribers about the impact of drugs on driving. Acknowledgments The authors would like to thank Anita van Oers, Shalana Beunen, Patrick van Schaaik, Leonie Löffler, Fabian van den Berg, Josiah Bonjer, and Elena Syurina for their assistance in data col- lection, Cees van Leeuwen, MD, for the medical supervision, Henk Brauers and Jo Gorissen for ensuring the safety of the participants during the highway driving test, and Irma Brauers for the logistic work. Author Contributions Conceived and designed the experiments: SJ AV EFV JGR JP. Performed the experiments: SJ JP. Analyzed the data: SJ JP EFV JGR AV. Contributed reagents/materials/analysis tools: SJ JP EFV JGR AV. Wrote the paper: SJ AV EFV JGR JP. 1. O’Hanlon JF, Haak TW, Blaauw GJ, Riemersma JB (1982) Diazepam impairs lateral position control in highway driving. Science 217(4554): 79–81. PMID: 7089544 2. O’Hanlon JF (1984) Driving performance under the influence of drugs: rationale for, and application of, a new test. Br J Clin Pharmacol 18 Suppl 1: 121–129. PMID: 6525328 3. Seppala T, Linnoila M, Mattila M (1979) Drugs, alcohol and driving. Drugs 17(5): 389. PMID: 37067 4. Vingilis E, Macdonald S (2002) Review: Drugs and Traffic Collisions. Traffic Inj Prev 3(1): 1–11. 5. Walsh JM, de Gier JJ, Christopherson AS, Verstraete AG (2004) Drugs and driving. Traffic Inj Prev 5 (3): 241–253. PMID: 15276925 Test Thus, overall performance at the ANT is sensitive to the impairing effects of sedation, while specific effects at the networks of attention are less sensitive to indicate impairment. Furthermore, no effects of an alcohol dose reaching a clinically relevant level of 0.5 g/L were found in a previous study [40]. In addition, the ANT has a relative long duration (i.e. 20 min). Thus, other tests with a shorter duration measuring reac- tion time could be preferred to screen for relevant impairment. The DSST, DT, and PBT were sensitive to the impairing effects of one night of sleep depri- vation, but effect sizes were only modest. In these tests, performance slightly improved in the morning, as effect sizes were lower after 26 hours of wakefulness compared with effect sizes after 20 hours of wakefulness. This finding of a slight improvement in the morning was previ- ously indicated in a cognitive psychomotor task [78]. This could be due to circadian effects, as assessing performance at 11:00 am is at a more favorable circadian phase compared to 5:00 am [79]. Therefore, the time of day should be considered when using these tests as screening tools to assess drug induced impairment. A potential limitation of the use of laboratory tasks to assess driving impairment is their lack of validity for measuring driving and predicting accident risk [80]. The present study found relatively high correlations between performance changes in the PVT and changes in the highway driving test due to sleep deprivation. In contrast, analysis of drug induced impairment found only modest correlations [8], [53]. The high correlations found in the present study could be explained by the type of task and the type of manipulation. Regarding the type of task, both the PVT and the highway driving task require the ability to sustain attention over a longer period. In addition, both tests are known to be very sensitive to the effects of sleep deprivation [24], [52]. Correlations tend to increase with stronger impairing effects, as shown by drug stud- ies [8], [53]. Drugs may have different or more subtle effects than a night of sleep deprivation. In addition, safe driving does not only depend on the ability to remain vigilant. Further studies PLOS ONE | DOI:10.1371/journal.pone.0117045 February 10, 2015 17 / 22 References 1. O’Hanlon JF, Haak TW, Blaauw GJ, Riemersma JB (1982) Diazepam impairs lateral position control in highway driving. Science 217(4554): 79–81. PMID: 7089544 2. O’Hanlon JF (1984) Driving performance under the influence of drugs: rationale for, and application of, a new test. 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Analysis of genetic networks regulating refractive eye development in collaborative cross progenitor strain mice reveals new genes and pathways underlying human myopia
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(2019) 12:113 (2019) 12:113 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 https://doi.org/10.1186/s12920-019-0560-1 Open Access Analysis of genetic networks regulating refractive eye development in collaborative cross progenitor strain mice reveals new genes and pathways underlying human myopia Tatiana V. Tkatchenko1, Rupal L. Shah2, Takayuki Nagasaki1 and Andrei V. Tkatchenko1,3* Tatiana V. Tkatchenko1, Rupal L. Shah2, Takayuki Nagasaki1 and Andrei V. Tkatchenko1,3* © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Population studies suggest that genetic factors play an important role in refractive error development; however, the precise role of genetic background and the composition of the signaling pathways underlying refractive eye development remain poorly understood. Methods: Here, we analyzed normal refractive development and susceptibility to form-deprivation myopia in the eight progenitor mouse strains of the Collaborative Cross (CC). We used RNA-seq to analyze gene expression in the retinae of these mice and reconstruct genetic networks and signaling pathways underlying refractive eye development. We also utilized genome-wide gene-based association analysis to identify mouse genes and pathways associated with myopia in humans. Results: Genetic background strongly influenced both baseline refractive development and susceptibility to environmentally-induced myopia. Baseline refractive errors ranged from −21.2 diopters (D) in 129S1/svlmj mice to + 22.0 D in CAST/EiJ mice and represented a continuous distribution typical of a quantitative genetic trait. The extent of induced form-deprivation myopia ranged from −5.6 D in NZO/HILtJ mice to −20.0 D in CAST/EiJ mice and also followed a continuous distribution. Whole-genome (RNA-seq) gene expression profiling in retinae from CC progenitor strains identified genes whose expression level correlated with either baseline refractive error or susceptibility to myopia. Expression levels of 2,302 genes correlated with the baseline refractive state of the eye, whereas 1,917 genes correlated with susceptibility to induced myopia. Genome-wide gene-based association analysis in the CREAM and UK Biobank human cohorts revealed that 985 of the above genes were associated with myopia in humans, including 847 genes which were implicated in the development of human myopia for the first time. Although the gene sets controlling baseline refractive development and those regulating susceptibility to myopia overlapped, these two processes appeared to be controlled by largely distinct sets of genes. (Continued on next page) (Continued on next page) * Correspondence: avt2130@cumc.columbia.edu Background in the retina, choroid, and sclera [39, 55–62]. Moreover, these studies suggested that similar biological processes underlie refractive development in animal models and humans [63]. Myopia is the most common ocular disorder worldwide [1]. The prevalence of myopia in the U.S. has increased from 25% to ~ 48% in the last 40 years [2–4]. The world- wide prevalence of myopia is predicted to increase from the current 25 to 50% in the next three decades [5], while the prevalence already exceeds 80% in several parts of Asia [6, 7]. Myopia often leads to serious blind- ing complications such as myopic maculopathy, retinal floaters, chorioretinal atrophy, retinoschisis, retinal tears, retinal detachment, and myopic macular degeneration [8–24]. It also represents a major risk factor for a num- ber of other serious ocular pathologies such as cataract and glaucoma [9, 10, 25–27]. Because of the increasing prevalence, myopia is rapidly becoming one of the lead- ing causes of vision loss in several parts of the world, and World Health Organization designated myopia as one of five priority health conditions [1, 8–10, 28]. Although these studies revealed important roles of genetic variation and gene expression in the develop- ment of refractive errors, the relationship between gen- etic background, gene expression and development of refractive errors remains unexplored. Here, we systemat- ically analyzed the role of genetic background in the regulation of retinal gene expression and signaling path- ways underlying refractive eye development in eight in- bred strains of mice and their association with human myopia using genome-wide gene expression profiling (RNA-seq) and gene-based genome-wide association analysis in the CREAM and UK Biobank human cohorts. We found that both the baseline refractive state of the eye and susceptibility to myopia are inherited as quanti- tative traits, demonstrating strong dependence on gen- etic background. Furthermore, genetic background strongly influenced expression of genes in the retina and modulated a well-defined set of signaling pathways highly conserved in chickens, mice, monkeys, and humans. Our data suggest that refractive eye develop- ment is regulated by hundreds to thousands of genes across ocular tissues and point to high evolutionary con- servation of signaling pathways underlying refractive de- velopment across vertebrate species. Development of myopia is controlled by both environ- mental and genetic factors [29–32]. (Continued from previous page) (Continued from previous page) Conclusions: Comparison with data for other animal models of myopia revealed that the genes identified in this study comprise a well-defined set of retinal signaling pathways, which are highly conserved across different vertebrate species. These results identify major signaling pathways involved in refractive eye development and provide attractive targets for the development of anti-myopia drugs. Keywords: Myopia, Refractive eye development, Genetic networks, Signaling pathways, Genetic variation, RNA-seq, Gene- based genome-wide association analysis, Evolutionary conservation of pathways Background Although environ- mental factors, such as reading and nearwork, play a very important role in the development of myopia [33–36], genetic studies suggest that the impact of environmental factors on refractive development is determined by genetic variation in “myopia-susceptibility genes” [37]. The role of genetic background in refractive eye development is also supported by animal studies, which revealed that the ex- tent of myopia experimentally induced in animal models is strongly influenced by genetic background [38–41]. Analysis of the size of ocular components in different strains of mice suggested a significant role of genetic back- ground in the regulation of refractive eye development [42, 43]. Wong and Brown [44] also described significant differences in visual detection, pattern discrimination and visual acuity among different strains of mice. The contri- bution of genetic factors to myopia has been estimated to be as high as 70–80% [45–50], and human genetic map- ping studies have identified over 270 chromosomal loci linked to myopia [51–54]. These loci implicate genes in- volved in multiple cellular and biological processes related to extracellular matrix organization, eye morphogenesis, retinal signaling, and visual perception [53, 54]. Gene ex- pression profiling studies also showed that development of myopia is accompanied by changes in gene expression Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Page 2 of 24 Analysis of form-deprivation myopia in mice and conducted according to the Declaration of Helsinki. All CREAM participants provided written informed con- sent. The UK Biobank received ethical approval from the National Health Service National Research Ethics Service (reference 11/NW/0382). Analysis of form-deprivation myopia in mice To examine the effect of genetic background on suscep- tibility to environmentally induced myopia in mice, we analyzed the extent of myopia induced by the diffuser- imposed retinal image degradation (visual form deprivation) in the eight strains of mice comprising Col- laborative Cross (129S1/svlmj, A/J, C57BL/6J, CAST/EiJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ mice). Visual input was degraded in one of the eyes by applying plastic diffusers, and refractive development of the treated eye was compared to that of the contralateral eye, which was not treated with a diffuser, as previously described [66, 67]. Diffusers represented low-pass optical filters, which degraded the image projected onto the ret- ina by removing high spatial frequency details. Frosted hemispherical plastic diffusers were hand-made from zero power rigid contact lenses made from OP3 plastic (diameter = 7.0 mm, base curve = 7.0 mm; Lens.com). Lenses were frosted using a fine sandpaper and inserted into a 3D-printed plastic frames (Proto Labs). On the first day of the experiment (P24), animals were anesthe- tized via intraperitoneal injection of ketamine and xyla- zine, and frames with diffusers were attached to the skin surrounding the right eye with six stitches using size 5– 0 ETHILON™microsurgical sutures (Ethicon) and rein- forced with Vetbond™glue (3 M Animal Care Products) (the left eye served as a control). Toenails were covered with adhesive tape to prevent mice from removing the diffusers. Animals recovered on a warming pad and were then housed under low-intensity constant light in trans- parent plastic cages for the duration of the experiment as previously described [66, 67]. Following 21 days of visual form deprivation (from P24 through P45), dif- fusers were removed and refractive status of both treated and control eyes was assessed using an automated Ethics approval and consent to participate t cs app o a a d co se t to pa t c pate Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were maintained as an in-house breed- ing colony. Food and water were provided ad libitum. All procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in ophthalmic and vision research and were approved by the Columbia University Institutional Animal Care and Use Committee. Animals were anes- thetized via intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) and were euthanized using CO2 followed by cervical dislocation. All human studies were approved by the relevant insti- tutional review boards and/or medical ethics committees Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Tkatchenko et al. BMC Medical Genomics Page 3 of 24 A B Fig. 1 Genetic background modulates refractive eye development and susceptibility to myopia in mice. a Refractive error is inherited as a quantitative trait in the founder strains of Collaborative Cross. Baseline refractive errors at P40 range from highly myopic to highly hyperopic depending on the genetic background in different strains. Horizontal red lines show mean refractive errors for each strain, while each dot corresponds to mean refractive errors of individual animals. b Susceptibility to form-deprivation myopia is inherited as a quantitative trait in the founder strains of Collaborative Cross. The extent of myopia induced by 21 days of visual form deprivation in different strains ranged from −5.5 ± 2.1 D in NZO/HlLtJ mice to −18.7 ± 3.1 D in CAST/EiJ mice. Horizontal red lines identify means of induced myopia for each strain, while each dot represents a mean interocular difference between the deprived eye and control contralateral eye for individual animals A B B Fig. 1 Genetic background modulates refractive eye development and susceptibility to myopia in mice. a Refractive error is inherited as a quantitative trait in the founder strains of Collaborative Cross. Baseline refractive errors at P40 range from highly myopic to highly hyperopic depending on the genetic background in different strains. Horizontal red lines show mean refractive errors for each strain, while each dot corresponds to mean refractive errors of individual animals. b Susceptibility to form-deprivation myopia is inherited as a quantitative trait in the founder strains of Collaborative Cross. Ethics approval and consent to participate The extent of myopia induced by 21 days of visual form deprivation in different strains ranged from −5.5 ± 2.1 D in NZO/HlLtJ mice to −18.7 ± 3.1 D in CAST/EiJ mice. Horizontal red lines identify means of induced myopia for each strain, while each dot represents a mean interocular difference between the deprived eye and control contralateral eye for individual animals Analysis of refractive state of the eyes in mice Outer circle shows gene ontology IDs for the biological processes; second circle shows up- or down-regulated genes in the myopic mice versus hyperopic mice; inner circle shows activation or suppression of the corresponding biological processes, while the size of the sector corresponds to statistical significance (larger sectors correspond to smaller P-values). c Top twenty-five canonical pathways affected by the genes whose expression correlates with the baseline refractive errors in the founder strains of Collaborative Cross. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways samples were homogenized at 4 °C in a lysis buffer using Bead Ruptor 24 tissue homogenizer (Omni). Total RNA was extracted from each tissue sample using miRNAeasy mini kit (QIAGEN) following the manufac- turer’s protocol. The integrity of RNA was confirmed by analyzing 260/280 nm ratios (Ratio260/280 = 2.11–2.13) on a Nanodrop (Thermo Scientific) and the RNA Integrity Number (RIN = 9.0–10.0) using Agilent Bioanalyzer. Illu- mina sequencing libraries were constructed from 1 μg of total RNA using the TruSeq Stranded Total RNA LT kit with the Ribo-Zero Gold ribosomal RNA depletion mod- ule (Illumina). Each library contained a specific index (bar- code) and were pooled at equal concentrations using the eccentric infrared photorefractor as previously described [64, 65]. The interocular difference in refraction between the treated and contralateral control eye served as an in- dication of the extent of induced myopia. Analysis of refractive state of the eyes in mice y y To examine the effect of genetic background on the re- fractive state of the eye in mice, we analyzed baseline re- fractive errors in the eight strains of mice comprising Collaborative Cross (129S1/svlmj, A/J, C57BL/6J, CAST/ EiJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ mice). The refractive state of both left and right eyes was determined on alert animals at P40 using an auto- mated eccentric infrared photorefractor as previously described [64, 65]. The animal to be refracted was immobilized using a restraining platform, and each eye was refracted along the optical axis in dim room light (< 1 lx), 20–30 min. after instilling 1% tropicamide ophthal- mic solution (Alcon Laboratories) to ensure mydriasis and cycloplegia. Five independent measurement series (~ 300–600 measurements each) were taken for each eye. The measurements were automatically acquired by the photorefractor every 16 msec. Each successful meas- urement series (i.e., Purkinje image in the center of the pupil and stable refractive error for at least 5 s.) was marked by a green LED flash, which was registered by the photorefractor software. Sixty individual measurements from each series, immediately preceding the green LED flash, were combined, and a total of 300 measurements (60 measurements × 5 series = 300 measurements) were collected for each eye. Data for the left and right eyes were combine (600 measurements total) to calculate mean re- fractive error and standard deviation for each animal. Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Page 4 of 24 A A B C C Fig. 2 Baseline refractive eye development in mice is regulated by a large number of genes via multiple retinal signaling pathways. a Expression of 2,302 retinal genes correlates with baseline refractive error in mice. Hierarchical clustering results show that genes, whose expression correlates with refractive error in the founder strains of Collaborative Cross, are organized in two clusters, i.e., one (top) cluster exhibiting increased expression in the highly hyperopic mice, and the second (bottom) cluster showing increased expression in the myopic mice. b Top fifteen biological processes affected by the genes whose expression correlates with the baseline refractive errors in the founder strains of Collaborative Cross. RNA extraction and RNA-seq Animals were euthanized following an IACUC-ap- proved protocol. Eyes were enucleated, the retinae were dissected from the enucleated eyes and the choroid/RPE removed. The retinae were washed in RNAlater (Thermo Fisher Scientific) for 5 min., fro- zen in liquid nitrogen, and stored at −80 °C until processed for this study. To isolate RNA, tissue Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Page 5 of 24 Fig. 3 Susceptibility to myopia in mice is regulated by a large number of genes via multiple retinal signaling pathways. a Expression of 1,917 retinal genes correlates with susceptibility to form deprivation myopia in mice. Hierarchical clustering results show that genes, whose expression correlates with susceptibility to myopia in the founder strains of Collaborative Cross, are organized in two clusters, i.e., one (top) cluster exhibiting increased expression in mice with high susceptibility to myopia, and the second (bottom) cluster showing increased expression in mice with low susceptibility to myopia. b Top fifteen biological processes affected by the genes whose expression correlates with susceptibility to myopia in the founder strains of Collaborative Cross. Outer circle shows gene ontology IDs for the biological processes; second circle shows up- or down- regulated genes in mice with high susceptibility to myopia versus mice with low susceptibility to myopia; inner circle shows activation or suppression of the corresponding biological processes, while the size of the sector corresponds to statistical significance (larger sectors correspond to smaller P-values). c Top twenty-five canonical pathways affected by the genes whose expression correlates with susceptibility to myopia in the founder strains of Collaborative Cross. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways randomized complete block (RCB) experimental design before sequencing on Illumina HiSeq 2500 sequencing system. The number of libraries per multiplexed sample was adjusted to ensure sequencing depth of ~ 70 million reads per library (paired-end, 2 × 100 nucleotides). The ac- tual sequencing depth was 76,773,554 ± 7,832,271 with read quality score 34.5 ± 0.4. package (version 7.0.18.1210, Partek), libraries were sepa- rated based on their barcodes, adapters were trimmed and remaining sequences were subjected to pre-alignment qual- ity control using Partek Flow pre-alignment QA/QC mod- ule. After the assessment of various quality metrics, bases with the quality score < 34 were removed (≤5 bases) from each end. RNA extraction and RNA-seq Sequencing reads were then mapped to the mouse reference genome Genome Reference Consortium Mouse Build 38 (GRCm38/mm10, NCBI) using the STAR aligner (version 2.5.2b) resulting in 95.0 ± 0.4% mapped reads per li- brary, which covered 35.4 ± 1.0% of the genome. Aligned Post-sequencing RNA-seq data validation and analysis The FASTQ raw data files generated by the Illumina se- quencing system were imported into Partek Flow software Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Page 6 of 24 A B C Fig. 4 Baseline refractive eye development and susceptibility to showing substantial overlap between genes underlying baseline Collaborative Cross progenitor strain mice. b Top fifteen biologic eye development (top panel) and susceptibility to myopia in mic processes; second circle shows up- or down-regulated genes in A B C Fig. 4 Baseline refractive eye development and susceptibility to myopia in mice are regulated via overlapping pathways. a Venn diagram showing substantial overlap between genes underlying baseline refractive eye development and genes regulating susceptibility to myopia in Collaborative Cross progenitor strain mice. b Top fifteen biological processes affected by the 714 genes associated with both baseline refractive eye development (top panel) and susceptibility to myopia in mice (bottom panel). Outer circle shows gene ontology IDs for the biological processes; second circle shows up- or down-regulated genes in the myopic mice versus hyperopic mice (top panel), or in mice with high susceptibility to form-deprivation myopia versus mice with low susceptibility to myopia (bottom panel); inner circle shows activation or suppression of the corresponding biological processes, while the size of the sector corresponds to statistical significance (larger sectors correspond to smaller P-values). c Top twenty-five canonical pathways affected by the 714 genes involved in the regulation of both baseline refractive eye development (top panel) and susceptibility to myopia (bottom panel) in the founder strains of Collaborative Cross. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways A B C Fig. 4 Baseline refractive eye development and susceptibility to myopia in mice are regulated via overlapping pathways. a Venn diagram showing substantial overlap between genes underlying baseline refractive eye development and genes regulating susceptibility to myopia in ll b b f f b l l ff d b h d h b h b l f A B C C A C B Fig. 4 Baseline refractive eye development and susceptibility to myopia in mice are regulated via overlapping pathways. a Venn diagram showing substantial overlap between genes underlying baseline refractive eye development and genes regulating susceptibility to myopia in Collaborative Cross progenitor strain mice. Post-sequencing RNA-seq data validation and analysis DAVID uses a powerful gene-enrichment algorithm and DAVID Gene Concept database to identify biological functions (gene ontology categories) affected by differen- tial genes, while GOplot integrates gene ontology infor- mation with gene expression information and predicts the effect of gene expression changes on biological pro- cesses. DAVID uses a modified Fisher’s exact test (EASE score) with a P-value threshold of 0.05 to estimate statis- tical significance of enrichment for specific gene ontol- ogy categories. IPA Pathways Activity Analysis module (QIAGEN) was used to identify canonical pathways effected by the genes involved in baseline refractive eye of genes with coordinate expression, differentially expressed transcripts were clustered using Partek Flow hierarchical clustering module using average linkage for the cluster dis- tance metric and Euclidean distance metric to determine the distance between data points. Each RNA-seq sample was analyzed as a biological replicate, thus, resulting in three biological replicates per strain. Post-sequencing RNA-seq data validation and analysis b Top fifteen biological processes affected by the 714 genes associated with both baseline refractive eye development (top panel) and susceptibility to myopia in mice (bottom panel). Outer circle shows gene ontology IDs for the biological processes; second circle shows up- or down-regulated genes in the myopic mice versus hyperopic mice (top panel), or in mice with high susceptibility to form-deprivation myopia versus mice with low susceptibility to myopia (bottom panel); inner circle shows activation or suppression of the corresponding biological processes, while the size of the sector corresponds to statistical significance (larger sectors correspond to smaller P-values). c Top twenty-five canonical pathways affected by the 714 genes involved in the regulation of both baseline refractive eye development (top panel) and susceptibility to myopia (bottom panel) in the founder strains of Collaborative Cross. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways reads were quantified to transcriptome using Partek E/M annotation model and the NCBI’s RefSeq Transcripts 80 an- notation file to determine read counts per gene/genomic re- gion. The generated read counts were normalized by the total read count and subjected to the analysis of variance (ANOVA) to detect genes whose expression correlates with either refractive error or susceptibility to myopia. Differen- tially expressed transcripts were identified using a P-value threshold of 0.05 adjusted for genome-wide statistical signifi- cance using Storey’s q-value algorithm [68]. To identify sets Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Page 7 of 24 Fig. 5 Summary of signaling pathways involved in regulation of baseline refractive eye development and susceptibility to myopia. Heatmap showing all statistically significant canonical pathways affected by the genes involved in baseline refractive development and the genes influencing susceptibility to myopia. Z-score shows activation or suppression of the corresponding pathways. The dots show statistical significance (P < 0.05) Fig. 5 Summary of signaling pathways involved in regulation of baseline refractive eye development and susceptibility to myopia. Heatmap showing all statistically significant canonical pathways affected by the genes involved in baseline refractive development and the genes influencing susceptibility to myopia. Z-score shows activation or suppression of the corresponding pathways. The dots show statistical significance (P < 0.05) for annotation, visualization and integrated discovery (DAVID) version 6.8 [69] and GOplot R package [70]. Identification of genes associated with refractive error in the human population using gene-based genome-wide association analysis development or regulating susceptibility to myopia, and predict the effect of gene expression changes in different strains on specific pathways. The activation z-score was employed in the IPA Pathways Activity Analysis module to predict activation or suppression of the canonical path- ways. The z-score algorithm is designed to reduce the chance that random data will generate significant predic- tions. The z-score provides an estimate of statistical quan- tity of change for each pathway found to be statistically significantly affected by the changes in gene expression. The significance values for the canonical pathways were calculated by the right-tailed Fisher’s exact test. The sig- nificance indicates the probability of association of mole- cules from a dataset with the canonical pathway by random chance alone. Pathways Activity Analysis module determines if canonical pathways, including functional end-points, are activated or suppressed based on the gene expression data in a dataset. Once statistically significant canonical pathways were identified, we subjected the data- sets to the Core Functional Analysis in IPA to compare the pathways and identify key similarities and differences in the canonical pathways underlying baseline refractive development and susceptibility to myopia. To identify genes associated with the development of re- fractive errors in humans among the genes whose expres- sion correlated with refractive eye development in mice, human homologs of candidate mouse genes were exam- ined for association with refractive error in the inter- national GWAS study of refractive error carried out by the Consortium for Refractive Error and Myopia (CREAM) [54] and the UK Biobank Eye and Vision con- sortium sample [72] using the Multi-marker Analysis of GenoMic Annotation (MAGMA) [73]. Human homologs of candidate mouse genes were obtained from Ensembl BioMart and were mapped according to gene definitions in the NCBI Entrez Gene database. Genes were defined according to NCBI build 37 (hg19/GRCh37) coordinates with 200 kb flanking regions appended to the transcription start/stop sites. LD patterns were estimated by MAGMA using the 1000 Genomes Phase 1, version 3 European an- cestry reference panel. As summary statistics were used as input, MAGMA gene-based analysis was performed using the default “snp-wise = mean” model. The CREAM sample included 148,485 individuals of European ancestry from 28 cohorts and 11,935 in- dividuals of Asian ancestry from eight studies. All participants included in this analysis from CREAM were 25 years of age or older. Gene ontology analysis and identification of canonical signaling pathways To identify biological functions (gene ontology categor- ies), which were significantly affected by the genes whose expression correlated with either baseline refractive er- rors or susceptibility to myopia, we used the database Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Page 8 of 24 Tkatchenko et al. BMC Medical Genomics Fig. 6 Genes localized within human myopia QTLs show functional overlap with genes underlying baseline refractive eye development and susceptibility to myopia in mice. 750 candidate genes localized within 279 human myopia QTLs exhibit statistically significant overlap with both genes involved in baseline refractive eye development and genes regulating susceptibility to myopia in Collaborative Cross progenitor strain mice Online Mendelian Inheritance in Man (OMIM) (McKu- sick-Nathans Institute of Genetic Medicine, Johns Hop- kins University) and NHGRI-EBI GWAS Catalog [71] databases. The LDlink’s LDmatrix tool (National Cancer Institute) was used to identify SNPs in linkage disequi- librium and identify overlapping chromosomal loci. We then used UCSC Table Browser to extract all genes lo- cated within critical chromosomal regions identified by the human linkage studies or within 200 kb (±200 kb) of the SNPs found by GWAS. The list of genes located within human QTLs was compared with the list of genes that we found to be associated with either baseline re- fractive errors or susceptibility to myopia in mice using Partek Genomics Suite (Partek). The statistical signifi- cance of the overlaps was estimated using probabilities associated with the hypergeometric distribution using Bioconductor software package GeneOverlap version 1.14.0 and associated functions. Fig. 6 Genes localized within human myopia QTLs show functional overlap with genes underlying baseline refractive eye development and susceptibility to myopia in mice. 750 candidate genes localized within 279 human myopia QTLs exhibit statistically significant overlap with both genes involved in baseline refractive eye development and genes regulating susceptibility to myopia in Collaborative Cross progenitor strain mice Identification of candidate genes for human myopia within known myopia QTLs To identify candidate genes for myopia in the QTLs pre- viously found to be linked to human myopia, we com- pared the genes that we found to be involved in refractive eye development in mice with a list of genes located within human myopia QTLs. We first compiled a list of all SNPs or markers exhibiting statistically sig- nificant association with myopia in the human linkage or genome-wide association studies (GWAS) using the UK Biobank is a large prospective study following the health and wellbeing of approximately 500,000 UK residents aged between 40 and 69 years-old at the baseline recruit- ment visit (during the period 2006–2010). One hundred thirty thousand five hundred twenty-one participants had non-cycloplegic autorefraction performed for at least one eye using the Tomey RC 5000 autorefractor-keratometer Page 9 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Fig. 7 Genes underlying baseline refractive eye development and susceptibility to myopia in mice are localized in human QTLs associated with myopia (part 1). Heatmap depicting genes and odds ratios for the overlaps between 109 human myopia QTLs and genes whose expression correlates with either baseline refractive errors, susceptibility to form deprivation myopia, or both in mice. Colors indicate odds ratios. Bold italic identifies genes found to be associated with refractive error in UK Biobank, CREAM, or both human samples by the gene-based genome-wide association analysis. Red identifies genes exhibiting correlation with both baseline refractive development and susceptibility to myopia in mice Page 10 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Fig. 8 Genes underlying baseline refractive eye development and susceptibility to myopia in mice are localized in human QTLs associated with myopia (part 2). Heatmap depicting genes and odds ratios for the overlaps between 109 human myopia QTLs and genes whose expression correlates with either baseline refractive errors, susceptibility to form deprivation myopia, or both in mice. Colors indicate odds ratios. Bold italic identifies genes found to be associated with refractive error in UK Biobank, CREAM, or both human samples by the gene-based genome-wide association analysis. Red identifies genes exhibiting correlation with both baseline refractive development and susceptibility to myopia in mice Fig. 8 Genes underlying baseline refractive eye development and susceptibility to myopia in mice are localized in human QTLs associated with myopia (part 2). Identification of candidate genes for human myopia within known myopia QTLs Heatmap depicting genes and odds ratios for the overlaps between 109 human myopia QTLs and genes whose expression correlates with either baseline refractive errors, susceptibility to form deprivation myopia, or both in mice. Colors indicate odds ratios. Bold italic identifies genes found to be associated with refractive error in UK Biobank, CREAM, or both human samples by the gene-based genome-wide association analysis. Red identifies genes exhibiting correlation with both baseline refractive development and susceptibility to myopia in mice Fig. 8 Genes underlying baseline refractive eye development and susceptibility to myopia in mice are localized in human QTLs associated with myopia (part 2). Heatmap depicting genes and odds ratios for the overlaps between 109 human myopia QTLs and genes whose expression correlates with either baseline refractive errors, susceptibility to form deprivation myopia, or both in mice. Colors indicate odds ratios. Bold italic identifies genes found to be associated with refractive error in UK Biobank, CREAM, or both human samples by the gene-based genome-wide association analysis. Red identifies genes exhibiting correlation with both baseline refractive development and susceptibility to myopia in mice Page 11 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 A A A B Fig. 9 (See legend on next page.) A B Fig. 9 (See legend on next page.) B Page 12 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 (See figure on previous page.) Fig. 9 Top biological processes associated with genes linked to refractive eye development in mice and localized in known human myopia QTLs. a Chord diagram showing key genes (left semicircle) and top biological processes (right semicircle) associated with genes correlated with baseline refractive development in mice and localized in known human QTLs linked to myopia. b Chord diagram showing key genes (left semicircle) and top biological processes (right semicircle) for genes correlated with susceptibility to myopia in mice and localized in known human QTLs linked to myopia. Colored bars underneath gene names show up- or down-regulation of corresponding genes in either myopic mice versus hyperopic mice (A), or mice with high susceptibility to myopia versus mice with low susceptibility to myopia (B) (Tomey Corp., Nagoya, Japan), with up to ten mea- surements taken for each eye. After the exclusion of unreliable readings, 130,459 participants had mea- sures for refractive astigmatism and spherical equiva- lent refractive error. Identification of candidate genes for human myopia within known myopia QTLs Participants of the UK Biobank and CREAM studies with conditions that might alter refraction, such as cataract sur- gery, laser refractive procedures, retinal detachment sur- gery, keratoconus, or ocular or systemic syndromes were excluded from the analyses. Refractive error was A B Fig. 10 Summary of key biological processes and pathways affected by genes underlying refractive eye development and found to be linked to myopia in UK Biobank and CREAM human cohorts. a Hierarchical clustering diagram showing top 30 biological processes affected by genes underlying baseline refractive eye development in humans. Outer circle shows hierarchical clusters of biological processes (identified by different colors) linked to baseline refractive development; inner circle shows clusters of the corresponding genes up- or down-regulated in myopic mice versus hyperopic mice. b Top twenty-five canonical pathways affected by the genes underlying baseline refractive eye development in humans. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways A B A B Fig. 10 Summary of key biological processes and pathways affected by genes underlying refractive eye development and found to be linked to myopia in UK Biobank and CREAM human cohorts. a Hierarchical clustering diagram showing top 30 biological processes affected by genes underlying baseline refractive eye development in humans. Outer circle shows hierarchical clusters of biological processes (identified by different colors) linked to baseline refractive development; inner circle shows clusters of the corresponding genes up- or down-regulated in myopic mice versus hyperopic mice. b Top twenty-five canonical pathways affected by the genes underlying baseline refractive eye development in humans. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways Fig. 10 Summary of key biological processes and pathways affected by genes underlying refractive eye development and found to be linked to myopia in UK Biobank and CREAM human cohorts. a Hierarchical clustering diagram showing top 30 biological processes affected by genes underlying baseline refractive eye development in humans. Outer circle shows hierarchical clusters of biological processes (identified by different colors) linked to baseline refractive development; inner circle shows clusters of the corresponding genes up- or down-regulated in myopic mice versus hyperopic mice. b Top twenty-five canonical pathways affected by the genes underlying baseline refractive eye development in humans. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways Tkatchenko et al. Identification of candidate genes for human myopia within known myopia QTLs BMC Medical Genomics (2019) 12:113 Page 13 of 24 A B Fig 11 Summary of key biological processes and pathways affected by genes regulating susceptibility to myopia and found to be linked to A A B Fig. 11 Summary of key biological processes and pathways affected by genes regulating susceptibility to myopia and found to be linked to myopia in UK Biobank and CREAM human cohorts. a Hierarchical clustering diagram showing top 30 biological processes affected by genes regulating susceptibility to myopia in humans. Outer circle shows hierarchical clusters of biological processes (identified by different colors) underlying regulation of susceptibility to myopia; inner circle shows clusters of the corresponding genes up- or down-regulated in mice with high susceptibility to myopia versus mice with low susceptibility to myopia. b Top twenty-five canonical pathways affected by the genes regulating susceptibility to myopia in humans. Horizontal yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways represented by measurements of refraction and analyzed as spherical equivalent (SphE = spherical refractive error + 1/ 2-cylinder refractive error). genetic differences between mice would have an im- pact on the baseline refractive state of the eye and susceptibility to myopia, we analyzed baseline refract- ive development and susceptibility to form-deprivation myopia in eight inbred strains of mice, which served as founder strains for the Collaborative Cross (CC) [74], i.e., 129S1/svlmj, A/J, C57BL/6 J, CAST/EiJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ mice. Results Genetic background modulates baseline refractive eye development and susceptibility to myopia in mice Genetic background was shown to influence the size of ocular components in mice. To investigate whether Genetic background modulates baseline refractive eye development and susceptibility to myopia in mice Genetic background modulates baseline refractive eye development and susceptibility to myopia in mice Genetic background was shown to influence the size of ocular components in mice. To investigate whether Page 14 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Table 1 Evolutionary conservation of retinal signaling pathways involved in refractive eye development Chicken Mouse Marmoset Human Canonical signaling pathways ● ● 4-hydroxyproline degradation I ● ● ● Actin cytoskeleton signaling ● ● ● Aldosterone signaling in epithelial cells ● ● Amyloid processing ● ● ● Amyotrophic lateral sclerosis signaling ● ● ● Androgen receptor signaling ● ● Antiproliferative role of somatostatin receptor 2 signaling ● ● ● ● Calcium signaling ● ● Choline biosynthesis III ● ● Chondroitin sulfate biosynthesis ● ● ● ● Circadian rhythm signaling ● ● ● Clathrin-mediated endocytosis signaling ● ● ● CREB signaling in neurons ● ● ● ● CXCR4 signaling ● ● Diphthamide biosynthesis ● ● Dopamine receptor signaling ● ● ● ● Dopamine-DARPP32 feedback signaling ● ● ● EIF2 signaling ● ● ● eNOS signaling ● ● ● Ephrin B signaling ● ● ● ● Ephrin receptor signaling ● ● ● ● Epithelial adherens junction signaling ● ● ERK/MAPK signaling ● ● ● ● Estrogen receptor signaling ● ● ● GABA receptor signaling ● ● ● Gap junction signaling ● ● ● ● Glucocorticoid receptor signaling ● ● ● ● Glutamate degradation/Glutamate receptor signaling ● ● ● Glutathione biosynthesis ● ● ● Glutathione redox reactions I ● ● ● Glutathione-mediated detoxification ● ● Glycoaminoglycan-protein linkage region biosynthesis ● ● ● Gαq signaling ● ● HIPPO signaling ● ● ● HMGB1 signaling ● ● ● ● Huntington’s disease signaling ● ● ● IGF-1 signaling ● ● ● ILK signaling ● ● ● Insulin receptor signaling ● ● ● Integrin signaling ● ● ● L-cysteine degradation I ● ● ● ● L-cysteine degradation III ● ● Mismatch repair in eukaryotes ● ● ● mTOR signaling Page 15 of 24 Tkatchenko et al. Genetic background modulates baseline refractive eye development and susceptibility to myopia in mice BMC Medical Genomics (2019) 12:113 Table 1 Evolutionary conservation of retinal signaling pathways involved in refractive eye development (Continued) Chicken Mouse Marmoset Human Canonical signaling pathways ● ● NF-κB signaling ● ● ● ● nNOS signaling ● ● ● NRF2-mediated oxidative stress response ● ● ● Oncostatin M signaling ● ● ● Phospholipase C signaling ● ● ● Phototransduction pathway ● ● ● ● PI3K/AKT signaling ● ● ● PPAR signaling ● ● ● ● PPARα/RXRα activation ● ● ● Production of nitric oxide and reactive oxygen species ● ● ● ● Protein kinase A signaling ● ● ● ● Protein ubiquitination pathway ● ● ● PTEN signaling ● ● Purine nucleotides de novo biosynthesis II ● ● ● Purine nucleotides degradation II (aerobic) ● ● RAN signaling ● ● ● RAR activation ● ● ● Regulation of eIF4 and p70S6K signaling ● ● ● Regulation of the epithelial-mesenchymal transition pathway ● ● ● Relaxin signaling ● ● ● ● RhoGDI signaling ● ● Semaphorin signaling in neurons ● ● ● ● Signaling by Rho family GTPases ● ● Sumoylation pathway ● ● Synaptic long-term depression ● ● ● ● Synaptic long-term potentiation ● ● ● TGF-β signaling ● ● Tight junction signaling ● ● ● tRNA splicing ● ● ● Wnt/Ca + pathway ● ● Xenobiotic metabolism signaling ● ● ● α-adrenergic signaling ● ● ● β-adrenergic signaling 1 Evolutionary conservation of retinal signaling pathways involved in refractive eye development (Continued) Mouse Marmoset Human Canonical signaling pathways We first measured baseline refractive errors in all eight strains at P40, i.e., when baseline refractions reach a plateau in mice [64]. We found that C57BL/6J mice were emmetropic on average (+ 0.3 ± 0.9 D). CAST/EiJ, NZO/ HlLtJ, PWK/PhJ, and WSB/EiJ mice exhibited various degrees of hyperopia ranging from + 10.6 ± 2.2 D to + 22 ± 4.0 D, whereas A/J, NOD/ShiLtJ, and 129S1/svlmj mice developed various degrees of myopia ranging from −3.5 ± 3.6 D to −21.2 ± 3.9 D (Fig. 1a, Additional file 1: Table S1). The differences between the strains were sta- tistically significant as revealed by ANOVA (F (7, 145) = 429.76, P < 0.00001). More importantly, the distribution of refractive errors in the CC mice was continuous, sug- gesting that refractive state of the eye in mice is inher- ited as a quantitative trait. Genetic background modulates baseline refractive eye development and susceptibility to myopia in mice We then analyzed susceptibility to myopia in the same strains of mice by evaluating the extent of induced form-deprivation myopia (Fig. 1b, Additional file 1: Table S2). Susceptibility to form-deprivation myopia was estimated by applying a diffuser to one eye at P24 and comparing the extent of induced myopia in the form-de- prived eye versus the contralateral control eye after 21 days of treatment. We found large differences in suscepti- bility to induced myopia between the strains (Fig. 1b, Page 16 of 24 Page 16 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Additional file 1: Table S2), which ranged from −5.5 ± 2.1 D in NZO/HlLtJ mice to −18.7 ± 3.1 D in CAST/EiJ mice. Other strains occupied intermediate positions between NZO/HlLtJ and CAST/EiJ mice and differences between the strains in the extent of induced myopia were statisti- cally significant as revealed by ANOVA (F (7, 48) = 9.8, P < 0.00001). The distribution of induced refractive errors was continuous, similar to baseline refractive errors, sug- gesting that susceptibility to myopia was also inherited as a quantitative trait in mice. Spearman’s rank-order correl- ation analysis showed that there was no statistically signifi- cant correlation between baseline refractive errors and the extent of induced myopia (rs = −0.60, P = 0.12). Collect- ively, these data suggest that genetic background plays an important role in baseline refractive development and sus- ceptibility to environmentally induced myopia in mice and that both baseline refractive error and susceptibility to myopia are inherited as quantitative traits. involved in the regulation of baseline refractive devel- opment ranged from regulation of neurogenesis and neuron migration to regulation of DNA methylation, visual perception, and synaptic vesicle endocytosis. Figure 2b shows the top 15 biological processes asso- ciated with these genes, including regulation of pro- tein kinase B, regulation of transcription and translation, covalent chromatin modification, insulin receptor signaling, dendrite morphogenesis, and re- sponse to oxidative stress, among others. Genes underlying baseline refractive development were also associated with multiple canonical signaling pathways in the retina (Fig. 2c, Additional file 2: Table S5). Negative refractive errors were associated with activa- tion of mTOR, EIF2, AMPK, β-adrenergic, and dopa- mine-DARPP32 feedback signaling pathways and suppression of HIPPO and RhoGDI signaling path- ways, among others. Taken together, these data sug- gest that refractive eye development is regulated by a large number of genes and pathways. Genetic background modulates baseline refractive eye development and susceptibility to myopia in mice In summary, the development of hyperopic and myopic refractive errors was associated with specific patterns of gene expression, and the activation or suppression of many retinal signaling pathways. Large number of genes are involved in regulation of baseline refractive error in mice via multiple retinal biological processes and signaling pathways g p g g p y To identify retinal genes influencing baseline refract- ive eye development in mice, we used RNA-seq to analyze gene expression in the retina of eight CC strains at P28 (an age when refractive development is progressing towards its stable plateau) (Fig. 2). We found that expression of 2,302 retinal genes strongly correlated with the baseline refractive state (Fig. 2a, Additional file 2: Table S3). Genes were organized in two distinct clusters. Expression of 793 genes com- prising the first cluster was positively correlated with hyperopia, i.e., expression was increased in the strains with positive refractive errors and decreased in the strains with negative refractive errors. Conversely, ex- pression of 1,509 genes comprising the second cluster was positively correlated with myopia, i.e., expression of these genes was increased in the mouse strains with negative refractive errors and decreased in the strains with positive refractive errors. We observed a clear transition from the “hyperopic” gene expression pattern in CAST/EiJ and PWK/PhJ mice with highly hyperopic refractive errors to the “myopic” gene ex- pression pattern in 129S1/svlmj mice with highly my- opic refractive errors. Other strains occupied intermediate positions between these two extremes and exhibited transitional patterns of gene expression, which correlated with average baseline refractive er- rors in these strains. Large number of genes are involved in regulation of susceptibility to myopia in mice via multiple retinal biological processes and signaling pathways We found that genetic background influences suscepti- bility to experimentally induced myopia in mice and that susceptibility to myopia appears to be controlled as a quantitative trait by multiple genes (Fig. 1b). To identify genes underlying inter-strain differences in susceptibility to myopia in mice, we analyzed gene expression in the retina of the eight CC strains upon induction of form- deprivation myopia at the whole-genome level, using RNA-seq (Fig. 3). We found that expression of 1,917 genes strongly correlated with the extent of form- deprivation myopia in different strains of mice (Fig. 3a, Additional file 3: Table S6). Similar to what we found with baseline refractive errors, these genes were organized in two clusters. Expression of 643 genes comprising the first cluster was positively correlated with the susceptibility to myopia, whereas expression of 1,274 genes in the second cluster was negatively corre- lated with the susceptibility to myopia. We found that there was a transition from a “high susceptibility” gene expression pattern in CAST/EiJ mice, which developed −18.7 ± 3.1 D of myopia, to a “low susceptibility” gene expression pattern in NZO/HlLtJ mice, in which only − 5.5 ± 2.1 D of myopia was induced by visual form deprivation. Other strains exhibited transitional gene expression patterns, which correlated with the extent of induced myopia. Gene ontology analysis revealed that the 2,302 genes whose expression correlated with baseline refractive error were associated with 116 biological processes, 77 cellular components, and 69 molecular functions in the retina (Additional file 2: Table S4). Biological processes Page 17 of 24 Page 17 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 pathways were also implicated in both baseline refractive development and susceptibility to myopia, including GP6 signaling pathway, melatonin signaling, RhoGDI signaling, PTEN signaling, opioid signaling pathway, PPARα/RXRα activation, PI3K/AKT signaling, estrogen receptor signaling, and tight junction signaling. Con- versely, synaptic long-term potentiation, α-adrenergic and β-adrenergic signaling, androgen and aldosterone signaling, ephrin receptor signaling, relaxin signaling, dopamine-DARPP32 feedback signaling, dopamine re- ceptor signaling, eNOS and nNOS signaling, somato- statin receptor 2 signaling, neurotrophin/TRK signaling, protein ubiquitination pathway, gap junction signaling, phototransduction pathway, and several other pathways were associated with baseline refractive development but not susceptibility to myopia. Large number of genes are involved in regulation of susceptibility to myopia in mice via multiple retinal biological processes and signaling pathways The amyloid processing, IGF-1 signaling, DNA methylation and transcriptional repression signaling, epithelial adherens junction signal- ing, iron homeostasis signaling pathway, RAR activation, RAN signaling, and several other pathways were associ- ated with susceptibility to myopia but not baseline re- fractive error. Thus, these data suggest that baseline refractive development and susceptibility to myopia are regulated by genetic networks with considerable overlap; however, the two genetic networks have substantial unique components, which may independently regulate either baseline refractive development or susceptibility to environmentally induced myopia. Gene ontology analysis suggested that 55 biological processes, 61 cellular components, and 41 molecular functions were associated with the 1,917 genes corre- lated with susceptibility to myopia (Additional file 3: Table S7). Figure 3b shows the top 15 biological pro- cesses involved in the regulation of susceptibility to my- opia, including regulation of signal transduction, cell-cell adhesion, transcription, translation, protein transport, and lysosome organization, among others. Analysis of the canonical pathways influenced by the genes corre- lated with susceptibility to myopia revealed that in- creased susceptibility to myopia was associated with suppression of mTOR signaling, EIF2 signaling, protein kinase A signaling, D-myo-inositol-5-phosphate metab- olism, cholesterol and choline biosynthesis, as well as with activation of amyloid processing, HIPPO signaling, PTEN signaling, and PPARα/RXRα signaling pathways (Fig. 3c, Additional file 3: Table S8). Collectively, these data implicate an elaborate retinal genetic network and multiple signaling pathways in the regulation of suscepti- bility to myopia in mice. Baseline refractive eye development and susceptibility to myopia in mice are regulated via overlapping but largely distinct retinal genetic networks g To estimate the relative contribution of genes whose ex- pression level correlated with baseline refractive error versus susceptibility to myopia, we analyzed the overlap between the two gene sets (Fig. 4). We found that 714 genes were correlated with both baseline refractive de- velopment and susceptibility to myopia (Fig. 4a, Add- itional file 4: Table S9). Gene ontology analysis revealed that these 714 genes were associated with 24 biological processes, 24 cellular components, and 14 molecular functions (Additional file 4: Table S10). Figure 4b shows the top 15 biological processes for the overlapping genes. Interestingly, the majority of these biological processes were suppressed in animals with high susceptibility to myopia (lower panel) and were activated in animals with negative baseline refractive errors (upper panel). A simi- lar trend was observed when we compared canonical signaling pathways affected by the overlapping genes (Fig. 4c, Additional file 4: Table S11). Signaling path- ways, which were activated in mice with highly negative baseline refractive errors, were suppressed in mice with high susceptibility to myopia, and vice versa. The top pathways associated with both baseline refractive devel- opment and susceptibility to myopia were EIF2 signal- ing, protein kinase A signaling, regulation of eIF4 and p70S6K signaling, mTOR pathway, HIPPO pathway, and axonal guidance signaling. Figure 5 shows a summary of all signaling pathways correlated with baseline refractive eye development and susceptibility to myopia. In addition to the pathways listed above, a number of other Many genes regulating baseline refractive eye development or susceptibility to myopia in mice are localized within chromosomal loci linked to human myopia with refractive error were identified with MAGMA (Multi-marker Analysis of GenoMic Annotation), using single-marker summary statistics from a genome-wide association study (GWAS) for refractive error and age- of-onset-of-spectacle-wear reported by the CREAM Consortium (N = 160,420 participants; [54]) or a GWAS for refractive error by the UK Biobank Eye & Vision Consortium (N = 88,005 participants; [72]) as input. Flanking regions of 200 kb upstream and downstream of transcription start and stop sites were included, in order to capture regulatory variants influencing the expression of nearby genes. g We next analyzed biological processes linked to the genes associated with refractive development in mice and local- ized within human myopia QTLs (Fig. 9, Additional file 8: Tables S17 and S18). Surprisingly, we found that although several biological processes, such as cell growth and prolif- eration, circadian regulation of gene expression, and regula- tion of neuron differentiation, were implicated in both baseline refractive development and regulation of suscepti- bility to myopia, many biological processes underlying base- line refractive development and susceptibility to myopia appeared to be different. Our data on the genes associated with baseline refractive development in mice and localized in human QTLs implicate several unique processes, includ- ing camera-type eye development, regulation of ion/cal- cium transport, regulation of membrane polarization during action potential and long-term synaptic potenti- ation, nitric oxide signaling, ephrin receptor signaling, glucocorticoid receptor signaling, regulation of glutamate secretion, and regulation of JAK-STATand MAPK signaling cascades (Fig. 9a, Additional file 8: Table S17). Conversely, the genes associated with susceptibility to myopia in mice and localized in human QTLs highlighted several different processes, including regulation of cell shape and cell migra- tion, dorsal/ventral pattern formation, vasculature morpho- genesis, photoreceptor cell function and development, cellular response to DNA damage, and small-GTPase-me- diated signal transduction (Fig. 9b, Additional file 8: Table S18). Collectively, these data suggest that there is a signifi- cant functional overlap between genes we found to be cor- related with refractive eye development in mice and genes causing myopia in humans. Moreover, our data suggest that development of refractive errors in humans is associated with genes that, in mice, regulate both baseline refractive development and susceptibility to refractive changes in- duced by the visual environment. Many genes regulating baseline refractive eye development or susceptibility to myopia in mice are localized within chromosomal loci linked to human myopia y Genes comprising genetic networks underlying important developmental and physiological processes often harbor mutations causing human diseases. Therefore, to identify genes associated with susceptibility to myopia in humans, we analyzed the overlap between the genes that we found to be associated with either baseline refractive develop- ment or susceptibility to myopia in mice and genes lo- cated within human quantitative trait loci (QTLs) linked to myopia (Figs. 6, 7 and 8, Additional file 5: Table S12). We found that 90 genes whose expression correlated with baseline refractive errors, 51 genes whose expression cor- related with susceptibility to myopia, and 23 genes whose expression correlated with both baseline refractive errors and susceptibility to myopia in mice were localized within human QTLs linked to myopia (Fig. 6, Additional file 6: Tables S13-S15). GeneOverlap analysis (Fig. 6) revealed that the overlaps for the genes involved in baseline refract- ive development and genes involved in the regulation of susceptibility to myopia were highly significant (OR = 2.75, P = 3.3 × 10−18; OR = 2.03, P = 1.2 × 10−07; respect- ively). The overlap between the genes located within Page 18 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 human QTLs and genes associated with both baseline re- fractive development and susceptibility to myopia in mice was weaker (OR = 1.62, P = 0.022); however, overall, these data suggest a functional association between genes asso- ciated with refractive eye development in mice and genes causing myopia in humans. GeneOverlap analysis revealed that a total of 164 mouse genes were located within 109 human QTLs, producing 1.5 candidate genes per QTL (Figs. 7 and 8, Additional file 7: Table S16). human QTLs and genes associated with both baseline re- fractive development and susceptibility to myopia in mice was weaker (OR = 1.62, P = 0.022); however, overall, these data suggest a functional association between genes asso- ciated with refractive eye development in mice and genes causing myopia in humans. GeneOverlap analysis revealed that a total of 164 mouse genes were located within 109 human QTLs, producing 1.5 candidate genes per QTL (Figs. 7 and 8, Additional file 7: Table S16). Many genes regulating baseline refractive eye development or susceptibility to myopia in mice are localized within chromosomal loci linked to human myopia y g Of the 2,302 genes associated with baseline refractive development in mice for which there were human ho- mologs, 277 genes in the CREAM dataset were associ- ated with refractive error in humans (FDR < 0.05), with 86 genes surviving Bonferroni correction (PBonferroni < 0.05) (Additional file 9: Table S19). Sixty-seven of these genes (including 43 genes with PBonferroni < 0.05) were lo- calized within previously identified human QTLs (Figs. 7 and 8), while 210 of the 277 genes (including 43 genes with PBonferroni < 0.05) have not previously been impli- cated in the development of refractive errors in the hu- man population (Additional file 9: Table S19). When the above analysis was repeated using the (independent) UK Biobank dataset, 560 genes were associated with refract- ive error (FDR < 0.05), including 156 genes with gen- ome-wide significance (PBonferroni < 0.05) (Additional file 9: Table S20). Four hundred eighty-eight of the five hun- dred sixty genes (including 104 genes with PBonferroni < 0.05) have not previously been implicated in the devel- opment of refractive errors in humans (Additional file 9: Table S20). Importantly, 190 genes associated with base- line refractive development in mice were replicated in both the CREAM and UK Biobank cohorts, including 69 genes which achieved genome-wide significance (PBonfer- roni < 0.05) (Additional file 10: Table S23). Of the 1,917 genes associated with the regulation of susceptibility to myopia in mice, gene-based analysis using the CREAM dataset identified 223 genes associ- ated with refractive error in humans (FDR < 0.05), in- cluding 72 genes at genome-wide significance (PBonferroni < 0.05) (Additional file 9: Table S21). Forty-six of these genes (including 33 genes with PBonferroni < 0.05) were localized within previously identified human QTLs (Figs. 7 and 8), whereas 177 of the 223 genes (including 39 genes with PBonferroni < 0.05) have not previously been implicated in the development of refractive errors in humans (Additional file 9: Table S21). MAGMA analysis of the 1,917 genes using the UK Biobank dataset revealed that 465 genes were associated with refractive error (FDR < 0.05), including 119 genes that withstood Bonfer- roni correction (PBonferroni < 0.05) (Additional file 9: Table S22). Discussion Human population studies and studies in animal models strongly suggest that both environmental and genetic factors play important roles in refractive eye develop- ment. Numerous linkage and genome-wide association studies in humans identified over 270 chromosomal loci linked to the development of myopia in humans [75]; however, very little is known about how genetic variation causing myopia affects gene expression and how changes in gene expression associated with differences in genetic backgrounds affect refractive eye development and sus- ceptibility to myopia. Analysis of the biological processes associated with the above genes linked to either baseline refractive develop- ment or regulation of susceptibility to myopia in humans (Figs. 10a and 11a, Additional file 10: Tables S25 and S26) revealed processes primarily related to calcium-medi- ated cell adhesion, synapse assembly, synaptic transmis- sion, protein translation, small GTPase-mediated signal transduction, and GABA receptor signaling. Several ca- nonical signaling pathways were also identified, including EIF2 and mTOR signaling, eIF4 and p70S6K signaling, epithelial adherence junction signaling, sumoylation path- way, and regulation of cellular mechanics by calpain pro- tease, among others (Figs. 10b and 11b, Additional file 10: Tables S27 and S28). Our data suggest that differences in genetic back- grounds play a very important role in refractive eye de- velopment and regulation of susceptibility to myopia. Moreover, we found that variations in genetic back- ground produce a continuous distribution of refractive errors and susceptibilities to myopia in a mouse popula- tion, characteristic of quantitative traits. Genetic varia- tions in different mouse strains also produce unique patterns of gene expression, which strongly correlate with either baseline refractive errors or susceptibility to induced myopia. Surprisingly, we found that the baseline refractive development and susceptibility to myopia are controlled by largely distinct sets of genes (Fig. 4a, Add- itional file 11: Tables S29 and S30, Additional file 12: Figure S1), which suggests that signaling pathways that regulate the trajectory of refractive eye development to- wards emmetropia, myopia, or hyperopia might be dif- ferent from the pathways that modulate the impact of optical defocus and other environmental factors on re- fractive development. Nevertheless, expression of 714 genes correlated with both baseline refractive errors and susceptibility to myopia, suggesting that at least some genes control both the pathways regulating the trajec- tory of refractive development and the impact of visual input on it. Many genes regulating baseline refractive eye development or susceptibility to myopia in mice are localized within chromosomal loci linked to human myopia Forty-nine of these genes (including 35 genes with Gene-based genome-wide association analysis of mouse genes identifies novel gene candidates for human myopia To build on the significant overlap observed above be- tween genes associated with refractive eye development in mice and human myopia QTLs, we examined whether the mouse genes whose expression level correlated with refractive error or myopia susceptibility were enriched for genetic variants associated with refractive error in humans. Human genes enriched for variants associated Page 19 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 regulation of the epithelial-mesenchymal transition, STAT3 signaling, regulation of stem cell pluripotency, VEGF and IGF-1 signaling, NRF2-mediated oxidative stress response, and PI3K/AKT signaling, among others, were involved in the regulation of susceptibility to my- opia (Fig. 11b, Additional file 10: Table S28). PBonferroni < 0.05) were localize within known human my- opia QTLs (Figs. 7 and 8), whereas 416 of the 465 genes (including 84 genes with PBonferroni < 0.05) were not previ- ously known to be associated with the development of re- fractive errors in the human population (Additional file 9: Table S22). One hundred and fifty-two genes involved in the regulation of susceptibility to myopia in mice were found to be linked to the development of refractive errors in both CREAM and UK Biobank cohorts, including 53 genes which reached genome-wide significance (PBonfer- roni < 0.05) in both samples (Additional file 10: Table S24). PBonferroni < 0.05) were localize within known human my- opia QTLs (Figs. 7 and 8), whereas 416 of the 465 genes (including 84 genes with PBonferroni < 0.05) were not previ- ously known to be associated with the development of re- fractive errors in the human population (Additional file 9: Table S22). One hundred and fifty-two genes involved in the regulation of susceptibility to myopia in mice were found to be linked to the development of refractive errors in both CREAM and UK Biobank cohorts, including 53 genes which reached genome-wide significance (PBonfer- roni < 0.05) in both samples (Additional file 10: Table S24). Discussion However, there were also substantial differences be- tween the two sets of human genes identified using ei- ther the mouse baseline refractive error or the mouse myopia susceptibility gene sets. The mouse baseline re- fractive error-derived human gene set was associated with nervous system development, post-embryonic cam- era-type eye development, retinal cone development, neuron migration, dendrite morphogenesis, regulation of glutamate metabolism, extracellular matrix organization, and beta-amyloid formation (Fig. 10a, Additional file 10: Table S25). This gene set was associated with several ca- nonical pathways distinct from those identified using the mouse susceptibility to myopia-derived human gene set. These pathways included integrin signaling, semaphorin signaling in neurons, glucocorticoid receptor signaling, phospholipase C signaling, synaptic long term potenti- ation, ephrin receptor signaling, nNOS signaling in neu- rons, and estrogen receptor signaling (Figs. 10b and 11b, Additional file 10: Tables S27 and S28). Genes that influenced either baseline refractive devel- opment or susceptibility to myopia affected a long list of biological and molecular functions; however, most note- worthy is the finding that over 29 canonical signaling pathways were involved in both baseline refractive devel- opment and regulation of susceptibility to myopia. Inter- estingly, the majority of these pathways were suppressed in animals with high susceptibility to myopia and acti- vated in animals with negative baseline refractive errors. The exceptions from this rule were sumoylation, The mouse susceptibility to myopia-derived human gene set, on the other hand, was associated with bio- logical processes related to developmental growth, neuron fate commitment, regulation of mesenchymal cell proliferation, potassium ion transmembrane trans- port, protein transport, response to estradiol and choles- terol, as well as cellular response to hypoxia (Fig. 11a, Additional file 10: Table S26). Analysis of canonical pathways suggested that pathways associated with TGF- β signaling, PPARα/RXRα activation, PTEN signaling, Page 20 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 Page 20 of 24 development appear to be highly conserved across species. Discussion These include camera-type eye development and post-embryonic eye morphogenesis, ephrin recep- tor signaling, glucocorticoid receptor signaling, regula- tion of circadian rhythms and circadian regulation of gene expression, glutamate signaling, regulation of neurogenesis and dendrite morphogenesis, regulation of nitric oxide biosynthesis, regulation of long-term synaptic potentiation, synapse assembly and chemical synapse transmission, calcium-dependent signaling, regulation of translation, small GTPase mediated sig- nal transduction, photoreceptor function and develop- ment, cell-cell adhesion, regulation of beta-amyloid formation, regulation of mesenchymal cell prolifera- tion, cellular response to hypoxia. We also found that many retinal signaling pathways involved in refractive development in mice are also subjected to genetic variation causing myopia in humans (Additional file 10: Tables S27 and S28). RhoGDI signaling, PTEN, and HIPPO signaling path- ways, which we found to be activated in animals with high susceptibility to myopia and suppressed in animals with negative baseline refractive errors. The observation that the same pathways were effected in the opposite di- rections in animals with high susceptibility to myopia and animals with highly negative baseline refractive er- rors may be explained by the role of optical defocus in the development of myopia. Considering that mice are housed in small cages and the exposure to distant vision is limited, animals with hyperopic refractive errors would be exposed to high levels of hyperopic optical de- focus (which was shown to cause myopia) compared to the animals with negative refractive errors; thus explain- ing why signaling pathways are effected in the same dir- ection in animals with high susceptibility to myopia and animals with hyperopic baseline refractive errors. Remarkably, we also found that many genes whose ex- pression correlated with either baseline refractive errors or susceptibility to myopia in mice were localized in the known human QTLs linked to myopia. The majority of these genes (90 genes) were exclusively involved in base- line refractive eye development, 51 genes were linked to the regulation of susceptibility to myopia, and 23 genes affected both baseline refractive development and sus- ceptibility to myopia. ) Interestingly, 27 genes that we found to be associated with refractive error development in mice were also among the genes differentially expressed in the retina of green monkeys with form-deprivation-induced myopia [39], and 233 genes were among the genes differentially expressed in the retina of marmosets exposed to positive and negative optical defocus [76]. Discussion There was also a 47- gene overlap with the genes found by Riddell et al. to be differentially expressed in the retina of chicks exposed to optical defocus [77], while 292 genes found in mice overlapped with genes found to be differentially expressed in the retina of chicks with lens-induced my- opia by Stone et al. [78]. Many of the genes that we found to be involved in refractive eye development in this study had been previously implicated in various physiological and pathological processes in the retina. For example, mutations in TTC21B gene, which we found to be involved in the regulation of susceptibility to myopia, were shown to cause a syndromic form of ret- inal dystrophy [79]. Mutations in EFEMP1 (gene in- volved in baseline refractive development) were found to be associated with Malattia Leventinese retinal dys- trophy characterized by the presence of RPE deposits [80]. Several studies linked a manganese transporter SLC30A10, which was found in this study to be involved in baseline refractive development, to parkinsonism, thus implicating this gene in the regulation of dopamine sig- naling [81, 82]. Interestingly, the expression of the GRM5 gene, which was found to be highly enriched in the dopaminergic amacrine cells of the retina [83], cor- related with susceptibility to myopia in mice. We also found that GRM5 was implicated in human myopia in the UK Biobank sample. Ephrin receptor A10 (EPHA10), which we found to be involved in the regulation of sus- ceptibility to myopia, was shown to influence cone Gene-based genome-wide association analysis of the genes we found in mice against CREAM and UK Bio- bank human samples revealed that 647 genes whose ex- pression correlated with baseline refractive errors in mice were also associated with refractive errors in humans, including 173 genes that withstood Bonferroni correction. Using gene-based analysis, we also found that 536 genes whose expression correlated with susceptibil- ity to myopia were associated with refractive errors in humans, including 138 genes which exhibited genome- wide significance (PBonferroni < 0.05). One hundred and ninety-eight of these genes were involved in both base- line refractive development and regulation of susceptibil- ity to myopia, including 34 genes which withstood Bonferroni correction. Discussion Although many of these genes were previously implicated in the development of human myopia, 572 genes (92 genes with PBonferroni < 0.05) whose expression correlated with baseline refractive de- velopment and 486 genes (79 genes with PBonferroni < 0.05) whose expression correlated with susceptibility to myopia were linked to human myopia for the first time, including 211 genes (54 genes with PBonferroni < 0.05) whose expression correlated with both baseline refract- ive development and susceptibility to myopia. Genes that we found to be involved in refractive error development in mice and humans affect a multitude of biological functions in the retina; how- ever, many biological functions underlying refractive Page 21 of 24 Page 21 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 underlying refractive error development. It appears that the number of genes involved in the regulation of re- fractive eye development may be as high as 3,505 in the retina alone, suggesting that refractive eye development may be regulated by hundreds to thousands of genes across ocular tissues. Interestingly, in spite of significant overlap between genes that control baseline refractive development and genes regulating susceptibility to my- opia, these two processes appear to be controlled by largely distinct sets of genes. The genes that we found to be involved in refractive eye development control, how- ever, a well-defined set of retinal signaling pathways that we begin to consistently find to be involved in refractive eye development across different species. This provides a solid framework for future studies and for the develop- ment of anti-myopia drugs. photoreceptor morphogenesis, implicating signaling at the level of photoreceptors in refractive eye development [84]. A GTP-binding protein GNL2, involved in baseline refractive development, was demonstrated to play an im- portant role in retinal neurogenesis [85]. Finally, AGRN gene, found by this study to be involved in baseline re- fractive development, was shown to interact with EGR1 (previously implicated in refractive eye development) and regulate synaptic physiology in the retina [86, 87]. Although many pathways that we found to under- lie refractive development in mice and humans in this study are novel, many of them are conserved across vertebrate species (Table 1). Seven out of 47 pathways found to be involved in optical defocus re- sponse in chickens by Riddell et al. Discussion [62] were also found to be involved in refractive eye development in this study (Additional file 2: Table S5, Additional file 3: Table S8, Additional file 10: Tables S27 and S28). We replicated 12 out of 20 canonical pathways identified by Stone et al. [78] in chickens with lens- induced myopia (Additional file 2: Table S5, Additional file 3: Table S8, Additional file 10: Tables S27 and S28). We also found that 35 out of 75 path- ways that we recently found to be involved in the development of hyperopia and myopia in marmosets [76] were also among the pathways which we found to be involved in refractive eye development in this study (Additional file 2: Table S5, Additional file 3: Table S8, Additional file 10: Tables S27 and S28). An important role of the amyloid signaling pathway, which we found to be involved in the regulation of susceptibility to myopia (Additional file 3: Table S8), is in agreement with our recent finding that a com- ponent of the amyloid signaling pathway APLP2 reg- ulates susceptibility to myopia in mice and humans [37]. Our data also suggest that the phototransduction pathway is involved in refractive eye development (Add- itional file 2: Table S5), in agreement with a recent GWAS study [54] and recent marmoset data [76]. Additional files Additional file 1 Table S1. Baseline refractive errors in Collaborative Cross mice measured at P40 (diopters). Table S2. Form-deprivation myopia in Collaborative Cross mice (deprived eye versus control eye, diopters). (XLSX 14 kb) Additional file 2 Table S3. List of genes whose expression correlates with baseline refractive error in Collaborative Cross mice. Table S4. Gene ontology categories significantly associated with genes whose expression correlates with baseline refractive error in Collaborative Cross mice (BP, biological process; CC, cellular component; MF, molecular function). Table S5. Canonical signaling pathways affected by genes whose expression correlates with baseline refractive error in Collaborative Cross mice. (XLSX 192 kb) Additional file 3 Table S6. List of genes whose expression correlates with susceptibility to myopia in Collaborative Cross mice. Table S7. Gene ontology categories significantly associated with genes whose expression correlates with susceptibility to myopia in Collaborative Cross mice (BP, biological process; CC, cellular component; MF, molecular function). Table S8. Canonical signaling pathways affected by genes whose expression correlates with susceptibility to myopia in Collaborative Cross mice. (XLSX 158 kb) Additional file 4 Table S9. List of genes whose expression correlates with both baseline refractive error and susceptibility to myopia in Collaborative Cross mice. Table S10. Gene ontology categories significantly associated with genes whose expression correlates with both baseline refractive error and susceptibility to myopia in Collaborative Cross mice (BP, biological process; CC, cellular component; MF, molecular Additional file 4 Table S9. List of genes whose expression correlates with both baseline refractive error and susceptibility to myopia in Collaborative Cross mice. Table S10. Gene ontology categories significantly associated with genes whose expression correlates with both baseline refractive error and susceptibility to myopia in Collaborative Cross mice (BP, biological process; CC, cellular component; MF, molecular function). Table S11. Canonical signaling pathways affected by genes whose expression correlates with both baseline refractive error and susceptibility to myopia in Collaborative Cross mice. (XLSX 110 kb) Conclusions Gene ontology categories significantly associated with genes localized within human myopia QTLs and whose expression correlates with susceptibility to myopia in mice (BP, biological process; CC, cellular component; MF, molecular function). (XLSX 15 kb) Authors’ contributions TVT and AVT conceptualized the study, analyzed refractive development in mice, performed RNA-seq, and analyzed the data. RLS performed gene- based genome-wide association analysis. TN contributed to the analysis of mouse data. AVT supervised the entire study, analyzed and validated data, and wrote the original draft of the manuscript. All authors read, edited, and approved the final version of the manuscript. Additional file 10 Table S23. List of mouse genes displaying significant association with refractive error in both UK Biobank and CREAM cohorts, and whose expression correlates with baseline refractive error in mice (Blue identifies PBonferroni < 0.05 in both cohorts). Table S24. List of mouse genes displaying significant association with refractive error in both UK Biobank and CREAM cohorts, and whose expression correlates with susceptibility to myopia in mice (Blue identifies PBonferroni < 0.05 in both cohorts). Table S25. Biological processes significantly associated with genes linked to refractive error in UK Biobank and CREAM cohorts, and whose expression correlates with baseline refractive error in mice (BP, biological process). Table S26. Biological processes significantly associated with genes linked to refractive error in UK Biobank and CREAM cohorts, and whose expression correlates with susceptibility to myopia in mice (BP, biological process). Table S27. Canonical signaling pathways significantly associated with genes linked to refractive error in UK Biobank and CREAM cohorts, and whose expression correlates with baseline refractive error in mice. Table S28. Canonical signaling pathways significantly associated with genes linked to refractive error in UK Biobank and CREAM cohorts, and whose expression correlates with susceptibility to myopia in mice. (XLSX 91 kb) Funding This work was supported by the National Institutes of Health grants R01EY023839 (AVT), P30EY019007 (Core Support for Vision Research received by the Department of Ophthalmology, Columbia University), and Research to Prevent Blindness (Unrestricted funds received by the Department of Ophthalmology, Columbia University). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests AVT is a named inventor on two US patent applications related to the development of a pharmacogenomics pipeline for anti-myopia drug development. The remaining authors declare that they have no competing interests. Ethics approval and consent to participate Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were maintained as an in-house breeding colony. All procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in ophthalmic and vision research and were approved by the Columbia University Institutional Animal Care and Use Committee. Animals were anesthetized via intraperitoneal injection of ketamine (90 mg/ kg) and xylazine (10 mg/kg) and were euthanized using CO2 followed by cer- vical dislocation. Additional file 11 Table S29. List of genes whose expression correlates with baseline refractive error in Collaborative Cross mice (molecular function). Table S30. List of genes whose expression correlates with susceptibility to myopia in Collaborative Cross mice (molecular function). (XLSX 163 kb) Additional file 12 Figure S1. Venn diagrams showing overlaps between different functional classes of genes underlying baseline refractive eye development and genes regulating susceptibility to myopia in Collaborative Cross progenitor strain mice. (EPS 1034 kb) All human studies were approved by the relevant institutional review boards and/or medical ethics committees and conducted according to the Declaration of Helsinki. All CREAM participants provided written informed consent. The UK Biobank received ethical approval from the National Health Service National Research Ethics Service (reference 11/NW/0382). Acknowledgements Additional file 9 Table S19. List of mouse genes displaying significant association with refractive error in CREAM cohort and whose expression correlates with baseline refractive error in mice (Blue identifies PBonferroni < 0.05 in both cohorts). Table S20. List of mouse genes displaying significant association with refractive error in UK Biobank cohort and whose expression correlates with baseline refractive error in mice (Blue identifies PBonferroni < 0.05 in both cohorts). Table S21. List of mouse genes displaying significant association with refractive error in CREAM cohort and whose expression correlates with susceptibility to myopia in mice (Blue identifies PBonferroni < 0.05 in both cohorts). Table S22. List of mouse genes displaying significant association with refractive error in UK Biobank cohort and whose expression correlates with susceptibility to myopia in mice (Blue identifies PBonferroni < 0.05 in both cohorts). (XLSX 172 kb) We are grateful to the CREAM Consortium, the UK Biobank Eye and Vision Consortium, and all the individuals who took part in the CREAM and UK Biobank studies, the personnel who recruited them, interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. This research has been conducted using the CREAM datasets and the UK Biobank Resource (application #17351). Special thanks to Jeremy Guggenheim who provided valuable feedback on an early version of the manuscript. Availability of data and materials All data generated or analyzed during this study are included in this article and its supplementary information files. Conclusions g p p function). Table S11. Canonical signaling pathways affected by genes whose expression correlates with both baseline refractive error and susceptibility to myopia in Collaborative Cross mice. (XLSX 110 kb) We have identified 2,302 genes which are involved in baseline refractive eye development and 1,917 genes which regulate susceptibility to myopia in mice. Our data suggest that at least 985 of these genes are sub- jected to genetic variation in the human population and are involved in refractive error development in humans. Eight hundred forty-seven of these genes were impli- cated in the development of human myopia for the first time (Additional file 9: Tables S19-S22). A large number of common genes and canonical pathways that we found to be involved in the regulation of refractive eye devel- opment in mice, chickens and humans suggests strong evolutionary conservation of the signaling pathways Additional file 5 Table S12. List of human myopia QTLs and candidate genes located within 200 kb of the lead SNP (or within critical region). (XLSX 28 kb) Additional file 6 Table S13. List of genes localized within human myopia QTLs and whose expression correlates with baseline refractive error in mice. Table S14. List of genes localized within human myopia QTLs and whose expression correlates with susceptibility to myopia in mice. Table S15. List of genes localized within human myopia QTLs and whose expression correlates with both baseline refractive error and susceptibility to myopia in mice. (XLSX 28 kb) Additional file 7 Table S16. List of human myopia QTLs showing overlap with genes exhibiting correlation with either refractive error or susceptibility to myopia in mice. (XLSX 16 kb) Page 22 of 24 Page 22 of 24 Tkatchenko et al. BMC Medical Genomics (2019) 12:113 protein; RAR: Retinoic acid receptor; RhoGDI: Rho GDP-dissociation inhibitor; RIN: RNA Integrity Number; RNA-seq: Massive parallel RNA sequencing; RPE: Retinal pigment epithelium; RXRα: Retinoid X receptor alpha; SNP: Single-nucleotide polymorphism; STAT3: Signal transducer and activator of transcription 3; TGF-β: Transforming growth factor beta; TRK: Tyrosine receptor kinase; TTC21B: Tetratricopeptide repeat domain 21B; VEGF: Vascular endothelial growth factor; Wnt: Wingless-related protein Additional file 8 Table S17. Gene ontology categories significantly associated with genes localized within human myopia QTLs and whose expression correlates with baseline refractive error in mice (BP, biological process; CC, cellular component; MF, molecular function). Table S18. Abbreviations AGRN A AK AGRN: Agrin; AKT: Protein kinase B; AMPK: AMP-activated protein kinase; ANOVA: Analysis of variance; CC: Collaborative Cross; CDK5: Cyclin- dependent kinase 5; CREAM: Consortium for Refractive Error and Myopia; CREB: cAMP response element binding protein; DARPP32: Dopamine- and cAMP-regulated phosphoprotein 32 kDa; DAVID: Database for annotation, visualization and integrated discovery; EFEMP1: EGF containing fibulin-like extracellular matrix protein 1; EGR1: Early growth response 1; EIF2: Eukaryotic initiation factor 2; eIF4: Eukaryotic initiation factor 4; eNOS: Endothelial nitric oxide synthase; EPHA10: Ephrin receptor A10; GABA: Gamma-aminobutyric acid; GNL2: Guanine nucleotide binding protein-like 2; GP6: Glycoprotein VI; GWAS: Genome-wide association study; IGF-1: Insulin-like growth factor 1; MAGMA: Multi-marker Analysis of GenoMic Annotation; mTOR: Mammalian target of rapamycin; nNOS: Neuronal nitric oxide synthase; NRF2: Nuclear factor erythroid 2-related factor 2; PI3K: Phosphatidylinositol 3-kinase; PPARα: Peroxisome proliferator-activated receptor alpha; PTEN: Phosphatase and tensin homolog; QTL: Quantitative trait locus; RAN: Ras-related nuclear References Surv Ophthalmol. 2014; 59(2):192–216. 13. Rumelt S, Sarrazin L, Averbukh E, Halpert M, Hemo I. Paediatric vs adult retinal detachment. Eye (Lond). 2007;21(12):1473–8. 42. Zhou G, Williams RW. Mouse models for the analysis of myopia: an analysis of variation in eye size of adult mice. Optom Vis Sci. 1999;76(6):408–18. 14. Brasil OF, Brasil MV, Japiassu RM, Biancardi AL, Souza DD, Oliveira RC, et al. Fundus changes evaluation in degenerative myopia. Arq Bras Oftalmol. 2006;69(2):203–6. 43. Puk O, Dalke C, Favor J, de Angelis MH, Graw J. Variations of eye size parameters among different strains of mice. Mamm Genome. 2006;17(8):851–7. 44. Wong AA, Brown RE. Visual detection, pattern discrimination and visual acuity in 14 strains of mice. Genes Brain Behav. 2006;5(5):389–403. 15. Ndiaye PA, Koffane RR, Wade A, Ndiaye CS, Gomez JC, Ndiaye MR. Frequency of retinal changes in myopia in a black population. J Fr Ophtalmol. 2001;24(9):927–9. 45. Peet JA, Cotch MF, Wojciechowski R, Bailey-Wilson JE, Stambolian D. 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Proc Natl Acad Sci U S A. 2003;100(16):9440–5. 69. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4(1):44–57. 70. Walter W, Sanchez-Cabo F, Ricote M. GOplot: an R package for visually combining expression data with functional analysis. Bioinformatics. 2015; 31(17):2912–4. 71. Buniello A, MacArthur JAL, Cerezo M, Harris LW, Hayhurst J, Malangone C, et al. The NHGRI-EBI GWAS catalog of published genome-wide association studies, targeted arrays and summary statistics 2019. Nucleic Acids Res. 2019;47(D1):D1005–D12. 72. Shah RL, Guggenheim JA, Eye UKB, Vision C. Genome-wide association studies for corneal and refractive astigmatism in UK biobank demonstrate a shared role for myopia susceptibility loci. Hum Genet. 2018;137(11–12):881–96. 73. de Leeuw CA, Mooij JM, Heskes T, Posthuma D. MAGMA: generalized gene- set analysis of GWAS data. PLoS Comput Biol. 2015;11(4):e1004219. 74. 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Tkatchenko TV, Troilo D, Benavente-Perez A, Tkatchenko AV. Gene expression in response to optical defocus of opposite signs reveals bidirectional mechanism of visually guided eye growth. References Menezo JL, Suarez-Reynolds R, Frances J, Vila E. Shape, number and localization of retinal tears in myopic over 8D, aphakic and traumatic cases of retinal detachment. An experience report. Ophthalmologica. 1977;175(1):10–8. 50. Lopes MC, Andrew T, Carbonaro F, Spector TD, Hammond CJ. Estimating heritability and shared environmental effects for refractive error in twin and family studies. Invest Ophthalmol Vis Sci. 2009;50(1):126–31. 51. Verhoeven VJ, Hysi PG, Wojciechowski R, Fan Q, Guggenheim JA, Hohn R, et al. Genome-wide meta-analyses of multiancestry cohorts identify multiple new susceptibility loci for refractive error and myopia. Nat Genet. 2013;45(3):314–8. 23. Kanski JJ. Giant retinal tears. Am J Ophthalmol. 1975;79(5):846–52. 24. 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Publisher’s Note PLoS Biol. 2018; 16(10):e2006021. 77. Riddell N, Crewther SG. Novel evidence for complement system activation in chick myopia and hyperopia models: a meta-analysis of transcriptome datasets. Sci Rep. 2017;7(1):9719. 77. Riddell N, Crewther SG. Novel evidence for complement system activation in chick myopia and hyperopia models: a meta-analysis of transcriptome datasets. Sci Rep. 2017;7(1):9719. 78. Stone RA, McGlinn AM, Baldwin DA, Tobias JW, Iuvone PM, Khurana TS. Image defocus and altered retinal gene expression in chick: clues to the pathogenesis of ametropia. Invest Ophthalmol Vis Sci. 2011;52(8):5765–77.
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Qeios, CC-BY 4.0 · Review, October 20, 2023 Qeios ID: Z4MSI3 · https://doi.org/10.32388/Z4MSI3 Review of: "Chronological simulation and Quantitative Evaluation of System Reliability in a Complex Restructured Power System" Shermila Crespo1 1 Anna University 1 Anna University Potential competing interests: No potential competing interests to declare. The authors provided details on the deployment of multiple FACTS devices in the system's most vulnerable zones. This deployment facilitated the calculation of EENS (Equivalent Energy Not Supplied) and, in tandem, enabled the evaluation of interconnected reliability metrics, including system frequency and duration. This comprehensive approach not only enhances overall system reliability but also fosters the efficient operation of both transmission and distribution networks. Qeios ID: Z4MSI3 · https://doi.org/10.32388/Z4MSI3 1/1
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To cite this version: Jakub Bijak, Daniel Courgeau, Robert Franck, Eric Silverman. Modelling in Demography: From Statistics to Simulations. Eric Silverman. Methodological Investigations in Agent-Based Modelling, 13, Springer, pp.167-187, 2018, Methodos series, ￿10.1007/978-3-319-72408-9_9￿. ￿hal-02079052￿ Distributed under a Creative Commons Attribution 4.0 International License Chapter 9 Modelling in Demography: From Statistics to Simulations Jakub Bijak, Daniel Courgeau, Robert Franck, and Eric Silverman HAL Id: hal-02079052 https://hal.science/hal-02079052v1 Submitted on 29 Jun 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1This chapter is based upon ideas from Courgeau et al. (2017), please see the original paper for some additional detail of some finer points related to demographic history and knowledge. © The Author(s) 2018 E. Silverman, Methodological Investigations in Agent-Based Modelling, Methodos Series 13, https://doi.org/10.1007/978-3-319-72408-9_9 © The Author(s) 2018 E. Silverman, Methodological Investigations in Agent-Based Modelling, Methodos Series 13, https://doi.org/10.1007/978-3-319-72408-9_9 167 9.1 An Introduction to Demography Demography, the study of population change, is a discipline with a lengthy and storied history. Most consider demography to have begun approximately 350 years ago, through the work of Graunt (1662), and the field continues to evolve today and incorporate new methods (Courgeau et al. 2017). As discussed in Parts I and II, the advent of agent-based modelling has brought with it the possibility of applying simulation methodologies to the social sciences, and in this respect demography is no exception. Recent work in demography has identified agent-based modelling in particular as a method with particular relevance for demographers; Billari et al. suggest the incorporation of these methods could result in the development of a new subfield of agent-based computational demography, or ABCD (Billari and Prskawetz 2003). ABMs have also been cited as a way to increase the theoretical relevance of demography, by inspiring demographers to delve deeper into social theory in their quest to design and parameterise more sophisticated models (Silverman et al. 2011; Burch 2002, 2003). In this chapter we will go further and propose that ABMs, beyond just herald- ing the birth of a new subfield, have the potential to push the demographic research agenda in a new direction.1 We will summarise the historical development of demography from Graunt’s time until the present day, and demonstrate the demography has displayed a penchant for incorporating new methodologies and 167 167 168 9 Modelling in Demography: From Statistics to Simulations building upon the work of years past. We will propose that ABMs and related simulation methods can form the foundations for a new model-based demography, a step forward for the progression of demography in the face of an ever more complex and changeable world. In Sect. 9.2 below, we will discuss the historical development of demographic methods. We will follow this in Sect. 9.3 with a description of the challenges faced by demography due to uncertainty and complexity in human populations. Sec- tion 9.4 will present the means by which demographers can incorporate simulation methods into the scientific programme, and Sect. 9.5 will bring this all together into a proposal for a model-based demography. 9.2 The Historical Development of Demography Demography, as mentioned above, is commonly thought to have begun with the work of Graunt in the seventeenth century (Graunt 1662). While the discipline itself may be ancient, over time the methods used in demography have evolved continuously, and demographers have incorporated a wide variety of approaches into their methodological toolbox. Note that throughout this chapter, we will refer to these methodological changes as ‘paradigms’, but we are using the term somewhat differently from the modern- day Kuhnian interpretation (Kuhn 1962). Here we use ‘paradigm’ to refer to the relationship between observed phenomena within a population and the key factors of mortality, fertility and migration which are used to explain population change in demography. We will identify four main paradigms over the course of demography’s history, and outline the primary differences between each and how these approaches have built on one another cumulatively over the centuries. Following Courgeau et al. (2017), we will recall Bacon’s seventeenth-century elaboration of the inductive method as we begin to discuss the origins of demo- graphic thought (Bacon 1863)[aphorism 19]: There are and can be only two ways of searching into and discovering truth. The one flies from the senses and particulars to the most general axioms, and from these principles, the truth of which it takes for settled and immovable, proceeds to judgement and to the discovery of middle axioms. And this way is now in fashion. The other derives from the senses and particulars, rising by a gradual and unbroken ascent, so that it arrives at the most general axioms at last of all. This is the true way, but as yet untried. Here Bacon sets up a contrast between the dominant methods in his time, and a second approach which became a foundational principle for modern scientific thought. In the former case, axioms are derived from human notions and intuitions, rather than observation of nature itself. Courgeau et al. argue that the ‘idols’ which derive from such an approach are still a problem in certain areas of science, even in the modern era (Courgeau et al. 2014). 9.2 169 The Historical Development of Demography Bacon’s alternative proposal requires detailed observation of the object of study and the development of axioms by induction. Through observation and experimen- tation, we can discover the principles governing the natural or social properties we seek to study. 9.2 The Historical Development of Demography As argued by Franck (2002b), induction in the Baconian method requires that these principles are such that if they were not present, ultimately the properties we observe would take an entirely different form. These foundational ideas were essential to the development of the first era of demography. Before Graunt, human events like births and deaths were not something to be studied or predicted, but were instead within the strict province of God’s plans for humanity. Graunt instead brought us the concept of the statistical individual – an important concept which we will revisit later in Part III – which would experience abstract events of fertility and mortality (Graunt 1662; Courgeau 2007). This key conceptual revolution allowed for the development of a science of human population, and thus to the advent of demography, epidemiology, and other related fields. Graunt also demonstrated the importance of probability in population studies, based upon the work of Huyghens (1657). He was able to use an estimation of the probability of death to estimate the population of the city of London, and in so doing was the first to use the concept of a statistical individual to examine population change scientifically. This inspired the work of contemporaries, such as Sir William Petty and his Political Arithmetick (Petty 1690), and was significant for the development of political economics in general. In the ensuing decades, and the following century, European thinkers continued to develop this school of thought. 9.2.1 Early Statistical Tools in Demography The concept of epistemic probability brought forth by Bayes (1763) and Laplace (1774, 1812), had a significant impact on demography. We will not go into a great deal of detail on the specifics here; for that, please turn to Courgeau (2012) and Courgeau et al. (2017) for a more in-depth summary. Speaking broadly, the advent of these techniques allowed demographers to answer more salient questions about human events through the use of prior probabilities, which had notable benefits for the growing field of population science. Similarly, the least-squares method drawn from astronomy began to be applied to demography as well, and over the course of the nineteenth century this method became quite widely used (Courgeau 2012). Censuses becoming more widely used meant that extensive data was also much more accessible. However, these early statistical tools assumed that the variables under inves- tigation displayed a certain mathematical structure, and these structures are not necessarily evident in the real world. This can lead to the ecological fallacy, meaning that aggregate, population-level data cannot be applied to the study of individual-level behaviour. The data being collected during this period was also entirely period-based or cross-sectional (Courgeau 2007). This cross-sectional 170 9 Modelling in Demography: From Statistics to Simulations paradigm implied that social factors influencing individuals are a result of aspects of the society surrounding them (i.e., political, economic, or social characteristics). As we will soon see, this separation of individuals and their social realities does not hold up under further scrutiny in many cases. 9.2.2 Cohort Analysis The development of the cohort analysis paradigm happened in the United States following World War II. Ryder (1951) was one of the early proponents, but Henry (1959) further developed and formalised the cohort approach. Courgeau summarises the core concept of the cohort analysis paradigm (Courgeau 2007, p. 36): The demographer can study the occurrence of only a single event, during the life of a generation or cohort, in a population that preserves all its characteristics and the same characteristics for as long as the phenomenon manifests itself. In order for these analyses to work well, however, we must assume that the pop- ulation is homogeneous and that any interfering phenomena must be independent. Such restrictions meant that demographers quickly sought out methods to study heterogeneous cohorts and interdependent phenomena. Thus Aalen developed a demographic application of a general theory of stochastic processes (Aalen 1975). 9.2.4 Multilevel Approaches In order to surpass these limitations, we must introduce the concept of social groupings, which can include families, social networks, workplaces, political affili- ations, and many more besides. Alongside these concepts demographers developed multilevel analyses to better link the individual with the social context around them (Mason et al. 1983; Goldstein 1987). This multilevel paradigm can resolve the gap between population-level models and individual event-histories: The new paradigm will therefore continue to regard a person’s behaviour as dependent on his or her past history, viewed in its full complexity, but this behaviour can also depend on external constraints on the individual, whether he or she is aware of them or not. (Courgeau 2007, pp. 79–80) Both the ecological and atomistic fallacies fade away under this new approach, given the direct links that are now possible between individuals and their social context: The ecological fallacy is eliminated, since aggregate characteristics are no longer regarded as substitutes for individual characteristics, but as characteristics of the sub-population in which individuals live and as external factors that will affect their behaviour. At the same time, we eliminate the atomistic fallacy provided that we incorporate correctly into the model the context in which individuals live. (Courgeau 2007, pp. 79–80) 9.2.3 Event-History Analysis The resultant event-history paradigm built upon these foundations allowed us to study the complex life histories of individuals (Courgeau and Lelièvre 1992). We are able to identify how both demographic and non-demographic factors affect individual behaviour. Of course, these analyses require extensive data; we need to follow individuals through their lives and collect information on their individual characteristics and the events which befall them. This means that longitudinal surveys become highly important in this type of demographic research. The event-history paradigm enforces a collective point of view, in which we estimate the parameters of a random process that affects all individuals, their trajec- tories through life, via analysis of a sample of individuals and their characteristics. This is perhaps conceptually difficult, but in essence we are seeking understanding of a process underlying all of these individual trajectories, rather than insight into the individuals themselves. Again we are studying statistical individuals, not real, observed individuals. However, in contrast to the ecological fallacy of the cross-sectional paradigm, here we may fall afoul of the atomistic fallacy, in which our focus on individual characteristics leads us to ignore the broader, societal context in which individual The Historical Development of Demography 171 9.2 behaviours develop. As described in Part II, individual behaviours are inextricably tied to the complex, multi-layered society in which they live, so isolating these processes can lead to misleading results and incorrect conclusions. behaviours develop. As described in Part II, individual behaviours are inextricably tied to the complex, multi-layered society in which they live, so isolating these processes can lead to misleading results and incorrect conclusions. 9.2.5 Cumulativity As demonstrated in the brief historical summary above, demography has advanced over the centuries due to a steady process of advancement through a series of paradigms. Each new paradigm has taken previous approaches as a starting point, identified their shortcomings and offered a means to overcome them. Having said that, the new paradigms have not eliminated the old; period, cross-sectional and cohort analyses remain relevant today, and are still used when the research question being posed would be suitably answered by one of those approaches. This is reminiscent of the situation in physics, where Newtonian physics is still perfectly relevant and useful in situations where relativistic effects have little or no impact. In demography, following Courgeau et al. (2017), we describe this as a cumu- lativity of knowledge, in which new paradigms have added new perspectives while retaining the insights provided by previous ones: 9 Modelling in Demography: From Statistics to Simulations 172 Table 9.1 Four successive paradigms in demography Paradigm Period Key focus Period 1662– Macro-level phenomena Cohort 1950s– Macro-level phenomena, measured along the cohort dimension Event history 1980s– Micro-level phenomena Multilevel 1980s– Macro-, micro-, and meso-level phenomena, measured from multiple perspectives Table 9.1 Four successive paradigms in demography Cumulativeness of knowledge seems self-evident throughout the history of population sciences: the shift from regularity of rates to their variation; the shift from independent phenomena and homogeneous populations to interdependent phenomena and heteroge- neous populations; the shift from dependence on society to dependence on the individual, ending in a fully multilevel approach. Each new stage incorporates some elements of the previous one and rejects others. The discipline has thus effectively advanced thanks to the introduction of successive paradigms. (Courgeau 2012, p. 239) Table 9.1 provides a summary of the four paradigms described above. Each of the four seeks to examine the key processes in population sciences – mortality, fertility and migration. However, each paradigm characterises the relationships between these processes differently. These paradigms are not static, either; each is undergoing continuous refinement over time. In addition, these four current paradigms still struggle to resolve certain types of research questions. For example, examining interactions between different elements of complex populations remains beyond the scope of even the cutting-edge event-history approach. 2We would go further, however, and note that sometimes the higher-level behaviours can go in the opposite direction from those at the lower level, producing what Boudon (1977) refers to as “perverse effects”. 9.2.5 Cumulativity This difficult problem, often referred to as the micro-macro link in demographic circles, is a familiar one from our perspective, forming one a significant motivation for deploying agent-based models and social simulations in general. Conte et al. offer a succinct description of the micro-macro link2: ...the loop process by which behaviour at the individual level generates higher-level structures (bottom-up process), which feedback to the lower level (top-down), sometimes reinforcing the producing behaviour either directly or indirectly. (Conte et al. 2012, p. 336) In addition, the micro-macro link is not necessarily uni-directional; higher- level actions, for example political decisions, can affect individual behaviours, which might then necessitate additional policy measures, and so forth. Multilevel approaches cannot cope with this kind of bidirectional effect. As we will see, a model-based demography may be better-placed to help demography cope with this complex aspects of population change. 173 9.3 Uncertainty, Complexity and Interactions in Population Systems 9.3.1 Uncertainty in Demographic Forecasts The three main processes of population change – mortality, fertility, and migration – all display significant amounts of uncertainty (Hajnal 1955; Orrell 2007). However, the relative levels of uncertainty differ between them; mortality is generally considered the least uncertain, and migration the most uncertain (National Research Council 2000). As demographers have come to accept the significant challenge posed by uncertainty, statistical demography has grown significantly in recent decades. Courgeau refers to this as the “return of the variance” to demography (Courgeau 2012). The limits of predictability in demographic forecasting has been a topic of signif- icant discussion within the demographic community (see Keyfitz 1981; Willekens 1990; Bijak 2010). Demographers have argued that forecasts should move from deterministic to probabilistic approaches, for example Alho and Spencer (2005). The field also acknowledges that predictions beyond a relatively short time horizon – a generation or so at most – have such high levels of uncertainty that scenario- based approaches to forecasting should be favoured (Orrell and McSharry 2009; Wright and Goodwin 2009). 9.3 Uncertainty, Complexity and Interactions in Population Systems As described in Part II, studying the complexities of human social interaction introduces a host of challenges for the modeller. These challenges are worth re- examining in the specific context of demography, as here they take on a somewhat different character than in other social sciences. Here we will identify the three primary epistemological challenges facing demography today, which will further inform our development of a model-based research agenda. Demography, while incorporating aspects of both individual- and population- level behaviour and their attendant complexities, benefits from having frequent access to very rich datasets, due to the inherent usefulness of those datasets for governments throughout the world. Demographic data also displays strong and persistent relationships, and much critical information on future population dynamics is already embedded in a population’s age structures. The long-term empirical focus of demography has allowed for these relationships to be examined in significant detail (Xie 2000; Morgan and Lynch 2001). 9.3.2 The Problem of Aggregation Here we refer once again to the ecological and atomistic fallacies described above. While the advent of the event-history approach and related methodologies like 174 9 Modelling in Demography: From Statistics to Simulations microsimulation has moved demography away from focusing exclusively on either the individual or the population, these methods are still relatively new (Willekens 2005; Courgeau 2007; Zinn et al. 2009). Microsimulation models are both multi- level and multi-state, meaning that individuals can move between states (such as health status, age group, socioeconomic class, etc.) according to transition probabilities estimated from survey data or census data. However, while these methods are certainly powerful, the challenge for demog- raphers has been their ever-increasing data requirements. The parameter space explodes in size as the ambition of these models grows, and thus demographers find themselves at the mercy of either data that is too limited to accommodate their research questions, or are simply unable to collect sufficient data in the first place due to prohibitive cost or organisational difficulties. We will examine this particular point in more detail in Sect. 9.8 when we discuss ‘feeding the beast’. 9.3.3 Complexity vs. Simplicity The third main epistemological issue for demographers is a direct consequence of the challenges of uncertainty and aggregation. While the temptation in demography today is to tend toward ever more complex and sophisticated models, whether these models are actually more powerful than their simpler neighbours is still an open question. Demography is fundamentally a discipline focused on predicting population change, and in that respect, there is no evidence to suggest that complex models outperform simpler ones (Ahlburg 1995; Smith 1997). Having said that, however, if we were to react too strongly to this revelation and throw aside complex models in favour of simpler ones, we may not achieve the results we desire. Developing detailed understanding of demographic processes may require coping with highly complex datasets and interactions. In those instances, simplicity may abstract away too many of the relevant factors for us to identify the key elements in the processes we wish to study. Prediction, after all, is a key goal in demography, but is far from the only goal; understanding and explanation are just as valid goals for us to pursue, and – unfortunately for us – often we cannot escape the impact of complexity in that context. 9.3.4 Addressing the Challenges While these three key challenges are quite significant, demography has moved forward in partnership with statistical innovations to develop techniques that can help us cope with these new realities. For example, recent developments in uncertainty quantification for complex models has made clear that models are themselves a source of uncertainty, right alongside the factors mentioned above Moving Toward a Model-Based Demography 9.4 175 (Raftery 1995). Bayesian statistics has presented us with several approaches to dealing with this aspect, such as including a term for model error or code error while building a model (Kennedy and O’Hagan 2001). The Bayesian perspective has also informed new approaches to mapping the relationships between model parameters and model outputs, even in highly complex computational models. Perhaps most accessible among these has been the Gaussian process emulator, a method for analysing the impact of model parameters on the final output variance (Kennedy and O’Hagan 2001; Oakley and O’Hagan 2002). While this approach has been most commonly used in highly complex computational simulations like global climate models, Gaussian process emulators have also been put to use in demographic projects3 in recent years (Bijak et al. 2013; Silverman et al. 2013a,b). Thus, as demography has continued to advance to cope with the challenges wrought by complexity, it has moved toward methods and perspectives more commonly associated with complexity science and related disciplines. The prospect of incorporating more exploratory modelling practices within the discipline has led some to seek a movement toward demography as a model-based science (Burch 2003; Courgeau et al. 2017), much like in population biology (Godfrey-Smith 2006; Levins 1966). 3We will examine the application of Gaussian process emulators to demography in significant detail later in Part III. 9.4 Moving Toward a Model-Based Demography Having established that demography, and the population sciences more broadly, have begun to move toward a model-based paradigm and incorporate insights from disciplines already inclined in that direction, we will revisit some concepts from Part II in order to start to bring together a coherent framework. In Chap. 5, we outlined a key distinction between two streams of modelling for the social sciences: social simulation and systems sociology. Systems sociology is fundamentally a more explanatory, and exploratory, form of modelling in which we focus on understanding foundational social theories that lead to the development and evolution of society. Demography, generally speaking, clearly leans more toward the social simulation stream, in which the focus is on modelling specific populations and developing powerful links with empirical data. Microsimulations, for example, fall under this category, given their dependence on transition probabilities derived from empirical population data (Zinn et al. 2009; Willekens 2005). Huneman (2014) suggests that we can further distinguish simulation approaches within that social simulation branch, between weak and strong simulations. The former aims for a scientific approach, looking to test a hypothesis even when data is hard to come by. The latter lies more in ‘opaque thought experiment’ territory, 176 9 Modelling in Demography: From Statistics to Simulations looking to explore simple models without being dependent on a specific theoretical basis. In the context of modelling for the social sciences there are strong similarities here of course to the systems sociology approach, in that in both cases we seek to step away from strong empirical ties and examine theories at a more foundational level. However we may characterise these more abstract models – as systems sociology models or strong simulations – demography has at its core a strong commitment to population data and empirically-relevant results. In that context, demographers seek empirically-derived foundations for modelling social mechanisms within a model; the exploratory, generative approach does not provide the kind of insight demographers find most valuable. In order to develop these foundations in a model- based demographic approach, we can bear in mind the suggestion of Conte et al. that: ...simulations must be accompanied by micro-macro-loop theories, i.e., theories of mech- anisms at the individual level that affect the global behavior, and theories of loop-closing downward effects or second-order emergence. (Conte et al. 2012, p. 9.4 Moving Toward a Model-Based Demography 342) Thus, a critical component of this modelling enterprise must be developing an understanding of this micro-macro link, and in the context of a simulation approach that suggests we must remain committed to a multilevel approach. This provides certain advantages as well, in that powerful tools already exist for multilevel modelling within demography; in a sense, then, we are simply updating the way in which these levels are being represented by putting them into simulation. 9.4.2 The Difficulties of Demographic Simulation While the prospect of a model-based demography offers many advantages, no approach comes free of drawbacks. As discussed in Part II, demography – and social science more generally – presents a difficult target for simulation modellers given the need for robust social theories to underpin their simulation efforts. Social theories are not difficult to find, but they are difficult to validate (Moss and Edmonds 2005). While demography differs from other social sciences in its applied focus and the rich population data from which it draws its insights (Xie 2000; Hirschman 2008), demographers interested in simulation must still rely on a solid theoretical backdrop in order to justify the conclusions drawn from their models. For demography to move forward as a model-based discipline, particularly with agent-based models, the discipline’s practical focus must be maintained. This means that simulations demographers build must be underpinned by population data, and, crucially, they must be constructed inductively. To do otherwise would be to construct social simulations that, while perhaps enlightening in terms of testing social theories, would have little to say about the core questions that have motivated demographers for these last 350 years. As Courgeau et al. suggest (2017), these tensions between the expansive explana- tory power of simulation and the focused empirical character of demography are not necessarily unresolvable. Following the example set by the historically cumulative progression of demographic knowledge outlined earlier, a model-based demography can build upon the power of the multilevel paradigm, incorporating the capabilities afforded by simulation approaches. In this way we establish a true model-based demography which retains the core empirical character of the discipline, while using simulation to enhance the explanatory power of demographic research. 9.4.1 The Explanatory Capacity of Simulation As we have discussed at length previously, agent-based models are uniquely positioned to provide greater explanatory power when applied to complex adaptive systems. This is just as attractive within a demographic context as it is for other social sciences (Burch 2003; Silverman et al. 2011). By allowing the modeller to represent the interactions between individuals and macro-level processes, agent- based models can grant us greater insight into how these different levels of activity influence one another (Chattoe 2003). However, taking advantage of this aspect requires that we develop a more sophisticated understanding of these interactions themselves; in the empirically-focused demographic context, simply creating behavioural rules for these interactions out of best-guess intuitions is not sufficiently rigorous. In order to delve more deeply into interactions between these levels of analysis, we may situate these interactions themselves as objects of scientific enquiry. By explicitly modelling these interactions in simulation, we can better represent the role of multiple, interacting systems in the final demographic outcomes we see in our empirical observations. This would shift demography more toward a model-based framework, and in so doing allow demographers to contribute more to theoretical Demography and the Classical Scientific Programme 177 9.5 advancements in the study of population change. To an extent this shift has already begun, as the incidence of demographic agent-based models influenced by theories of social complexity has increased since the turn of the century (Kniveton et al. 2011; Bijak et al. 2013; Silverman et al. 2013a; Willekens 2012; Geard et al. 2013). 9.5 Demography and the Classical Scientific Programme Returning once again to the pioneering, foundational work of Bacon, Graunt and others, we can revisit the classical scientific programme of research and illustrate how a model-based demography enhances this approach. In the natural sciences this approach is very much still in evidence, but in the social sciences we see it less frequently. In essence, in the classical scientific approach we use observations of some natural property, and from those observations attempt to determine the axioms or 9 Modelling in Demography: From Statistics to Simulations 178 laws driving the process behind this property (Courgeau et al. 2017; Franck 2002a). The classical programme is thus functional-mechanistic, in that we are investigating the process generating the property we are interested in by modelling the functions underlying that process. In the context of demography, for example, we set out to understand the process generating patterns of population change, which at the core are controlled by the three functions of mortality, fertility and migration. Courgeau et al. (2017, p. 42, original emphasis) identify some key examples of this functional- mechanistic approach to social sciences in the past: The ‘law’ of supply and demand, as another example, is the ‘first’ structure of functions which was inferred (induced) by Adam Smith from the observation of markets: it rules the process of social exchanges generating the market. Karl Marx inferred the general structure of functions ruling the process that generates industrial production from a thorough historical study of the technical and social organisation: this ‘first’ principle consists of separating labour and capital. Finally, Durkheim inferred the integration theory from a sustained statistical analysis of the differences in suicide rates between several social milieus: the social process which generates suicides, whichever their causes, is ruled by the integration of the individual agents. The application of the classical programme led to these prominent theoretical results at the height of social sciences. In these examples we see that significant theoretical advances in social sciences have come about thanks to the considered application of the classical scientific programme. Smith, Marx and Durkheim chose a social property to focus on – the market, industrial production, and suicide, respectively – and in each case used thorough observations to infer the functional structure underlying these social properties. 9.5 Demography and the Classical Scientific Programme Of course the impact of these inductive scientific efforts should not be underestimated; Marx’s Capital, for example, remains perhaps the most influential critique of capitalist modes of production ever written, while Adam Smith is memorialised in the names of free-market thinktanks the world over. Demography, as pointed out above, has adhered to a largely similar programme over its history. The observations of populations over the centuries from Graunt onwards has identified mortality, fertility and migration as the primary functions ruling the process of demographic change. Identifying these core functions has helped in turn to focus demographers on those social factors which contribute to these functions, which in turn helps identify those specific demographic variables which may be of greatest interest for further refinement of our understanding of those three functions. However, in recent years some have proposed that demography has strayed from its scientific lineage (Courgeau and Franck 2007). The power of demographic methods, and their widespread acceptance amongst policy-makers, has led to a reduced focus on theoretical innovation in the discipline (Silverman et al. 2011). Yet we cannot declare an ‘end of history’ in demography; the surge in interest in complexity and agent-based approaches since the early 2000s makes that clear. Demography should continue to evolve cumulatively to adapt to new challenges, as it has done in the past, and here we suggest that a model-based demography rooted in that classical scientific programme should be the next step in that evolution. Stages of Model-Based Demographic Research 179 9.6 9.6 Stages of Model-Based Demographic Research Figure 9.1 below illustrates the key stages of a functional-mechanistic approach to demography. We start, as has been the case historically, with observations of the properties of demographic processes (mortality, fertility and migration). Through detailed analysis of this data, we proceed to the second step, and attempt to infer the functional structure underlying those processes. From there, we seek to identify the social factors which contribute to those functions, which then leads us to be able to model that structure in detail. Model-based demography then uses this process as a basis for the next two stages. Conceptual modelling allows us to develop and construct simulations of the interactions at play in the demographic system of interest. The results produced by these simulations can help us to identify areas in which further data collection would be advantageous, and at that point we can start the cycle again. Thus we see model-based demography as an iterative process, in which each trip through this cycle allows us to further refine both the empirical processes and the simulation design and development. g p Courgeau et al. (2017, p. 44) characterise this new research programme as consisting of three main pillars: 1. Adherence to the classical programme of scientific enquiry 2. Enhancement of the ways in which demographic phenomena are measured and inter- preted p 3. The use of formal models, based on the functional-mechanistic principles, as fully- fledged tools of population enquiries. Thus the focus is on integrating functional-mechanistic models directly into the practice of demography, as a cumulative enhancement of previous paradigms. These models are not intended to become a replacement for previous methods, Fig. 9.1 The inductive functional-mechanistic approach in model-based demography (See Courgeau et al. 2017, p. 43) Fig. 9.1 The inductive functional-mechanistic approach in model-based demography (See Courgeau et al. 2017, p. 43) 180 9 Modelling in Demography: From Statistics to Simulations nor an object of interest in and of themselves; instead, they are part and parcel of the demographic research process, both informing and being informed by the observations that form the empirical heart of the discipline. Courgeau et al. further suggest that demography cannot rely on other social science disciplines to provide key innovations (Courgeau et al. 2017). Indeed, as we have seen in Part II, the difficulties we encounter in the simulation of social systems are common to the field at large. 9.6 Stages of Model-Based Demographic Research Demography has a certain empirical advantage over most other social science disciplines, as well, so taking on board theories and methods from less empirically-focused social sciences could instead reduce demographers’ ability to benefit from the data-rich nature of population data. The discipline thus presents an intriguing example of the challenges we must face when developing a model-based approach that is amenable to simulation. While agent-based models can offer substantial power and flexibility to answer appropriately-posed research questions, there is a balance to be struck between embracing that power and ensuring that the core empirical basis and theoretical backdrop of a given discipline are maintained. We need to consider carefully how a model-based approach may move us closer to solving the core epistemological difficulties in demography; transforming demography into a social simulation discipline wholesale might help us shift away from those responsibilities, but it may not help us actually address them. 9.7 Overcoming the Limitations of Demographic Knowledge As discussed in Sect. 9.3 above, demography faces some key limitations in its ability to explain demographic phenomena. One measure of the success of our proposed model-based demography is whether it could allow us to overcome these limitations, and bolster both the theoretical and explanatory capacity of demography beyond the limits of its current statistically-focused methodological foundation. The problem of uncertainty in demography has led to the emergence of new statistical methods within the discipline, and a general agreement that demographic predictions become too uncertain to be useful beyond a generation or so (Keyfitz 1981). The use of simulation within a model-based demography could help us to circumvent this limitation by facilitating the use of computational models for scenario generation. Simulations can be used to explore the parameter space in which they operate, investigating how different scenarios might affect the behaviour of the simulated population at both the individual and population levels. While these scenarios would not magically present us with enhanced predictive power, they would enable us to present possible ways in which populations may change beyond the one-generation time horizon, given certain assumptions about which parameters are most susceptible to variation. Model-based demography may also be able to help demographers cope with the aggregation problem. While representing both the micro and macro levels of analysis within a simulation is far from simple, some simulation projects have The Pragmatic Benefits of Model-Based Demography 181 9.8 allowed for feedbacks between these levels (Billari and Prskawetz 2003; Murphy 2003; Silverman and Bryden 2007). Representing these multiple levels in a single simulation, as well as the interactions between those levels, allows us to avoid the aggregation problem. However, in this context the question of which observations are most useful in such a complex model becomes more critical; we will revisit this issue in more detail in the next section. Finally, the problem of simplicity can also be addressed by well-considered simulation methods, particularly agent-based modelling. Statistical demographic models easily provoke a tendency toward the inclusion of ever-increasing amounts of data. However, agent-based simulations exhibit behaviour that is more driven by the choice and values of simulation parameters rather than the data which is fed into them. As we have discussed in Part II, in some social simulations data is of little or no importance (as in the case of Schelling’s residential segregation model Schelling 1978). 9.7 Overcoming the Limitations of Demographic Knowledge Demography by its nature and its empirical focus requires more data input than most areas of social science, but the widespread use of agent-based approaches would necessitate a more careful approach to the integration and use of data. Failure to do so would see us struggling with issues of tractability as models became increasingly unwieldy and difficult to analyse; here we may wish to use the insights drawn in Part II from Lars-Erik Cederman (2002) and Levins (1966) to consider the appropriate balance of tractability versus the levels of generality, precision and realism required for the research question at hand. 9.8 The Pragmatic Benefits of Model-Based Demography – But you are paying a lot of money for the dragon! – And what, should we just give it to the citizens instead? [...] I see you know nothing about the principles of economics! Export credit warms up the economy and increase the global turnover. – And what, should we just give it to the citizens instead? [...] I see you know nothing about the principles of economics! Export credit warms up the economy and increases the global turnover. g – But it also increases the dragon as such – I stopped him. – The more intensely you feed him, the bigger he gets; and the bigger he gets, the higher his appetite. What kind of a calculation is it? He will finally devour you all! – But it also increases the dragon as such – I stopped him. – The more intensely you feed him, the bigger he gets; and the bigger he gets, the higher his appetite. What kind of a calculation is it? He will finally devour you all! Stanisław Lem, Po˙zytek ze smoka [The Use of a Dragon] (1983/2008: 186) Stanisław Lem, Po˙zytek ze smoka [The Use of a Dragon] (1983/2008: 186) As alluded to above, a common problem faced by statistical demographers is the pressure to bolster the empirical power of demography, or perhaps more properly the perceived empirical power of demography, by including ever-larger amounts of population data (Silverman et al. 2011). The rise of multilevel modelling and microsimulation approaches has made the problem even more evident, as the laudable goals of reducing uncertainty and unravelling the micro-macro link leads to an explosive growth in data requirements. This tendency has effects beyond just creating large and unwieldy models. The process of population data collection itself is both time-consuming and expensive, requiring the design, distribution and collection and increasingly complex surveys. As these surveys grow more complicated, so does the data analysis process, and 182 9 Modelling in Demography: From Statistics to Simulations designing the subsequent statistical or microsimulation models becomes ever more difficult. Silverman, Bijak and Noble call this process ‘feeding the beast’ (Silverman et al. 2011), in which demographers get caught in a vicious cycle of sorts, attempting to feed data-hungry models with increasing amounts of data, only to feel pressure to further ‘improve’ these models next time around with yet another injection of observations. While this process is a result of fundamentally positive motivations, evidence suggests that complex models do not necessarily demonstrate better predictive capacity than their simpler fellows, though complex models due require more costly data collection and would tend to have a longer turn-around time between new versions. Silverman and Bijak cite a couple of examples of this phenomenon: • Weidlich and Haag (1988) developed an ambitious system dynamics model of migration which attempted to address the micro-macro link; however, the model had very significant data requirements and did not fully address some of the complexities of the migration process itself due to the lack of individual agency in the model. • The MicMac project (Willekens 2005; Zinn et al. 2009) proposed a new method of dynamic microsimulation which consists of a macro portion (‘Mac’) and a micro-level model (‘Mic’). However, this modelling method is likewise very hungry for data; the ‘Mac’ portion needs detailed data on transition rates, while the ‘Mic’ portion requires a number of variables to be specified at the individual level. Stanisław Lem, Po˙zytek ze smoka [The Use of a Dragon] (1983/2008: 186) With this in mind, our proposed model-based demography should proceed with awareness of the problem posed by the data-hungry ‘beast’, and offer solutions that protect the empirical focus of demography while helping us to build models that – in the words of John Hajnal – “involve less computation and more cognition than has generally been applied” (Hajnal 1955, p. 321). In Chap. 10, we will begin to present some demographic models which attempt to apply these principles, avoiding ‘the beast’ while maintaining the empirical focus expected in the discipline. 9.9 Benefits of the Classical Scientific Approach Even if we accept that simulation approaches to demography can provide us signif- icant benefits, both theoretical and more pragmatic, there is a danger that we may exchange some strengths of demography for weaknesses of simulation. We propose that a fully fleshed-out model-based paradigm connected to the classical scientific programme in demography would alleviate at least some of these concerns. A significant problem in the simulation approach, as outlined in Part II, is the complexity of social systems and thus the inherent difficulty in selecting which components of those systems are to be represented in simulation. Selecting these components generally comes about through the selection of a favoured social theory, 9.10 Conclusions Conclusions 183 or a core set of assumptions about the functioning of the social processes under examination. The classical scientific programme helps in our drive to select the most relevant structures and functions which should be replicated in simulation. When under- taking an examination of some social property under the classical programme, we narrow our focus to those processes which generate that property. Our observations focus on that one particular social property, and using those observations we then seek to infer (induce) those functions which generate that property. In this way proceeding via the classical scientific programme helps reduce complexity in our modelling enterprise; our observations focus our enquiries on processes and functions plausibly connected to the property of interest, and this in turn provides clearer guidance on which particular variables must be parameterised and instantiated in simulation. This inductive process thus helps us to avoid the problem of complexity in demographic research. Another advantage of the classical scientific programme is its ability to generalise social models. The classical scientific focus on functional-mechanistic explanations means that we are examining social systems in an analogous way to natural and biological systems, in an attempt to reverse engineer the means by which a social process is generated (Franck 2002a). From this viewpoint, when we see a social process replicated in another population, for example, we can reasonably posit that the same generative process, and thus the same functional structure, should be present. 9.9 Benefits of the Classical Scientific Approach In this way we are developing functional-mechanistic explanations for social processes which can be validated in the real world – due to the inductive process underlying these explanations which relies upon empirical observations in the first place – and which can be generalised, assuming that the iterative process of model refinement and data collection confirms our conclusions about the generative process we identified. 9.10 Conclusions This chapter has been, in a sense, a whirlwind tour of the discipline of demography, its strengths and weaknesses, and its prospects for the future. As we have seen, demography is a storied discipline, centuries old and tied deeply into local, national and global institutions of politics and policy-making. Understanding and forecasting human population change is of vital relevance to any modern society, after all; without a clear picture of our society and where it is headed, planning for social policies, immigration, health services, tax structures, and so many more aspects of our governance become far more difficult. That real-world, empirical focus in demography is clearly its greatest strength; the rich nature of population data has allowed demography to develop into a methodical, statistically advanced discipline quite unlike most social sciences. However, these strengths have brought their own challenges, and in particular the three epistemological limitations in demography of uncertainty, aggregation and 184 9 Modelling in Demography: From Statistics to Simulations complexity have led to significant debate within the field about the constraints of demographic enquiry and how to proceed (Keyfitz 1981; Xie 2000; Silverman et al. 2011). Here we have outlined a model-based demographic research programme, taking inspiration once again from population biology developing the social simulation stream of research into a form that maintains the empirical richness of demogra- phy. The model-based programme builds upon the four existing methodological paradigms in demography, enhancing the power and flexibility of multilevel mod- elling approaches. The model-based programme is intended to be an integrated part of the demographic research process, allowing models to influence and in turn be influenced by developments in data collection and analysis. Model-based demography also allows us to extend our predictive horizon in demography, using scenario-based simulation approaches to explore areas of the parameter space beyond the notional one-generation time horizon. As we will see in subsequent chapters, exploring this parameter space in detail using methods like Gaussian process emulators further enables us to understand the behaviour of our simulations, and identify scenarios that may be of particular interest to policy-makers looking to plan for policy spillover effects or unexpected shifts in the population (Silverman et al. 2013a). The incorporation of multiple, interacting levels of social processes in our models can allow us to avoid the ecological and atomistic fallacies (Silverman et al. 9.10 Conclusions 2011), and better understand the interactions between social processes that generate key effects at the population level. The ability of agent-based simulations to incorporate individual-level behaviours means that we can also incorporate qualitative data into our models in both the design and implementation phases (Polhill et al. 2010), adding another avenue of empirical relevance to our arsenal. Next we will analyse some examples of simulation modelling in the demographic context, in order to further develop the model-based programme and identify productive avenues for simulation approaches to population change. In so doing we will discuss aspects of model analysis and uncertainty quantification and how they can help us avoid the problem of complex social models becoming intractable and impenetrable. Ultimately, demography gives us an exciting example of how a fundamentally classical, empirical discipline can use those strengths to its advantage when adopting a methodology most commonly associated with generative, theoretical explanations of social processes. This should serve as a useful model for other disciplines wishing to expand into the simulation arena while maintaining a focus on empirically-driven, policy-relevant research. Acknowledgements Jakub Bijak and Eric Silverman wish to thank the Engineering and Physical Sciences Research Council (EPSRC) grant EP/H021698/1 “Care Life Cycle” for supporting this research. We also thank Frans Willekens, Anna Klabunde and Jason Hilton for valuable input and discussion. 185 References References Aalen, O. O. (1975). Statistical inference for a family of counting processes. PhD thesis. University of California, Berkeley Ahlburg, D. A. (1995). Simple versus complex models: Evaluation, accuracy and combining. 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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
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Intravenous methylprednisolone or immunoglobulin for anti-glutamic acid decarboxylase 65 antibody autoimmune encephalitis: which is better?
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Li et al. BMC Neurosci (2020) 21:13 https://doi.org/10.1186/s12868-020-00561-9 Li et al. BMC Neurosci (2020) 21:13 https://doi.org/10.1186/s12868-020-00561-9 BMC Neuroscience Open Access © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background:  Patients positive for anti-glutamic acid decarboxylase 65 (GAD65) antibodies have attracted increas‑ ing attention. Their clinical manifestations are highly heterogeneous and can be comorbid with tumors. Currently, there is no consensus on the therapeutic regimen for anti-GAD65-associated neurological diseases due to the clinical complexity, rarity and sporadic distribution. We reported six anti-GAD65 autoimmune encephalitis (AE) patients who received intravenous methylprednisolone (IVMP) or immunoglobulin (IVIG) or both. Then, we evaluated the therapeu‑ tic effect of both by summarizing results in previous anti-GAD65 AE patients from 70 published references. Results:  Our six patients all achieved clinical improvements in the short term. Unfortunately, there was no significant difference between IVMP and IVIG in terms of therapeutic response according to the previous references, and the effectiveness of IVMP and IVIG was 45.56% and 36.71%, respectively. We further divided the patients into different subgroups according to their prominent clinical manifestations. The response rates of IVMP and IVIG were 42.65% and 32.69%, respectively, in epilepsy patients; 60.00% and 77.78%, respectively, in patients with stiff-person syndrome; and 28.57% and 55.56%, respectively, in cerebellar ataxia patients. Among 29 anti-GAD65 AE patients with tumors, the response rates of IVMP and IVIG were 29.41% and 42.11%, respectively. There was no significant difference in effective‑ ness between the two regimens among the different subgroups. Conclusion:  Except for stiff-person syndrome, we found that this kind of AE generally has a poor response to IVMP or IVIG. Larger prospective studies enrolling large numbers of patients are required to identify the optimal therapeutic strategy in the future. Keywords:  Glutamic acid decarboxylase 65, Autoimmune encephalitis, Methylprednisolone, Immunoglobulin and the presynaptic terminal of inhibitory neurons, is becoming increasingly important both clinically and experimentally [1–4]. Patients with high serum and cer- ebrospinal fluid (CSF) titers of anti-GAD65 antibodies (Abs) have been proven to be clinically heterogeneous, including those with stiff-person syndrome (SPS), cer- ebellar ataxia (CA), limbic encephalitis (LE), nonlimbic autoimmune encephalitis (AE), autonomic neuropathy and other multifarious neurological disorders [5, 6]. Fur- thermore, tumors have been identified in several cases, Intravenous methylprednisolone or immunoglobulin for anti‑glutamic acid decarboxylase 65 antibody autoimmune encephalitis: which is better? Tao‑Ran Li1,2, Yu‑Di Zhang1,3, Qun Wang1, Xiao‑Qiu Shao1, Zhi‑Mei Li1 and Rui‑Juan Lv1* Background Glutamic acid decarboxylase 65 (GAD65), an intracellu- lar antigen that is highly expressed in pancreatic β-cells *Correspondence: lvruijuan1981@126.com 1 Department of Neurology, Beijing Tiantan Hospital, Capital Medical University; China National Clinical Research Center for Neurological Diseases, 119 South Fourth Ring West Road, Fengtai District, Beijing 100070, People’s Republic of China Full list of author information is available at the end of the article *Correspondence: lvruijuan1981@126.com 1 Department of Neurology, Beijing Tiantan Hospital, Capital Medical University; China National Clinical Research Center for Neurological Diseases, 119 South Fourth Ring West Road, Fengtai District, Beijing 100070, People’s Republic of China Full list of author information is available at the end of the article *Correspondence: lvruijuan1981@126.com 1 Department of Neurology, Beijing Tiantan Hospital, Capital Medical University; China National Clinical Research Center for Neurological Diseases, 119 South Fourth Ring West Road, Fengtai District, Beijing 100070, People’s Republic of China Full list of author information is available at the end of the article Li et al. BMC Neurosci (2020) 21:13 Li et al. BMC Neurosci (2020) 21:13 Page 2 of 11 Page 2 of 11 indicating that anti-GAD65 Abs are associated with paraneoplastic neurological syndrome [7]. Even more perplexing, anti-GAD65 Abs are frequently comorbid with one or more systemic autoimmune diseases and other Abs [5, 8, 9]. Because of the clinical heterogeneity, together with highly variable complexity, low prevalence and sporadic distribution, performing large-scale clini- cal trials are challenging. Therefore, there is currently no global consensus on the therapeutic regimen, mainly including corticosteroids, immunoglobulin, plasma exchange and other immunosuppressant drugs; all of the available experience is based on case reports. However, management may vary widely among different medical centers, even when opposing recommendations are made [9–11]. basis of their relevance to the inclusion criteria. In addi- tion, a back-search of reference lists from retrieved pub- lications was also conducted to identify other potentially relevant articles. As a result, 21 other articles, including 13 case (series) reports and 8 research articles, were also included. We eliminated many references for the following rea- sons. First, we excluded studies for which it was difficult to assess the efficacy of methylprednisolone, immuno- globulin or a combination of methylprednisolone and immunoglobulin because patients received immunosup- pressive therapy in addition to IVMP or IVIG meanwhile, such as plasma exchange [14–16], immunoadsorption [17], azathioprine [18], rituximab [19], mycophenolate mofetil [20] or others [21]. Second, we excluded stud- ies for which we could not obtain the patients’ response to IVMP or IVIG or their combination because of the ambiguous descriptions or the inaccessible follow-up information in the original literature [22–27]. Third, we excluded studies for which the different clinical manifes- tations of patients responded inconsistently to treatment; for example, in patient D in the literature [28], seizure frequency did not respond to IVMP, while CA improved. Fourth, we excluded studies for which patients took pred- nisone or dexamethasone only orally [19, 29–33]. Fifth, we excluded those patients with concurrent Rasmussen encephalitis [34, 35].h In this study, we retrospectively analyzed the clinical characteristics of six anti-GAD65 AE patients in our ter- tiary epilepsy center who presented with different clinical injury sites and severity. To date, no study has indicated which treatment is better for anti-GAD65 AE patients when comparing intravenous methylprednisolone (IVMP) and intravenous immunoglobulin (IVIG). Patients and Methods Six subjects from Beijing Tiantan Hospital gave written informed consent for participation and written consent to permit the publication of clinical details. The study was approved by the Medical Ethics Committee of Bei- jing Tiantan Hospital, Capital Medical University, and was carried out in accordance with the Declaration of Helsinki. Thus, we analyzed the chosen treatments for all anti-GAD65 AE patients by searching PubMed, and we compared the efficacy of IVMP with that of IVIG. The purpose of the study is to raise the awareness of this disease and guide clinical therapies. The 70 references included in the statistics are listed in Additional file  1. We marked the references containing patients who presented with seizures, SPS or CA and ref- erences containing patients who had coexisting tumors or received combination therapy. Statistical analysis We counted the number of patients who responded to IVMP or IVIG or a combination of both in the literature. At the same time, we also counted the number of patients who did not respond to IVMP, IVIG or a combination of both. The clinical treatment for anti-GAD65 AE patients was diverse and complex, and most patients underwent a variety of treatment options. In many cases, patients changed into the second treatment plan since the first was ineffective or because of illness relapse; therefore, the same patient was likely to be assigned two statistical data points. For example, if one patient made no response to IVMP while responding to IVIG, we counted once that IVIG was effective and counted once that IVMP was inef- fective. If one patient responded to combination therapy (not including that there was a time interval between IVMP and IVIG), we counted that combination therapy was effective once instead of calculating once separately that both IVMP and IVIG were effective. If one patient was unresponsive to combination therapy, we counted We conducted a search on PubMed for articles up to April 2019 and used the title/abstract search terms “encephalitis” and “GAD” or “glutamic acid decarboxy- lase”. A total of 133 references were retrieved. The criteria for enrollment were as follows: patients with anti-GAD65 Abs received a therapy of IVMP or IVIG or a combina- tion of both, and we were able to obtain the response to the treatment. We were not particularly concerned about whether patients used antiepileptic drugs (AEDs) because the effect of AEDs was very limited [12, 13]. IVMP or IVIG meant that the patients received high- dose methylprednisolone or immunoglobulin pulse ther- apy, respectively, but the duration was variable, often by more than 5 days. Reference screening was conducted by two experienced neurological doctors, Tao-Ran Li and Rui-Juan Lv. Forty-nine articles, including 38 case (series) reports and 11 research articles, were included on the Li et al. BMC Neurosci (2020) 21:13 Page 3 of 11 the cell-based transfection immunofluorescence assay method, showed only positive anti-GAD65 Abs. Brain magnetic resonance imaging (MRI) showed abnormal signals of the right medial temporal lobe (Fig. 1a). The long-term video electroencephalogram (EEG) showed significant frequent sharp waves, slow waves and sharp- slow complex waves in the right frontal and temporal regions during the interictal phase (Fig. 2). Patient 2: SPS, CA and intractable epilepsyh , p p y The patient is a 35-year-old male. His mother had suf- fered hyperthyroidism. He was also diagnosed with hyperthyroidism at 15 years old and developed noctur- nal GTCSs 2  years later. These attacks lasted 4–5  min with a frequency of twice per month. At 24  years of age, he gradually developed barylalia and gait instabil- ity, and these symptoms progressively deteriorated. The second form of seizure occurred at 25 years of age and manifested as right-side deflection of the mouth, eyeball and head in an unconscious state, accompanied by oro- pharyngeal automatism and groping action. The seizure lasted 2–3 min and also occurred at night, with 1–2 sei- zures/year. Three years later, the frequency of the second type increased to once per week. He was diagnosed with late-onset type one diabetes at the age of 31, and blood sugar was well controlled. Three years ago, he under- went an excision of the epileptogenic focus in the left frontal lobe due to intractable epilepsy. After the opera- tion, he received adequate doses of levetiracetam, clon- azepam and carbamazepine, and although the first type of seizure disappeared, no improvement was achieved in the second type. Two months ago, the patient appeared to have a third form of seizure, which was character- ized as a sudden trance and interruption of actions, last- ing 2–20 min, with a frequency of one per day. In recent days, he suffered paroxysmal muscle stiffness and limb rigidity, accompanied by soreness of the lumbar muscles Statistical analysis During the ictal phase, we found that the EEG rhythmic changes first appeared in the right central, parietal, posterior tempo- ral and midline regions, accompanied by clinical seizures approximately 1 s later, manifested as a nauseous feeling and oropharyngeal automatism with impaired aware- ness, and the seizures lasted approximately 50 s (Fig. 3). The frequency of the above episodes was 2–3 times/day. Therefore, the patient was diagnosed with anti-GAD65 LE, and a 5-day course of IVMP, 500 mg/day, was initi- ated and gradually reduced. Subsequently, she took prednisone (1  mg/kg/day) and levetiracetam (750  mg/ q12h) for maintenance therapy. The clinical improve- ment was remarkable. Four months later, she received a 5-day course of IVIG, 400 mg/kg/day, for consolidation therapy. Currently, she takes only levetiracetam and feels nauseous occasionally. once separately that both IVMP and IVIG were ineffec- tive. We completely followed the information obtained in the literature and objectively collected data according to the original authors’ standpoints. Statistical analyses were performed with SPSS 22.0 software (SPSS, Chicago, IL, USA). Differences were evaluated by the Chi squared test or Fisher’s exact test. P < 0.05 was considered to be statistically significant. Patients from our center Six illustrative anti-GAD65 AE cases from our center are comparatively described in Table 1, and we include two typical examples for a detailed description below owing to spatial confinement. The six patients were all right- handed and were born at term to nonconsanguineous Chinese parents, with no abnormal antenatal or postna- tal issues of note, and reached developmental milestones appropriately. Except for patients 2, 5 and 6, their past medical histories were unremarkable, and their family histories of seizures or neurological and immune disor- ders were also not special except that of patient 2. They all presented seizures as the initial symptom; however, their intracranial lesions and subsequent clinical mani- festations were different as follows: patient 1 was charac- terized as having typical LE, patient 2 had obvious brain atrophy and features of multiple different clinical syn- dromes due to a long-term disease course, the remaining four patients mainly presented with medial temporal lobe injury in imaging (except patient 3), and all patients were characterized as having drug-resistant epilepsy (patient 4 also had SPS). The average time from onset to immuno- therapy for the six patients was approximately 3 months, 18  years, 20  months, 19  years, 10  years and 5  years, respectively. They received different treatment regimens, all showing various degrees of improvement in the short term. Patient 1: typical LE A 35-year-old female reported sudden onset of noctur- nal generalized tonic–clonic seizures (GTCSs) 3 months ago; these GTCSs lasted approximately 1 min, with 5 epi- sodes in total. In addition, she also had an inexplicable sense of fear, several times per day. She received adequate doses of levetiracetam, and the effect was not obvious. During the past 2  months, she experienced 3–4 con- scious nauseous sensations per week. Initial neurologi- cal examination revealed only a decrease in calculation capacity. The Montreal Cognitive Assessment (MoCA) score was 22 (primary school degree) on a scale ranging from 0 to 30. Routine examinations of blood and CSF were normal. Comprehensive onconeural and neuronal surface Abs screening in the serum and CSF, detected by Page 4 of 11 Li et al. BMC Neurosci (2020) 21:13 Table 1  Clinical characteristics of six GAD65 positive-AE patients from our center GAD65 glutamic acid decarboxylase 65, AE autoimmune encephalitis, F female, M male, SPS simple partial seizures, CPS complex partial seizures, sGTCS secondary generalized tonic–clonic seizures, MoCA montreal cognitive assessment, CI cognitive impairment, SPS* stiff person syndrome, CA cerebellar ataxia, NBC neurobehavioral changes, AEDs antiepileptic drugs, LEV levetiracetam, CBZ carbamazepine, CZP clonazepam, OXC oxcarbazepine, MRI magnetic resonance imaging, L left, R right, B bilateral, (M)TL (medial) temporal lobe, Amy amygdala, Hipp hippocampus, FL frontal lobe, 18F-FDG-PET/CT 18-fluoro-deoxyglucose-positron emission tomography/computed tomography, Tha thalamus, LADA late-onset type one diabetic, MG myasthenia gravis, abs antibodies, ANA antinuclear antibodies, TPA thyroid peroxidase antibody, TGA​ thyroglobulin antibody, CSF cerebrospinal fluid, IVMP intravenous methylprednisolone, IVIG intravenous immunoglobulin, MF mycophenolate mofetil, NA not available Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Age/gender 35/F 35/M 29/F 49/F 27/F 33/F Past history Unremarkable Excision of epileptogenic focus in 2015 Unremarkable Unremarkable Excision of thymoma in 2013 L Thyroidectomy Family history Unremarkable Hyperthyroidism of his mother Unremarkable Unremarkable Unremarkable Unremarkable Symptom onset 2018/08 2000/? 2016/06 2000/? 2009/? 2014/? Patient 1: typical LE Initial symptoms Seizure Seizure Seizure Seizure Seizure Seizure Seizure types SPS; CPS; sGTCS sGTCS; CPS SPS CPS; sGTCS CPS; sGTCS CPS Cognition/MoCA Impaired/22 Impaired/22 Impaired/NA Impaired/19 Impaired/23 Impaired/23 Other Symptoms CI CI, ­SPS*, CA, NBC CI CI, ­SPS* CI CI AEDs LEV, 1000 mg/day LEV, 1000 mg/day; CBZ, 600 mg/day; CZP, 1 mg/ day CBZ, 500 mg/day CZP, 4 mg/q8h; CBZ, 400 mg/qd OXC, 300 mg/q12h; LEV, 250 mg/q12h LEV, 625 mg/q12h; discon‑ tinuation by herself later Seizure Frequency at the time of Immunotherapy CPS, 2–3/day; sGTCS, 1–2/ month sGTCS, zero; CPS, 1/day SPS, 2–6/week CPS, 2–4/week; sGTCS, 1–2/month CPS, 1–2/day; sGTCS, 1–2/ year CPS, 2–13/day MRI lesions R MTL, Amy, Hipp Atrophy of L MTL, Hipp and B cerebellum B FL B MTL B Hipp L Hipp and Amy 18F-FDG-PET/CT Hypometabolism of R TL NA Hypometabolism of R Tha, L Hipp and TL Hypometabolism of L TL Hypometabolism of L TL Hypometabolism of L TL Others Thymus residue No Thymus residue No Thymoma No Immunotherapy 2018/11 2018/11 2018/02 2019/03 2019/07 2019/04 Comorbidities No Hyperthyroidism, LADA No No MG; hyperthyroidism No Serum examination show‑ ing positive Abs No ANA, anti-mitochondrial M2 subtype abs, anti- Ro-52 abs, TPA, TGA, anti- cardiolipin abs TPA, TGA​ No TPA No Anti-GAD65 Abs, serum 1:10 positive 1:100 positive NA, positive 1:320 positive 1:100 positive 1:100 positive Anti-GAD65 Abs, CSF 1:100 positive 1:100 positive NA, positive 1:320 positive 1:320 positive 1:100 positive Immunotherapy IVMP and prednisone tapered; IVIG IVIG twice, and MF IVIG and prednisone tapered IVMP and IVIG, medrol tapered IVMP and IVIG, prednisone tapered IVMP and IVIG, prednisone tapered Outcome (2019.07) CPS, 2–3/month CPS, 3–4/month NA no more seizures; ­SPS* improved no more CPS and sGTCS CPS, 1–2/day Page 5 of 11 Li et al. BMC Neurosci (2020) 21:13 Fig. 1  Axial magnetic resonance imaging (MRI) findings of patient 1 and patient 2 in fluid-attenuated inversion recovery (FLAIR) sequence. a Patient 1, a 35-year-old Chinese female, was diagnosed with typical limbic encephalitis, and MRI showed increased FLAIR signals of the right medial temporal lobe, including the amygdala and hippocampus. b–d Patient 2, a 35-year-old male, was characterized by stiff-person syndrome, cerebellar ataxia and intractable epilepsy. MRI indicated postoperative changes in the left frontal lobe, volume reduction in the left temporal lobe and hippocampus, and encephalatrophy, especially in the bilateral cerebellum Fig. Patient 1: typical LE 1  Axial magnetic resonance imaging (MRI) findings of patient 1 and patient 2 in fluid-attenuated inversion recovery (FLAIR) sequence. a Patient 1, a 35-year-old Chinese female, was diagnosed with typical limbic encephalitis, and MRI showed increased FLAIR signals of the right medial temporal lobe, including the amygdala and hippocampus. b–d Patient 2, a 35-year-old male, was characterized by stiff-person syndrome, cerebellar ataxia and intractable epilepsy. MRI indicated postoperative changes in the left frontal lobe, volume reduction in the left temporal lobe and hippocampus, and encephalatrophy, especially in the bilateral cerebellum that he had multiple kinds of autoimmune Abs simul- taneously (seen in Table  1). Routine CSF analysis was normal. The onconeural and neuronal surface Abs test showed positive anti-GAD65 Abs in both serum and CSF. The brain MRI indicated postoperative changes and encephalatrophy (Fig. 1b–d). Electromyography showed continuous release of motor unit action potentials of the paraspinal muscles and rectus abdominis in a quiet state, which were obviously inhibited after intravenous diaze- pam, supporting the diagnosis of SPS. and difficulty in lying flat; these symptoms were prone to occur when stimulated by environmental factors. In addi- tion, he occasionally developed palpitations, sweats and breathing difficulties and became irritable and depressed. and difficulty in lying flat; these symptoms were prone to occur when stimulated by environmental factors. In addi- tion, he occasionally developed palpitations, sweats and breathing difficulties and became irritable and depressed. On neurological examination, he exhibited bilateral exophthalmos, dysarthria and left blepharospasm. The muscular tension and tendon reflex of the four limbs were decreased. He could not stand still and complete the finger-nose tests. The MoCA score was 22 (under- graduate degree). Routine blood examinations suggested Li et al. BMC Neurosci (2020) 21:13 Page 6 of 11 Fig. 2  Interictal phase electroencephalogram of patient 1. There were frequent sharp waves and slow waves in the right temporal regions in the interictal phase Fig. 2  Interictal phase electroencephalogram of patient 1. There were frequent sharp waves and slow waves in the right temporal regions in the interictal phase A 5-day course of IVIG, 400 mg/kg/day, was initiated; subsequently, he took mycophenolate mofetil (gradu- ally increased to 1500 mg/q12h), levetiracetam (250 mg/ q12h), clonazepam (1  mg/q12h) and carbamazepine (200 mg/q12h) for long-term maintenance therapy. The above symptoms improved significantly. One month later, he received another 5-day course of IVIG for con- solidation therapy. Patient 1: typical LE Three months later, the SPS had almost disappeared, the ataxia improved, he could walk by himself, and the number of seizures was reduced by more than 50%. We further divided patients according to their typi- cal clinical manifestations. In Table  3, we present data on the patients who manifested seizures, 42.65% (29/68) of whom were responsive to IVMP and 32.69% (17/52) responded to IVIG (in comparison with Table  2, in Table 3, we added an extra patient, whose seizures were not improved with IVMP, while IVMP was beneficial for CA) [28]. Among patients with SPS, 60.00% (3/5) ben- efited from IVMP, and 77.78% (7/9) gained ameliora- tion from IVIG. In patients with CA, the effectiveness of IVMP and IVIG was 28.57% (2/7) and 55.56% (5/9), respectively (in Table  3, an extra patient was added in comparison with Table  2 [28]). In these above patients from different subgroups, there was no difference in therapeutic effect between the two kinds of treatment. Furthermore, in these AE patients who manifested sei- zures or CA, the effectiveness of combination therapy was 44.83% (13/29) and 25.00% (1/4), respectively. In our analysis, only one SPS patient received combination ther- apy but acquired no benefits [7].i Patients from previous literaturesi Seventy references were identified, and the included patients were all diagnosed with anti-GAD65 AE. In Table 2, we present the gathered data of all patients with different clinical manifestations and found that the effec- tiveness of IVMP was 45.56% (41/90) and that of IVIG was 36.71% (29/79). There was no statistically signifi- cant difference between IVMP and IVIG. In addition, 35 patients received IVMP and IVIG combination therapy, but only 42.86% (15/35) experienced an improvement in their clinical symptoms. i As shown in Table  3 and Additional file  2: Table  S1, of the 29 anti-GAD65 AE patients with tumors, 16 (55.17%) were male, and the median age was 61.8 years Li et al. BMC Neurosci (2020) 21:13 Page 7 of 11 Fig. 3  Ictal phase electroencephalogram of patient 1. One clinical seizure was detected, which presented as a nauseous feeling and oropharyngeal automatism with impaired awareness, and the seizure lasted approximately 50 s second before sudden movement interruption of the patient, EEG showed widespread low voltage in the right leads, followed by rapid rhythmic changes with low amplitude in the right middle-posterior temporal region initially, and spreading to the right middle temporal region 2 s later. The amplitude increased and the frequency decreased gradually and then spread to adjacent leads, accompanied by electromyogram interference and motion artifacts Fig. 3  Ictal phase electroencephalogram of patient 1. One clinical seizure was detected, which presented as a nauseous feeling and oropharyngeal automatism with impaired awareness, and the seizure lasted approximately 50 s second before sudden movement interruption of the patient, EEG showed widespread low voltage in the right leads, followed by rapid rhythmic changes with low amplitude in the right middle-posterior temporal region initially, and spreading to the right middle temporal region 2 s later. The amplitude increased and the frequency decreased gradually and then spread to adjacent leads, accompanied by electromyogram interference and motion artifacts Table 2  IVMP and  IVIG effectiveness of  total patients in the previous 70 references Differences were evaluated by Chi square test IVMP intravenous methylprednisolone, IVIG intravenous immunoglobulin Effective number Ineffective number p IVMP 41 49 IVIG 29 50 0.244 (IQR: 55.0–73.0  years). It should be pointed out that a 5-year-old girl was diagnosed with AE 13  months after hematopoietic stem cell transplantation (because of pineoblastoma); thus, the tumor and the AE may not have been directly related [36]. Patients from previous literaturesi The median age of the remaining 28 patients increased to 63.9  years (IQR: 57.0–73.0  years), much higher than the median age of 48 years for the 106 anti-GAD65 patients without tumors Table 2  IVMP and  IVIG effectiveness of  total patients in the previous 70 references Table 3  IVMP and IVIG effectiveness of patients with different clinical manifestations in the previous 70 references Differences were evaluated by Chi square test or Fisher’s exact test* EN effective numbers, IEN ineffective numbers, IVMP intravenous methylprednisolone, IVIG intravenous immunoglobulin Patients with seizures Patients with SPS Patients with CA Patients with tumors EN IEN p EN IEN p EN IEN p EN IEN p IVMP 29 39 3 2 2 5 5 12 IVIG 17 35 0.266 7 2 0.580* 5 4 0.358* 8 11 0.429 Table 3  IVMP and IVIG effectiveness of patients with different clinical manifestations in the previous 70 references Differences were evaluated by Chi square test or Fisher’s exact test* EN effective numbers, IEN ineffective numbers, IVMP intravenous methylprednisolone, IVIG intravenous immunoglobulin Patients with seizures Patients with SPS Patients with CA Patients with tumors EN IEN p EN IEN p EN IEN p EN IEN p IVMP 29 39 3 2 2 5 5 12 IVIG 17 35 0.266 7 2 0.580* 5 4 0.358* 8 11 0.429 veness of patients with different clinical manifestations in the previous 70 references ble 3  IVMP and IVIG effectiveness of patients with different clinical manifestations in the previous 7 effective numbers, IEN ineffective numbers, IVMP intravenous methylprednisolone, IVIG intravenous immunoglobulin Li et al. BMC Neurosci (2020) 21:13 Page 8 of 11 potassium channel Abs, researchers have verified that the former tend to be seriously resistant to AEDs and immu- notherapy, resulting in a chronic situation [12, 40, 43]. in a previous study [7]. The most common symptom was SPS (11/29, 37.93%), followed by LE and nonlimbic AE (8/29 and 4/29, respectively). Among these patients, the therapeutic effect was dismal, with 29.41% and 42.11% improvement rates in symptoms for IVMP and IVIG, respectively, and no significant difference was found. Moreover, the combination therapy was effective for only 20.00% (2/10) of patients. In addition, 21 patients (72.41%) had oncological treatment (surgery, chemother- apy, and radiotherapy alone or combined). Other clinical data are listed in Additional file 2: Table S1. py g [ ] Gagnon et al. Patients from previous literaturesi compiled an excellent review of 31 arti- cles, describing the detailed treatment regimens of patients [8]. In contrast, our study considered only the response of patients to IVMP or IVIG due to the com- plexity of clinical treatments, and we attempted to analyze the effectiveness of the chosen treatments for patients from 70 previous articles. In patients who received IVMP, the effective rate was 45.56% (41/90), which was slightly higher than that of 36.71% (29/79) for patients who received IVIG. In the study of Gagnon et al. [8], the favorable outcome rates were 46.15% (6/13) for IVMP and 50.00% (2/4) for IVIG, and the effective rate for IVMP was similar to ours; however, the number of patients in our study was much more than that in their study because we searched more comprehensive refer- ences and included more patients. In patients with sei- zures, the response rates of IVMP and IVIG were 42.65% (29/68) and 32.69% (17/52), respectively, which were similar when counting all patients. Although we cannot recommend which method is more effective because no significant difference between IVMP and IVIG was found, importantly, it is very difficult for these patients to become seizure free from only AEDs [12, 13], and immu- notherapy is currently still indispensable. In patients with SPS or CA, IVIG seemed to have a slightly better thera- peutic effect than IVMP (77.78% vs. 60.00%; 55.56% vs. 28.57%). Consistent with our results, in a randomized, double-blinded, placebo-controlled crossover trial in anti-GAD65-Ab-positive SPS patients, IVIG resulted in significant improvements in objective stiffness param- eters and activities of daily living [44], and corticosteroids or other therapies may be very disappointing [45]. How- ever, as for CA, a study of 118 CA patients (including 41 anti-GAD65-Ab-positive cases) drew a different conclu- sion from ours, suggesting that corticosteroids may be the best regimen [6]. Considering the small sample size and the heterogeneity of subjects, further research is needed. Theoretically, combined therapy should be bet- ter than monotherapy, but the effective rate was disap- pointing in our research, with only 15/35 (42.86%) in all patients and 13/29 (44.83%), 0/1 (0%), and 1/4 (25.00%) in epilepsy, SPS and CA patients, respectively. A similar conclusion was obtained in the study of Gagnon et  al. where steroids plus IVIG or plasma exchange had benefi- cial effects on only 6/18 (33.33%) anti-GAD65 LE patients [8]. Patients from previous literaturesi One important reason contributing to the results is that patients who received combined immunotherapy were usually in serious conditions. Although we cannot make comparisons with monotherapy due to statistical i It should be mentioned that we cannot compare the efficiency of combination therapy with monotherapy because of their statistical overlaps. Discussion Identical  conclusions  were  obtained in 12 other patients [28, 48], making the routine use of immunotherapy debatable. Nevertheless, anti-GAD65- associated SPS is likely to be an exception because more than 70% of patients can obtain relief from IVIG, and notably, the abovementioned clinical trial further clarifies the validity of IVIG [44]. y g In our study, we also observed 29 anti-GAD65 AE patients who had coexisting tumors; in general, their reactions to immunotherapy were unsatisfactory. Ten of them received combined treatment of IVMP and IVIG, but only two benefited. Among the remaining 19 patients, the effectiveness of IVMP and IVIG was 55.56% (5/9) and 72.73% (8/11), respectively (one patient received IVMP and IVIG successively, not in combination); how- ever, when calculated together, the improvement rates of IVMP and IVIG slumped to 29.41% (5/17) and 42.11% (8/19), respectively. Interestingly, a good outcome may be predicted for patients who had coexisting thymoma; only one out of 8 did not respond to immunotherapy, but the symptoms could be relieved by benzodiazepines or baclofen [46]. In contrast, only two out of 11 lung cancer patients acquired temporary improvement with immu- notherapy, and one died in a short period of time [47]. The results are consistent with a previous observation, which found that 46.67% (7/15) of anti-GAD65-asso- ciated paraneoplastic neurological syndrome patients acquired clinical improvement or stability under various immunotherapy treatments, and amelioration occurred in only 4 thymic tumor patients, while only 1/6 of lung cancer patients was stable, and the rest were all dete- riorated [7]. In addition to the older age compared with tumor-negative anti-GAD65 AE patients, which probably led to poor prognosis, tumor treatment is also a factor that needs to be considered. Among patients undergo- ing active treatment of primary tumors, 13/21 (61.90%) gained benefits from immunotherapy, while no patients improved if the tumor was not managed. Considering the adverse influences of particular tumor types, old age and passive tumor treatment on prognosis, the underlying tumors should be screened in some conditions early [7], and elaborate studies involving various factors are criti- cally needed to guide future therapy.h i Our study also had some limitations. First, the retro- spective analysis of the literature had the following inher- ent limitations: the diverse vocabularies could have led to a certain degree of misinterpretation; the description of clinical features, investigations, and outcomes also dif- fered from one article to another. Discussion Second, we abandoned some related literature because we could not obtain the prognosis after immunotherapy, leading to statisti- cal bias. Third, different hospitals had their own experi- ence in choosing IVMP, IVIG or others, and the absence of IVMP or IVIG therapy did not mean that the patient was insensitive to it. Fourth, we excluded many patients who received combination therapy (IVMP/IVIG and oth- ers) because we could not judge which drug had an effect. Fifth, there was a certain ineluctable subjectivity in our judgment of reactivity to therapy. Sixth, there were sig- nificant differences in the follow-up time of patients, and we focused only on whether the clinical symptoms were improved after immunotherapy, leading to the inability to judge the effective time of IVIG and IVMP treatment. Although many patients were initially sensitive to immu- notherapy, they will relapse inevitably after a period of time [13, 24, 36, 41, 48, 50–54], which we considered may be a short-term outcome. Considering the shortcomings of our research and limitations in this field, therapeutic strategies cannot be reasonably recommended currently except IVIG for anti-GAD65-associated SPS, and long- term immunotherapy may be the best option. There was no denying that some anti-GAD65 patients completely recovered without immunotherapy; how- ever, they were reported just in scattered case reports [12, 19, 23, 48, 49]. We firmly believe that this AE was characterized as a form of chronic, non-remitting dis- order in most cases, which  was consistent  with  the standpoint from other researchers [8, 11, 40]. Several medical centers also tried to summarize their own treatment experiences and expected to offer the best treatment plan, but all ended in failure [9, 11, 28, 40, 48]. Seven anti-GAD65 LE patients received monthly high-intensity immunotherapy, with a median total methylprednisolone equivalent dose up to 18  g, but none became seizure free [40]. Malter et al. retrospec- tively analyzed the complex treatment regimens of Discussion Screening for anti-GAD65 Abs has been widely reported among type 1 diabetic patients. However, increased awareness of neurologists in considering patients with anti-GAD65 Abs remains challenging. Among adult epi- lepsy patients with unknown etiology, three recent stud- ies found that 1.7% (7/416) [37], 16.1% (18/112) [38], and 21.7% (5/23) of patients were positive for serum anti- GAD65 Abs [39]. The anti-GAD65-Ab positive rate was 17.0% (9/53) in LE patients [40]. Hence, the incidence of anti-GAD65 diseases may be seriously underestimated in practice. Here, we have described the clinical informa- tion of six anti-GAD65 AE patients in our cohort, five of whom were diagnosed after years of repeated medi- cal consultations. Therefore, it should be noted that the time from symptom onset to immunotherapy was gen- erally very long due to the lack of awareness, and some patients, including one of ours, even underwent brain tissue resection for drug-resistant epilepsy, which usually led to unsatisfactory outcomes [11, 41, 42]. Currently, there is no consensus on the treatment of anti-GAD65 AE. However, we believe that anti-GAD65 Abs are unique compared with other onconeural Abs. Patients with neurological syndromes associated with anti-GAD65 Abs are not typically considered at very high risk for cancer [7], and their prognosis seems to be very different from that of other traditional onconeu- ral Abs [6, 25]. Jones et al. proved that the detection of anti-GAD65 Abs in adult autoimmune CA patients may predict relatively better immunotherapy response and neurological outcomes, similar to those of patients sero- positive for neuronal surface Abs [6]. In contrast, the beneficial effects of immunotherapy on seizure frequency and cognition can be acquired only in paraneoplastic Ab- positive LE patients but not obviously in anti-GAD65 LE patients [25]. Furthermore, according to considerable research efforts devoted to the comparison of AE patients with anti-GAD65 Abs and those with anti-voltage gated Li et al. BMC Neurosci (2020) 21:13 Page 9 of 11 overlaps, the method is still highly recommendable and very promising. 13 anti-GAD65 epilepsy patients and found that the seizure and cognitive response to immunotherapies were poor, with the most frequently achieved seizure response (≥ 50% reduction) occurring in only 45% of patients under intensive corticosteroid treatment (median 19 g methylprednisolone equivalent) [11]. Pit- tock et al. found that the benefits from a trial of immu- notherapies in 27 patients with anti-GAD65 Abs were temporary [9]. Conclusions Here, we reported six anti-GAD65 AE patients and found that they all achieved clinical improvements in a short period of time after immunotherapy. However, by sum- marizing the therapeutic effects of previous patients, we have confirmed that this AE usually has a poor response to IVMP or IVIG, except anti-GAD65-associated SPS. Larger prospective studies enrolling large numbers of Page 10 of 11 Page 10 of 11 Li et al. BMC Neurosci (2020) 21:13 Li et al. BMC Neurosci (2020) 21:13 patients are required to identify the optimal therapeutic strategy. patients are required to identify the optimal therapeutic strategy. South Fourth Ring West Road, Fengtai District, Beijing 100070, People’s Repub‑ lic of China. 2 Department of Neurology, Xuanwu Hospital of Capital Medical University, 45 Chang Chun Road, Xicheng District, Beijing 100053, People’s Republic of China. 3 Department of Neurology, the Second Hospital of Hebei Medical University, Hebei Medical University, 215 Heping West Road, Xinhua District, Shijiazhuang 050000, People’s Republic of China. Consent for publication 15. Sinnreich M, Assal F, Hefft S, Magistris MR, Chizzolini C, Landis T, Burkhard PR. Anti-GAD antibodies and breast cancer in a patient with stiff-person syndrome: a puzzling association. Eur Neurol. 2001;46(1):51–2. All participants provided written consent for publication for the publication of clinical details. 16. Liu B, Zhou Y, Meng L, Skinner H. A survival case of super-refractory status epilepticus due to glutamic acid decarboxylase antibodies-associated limbic encephalitis. Cureus. 2018;10(8):e3125. Funding h k This work was supported by grants from the Application Research of Capital Clinical Characteristics (No. Z181100001718082), the Beijing Dongcheng District Outstanding Talent Funding Project (No. 2019DCT-M-18), the National Natural Science Foundation of China (No. 81301119), the Beijing Municipal Administration of Hospitals’ Youth Programme (No. QML20160504), the China Association Against Epilepsy-UCB Research Fund (No. CAAE- UCB2016008) and the Natural Science Foundation of Capital Medical University (No. PYZ2017055). The funding bodies had no influence on the design of the study, collection, analysis, and interpretation of data or writing of the manuscript. 9. Pittock SJ, Yoshikawa H, Ahlskog JE, Tisch SH, Benarroch EE, Kryzer TJ, Lennon VA. Glutamic acid decarboxylase autoimmunity with brain‑ stem, extrapyramidal, and spinal cord dysfunction. Mayo Clin Proc. 2006;81(9):1207–14. 10. Korff CM, Parvex P, Cimasoni L, Wilhelm-Bals A, Hampe CS, Schwitzgebel VM, Michel M, Siegrist CA, Lalive PH, Seeck M. Encephalitis associated with glutamic acid decarboxylase autoantibodies in a child: a treatable condition. Arch Neurol. 2011;68(8):1065–8. 11. Malter MP, Frisch C, Zeitler H, Surges R, Urbach H, Helmstaedter C, Elger CE, Bien CG. Treatment of immune-mediated temporal lobe epilepsy with GAD antibodies. Seizure. 2015;30:57–63. Availability of data and materials The datasets supporting the conclusions of this article are available when contacting authors. 12. Feyissa AM, López Chiriboga AS, Britton JW. Antiepileptic drug therapy in patients with autoimmune epilepsy. Neurol Neuroimmunol Neuroin‑ flamm. 2017;4(4):e353. Acknowledgements Not applicable. 6. Jones AL, Flanagan EP, Pittock SJ, Mandrekar JN, Eggers SD, Ahlskog JE, McKeon A. Responses to and outcomes of treatment of autoimmune cerebellar ataxia in adults. JAMA Neurol. 2015;72(11):1304–12. Ethics approval and consent to participate The study was approved by the Medical Ethics Committee of Beijing Tiantan Hospital, Capital Medical University, and was carried out in accordance with the Declaration of Helsinki. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. All participants gave written informed consent to participate. 13. Farooqi MS, Lai Y, Lancaster E, Schmitt SE, Sachais BS. Therapeutic plasma exchange and immunosuppressive therapy in a patient with anti-GAD antibody-related epilepsy: quantification of the antibody response. J Clin Apher. 2015;30(1):8–14. 14. Olson JA, Olson DM, Sandborg C, Alexander S, Buckingham B. Type 1 diabetes mellitus and epilepsia partialis continua in a 6-year-old boy with elevated anti-GAD65 antibodies. Pediatrics. 2002;109(3):E50. Authors’ contributions RJL, YDZ, QW, XQS and ZML provided the clinical data. TRL and RJL acquired the literature data. TRL drafted the manuscript. RJL critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript. 7. Ariño H, Höftberger R, Gresa-Arribas N, Martínez-Hernández E, Armangue T, Kruer MC, Arpa J, Domingo J, Rojc B, Bataller L, et al. Paraneoplastic neurological syndromes and glutamic acid decarboxylase antibodies. JAMA Neurol. 2015;72(8):874–81. 8. Gagnon MM, Savard M. Limbic encephalitis associated with GAD65 antibodies: brief review of the relevant literature. Can J Neurol Sci. 2016;43(4):486–93. Received: 4 October 2019 Accepted: 21 March 2020 Additional file 1. References included in the statistics. We conducted a search on PubMed for articles up to April 2019 and finally included 70 references. The article type, clinical characteristics of each patient, and whether the patient received combined therapy are noted. References 1. Daif A, Lukas RV, Issa NP, Javed A, VanHaerents S, Reder AT, Tao JX, Warnke P, Rose S, Towle VL, et al. Antiglutamic acid decarboxylase 65 (GAD65) antibody-associated epilepsy. Epilepsy Behav. 2018;80:331–6. Additional file 2: Tabe S1. Clinical data of 29 patients who had coexist‑ ing tumors in previous references. The clinical information, concomitant tumor, tumor therapy and response to immunotherapy of each patient are listed. 2. Dubey D, Pittock SJ, Kelly CR, McKeon A, Lopez-Chiriboga AS, Lennon VA, Gadoth A, Smith CY, Bryant SC, Klein CJ, et al. Autoimmune encephalitis epidemiology and a comparison to infectious encephalitis. Ann Neurol. 2018;83(1):166–77. Competing interests On behalf of all authors, the corresponding author confirms no conflict of interest. 17. Hagiwara H, Enomoto-Nakatani S, Sakai K, Ugawa Y, Kusunoki S, Kanaz‑ awa I. Stiff-person syndrome associated with invasive thymoma: a case report. J Neurol Sci. 2001;193(1):59–62. Abbreviations 3. Hackert JK, Müller L, Rohde M, Bien CG, Köhling R, Kirschstein T. Anti- GAD65 containing cerebrospinal fluid does not alter GABAergic transmis sion. Front Cell Neurosci. 2016;10:130. 3. Hackert JK, Müller L, Rohde M, Bien CG, Köhling R, Kirschstein T. Anti- GAD65 containing cerebrospinal fluid does not alter GABAergic transmis‑ sion. Front Cell Neurosci. 2016;10:130. GAD65: Glutamic acid decarboxylase 65; Abs: Antibodies; CSF: Cerebrospinal fluid; SPS: Stiff-person syndrome; CA: Cerebellar ataxia; LE: Limbic encephalitis; AE: Autoimmune encephalitis; IVMP: Intravenous methylprednisolone; IVIG: Intravenous immunoglobulin; AEDs: Antiepileptic drugs; GTCSs: Generalized tonic–clonic seizures; MoCA: Montreal Cognitive Assessment; MRI: Magnetic resonance imaging; EEG: Electroencephalogram. 4. Gresa-Arribas N, Ariño H, Martínez-Hernández E, Petit-Pedrol M, Sabater L, Saiz A, Dalmau J, Graus F. Antibodies to inhibitory synaptic proteins in neurological syndromes associated with glutamic acid decarboxylase autoimmunity. PLoS ONE. 2015;10(3):e0121364. 5. McKeon A, Tracy JA. GAD65 neurological autoimmunity. Muscle Nerve. 2017;56(1):15–27. 5. McKeon A, Tracy JA. GAD65 neurological autoimmunity. Muscle Nerve. 2017;56(1):15–27. Supplementary information accompanies this paper at https​://doi. org/10.1186/s1286​8-020-00561​-9. Supplementary information accompanies this paper at https​://doi. org/10.1186/s1286​8-020-00561​-9. Received: 4 October 2019 Accepted: 21 March 2020 Author details 1 1 Department of Neurology, Beijing Tiantan Hospital, Capital Medical Univer‑ sity; China National Clinical Research Center for Neurological Diseases, 119 18. Sharma CM, Pandey RK, Kumawat BL, Khandelwal D, Gandhi P. A unique combination of autoimmune limbic encephalitis, type 1 diabetes, and Page 11 of 11 Page 11 of 11 Page 11 of 11 Li et al. BMC Neurosci (2020) 21:13 Li et al. BMC Neurosci (2020) 21:13 Stiff person syndrome associated with GAD-65 antibody. Ann Indian Acad Neurol. 2016;19(1):146–9. Stiff person syndrome associated with GAD-65 antibody. Ann Indian Acad Neurol. 2016;19(1):146–9. 38. Dubey D, Alqallaf A, Hays R, Freeman M, Chen K, Ding K, Agostini M, Vernino S. Neurological autoantibody prevalence in epilepsy of unknown etiology. JAMA Neurol. 2017;74(4):397–402. 19. Petit-Pedrol M, Armangue T, Peng X, Bataller L, Cellucci T, Davis R, McCracken L, Martinez-Hernandez E, Mason WP, Kruer MC, et al. Encephalitis with refractory seizures, status epilepticus, and antibodies to the GABAA receptor: a case series, characterisation of the antigen, and analysis of the effects of antibodies. Lancet Neurol. 2014;13(3):276–86. 39. Falip M, Carreño M, Miró J, Saiz A, Villanueva V, Quílez A, Molins A, Barceló I, Sierra A, Graus F. Prevalence and immunological spectrum of temporal lobe epilepsy with glutamic acid decarboxylase antibodies. Eur J Neurol. 2012;19(6):827–33. 40. Malter MP, Helmstaedter C, Urbach H, Vincent A, Bien CG. Antibodies to glutamic acid decarboxylase define a form of limbic encephalitis. Ann Neurol. 2010;67(4):470–8. 20. Quek AM, Britton JW, McKeon A, So E, Lennon VA, Shin C, Klein C, Watson RE, Kotsenas AL, Lagerlund TD, et al. Autoimmune epilepsy: clinical characteristics and response to immunotherapy. Arch Neurol. 2012;69(5):582–93. 41. Hansen N, Ernst L, Rüber T, Widman G, Becker AJ, Elger CE, Helmstaedter C. Pre- and long-term postoperative courses of hippocampus-associated memory impairment in epilepsy patients with antibody-associated limbic encephalitis and selective amygdalohippocampectomy. Epilepsy Behav. 2018;79:93–9. 21. Rakocevic G, Hussain A. Stiff person syndrome improvement with chemotherapy in a patient with cutaneous T cell lymphoma. Muscle Nerve. 2013;47(6):938–9. 22. Ances BM, Vitaliani R, Taylor RA, Liebeskind DS, Voloschin A, Houghton DJ, Galetta SL, Dichter M, Alavi A, Rosenfeld MR, et al. Treatment-responsive limbic encephalitis identified by neuropil antibodies: MRI and PET cor‑ relates. Brain. 2005;128(Pt 8):1764–77. 42. Bonello M, Larner AJ, Marson AG. Profound amnesia after temporal lobec‑ tomy: an autoimmune process resembling patient hm. Case Rep Neurol. 2014;6(3):251–5. 23. Author details 1 Haberlandt E, Bast T, Ebner A, Holthausen H, Kluger G, Kravljanac R, Kröll- Seger J, Kurlemann G, Makowski C, Rostasy K, et al. Limbic encephalitis in children and adolescents. Arch Dis Child. 2011;96(2):186–91. 43. Frisch C, Malter MP, Elger CE, Helmstaedter C. Neuropsychological course of voltage-gated potassium channel and glutamic acid decarboxylase antibody related limbic encephalitis. Eur J Neurol. 2013;20(9):1297–304. y p 44. Dalakas MC, Fujii M, Li M, Lutfi B, Kyhos J, McElroy B. High-dose intra‑ venous immune globulin for stiff-person syndrome. N Engl J Med. 2001;345(26):1870–6. 24. Kojima G, Inaba M, Bruno MK. PET-positive extralimbic presentation of anti-glutamic acid decarboxylase antibody-associated encephalitis. Epileptic Disord. 2014;16(3):358–61. p p 25. Hansen N, Widman G, Witt JA, Wagner J, Becker AJ, Elger CE, Helmstaed‑ ter C. Seizure control and cognitive improvement via immunotherapy in late onset epilepsy patients with paraneoplastic versus GAD65 autoanti‑ body-associated limbic encephalitis. Epilepsy Behav. 2016;65:18–24. 45. Alexopoulos H, Dalakas MC. Immunology of stiff person syndrome and other GAD-associated neurological disorders. Expert Rev Clin Immunol. 2013;9(11):1043–53. 46. Thomas S, Critchley P, Lawden M, Farooq S, Thomas A, Proudlock FA, Constantinescu CS, Gottlob I. Stiff person syndrome with eye move‑ ment abnormality, myasthenia gravis, and thymoma. J Neurol Neurosurg Psychiatry. 2005;76(1):141–2. 26. Ben Achour N, Ben Younes T, Rebai I, Ben Ahmed M, Kraoua I, Ben Youssef- Turki I. Severe dysautonomia as a main feature of anti-GAD encephalitis: report of a paediatric case and literature review. Eur J Paediatr Neurol. 2018;22(3):548–51. 47. Spitz M, Ferraz HB, Barsottini OG, Gabbai AA. Progressive encephalomyeli‑ tis with rigidity: a paraneoplastic presentation of oat cell carcinoma of the lung. Case report. Arq Neuropsiquiatr. 2004;62(2B):547–9. 27. Finelli PF. Autoimmune limbic encephalitis with GAD antibodies. Neuro‑ hospitalist. 2011;1(4):178–81. 28. Lilleker JB, Biswas V, Mohanraj R. Glutamic acid decarboxylase (GAD) anti‑ bodies in epilepsy: diagnostic yield and therapeutic implications. Seizure. 2014;23(8):598–602. 48. Mäkelä KM, Hietaharju A, Brander A, Peltola J. Clinical management of epilepsy with glutamic acid decarboxylase antibody positivity: the inter‑ play between immunotherapy and anti-epileptic drugs. Front Neurol. 2018;9:579. 29. Akaishi T, Jin K, Kato K, Itabashi H, Misu T, Tateyama M, Iwasaki M, Aoki M, Nakasato N. Clinical characteristics of four patients with temporal lobe epilepsy associated with elevated anti-GAD antibodies. Rinsho Shinkei‑ gaku. 2015;55(11):804–9. 49. Graus F, Saiz A, Lai M, Bruna J, López F, Sabater L, Blanco Y, Rey MJ, Ribalta T, Dalmau J. Author details 1 Neuronal surface antigen antibodies in limbic encephalitis: clinical-immunologic associations. Neurology. 2008;71(12):930–6. 30. Specchio N, Fusco L, Claps D, Vigevano F. Epileptic encephalopathy in children possibly related to immune-mediated pathogenesis. Brain Dev. 2010;32(1):51–6. 50. Akman CI, Patterson MC, Rubinstein A, Herzog R. Limbic encephalitis associated with anti-GAD antibody and common variable immune deficiency. Dev Med Child Neurol. 2009;51(7):563–7. 31. Knudsen A, Bredholt G, Storstein A, Oltedal L, Davanger S, Krossnes B, Honnorat J, Vedeler CA. Antibodies to CRMP3-4 associated with limbic encephalitis and thymoma. 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Ganor Y, Goldberg-Stern H, Amrom D, Lerman-Sagie T, Teichberg VI, Pelled D, Futerman AH, Zeev BB, Freilinger M, Verheulpen D, et al. Autoimmune epilepsy: some epilepsy patients harbor autoantibodies to glutamate receptors and dsDNA on both sides of the blood-brain barrier, which may kill neurons and decrease in brain fluids after hemispherotomy. Clin Dev Immunol. 2004;11(3–4):241–52. 54. Markakis I, Alexopoulos H, Poulopoulou C, Akrivou S, Papathanasiou A, Katsiva V, Lyrakos G, Gekas G, Dalakas MC. Immunotherapy-responsive limbic encephalitis with antibodies to glutamic acid decarboxylase. J Neurol Sci. 2014;343(1–2):192–4. 35. Ganor Y, Freilinger M, Dulac O, Levite M. Monozygotic twins discordant for epilepsy differ in the levels of potentially pathogenic autoantibodies and cytokines. Autoimmunity. 2005;38(2):139–50. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 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Multicenter Study of Lumen-apposing Metal Stents With or Without Pigtail in Endoscopic Ultrasound-guided Biliary Drainage for Malignant Obstruction- Bampi Trial: an Open-label, Randomized Controlled Trial Protocol.
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Multicenter Study of Lumen-apposing Metal Stents With or Without Pigtail in Endoscopic Ultrasound- guided Biliary Drainage for Malignant Obstruction- Bampi Trial: an Open-label, Randomized Controlled Trial Protocol. Silvia Salord  Hospital Universitari de Bellvitge Berta Laquente  Hospital Duran i Reynals: Institut Catala d'Oncologia Cristian Tebe  Bellvitge Institute for Biomedical Research: Institut d'Investigacio Biomedica de Bellvitge Sebastia Videla  Bellvitge University Hospital: Hospital Universitari de Bellvitge Manuel Perez-Miranda  Hospital Universitario Rio Hortega de Valladolid: Hospital Universitario Rio Hortega Joan b Gornals  (  jgornals@bellvitgehospital.cat ) Hospital Universitari de Bellvitge https://orcid.org/0000-0001-8857-3556 Multicenter Study of Lumen-apposing Metal Stents With or Without Pigtail in Endoscopic Ultrasound- guided Biliary Drainage for Malignant Obstruction- Bampi Trial: an Open-label, Randomized Controlled Trial Protocol. Page 1/23 guided Biliary Drainage for Malignant Obstr Bampi Trial: an Open-label, Randomized Co Trial Protocol. Albert Garcia-Sumalla  Hospital Universitari de Bellvitge Carme Loras  Hospital Universitari MutuaTerrassa Vicente Sanchiz  Hospital Clínico Universitario de Valencia Rafael Pedraza-Sanz  Hospital General Universitari de Castelló: Hospital General Universitari de Castello Enrique Vazquez-Sequeiros  Hospital Ramon y Cajal: Hospital Universitario Ramon y Cajal Jose Ramon Aparicio  Hospital General Universitario de Alicante: Hospital General Universitari d'Alacant Carlos De la Serna-Higuera  Rio Hortega University Hospital: Hospital Universitario Rio Hortega Daniel Luna-Garcia  Hospital Universitari de Bellvitge Xavier Andujar  Hospital Universitari MútuaTerrassa: Hospital Universitari MutuaTerrassa Maria Capilla  Hospital Clínico Universitario de Valencia Tatiana Barbera  Hospital General Universitari de Castelló: Hospital General Universitari de Castello Jose Ramon Foruny-Olcina  Hospital Ramon y Cajal: Hospital Universitario Ramon y Cajal Belen Martinez-Moreno  Hospital General Universitario de Alicante: Hospital General Universitari d'Alacant Miguel Dura  Hospital Universitario Rio Hortega de Valladolid: Hospital Universitario Rio Hortega Carme Loras  Hospital Universitari MutuaTerrassa Vicente Sanchiz  Hospital Clínico Universitario de Valencia Daniel Luna-Garcia  Hospital Universitari de Bellvitge Page 1/23 Daniel Luna Garcia  Hospital Universitari de Bellvitge Xavier Andujar  Hospital Universitari MútuaTerrassa: Hospital Universitari MutuaTerrassa Maria Capilla  Hospital Clínico Universitario de Valencia Tatiana Barbera  Hospital General Universitari de Castelló: Hospital General Universitari de Castello Jose Ramon Foruny-Olcina  Hospital Ramon y Cajal: Hospital Universitario Ramon y Cajal Belen Martinez-Moreno  Hospital General Universitario de Alicante: Hospital General Universitari d'Alacant Miguel Dura  Hospital Universitario Rio Hortega de Valladolid: Hospital Universitario Rio Hortega Silvia Salord  Hospital Universitari de Bellvitge Berta Laquente  Hospital Duran i Reynals: Institut Catala d'Oncologia Cristian Tebe  Bellvitge Institute for Biomedical Research: Institut d'Investigacio Biomedica de Bellvitge Sebastia Videla  Bellvitge University Hospital: Hospital Universitari de Bellvitge Manuel Perez-Miranda  Hospital Universitario Rio Hortega de Valladolid: Hospital Universitario Rio Hortega Joan b Gornals  (  jgornals@bellvitgehospital.cat ) Hospital Universitari de Bellvitge https://orcid.org/0000-0001-8857-3556 Research Article Keywords: Biliary drainage, Choledochoduodenostomy, Endoscopic ultrasound, Lumen-apposing metal stent, Malignant biliary obstruction, Plastic stent, Trial Posted Date: February 4th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1286894/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Trials on February 25th, 2022. See the published version at https://doi.org/10.1186/s13063-022-06106-1. Research Article Posted Date: February 4th, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Version of Record: A version of this preprint was published at Trials on February 25th, 2022. See the published version at https://doi.org/10.1186/s13063-022-06106-1. Page 2/23 Page 2/23 Page 2/23 Page 2/23 Abstract Background: It is unclear whether the insertion of an axis-orienting double-pigtail plastic stent (DPS) through biliary lumen-apposing meal stent (LAMS) in EUS-guided choledochoduodenostomy (CDS) improves the stent patency. The aim of this study is to determine whether this technical variant offers a clinical benefit in EUS-guided biliary drainage (BD) for the management of distal malignant biliary obstruction. Methods/design: This is a multicenter open-label, randomized controlled trial with two parallel groups. Eighty-four patients with malignant biliary obstruction will undergo EUS-BD (CDS type) using LAMS in 7 tertiary hospitals in Spain and will be randomized to the LAMS and LAMS plus DPS groups. The primary endpoint is the rate of recurrent biliary obstruction, as a stent dysfunction parameter, detected during follow-up. Secondary endpoints: technical and clinical success (reduction in bilirubin >50% within 14 days of stent placement), safety, and others (number of reinterventions, time to biliary obstruction, prognostic factors, survival rate). Discussion: The BAMPI trial has been designed to determine whether the addition of a coaxial axis- orienting DPS through LAMS is superior to LAMS alone to prevent stent dysfunction. Trial registration: ClinicalTrials.gov, NCT04595058. Registered on October 14, 2020. Trial registration: ClinicalTrials.gov, NCT04595058. Registered on October 14, 2020. Background Transpapillary stenting by endoscopic retrograde cholangiopancreatography (ERCP) remains the gold standard treatment for malignant biliary obstruction (MBO), but may fail in up to 15% of cases, and it carries the risks of post-ERCP pancreatitis and stent dysfunction secondary to tumor ingrowth and/or overgrowth [1, 2]. For these reasons, since the emergence of Endoscopic Ultrasound-guided biliary drainage (EUS-BD) using electrocautery-enhanced (EE) lumen-apposing metal stents (LAMS), by creating a choledochoduodensotomy (CDS), it has been proposed as a viable alternative to ERCP [3, 4]. Firstly, as a rescue strategy after failed ERCP, in palliative scenarios, or even as a bridge to surgery [5, 6], and then recently, as a first-line modality for MBO in controlled trials [2, 7–9]. Although recent metanalyses and systematic reviews [10–14] have postulated that EUS-BD using LAMS has high technical and clinical success rates, some concerns exist regarding its safety, as non-negligible rates of adverse events (AE) have been reported [15, 16]. Recurrent biliary obstruction (RBO), as a stent dysfunction parameter, is a major issue to consider after EUS-BD using LAMS. Several cases of stent dysfunction (e.g., food or lithiasis impaction, sump syndrome, stent migration) have been documented due to a possible limitation in LAMS design, as the short length of the stent causes the distal flange to become impacted against the opposite biliary wall [10, 17, 18]. The insertion of a coaxial double-pigtail plastic stent (DPS) has been demonstrated to improve LAMS patency and bile flow, by maintaining a vertical orientation in the bile duct. But a recent retrospective study by our group did not encounter sufficient evidence to recommend its routine use [19]. Page 3/23 For these reasons, a multicenter randomized controlled trial has been designed to assess whether a coaxial DPS within a biliary LAMS is superior to a single LAMS in EUS-BD (CDS type) for MBO. For these reasons, a multicenter randomized controlled trial has been designed to assess whether a coaxial DPS within a biliary LAMS is superior to a single LAMS in EUS-BD (CDS type) for MBO. Methods/design The BAMPI (Biliary Apposing Metal PIgtail) trial is a multicenter, open-label, randomized controlled clinical trial with two parallel groups, and with a 1:1 allocation ratio. Eighty patients with MBO will be scheduled for EUS-BD in seven tertiary Spanish hospitals and will be randomized to the LAMS alone or the LAMS plus DPS group. We hypothesize the addition of a prophylactic DPS through LAMS to be associated with fewer episodes of RBO and, consequently, fewer biliary reinterventions (BRI). Central ethical approval of the study protocol has been confirmed by the Comité Ético de Investigación Clínica (CEIC) del Hospital Universitari de Bellvitge-IDIBELL (ref approval no. ICPS024/20, on November, 2020) and we will not begin recruiting at other centers in the trial until local ethical approval has been obtained. A checklist with the recommendations for Interventional Trials (SPIRIT) is attached as Additional file 1. SPIRIT-figure with schedule of enrolment, interventions, and assessments in the BAMPI trial (figure 1). Patients with any of the following will be excluded - Pregnancy or breast-feeding. - Pregnancy or breast-feeding. - Another type of biliary drainage at the time of the procedure (cholecystostomy, percutaneous drainage, etc.). - Failure to sign informed consent form. - Intellectual handicap and unable to understand the nature and possible consequences of the study, unless there is a competent legal representative. - Unable to adhere to subsequent follow-up requirements. - Unable to adhere to subsequent follow-up requirements. CBD common bile duct; EUS endoscopic ultrasound; INR international normalized ratio. Recruitment Principal investigators from each center will have the task of presenting strategies to promote enrolment and ensure the target sample size. Prior to the start of the study, a meeting with oncology and digestive surgery teams will be organized with the aim of creating collaboration bridges. Study population All patients admitted with distal MBO and clinical criteria that justify EUS-BD will be considered for consent. The investigator at each center will assess the inclusion of the patient in the study, and eligibility either as a first-line BD method, or as a rescue method after failed ERCP. The patient will be correctly informed by personnel knowledgeable about the specifics of the study, who will help to resolve any questions that may arise. The informed consent form will be signed prior to the procedure and a signed copy will be given to the patient. The patient has the right to opt out of the study at any time. The inclusion and exclusion criteria are listed in Table 1. Page 4/23 Page 4/23 Table 1 Inclusion and exclusion criteria. Inclusion criteria: Patients eligible for the trial must fulfil all the following at randomization - Age 18 years or more - Malignant biliary obstruction with clinical criteria that justifies EUS-guided biliary drainage. - Capable of understanding and signing informed consent form. - Understanding the type of study and complying with the follow-up of complementary tests during the study’s duration. Exclusion criteria: General description of the technique General description of the technique Treatment allocation is to EUS-BD with LAMS alone vs. LAMS plus DPS. All procedures will be performed by experienced endoscopists in EUS-guided transmural stenting. Procedures will be performed under deep sedation or tracheal intubation, in accordance with the directives of each center. Coagulation disorders will be corrected prior to the procedure. In case of INR>1.5 this will be corrected with the protocol of each institution. In all interventional procedures, CO2 will be used as an insufflation agent. Prophylactic antibiotic therapy will be given in accordance with institutional protocols. Randomization and masking Patients will be enrolled in this trial by gastroenterologists, surgeons, oncologists, and endoscopists who will evaluate cases in the inpatient wards or outpatient consultation areas. Investigators of each center will be responsible for entering all necessary criteria to an online platform that will generate the randomization sequence, and participants will be randomized with an arbitrary number. Page 5/23 A code list will be generated by our biostatistics department, using R software (v 3.6.3) by randomization with a 1:1 randomization ratio, by blocks, stratified by age (<65 years-old / >65 years-old) and by the presence of liver metastases. The assumption is made that liver metastasis may elevate bilirubin and cholestasis parameters without involving bile duct obstruction. Everyone will be assigned a randomization code along with the treatment that corresponds to it. Once the patient meets the eligibility criteria and has provided informed consent, we will proceed to the allocation of each participant centrally, ensuring allocation concealment, and based on the randomization list. To prevent different subject recruitment rates at the various hospitals from interfering in the development of the study, the entire population will be randomized in blocks of four between the two treatment possibilities. Qualification of centers This clinical trial will be performed at the endoscopy unit of seven referral centers in Spain. Endoscopists will all be experienced in endoscopic intervention and therapeutics, such as stenting and EUS-guided transmural drainage. To avoid biases derived from the learning curve, only those centers with proven experience using biliary EE-LAMS (EUS-guided CDS) have been invited to participate. To objectify usage data, the database “NRPAL: National Registry of Lumen Apposing Metal Stents Incidences, NCT04059926”, which collects data from all LAMS placed for one year, was checked. Minimum requirements were at least 12 LAMS placed in one year for any indication and a minimum experience of 7 EUS-BD using EE-LAMS. All the experienced endoscopists are members of the Spanish Society of Digestive Endoscopy (SEED). Gallbladder option When a CDS is not possible or too risky, a BD rescue by EUS-guided gallbladder transmural drainage may be considered. It is important to note that this should only be offered in cases without an optimal endosonographic window for the creation of CDS (e.g., interposal vessels, CBD <10mm, excessive GI tract-CBD distance, scope instability, others). It may be randomized in the same way (LAMS vs. LAMS- DPS group) and followed up according to the protocol. This additional subgroup will be analyzed and documented, but outside of the trial’s main outcomes. Additional interventions After the LAMS deployment, intra-stent dilation is permitted using balloons at the endoscopist’s discretion. Other endoscopic procedure such as EUS-guided TA, EUS-guided gastro-enterostomy, stenting (esophageal, duodenal), and EUS-guided celiac plexus blockage/neurolysis performed during the index procedure will be meticulously noted in a specific section in the electronic case report form (eCRF). LAMS plus coaxial DPS procedure In those patients allocated in the LAMS-DPS cohort, a DPS (preferably 7Fr x 3, 5 or 7cm, Advanix, Boston Sc) will be placed coaxially through the LAMS, preferably with upward (intra-hepatic) orientation. This selection of small DPS sized should ease the advance and release by offering less friction. Selection of LAMS size will be based on bile duct diameter and availability. Selection of LAMS size will be based on bile duct diameter and availability EUS-BD with EE-LAMS Each selected case will ensure a conclusive diagnosis of MBO. If necessary EUS-guided tissue acquisition (TA) will be performed previously to or immediately after the transmural drainage. The TA technique will be at the endoscopist’s choice. Firstly, EUS oversight will be done with the purpose of ruling out features that could jeopardize a transmural BD (such as ascites, long distance common bile duct (CBD)- transducer, or intervening vessels). The extrahepatic bile duct will be identified by using a linear echoendoscope (Fujifilm EG-580-UT, Olympus GF-UCT180), selecting an optimal point for carrying out the EUS-guided intervention. The long scope position will preferably be used to maintain stability and, if necessary, to ease advancing of a guidewire to intrahepatic direction. A small-medium sized LAMS (6, 8, Page 6/23 10-mm in diameter, and 8 or 10-mm in length, Hot-AXIOS™ stent with an electrocautery-enhanced delivery system, Boston Scientific, Marlborough, MA, USA) will be used in all included cases. 10-mm in diameter, and 8 or 10-mm in length, Hot-AXIOS™ stent with an electrocautery-enhanced delivery system, Boston Scientific, Marlborough, MA, USA) will be used in all included cases. The access step will be standardized in two different techniques, as explained in our previous report [19]: (i) freehand style (optional preloaded guidewire), with direct access to the bile duct, and (ii) classic technique, with initial puncture of the extrahepatic bile duct using a 19-gauge needle and advancing a standard guidewire, after which a LAMS is advanced over the guidewire. In all cases, the biliary LAMS is inserted using the cautery system, and deployed under EUS-guidance without tract dilation. The use of fluoroscopy will be decided upon based on technical considerations and the endoscopist’s opinion. Clinical evaluation and follow-up Data collection and calendar. The collecting of clinical information of the patients will begin at the outset (baseline) and will continue with follow-up as established and defined in the study. Data will be collected at baseline visit, indexing procedure 24 hours later, and at days 14, 30, 90, 180, 270, and 1-year post-randomization. Collected data include primary, secondary, and additional endpoint data, demographics, comorbidities, oncological data, laboratory test findings, technical details, and clinical data during follow-up ((Figure 1 and 2). AEs will be noted from the beginning of the test until the conclusion of follow-up by means of scheduled controls and will be handled and treated in accordance with the directives of the patient’s medical team. All additional tests and interventions will be duly documented. Any instances of death during the follow- up will be investigated to rule out possible relation to the endoscopic procedure. Such occurrences will also be recorded in the CRF. If there is clinical suspicion of obstruction or migration of the stent, an upper endoscopy will be carried out. Based on the findings of this procedure, the problem will be resolved in accordance with the directives of the intervening endoscopist. Any additional procedure or endoscopic intervention will be duly documented. All data will be collected by research personnel knowledgeable in the use of the eCRF (data capturing software, redCAP). Collected patient data will be de-identified, meaning that personal information will not be stored in the main database. All data will be stored for 7 years after publication or as requested in standard operating procedure from individual sites. Only data relevant to the study as outlined in this protocol will be collected by the research team. Due to the social and health crisis arising from the SARS-CoV-2 pandemic and taking into account the likely frailty of the candidates to be enrolled in the study, the protocol contemplates restrictions on physical presence visits. Only the indexing procedure and the 24 hours’ post-procedure visits will require an on-site visit since a minimum one-day hospital admission is mandatory. A BAMPI trial flowchart is provided (figure 2). Additional comments No removal of the stents is contemplated due to the malignant condition of the participants. Non- inpatients, they will remain in the center for a minimum of 24 h for clinical observation under clinical supervision. In case of technical failure for any reason, alternative strategies will be decided upon with the aim of offering the best possible treatment to the patient. Page 7/23 Definitions Technical success is defined as successful placement of the stents (LAMS, DPS or both) between extrahepatic bile duct and gastrointestinal lumen by endoscopy, endosonography, and, optionally, fluoroscopy. Clinical success is defined according to the TOKYO criteria as a 50% decrease in or normalization of total serum bilirubin level within 14 days of index procedure (stent placement). For cholangitis without obstructive jaundice (e.g., segmental cholangitis, cholecystitis), success can be defined as cessation of Page 8/23 Page 8/23 antibiotics or a 50% decrease in or normalization of levels of blood inflammatory markers within 14 days of stent insertion [20]. RBO is defined as a composite endpoint of stent dysfunction (either occlusion or migration). See Table 2 (outcomes) for complete information [20]. Table 2 Primary and secondary endpoints. Primary endpoint: - Rate of recurrent biliary obstruction (RBO) between the two strategies (LAMS with and without coaxial DPS) *RBO is associated with a stent dysfunction (an endpoint of either occlusiona or migrationb). Tokyo criteria - Clinical recurrence (jaundice, fever, suspicious colangitis, pruritus). - Recurrence of cholestasis parameters (Any increase in GGT/ALP or bilirubin from its lowest level post-index procedure). Both WITH evidence of biliary obstruction on imaging (dilation on US/CT/MRI) or endoscopic findings suggesting it c. Secondary endpoints: - Technical success defined as successful stents (LAMS, DPS or either) between the extrahepatic biliary duct and the upper gastrointestinal tract determined by endoscopy, endosonography or fluoroscopy. - Clinical success defined as >50% decrease in bilirubin at 14 days from stent placement. For cholangitis, clinical success is defined as cessation of antibiotics or normalization of levels of blood inflammatory markers within 14 days of stent placement. - Safety, as defined per the ASGE lexicon/Tokyo criteria for endoscopic AEs and divided into early adverse events (within 14 days of index procedure) and delayed AE (>14 days). - Additional outcomes: t-RBO, BRI, procedure time and survival/mortality rates. AE Adverse Event; ALP alkaline phosphatase; ASGE American Society of Gastrointestinal Endoscopy; BRI biliary reintervention; CT computed tomography; ERCP endoscopic retrograde cholangio- pancreatography; EUS endoscopic ultrasound; GGT gamma-glutamyl transferase; RBO recurrent biliary obstruction; US ultrasound; MRI magnetic resonance imaging. a Stent occlusion defined as elevation of inflammatory parameters/cholestasis evidence along with biliary dilation on imaging studies or endoscopic findings. Tokyo criteria [20]. b Stent migration defined as presence of completely/partially migrated stent at the time of Primary and secondary endpoints. Definitions Time to RBO (t-BO) or stent patency is defined as the time from stent placement until the point when symptoms associated with occlusion or migration are observed. BRI is defined as the need to perform a new therapeutic maneuver on the bile duct due to RBO. A distinction must be made between: BRI is defined as the need to perform a new therapeutic maneuver on the bile duct due to RBO. A distinction must be made between: - Endoscopic biliary reintervention (e-BRI): endoscopic procedure with the aim of optimizing the transmural BD. It includes stent cleaning, stent change, additional stent insertion, or any other stent- related endoscopic maneuver. For specific information, see Rescue options below. - Endoscopic biliary reintervention (e-BRI): endoscopic procedure with the aim of optimizing the transmural BD. It includes stent cleaning, stent change, additional stent insertion, or any other stent- related endoscopic maneuver. For specific information, see Rescue options below. - Radiological biliary reintervention (r-BRI): interventional radiological procedure with percutaneous access, with the aim of repermeabilizing the obstructed endoscopic BD. - Radiological biliary reintervention (r-BRI): interventional radiological procedure with percutaneous access, with the aim of repermeabilizing the obstructed endoscopic BD. Definitions Primary endpoint: - Rate of recurrent biliary obstruction (RBO) between the two strategies (LAMS with and w coaxial DPS) *RBO is associated with a stent dysfunction (an endpoint of either occlusiona or migratio criteria - Clinical recurrence (jaundice, fever, suspicious colangitis, pruritus). - Recurrence of cholestasis parameters (Any increase in GGT/ALP or bilirubin from its low post-index procedure). Both WITH evidence of biliary obstruction on imaging (dilation on US/CT/MRI) or endosc suggesting it c. Secondary endpoints: - Technical success defined as successful stents (LAMS, DPS or either) between the extra biliary duct and the upper gastrointestinal tract determined by endoscopy, endosonograp fluoroscopy. - Clinical success defined as >50% decrease in bilirubin at 14 days from stent placement. cholangitis, clinical success is defined as cessation of antibiotics or normalization of lev inflammatory markers within 14 days of stent placement. - Safety, as defined per the ASGE lexicon/Tokyo criteria for endoscopic AEs and divided i adverse events (within 14 days of index procedure) and delayed AE (>14 days). - Additional outcomes: t-RBO, BRI, procedure time and survival/mortality rates. AE Adverse Event; ALP alkaline phosphatase; ASGE American Society of Gastrointestinal BRI biliary reintervention; CT computed tomography; ERCP endoscopic retrograde cholan pancreatography; EUS endoscopic ultrasound; GGT gamma-glutamyl transferase; RBO re biliary obstruction; US ultrasound; MRI magnetic resonance imaging. a Stent occlusion defined as elevation of inflammatory parameters/cholestasis evidence biliary dilation on imaging studies or endoscopic findings. Tokyo criteria [20]. b Stent migration defined as presence of completely/partially migrated stent at the time o endoscopic reintervention with evident stent dysfunction. c Causes of stent occlusions: tumor ingrowth/overgrowth, biliary sludge, food impaction, kinking of CBD to stent). Tokyo criteria [20]. - Safety, as defined per the ASGE lexicon/Tokyo criteria for endoscopic AEs and divided into early adverse events (within 14 days of index procedure) and delayed AE (>14 days). - Additional outcomes: t-RBO, BRI, procedure time and survival/mortality rates. Additional outcomes: t-RBO, BRI, procedure time and survival/mortality rates AE Adverse Event; ALP alkaline phosphatase; ASGE American Society of Gastrointestinal Endoscopy; BRI biliary reintervention; CT computed tomography; ERCP endoscopic retrograde cholangio- pancreatography; EUS endoscopic ultrasound; GGT gamma-glutamyl transferase; RBO recurrent biliary obstruction; US ultrasound; MRI magnetic resonance imaging. Page 9/23 Time to RBO (t-BO) or stent patency is defined as the time from stent placement until the point when symptoms associated with occlusion or migration are observed. Safety reporting (AEs) Additional file 2 includes all AE definitions based on the MEDDEV 2.12 guidelines (rev 8, July 2019) “Guidelines on medical devices: Clinical investigations: Serious Adverse event reporting under Directives 93/42 / EEC and 90/385 / EEC”, regarding safety in clinical research with medical devices, which are determined according to the causal relationship and/or severity of AEs. To clarify the causal relationship and grading of AEs, MEDDEV guidelines will be consulted. Additional file 2 includes all AE definitions based on the MEDDEV 2.12 guidelines (rev 8, July 2019) “Guidelines on medical devices: Clinical investigations: Serious Adverse event reporting under Directives 93/42 / EEC and 90/385 / EEC”, regarding safety in clinical research with medical devices, which are determined according to the causal relationship and/or severity of AEs. Considering a 5% dropout rate, a final sample of 84 patients (42 patients in each arm) is needed. Considering a 5% dropout rate, a final sample of 84 patients (42 patients in each arm) is needed. Statistical analysis Outcomes The primary outcome is the rate of RBO after index procedure. As noted above (Definition section), this is a direct indicator of stent dysfunction, and it is defined as a composite point of either biliary occlusion or stent migration. The secondary outcomes include technical and clinical success, safety data and other patient-relevant outcomes (time to RBO, number of reinterventions, procedural time, and mortality rates). Safety reporting (AEs) AE is any unwanted medical event, injury or clinical (e.g., signs, symptoms, or abnormal laboratory results) suffered by patients during the study, whether related to the endoscopic procedure or stent. All AEs (reported by medical staff or patients) must be documented. All serious adverse events (SAEs) must be notified to the principal investigator within 3 days. In the event of a death, this notification will be made within less than 24 hours. AEs will be rated as mild (hospitalization for 1-3 days), moderate (hospitalization for 4-10 days, ICU admission for 1 night, endoscopic/radiological intervention), severe (hospitalization >10 days, ICU admission >1 night, or necessity for surgery), or fatal, in accordance with the nomenclature for AEs in endoscopy (American Society for Gastrointestinal Endoscopy (ASGE) Workshops 2010) [21]. AEs associated with endoscopic biliary stents (e.g., pancreatitis, non-occlusion cholangitis, cholecystitis, bleeding, perforation, bile leakage) are defined by the standardized reporting system for endoscopic biliary stent placement (Tokyo criteria, 2018) [20, 21]. It is important to note that RBO is not classified as an AE, but as consequence of stent occlusion or migration. Non-occlusion cholangitis will be defined as fever which continues longer than 24h without dilation of the drained duct. It may require medication, hospitalization, or intervention. Non-occlusion cholangitis will be defined as fever which continues longer than 24h without dilation of the drained duct. It may require medication, hospitalization, or intervention. The determination as to what is an AE related to the procedure or the medical device (procedure-related and stent-related) and what is not will be made by the medical team and local investigator, with final approval of the principal investigator (JG). The AE will be chronologically differentiated as pre-procedure, intra-procedure, post-procedure, or early AE (up to 14 days from the index procedure), and, finally, late AE (after 14 days) according the ASGE lexicon guideline [21]. Additionally, another phone call has been added at 30 days, to ensure that all stent-related AEs will be detected, as suggested by Tokyo criteria [20]. Page 10/23 Page 10/23 Page 10/23 To clarify the causal relationship and grading of AEs, MEDDEV guidelines will be consulted. Sample size calculation The sample size calculation is based on the primary hypothesis of detecting significant differences in the rates of RBO and BRI between the LAMS and LAMS plus DPS groups (assessing for superiority). Null hypothesis (H0): πLAMS = πLAMS-DPS Alternative hypothesis (H1): πLAMS ≠ πLAMS-DPS Alternative hypothesis (H1): πLAMS ≠ πLAMS-DPS Based on recent published data, we estimate a recurrent biliary obstruction rate of 20% vs. 1% for the LAMS alone vs. LAMS-DPS groups, respectively up during follow-up period. To achieve a statistical power of 80% with a two-sided type I error of 5%, a total of 80 patients (40 in each arm) is required to reject the null hypothesis. https://www.stat.ubc.ca/~rollin/stats/ssize/b2.html Based on recent published data, we estimate a recurrent biliary obstruction Considering a 5% dropout rate, a final sample of 84 patients (42 patients in each arm) is needed. Data management Throughout the study the promoters will monitor the quality of the trial with special attention to protocol deviations and the quality of the data entered in the database. Scheduled video call will be performed by the primary investigator (JG) and/or research fellow (AGS). These audits will occur at 6 months post- enrollment and at the end of the study period. At the end of the trial, a meeting will be held to consider the data management report. This report will describe the deviations from the protocol identified in each of the patients. These deviations will be classified as major or minor, and those patients with major deviations will be excluded from the protocol analysis. After the meeting, the suitability of the database for analysis will be considered, and the database will then be closed. Statistical analysis Statistical analysis will be carried out with the R statistical software (version 3.6.2) supervised by the Biostatistics team of our research institute (IDIBELL). Intention-to-treat and per-protocol analysis will be carried out. All study variables will be presented for stent groups and in total, using descriptive statistics in accordance with the nature of the variable. Thus, continuous variables will be described indicating the number of non-missing observations, mean, standard deviation, minimum, first quartile, median, third quartile, and maximum. The categorical variables will be described indicating the number of non-missing observations and the percentages of the different categories. Page 11/23 Realization of the aim of the study will be analyzed by comparing the cumulative incidence of RBO (as a stent dysfunction parameter) in the LAMS cohort versus LAMS-DPS cohort using a Chi-Square test. The magnitude of the effect will be estimated through an incidence ratio and relative risk with 95% confidence interval. The main analysis will be replicated in an adjusted way using a binomial regression model. Age, sex, oncological status, and comorbidities will be taken as adjustment variables. Safety will also be evaluated by describing the number and type of AE in each study group, and the incidence will be compared using a Chi-square test. In addition, the survival of patients will be analyzed according to the two proposed strategies until the end of follow-up, describing the Kaplan-Meyer curves and comparing them using the LogRank test. The level of statistical significance is established at p-value <0.05. Subgroup analysis The main analysis will also be carried out in the following subgroups: Age <65 vs. Age >65. Age <65 vs. Age >65. Age <65 vs. Age >65. Presence of liver metastasis vs. Absence of liver metastasis. Presence of liver metastasis vs. Absence of liver metastasis. Presence of liver metastasis vs. Absence of liver metastasis Statistical analysis plan The statistical analysis plan will be finalized before the close of the database. This plan will include all the analysis described and others, mainly on the sensitivity of the results and the management of the missing data. If there is some deviation in the plan of statistical analysis regarding the main variable, an addendum to the protocol will be made. No changes will be made to the original analysis plan once the database is closed. No interim analysis is planned given the short accrual time, relatively small sample size, and short follow- up period. Withdrawal Withdrawal from the study will result from any AE or other clinical condition of the patient which, at the clinician’s discretion, warrants such, or from pregnancy, or at the expressed wishes of the patient. Withdrawal from treatment will not mean suspension of the study, given that follow-up will be maintained until the end of the study in accordance with the protocol. Ethical aspects and confidentiality The protocol will be approved by the CEIC of each participating hospital as well as that of the coordinating center (Hospital Universitari de Bellvitge). The study researchers will carry out their tasks in compliance with ethical principles of clinical research established in the Declaration of Helsinki, and with the norms of Good Clinical Practices. The study is considered a clinical trial with a low intervention level, since it compares two standard treatments with each other. Furthermore, all the medical devices used have CE marking with authorization for their use in endoscopic biliary drainage. For these reasons, the contracting of a specific civil liability policy is not required in accordance with the provisions of current legal regulations, complying with the assumptions established for coverage by the assistance policy. Before inclusion of the patient in the trial, written informed consent will be requested. In relation to the study data, we will follow the provisions of Organic Law 3/2018 of December 5th on “Protection of Personal Data”. Personal Data”. Publication of results There is a commitment to publish the results of this study in high impact international journals, should the results be of sufficient scientific interest. No patient names will appear in any article, and no one, with the exception of the researchers in this study and the members of the hospital ethical committees, will have access to the data, in accordance with the Law on the Protection of Data of a Personal Nature. Other considerations Rescue by e-BRI and cross-over: Rescue by e-BRI and cross-over: Rescue by e-BRI and cross-over: Page 12/23 Page 12/23 Depending on the initial endoscopic treatment carried out, in case of stent dysfunction or RBO, cross-over rescue treatment may be considered when the initial per protocol strategy fails: In LAMS cohort: adding a co-axial DPS through LAMS. In LAMS-DPS cohort: removing DPS and leaving LAMS or adding another coaxial stent, either fully- covered self-expandable metal stent or another DPS. In LAMS-DPS cohort: removing DPS and leaving LAMS or adding another coaxial stent, either fully- covered self-expandable metal stent or another DPS. These potential cross-over options are considered in the e-BRI, described in a previous section (see definitions). In all these situations, the follow-up will be maintained until the end of the study in accordance with the protocol. Alternatively, if an endoscopic technique is not possible, other options (such as radiological intervention) will be offered. Discussion Page 13/23 Page 13/23 Currently, in vogue in the recent literature is reporting that EUS-BD (CDS type) using EE-LAMS is an effective and safe technique for biliary decompression in patients who failed ERCP [5, 6, 10–12, 23]. Some prospective studies or protocol trials have even been designed to assess CDS using LAMS as primary treatment modality for distal MBO over ERCP [2, 7–9]. In fact, if it is shown that EUS-CDS is superior to ERCP, in terms of stent patency and safety, for the first- line drainage of MBO, it is expected that the first-line BD method will be changed from ERCP to EUS-CDS, and that interruption of chemotherapy due to stent dysfunction could be avoided [9]. Additionally, even though percutaneous transhepatic BD (PTBD) may be considered as another alternative option after failed ERCP, some recent reports have claimed that EUS-BD with LAMS is superior to PTBD in terms of clinical success, safety, cost, and overall survival [24]. The introduction of these specific biliary LAMS represents a great technical improvement in EUS-BD of distal MBO [3, 4]. The cautery-assisted stent eases the transmural technique, avoiding complexity, and failure risk linked to the devices exchanging or ostomy dilation, even without fluoroscopy guidance. Even so, in two recent papers, including a meta-analysis, showing that LAMS and SEMS are comparable in terms of efficacy, safety, and reintervention rates for EUS-CDS, the authors concluded that further investigation is required, above all in adverse events [13, 23]. Stent dysfunction of biliary LAMS in EUS-CDS is one of the main concerns, and it has been reported in up to 26.3% of cases [15, 17, 18]. Unlike in pancreatic fluid collection [26], the prophylactic placement of a DPS within a biliary LAMS could offer two hypothetical advantages to prevent biliary AEs as RBO: (i) as an axis-orienting stent to maintain a non-perpendicular axis of the LAMS in the CBD; (ii) decreasing the risk of migration, limiting the possibility of LAMS slipping out. In a U.S. multicentre retrospective study, El Chafic et al. proposed this technical variant, because RBO developed in significantly more patients with LAMS alone compared to LAMS plus DPS (50% vs. 11.8%; p=0.02) [23]. Similarly, two recent papers, including a large cohort from the UK and Ireland, revealed lower rates of cholangitis and BRI (0% vs 12.2%, p .03) in the DPS-LAMS group [22, 27]. b- BAMPI, November 2020, amendment nº 1: version 1.1. - Main amendments: (i) to clarify: The study is considered a clinical trial with a low intervention level. To contract a specific civil liability policy is not required in accordance with the provisions of current legal regulations. - Main amendments: (i) to clarify: The study is considered a clinical trial with a low intervention level. To contract a specific civil liability policy is not required in accordance with the provisions of current legal regulations. Discussion None of these previous studies were specifically designed for comparing the usefulness of the coaxial stent. Recently, our group published a retrospective comparative study assessing the hypothetical benefit of a DPS within a biliary EE-LAMS in EUS-CDS. The DPS plus LAMS group had a higher clinical success rate, non-significantly lower AE rate, and longer significant procedural time. Curiously, the RBO rate was a little bit higher in the DPS-LAMS group [19]. Therefore, these results did not support this technical variant being used routinely, and it was a time-consuming approach. Why the need to carry out a larger prospective and randomized study? In case of confirmation that inserting a DPS within a LAMS during a EUS-CDS reduces the need for re- interventions, improves the biliary stent patency and prevents delays in the oncological management, this Page 14/23 Page 14/23 would surely have an impact on the oncological outcomes and survival rates. To date there is no prospective data about this issue. would surely have an impact on the oncological outcomes and survival rates. To date there is no prospective data about this issue. The BAMPI trial will include seven referral centers, experts in EUS-guided transmural drainage, with proven experience in EUS-BD using EE-LAMS. Hospital Universitari de Bellvitge has leadership role in centralizing decisions in case of controversies, and in limiting heterogeneity. In conclusion, this is the first multicentre randomized controlled trial designed to clarify whether the DPS plus LAMS strategy should be recommended routinely during a EUS-CDS in distal MBO. The results of this trial will have an impact on the clinical practice in treating patients with biliopancreatic malignancies. c- BAMPI November, 2021, amendment nº2: version 1.2. - Minor changes: addition of a new rescue variant technique. If CDS is not possible (due to interposing vessels, significant distance), gallbladder drainage will be considered. - Minor changes: addition of a new rescue variant technique. If CDS is not possible (due to interposing vessels, significant distance), gallbladder drainage will be considered. Protocol of submitted version, number and date: Number 1.3; date December, 2021. Recruitment: Start date November 17, 2020 and recruitment will be completed by October, 2022. Revision chronology: Recruitment: Start date November 17, 2020 and recruitment will be completed by October, 2022. Revision chronology: a- BAMPI, August 2020, original: version 1.0, first draft of the study protocol. a- BAMPI, August 2020, original: version 1.0, first draft of the study protocol. b- BAMPI, November 2020, amendment nº 1: version 1.1. TRIAL STATUS Protocol of submitted version, number and date: Number 1.3; date December, 2021. Protocol of submitted version, number and date: Number 1.3; date December, 2021. d- BAMPI December 2021, amendment nº3: version 1.3 – definitive. - Minor changes: extension of the recruitment period (until October 2022). - Minor changes: extension of the recruitment period (until October 2022). Page 15/23 List Of Abbreviations AE: Adverse event ASA: American Society of Anesthesiologists’ classification BD: Biliary drainage BRI: Biliary reintervention List Of Abbreviations SEED: Spanish Society of Digestive Endoscopy TA: Tissue acquisition Authors' contributions: AGS, CT, and JBG drafted the manuscript. AGS, CL, VS, RPS, EVS, JRA, CSH, DLG, XA, MC, TB, JRF, BM, CC, SS, BL, MPM, and JBG provided a critical review, and, as investigators at each institution, will promote enrolment of patients. JBG and AGS registered the study. AGS, JBG, and CT contributed to the statistical analysis. SV offered scientific advice on the publication of a protocol. AGS, JBG, MPM, and CLA participated in the design of the study. JBG conceived the project. AGS and JBG designed the study, revised the manuscript, and approved the final submission. All authors read and approved the final manuscript. Competing interests: - A Garcia-Sumalla is a PhD student at the Faculty of Medicine and Health Sciences, University of Barcelona, and this paper is part of his doctoral project. - A Garcia-Sumalla is a PhD student at the Faculty of Medicine and Health Sciences, University of Barcelona, and this paper is part of his doctoral project. - JB Gornals acts as a PhD Director; is a consultant for Boston Scientific and has received a research grant from this company. - M Pérez-Miranda is consultant and speaker for Medtronic, Olympus, Taewoong, and M.I.Tech - E Vazquez-Sequeiros is a consultant for Boston Scientific - JR Aparició is a consultant for Boston Scientific. - The other authors declare that they have no competing interests. Declarations Ethics approval and consent to participate: Ethical approval was obtained from the Comité Ético de Investigación Clínica del Hospital Universitari de Bellvitge --IDIBELL (Barcelona; ref approval no. ICPS024/20), on November 5, 2020. Written and oral informed consent will be obtained from all participants before enrolment in the study. Data collected during the course of the research will be kept strictly confidential and only accessed by investigators. An individual trial identification number and participant details will be stored on a secure database. Any subsequent amendments of the protocol need to be approved by the relevant ethical bodies before implementation. Participants will also be asked for permission for the research team to share relevant data with investigators from universities taking part in the research or from regulatory authorities, where relevant. Ethics approval and consent to participate: Ethical approval Investigación Clínica del Hospital Universitari de Bellvitge --ID ICPS024/20), on November 5, 2020. Written and oral inform participants before enrolment in the study. Data collected du kept strictly confidential and only accessed by investigators. and participant details will be stored on a secure database. A protocol need to be approved by the relevant ethical bodies b also be asked for permission for the research team to share universities taking part in the research or from regulatory aut Ethics approval and consent to participate: Ethical approval was obtained from the Comité Ético de Investigación Clínica del Hospital Universitari de Bellvitge --IDIBELL (Barcelona; ref approval no. ICPS024/20), on November 5, 2020. Written and oral informed consent will be obtained from all participants before enrolment in the study. Data collected during the course of the research will be kept strictly confidential and only accessed by investigators. An individual trial identification number and participant details will be stored on a secure database. Any subsequent amendments of the protocol need to be approved by the relevant ethical bodies before implementation. Participants will also be asked for permission for the research team to share relevant data with investigators from universities taking part in the research or from regulatory authorities, where relevant. Consent for publication: not applicable. Availability of data and material: minimal dataset necessary to interpret the findings available from the corresponding author on reasonable request. Availability of data and material: minimal dataset necessary to interpret the findings available from the corresponding author on reasonable request. Page 16/23 Page 16/23 Page 16/23 Funding: This study received a grant (Fundacion SEED) from the Sociedad Española de Endoscopia Digestiva in 2020, and a grant (beca d’iniciació) from the Societat Catalana de Digestologia in 2021. The funding bodies have no role in the trial design or interpretation of the data. Steering Committee: Manuel Perez-Miranda, Hospital Universitario Rio Ortega, Valladolid, Spain. Manuel Perez-Miranda, Hospital Universitario Rio Ortega, Valladolid, Spain. Carme Loras, Hospital Mutua Terrassa, Catalonia, Spain. Joan B Gornals, Hospital Universitari de Bellvitge, Barcelona, Catalonia, Spain. Joan B Gornals, Hospital Universitari de Bellvitge, Barcelona, Catalonia, Spain. Acknowledgements: The Asociación Española de Gastroenterología (AEG) for providing us with REDCap platform. This platform is free of charge, and is a secure web-based application designed to support data capture for research studies. Enric Sospedra, secretary of the Comité Ètic i d’Investigació Clínica (CEIC) de l’Hospital Universitari de Bellvitge, for his generous advice on drafting the responses to co-centers CEIm amendments. The institutions and scientific societies that supported this project with grants (Sociedad Española de Endoscopia Digestiva and Societat Catalana de Digestologia). Page 17/23 Sandra Maisterra, Julio G Velasquez-Rodriguez, and Claudia F Consiglieri of Hospital Page 17/23 Sandra Maisterra, Julio G Velasquez-Rodriguez, and Claudia F Consiglieri of Hospital Page 17/23 Universitari de Bellvitge, who will assist in the enrolment of patients. References 1. Puga M, Pallarès N, Velásquez-Rodríguez J, García-Sumalla A, Consiglieri CF, Busquets J, Laquente B, Calvo M, Fabregat J, Castellote J, Gornals JB. Endoscopic biliary drainage in unresectable biliary obstruction: the role of endoscopic ultrasound-guidance in a cohort study. Rev Esp Enferm Dig 2019; 111: 683-689. 2. Chen YI, Callichurn K, Chatterjee A, Desilets E, Fergal D, Forbes N, Gan I, Kenshil S, Khashab MA, Kunda R, Lam E, May G, Mohamed R, Mosko J, Paquin SC, Sahai A, Sandha G, Teshima C, Barkun A, Barkun J, Bessissow A, Candido K, Martel M, Miller C, Waschke K, Zogopoulos G, Wong C; ELEMENT trial and for the Canadian Endoscopic Research Collaborative (CERC). ELEMENT TRIAL: study protocol for a randomized controlled trial on endoscopic ultrasound-guided biliary drainage of first intent with a lumen-apposing metal stent vs. endoscopic retrograde cholangio-pancreatography in the management of malignant distal biliary obstruction. Trials. 2019; 20: 696. 3. Itoi T, Binmoeller KF. EUS-guided choledochoduodenostomy by using a biflanged lumen-apposing metal stent. Gastrointest Endosc. 2014; 79: 715. 3. Itoi T, Binmoeller KF. EUS-guided choledochoduodenostomy by using a biflanged lumen-apposing metal stent. Gastrointest Endosc. 2014; 79: 715. 4. Gornals JB, Consiglieri C, Gallarreta V, Busquets J, Catala I, Laquente B. Single-session fluoroless endoscopic ultrasound-guided fine-needle aspiration and choledochoduodenostomy with a biliary lumen-apposing stent. Endoscopy. 2015;47:418-419. 4. Gornals JB, Consiglieri C, Gallarreta V, Busquets J, Catala I, Laquente B. Single-session fluoroless endoscopic ultrasound-guided fine-needle aspiration and choledochoduodenostomy with a biliary lumen-apposing stent. Endoscopy. 2015;47:418-419. 5. Anderloni A, Anderloni A, Fugazza A, Troncone E, Auriemma F, Carrara S, Semeraro R, Maselli R, Di Leo M, D'Amico F, Sethi A, Repici A. Single-stage EUS-guided choledochoduodenostomy using a lumen-apposing metal stent for malignant distal biliary obstruction. Gastrointest Endosc. 2019; 89: 69-76. 6. Jacques J, Privat J, Pinard F, Fumex F, Valats JC, Chaoui A, Cholet F, Godard B, Grandval P, Legros R, Kerever S, Napoleon B. Endoscopic ultrasound-guided choledochoduodenostomy with electrocautery- enhanced lumen-apposing stents: A retrospective analysis. Endoscopy. 2019; 51: 540–547. 7. Paik WH, Lee TH, Park DH, Choi JH, Kim SO, Jang S, Kim DU, Shim JH, Song TJ, Lee SS, Seo DW, Lee SK, Kim MH. EUS-guided biliary drainage versus ERCP for the primary palliation of malignant biliary obstruction: a multicenter randomized clinical trial. American Journal of Gastroenterology. 2018; 113: 987–97. Page 18/23 8. References Park JK, Woo YS, Noh DH, Yang J il, Bae SY, Yun HS, Lee JK, Lee KT, Lee KH. Efficacy of EUS-guided and ERCP-guided biliary drainage for malignant biliary obstruction: prospective randomized controlled study. Gastrointestinal Endoscopy. 2018; 88: 277–82. 9. Itonaga M, Kitano M, Yoshikawa T, Ashida R, Yamashita Y, Hatamaru K, Takenaka M, Yamazaki T, Ogura T, Nishioka N, Sakai A, Masuda A, Shiomi H, Shimokawa T. Comparison of endoscopic ultrasound-guided choledochoduodenostomy and endoscopic retrograde cholangiopancreatography in first-line biliary drainage for malignant distal bile duct obstruction: A multicenter randomized controlled trial. Medicine (Baltimore). 2021 Mar 26;100(12):e25268. 10. Krishnamoorthi R, Dasari CS, Thoguluva V, Priyan H, Jayaraj M, Law J, Larsen M, Kozarek R, Ross A, Irani S. Effectiveness and safety of EUS‑guided choledochoduodenostomy using lumen‑apposing metal stents ( LAMS): a systematic review and meta‑analysis. Surg Endosc 2020; 34: 2866-2877. 11. Jain D, Shah M, Patel U, Sharma A, Singhal S. Endoscopic ultrasound guided choledocho- enterostomy by using lumen apposing metal stent in patients with failed endoscopic retrograde cholangiopancreatography: a literature review. Digestion. 2018; 98:1–10. 12. Mohan BP, Shakhatreh M, Garg R, Ponnada S, Navaneethan U, Adler DG. Efficacy and safety of endoscopic ultrasound-guided choledochoduodenostomy: a systematic review and metanalysis. Journal of Clinical Gastroenterology. 2019; 53: 243–50. 12. Mohan BP, Shakhatreh M, Garg R, Ponnada S, Navaneethan U, Adler DG. Efficacy and safety of endoscopic ultrasound-guided choledochoduodenostomy: a systematic review and metanalysis. Journal of Clinical Gastroenterology. 2019; 53: 243–50. 13. Amato A, Sinagra E, Celsa C, Enea M, Buda A, Vieceli F, Scaramella L, Belletrutti P, Fugazza A, Cammà C, Radaelli F, Repici A, Anderloni A. Efficacy of lumen-apposing metal stents or self-expandable metal stents for endoscopic ultrasound-guided choledochoduodenostomy: a systematic review and meta- analysis. Endoscopy. 2021; 53: 1037-1047. 13. Amato A, Sinagra E, Celsa C, Enea M, Buda A, Vieceli F, Scaramella L, Belletrutti P, Fugazza A, Cammà C, Radaelli F, Repici A, Anderloni A. Efficacy of lumen-apposing metal stents or self-expandable metal stents for endoscopic ultrasound-guided choledochoduodenostomy: a systematic review and meta- analysis. Endoscopy. 2021; 53: 1037-1047. 14. Han SY, Kim SO, So H, Shin E, Kim DU, Park DH. EUS-guided biliary drainage versus ERCP for first-line palliation of malignant distal biliary obstruction: A systematic review and meta-analysis. Scientific Reports. 2019 Dec 1;9(1). 14. Han SY, Kim SO, So H, Shin E, Kim DU, Park DH. References EUS-guided biliary drainage versus ERCP for first-line palliation of malignant distal biliary obstruction: A systematic review and meta-analysis. Scientific Reports. 2019 Dec 1;9(1). 15. Tsuchiya T, Teoh AYB, Itoi T, Yamao K, Hara K, Nakai Y, Isayama H, Kitano M. Long-term outcomes of EUS-guided choledochoduodenostomy using a lumen-apposing metal stent for malignant distal biliary obstruction: a prospective multicenter study. Gastrointestinal Endoscopy [Internet]. 2018; 87: 1138–1146. 16. Garcia-Sumalla A, Bazaga S, Gornals JB. EUS-guided biliary rendezvous as an emergent rescue after failed choledochoduodenostomy using a lumen-apposing metal stent. VideoGIE. 2021 Jun 1; 6: 263–5. 16. Garcia-Sumalla A, Bazaga S, Gornals JB. EUS-guided biliary rendezvous as an emergent rescue after failed choledochoduodenostomy using a lumen-apposing metal stent. VideoGIE. 2021 Jun 1; 6: 263–5. 17. Mosquera-Klinger G, de la Srna C, Perez-Miranda M. Endoscopic sump syndrome secondary to EUS- guided choledochoduodenostomy with a lumen-apposing metal stent. Rev Esp Enferm 111:74-76. 17. Mosquera-Klinger G, de la Srna C, Perez-Miranda M. Endoscopic sump syndrome secondary to EUS- guided choledochoduodenostomy with a lumen-apposing metal stent. Rev Esp Enferm 111:74-76. 18. Gornals JB, Consiglieri CF, Bergamino MA. Double pigtail for preventing ascending cholangitis after endoscopic ultrasonography-guided choledochoduodenostomy with lumen-apposing metal stent. Dig Endosc. 2016; 28: 100. 19. Garcia-Sumalla A, Loras C, Guarner-Argente C, Velasquez-Rodriguez JG, Andujar X, Salord S, Busquets J, Tebe C, Laquente B, Gornals JB. Is a coaxial plastic stent within a lumen-apposing metal 19. Garcia-Sumalla A, Loras C, Guarner-Argente C, Velasquez-Rodriguez JG, Andujar X, Salord S, Busquets J, Tebe C, Laquente B, Gornals JB. Is a coaxial plastic stent within a lumen-apposing metal Page 19/23 Page 19/23 stent useful for the management of distal malignant biliary obstruction? Surg Endosc. 2021; 35: 4873-4881. stent useful for the management of distal malignant biliary obstruction? Surg Endosc. 2021; 35: 4873-4881. 20. Hamada T, Nakai Y, Isayama H. TOKYO criteria: Standardized reporting system for endoscopic biliary stent placement. Gastrointestinal Interv. 2018; 7: 46–51. DOI:10.18528/gii180016. 20. Hamada T, Nakai Y, Isayama H. TOKYO criteria: Standardized reporting system for endoscopic biliary stent placement. Gastrointestinal Interv. 2018; 7: 46–51. DOI:10.18528/gii180016. 21. Cotton PB, Eisen GM, Aabakken L, Baron TH, Hutter MM, Jacobson BC, Mergener K, Nemcek A Jr, Petersen BT, Petrini JL, Pike IM, Rabeneck L, Romagnuolo J, Vargo JJ. A lexicon for endoscopic adverse events: report of an ASGE workshop. Gastrointestinal Endoscopy. 2010 ;71:446–454. 21. References Cotton PB, Eisen GM, Aabakken L, Baron TH, Hutter MM, Jacobson BC, Mergener K, Nemcek A Jr, Petersen BT, Petrini JL, Pike IM, Rabeneck L, Romagnuolo J, Vargo JJ. A lexicon for endoscopic adverse events: report of an ASGE workshop. Gastrointestinal Endoscopy. 2010 ;71:446–454. 22. On W, Paranandi B, Smith AM, Venkatachalapathy S v., James MW, Aithal GP, Varbobitis I, Cheriyan D, McDonald C, Leeds JS, Nayar MK, Oppong KW, Geraghty J, Devlin J, Ahmed W, Scott R, Wong T, Huggett MT. EUS-guided choledochoduodenostomy with electrocautery-enhanced lumen-apposing metal stents in patients with malignant distal biliary obstruction: multicenter collaboration from the United Kingdom and Ireland. Gastrointestinal Endoscopy. 2021 Oct 9; S0016-5107(21)01702-8. doi: 10.1016/j.gie.2021.09.040. 23. el Chafic AH, Shah JN, Hamerski C, Binmoeller KF, Irani S, James TW, Baron TH, Nieto J, Romero RV, Evans JA, Kahaleh M. EUS-guided choledochoduodenostomy for distal malignant biliary obstruction using electrocautery-enhanced lumen-apposing metal stents: First US, multicenter experience. Dig Dis Sci. 2019; 64: 3321–3327. 23. el Chafic AH, Shah JN, Hamerski C, Binmoeller KF, Irani S, James TW, Baron TH, Nieto J, Romero RV, Evans JA, Kahaleh M. EUS-guided choledochoduodenostomy for distal malignant biliary obstruction using electrocautery-enhanced lumen-apposing metal stents: First US, multicenter experience. Dig Dis Sci. 2019; 64: 3321–3327. 24. Ginestet C, Sanglier F, Hummel V, Rouchaud A, Legros R, Lepetit H, Dahan M, Carrier P, Loustaud-Ratti V, Sautereau D, Albouys J, Jacques J, Geyl S. EUS-guided biliary drainage with electrocautery- enhanced lumen-apposing metal stent placement should replace PTBD after ERCP failure in patients with distal tumoral biliary obstruction: a large real-life study. Surg Endosc. 2021 Oct 4. doi: 10.1007/s00464-021-08653-1. 24. Ginestet C, Sanglier F, Hummel V, Rouchaud A, Legros R, Lepetit H, Dahan M, Carrier P, Loustaud-Ratti V, Sautereau D, Albouys J, Jacques J, Geyl S. EUS-guided biliary drainage with electrocautery- enhanced lumen-apposing metal stent placement should replace PTBD after ERCP failure in patients with distal tumoral biliary obstruction: a large real-life study. Surg Endosc. 2021 Oct 4. doi: 10.1007/s00464-021-08653-1. 25. de Benito Sanz M, Nájera-Muñoz R, de la Serna-Higuera C, Fuentes-Valenzuela E, Fanjul I, Chavarría C, García-Alonso FJ, Sanchez-Ocana R, Carbajo AY, Bazaga S, Perez-Miranda M. Lumen apposing metal stents versus tubular self-expandable metal stents for endoscopic ultrasound-guided choledochoduodenostomy in malignant biliary obstruction. Surg Endosc. 2021; 35: 6754-6762. 25. References de Benito Sanz M, Nájera-Muñoz R, de la Serna-Higuera C, Fuentes-Valenzuela E, Fanjul I, Chavarría C, García-Alonso FJ, Sanchez-Ocana R, Carbajo AY, Bazaga S, Perez-Miranda M. Lumen apposing metal stents versus tubular self-expandable metal stents for endoscopic ultrasound-guided choledochoduodenostomy in malignant biliary obstruction. Surg Endosc. 2021; 35: 6754-6762. 26. Puga M, Consiglieri CF, Busquets J, Pallarès N, Secanella L, Peláez N, Fabregat J, Castellote J, Gornals JB. Safety of lumen-apposing stent with or without coaxial plastic stent for endoscopic ultrasound-guided drainage of pancreatic fluid collections: a retrospective study. Endoscopy. 2018; 50: 1022-1026. 26. Puga M, Consiglieri CF, Busquets J, Pallarès N, Secanella L, Peláez N, Fabregat J, Castellote J, Gornals JB. Safety of lumen-apposing stent with or without coaxial plastic stent for endoscopic ultrasound-guided drainage of pancreatic fluid collections: a retrospective study. Endoscopy. 2018; 50: 1022-1026. 27. Chin JY-L, Seleq S, Weilert F. Safety and outcomes of endoscopic ultrasound-guided drainage for malignant biliary obstruction using cautery-enabled lumen-apposing metal stent. Endoscopy International Open. 2020;08: E1633–8. 27. Chin JY-L, Seleq S, Weilert F. Safety and outcomes of endoscopic ultrasound-guided drainage for malignant biliary obstruction using cautery-enabled lumen-apposing metal stent. Endoscopy International Open. 2020;08: E1633–8. 27. Chin JY-L, Seleq S, Weilert F. Safety and outcomes of endoscopic ultrasound-guided drainage for malignant biliary obstruction using cautery-enabled lumen-apposing metal stent. Endoscopy International Open. 2020;08: E1633–8. Figures Page 20/23 Figure 1 nrollment, interventions, and assessment in the BAMPI trial (SPIRIT-Figure). Enrollment, interventions, and assessment in the BAMPI trial (SPIRIT-Figure). ª Mandatory only in case of NOT meeting clinical success criteria on previous visit (14 day).BRI biliary reintervention; d days; EUS-BD, endoscopic ultrasound-guided biliary drainage. ª Mandatory only in case of NOT meeting clinical success criteria on previous visit (14 day).BRI biliary reintervention; d days; EUS-BD, endoscopic ultrasound-guided biliary drainage. Page 21/23 Page 21/23 Figure 2 BAMPI trial Flowchart. DPS double-pigtail plastic stent, ERCP endoscopic retrograde cholangio-pancreatography, EUS-BD endoscopic ultrasound guided biliary drainage, EE-LAMS, electrocautery-enhanced, lumen-apposing metal stent. Page 22/23 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. appendix1SPIRITchecklist.BAMPIdef.doc appendix1SPIRITchecklist.BAMPIdef.doc appendix2BAMPISafetydefinitionsAE.docx appendix2BAMPISafetydefinitionsAE.docx Page 23/23
W1574249369.txt
https://dergipark.org.tr/tr/download/article-file/619190
tr
Okul kütüphanesi personelinin eğitiminde kuram ve uygulama
DergiPark (Istanbul University)
1,996
cc-by
2,673
Hacettepe Oniversits$i Edebiyat FakiJIIesi Dergisi Cilt 131Sayı ~2.Aralık 1996/8. 87-100 OKUL KÖ1'ÜPJL\NE8İ PERSONlttJNt:N EGtTbdNDIt KURALı VE' UYG1JLAMA Yrd. DoÇ. Dr. IL incı Öl'fAL(.} GiRiş. Topluİnsal geUşınenin gereksınımıerini ashna uygun olarak yansıtan "kuraro" ve bu.kuraının anJaŞıIınaSını sagiayan "uygulama" birbirlerini sürekli etlQleyerek yeni gelişimlere ve degış1mlere yol açarlar. Kuram. uygulanınca her ikisi de birbirlerini gelişttrmeye başlarıar. İnsan önce yaşamış ve sonra yaşadıklamun düşüncesini edinmiŞ 01dl:ıgundan uyguIamanıİ1. kuramdan Once geldiAt de dilşilnOlilr. Hangtstntn önce geldigıtartıŞ1lm"kh. birlikte. teme~ olan: Uygul"m"mn kuramı zengln1eştirirken. kuramın da uygulamayı güçlendirmesidiİ' (Hançerlioglu 1977 :281. 345), , , Kuram ve uygulama ilişkisini okul kütilphanesi personelintn egitiminde inceleyen bu araştumayıyapa:rken amacımız : Kuramsal ge1tşıneleıi Izleyerek ve Tiirktye'deki uyguIamaIıuı ortaya çıkararak. okul kütilphanesi personeUntn egtttıngerekstntmlerlni vurgul"m"ıq'1". çalışmamız içtn gereken araştırmalarda "betimleme yöntenli" kullamImıştır. Mevcut olayların daha önceki olay ve koşullarla illşkileıi dikkate alınarak dUrumlar aıasmdaki etkileştııı açik1anmaya çalışı)mıştır. Yayın inceleJIlesi yapılmıştır. Bettnı1eme yOntenıtne uygun olarak; veli toplama teknlkleıinden anket ve görüşıne lle gerekli veıiler topIannııştır.' .' ç"ıışm"m'7.1n ilk hOliimünde;okul sonel egtttmi içtn geliştlrilenkuramve lditilphanesi örgiltünde peruygııı"m<>lıor incelenmtştlr. İkinci hOliimde; okul kütQphaneslnde çahşan ve çalışacakpersoneUn . Tiirktye'de nasıl egttildiglni gösteren araş~ aktanImıştır. Diger bır deyişle. konunun Türkiye'deki kuram ve uygulaması ortaya ÇIkanlmıştır. 16 TenımUZ 1990 - 3 AgUstos 1990 taıihleıi arasında lL. Okul Kiltüphanecillgt kUrsuna katılan 129 kişiye kurs ı>aŞiangıcında (') HaC8\lepe Oniv8rsi1asi, Edebiyat FakOIlosi,KOtiJpIıan8cii< BOIOmO 87 --- -- -- Inciona! ve bltlmInde anket uygularımıştır. Adı geçen bu kursa katılan. TürkIye'nın çeşitli il ve ilçelerinde çahŞnı91rt..olan okul küt(lphanesi personeli Ile Agı.ıstos 1990'dan Şubat 1996'ya d~ görüşmelerin yapılmiısı ve b~ sürdilı'iihneslyle verilen kursun etkılerı saptanmıştır. Uçüncü bölümde; degerlendirmelerle sdnuca vanlmış, önerIlerde bulunulınuştur. I. OKUL Kt!TOPIWm8i OJlOOTONJ)B PBB8OL'IEL EÖiTiMt içiN _ GELişTiıdı.EN KURAM VE UYGtILAMALAR . - Düşünce alanındaki bilgi "kuram". eylemsel çabalar "uygulama" olarak tanmıJanınak:Qı (Hançerlioglu 1977 : 344). EgItim örgütlerin! Inceleyen çahşniaIar kuramın uygulamayla en az üç yönden ilişkili oldugunu göstennektedlr. Teıne1ll1şk11er : 1- Kuranun uygulayıcı ıçın kabul edilebilir olmasında; 2- Sorunların çözQmü IÇin genel yöntemin sagIanmasında; 3- Kuranun uygulama IÇin rehberlik etInesJııde toplanm~ Irt"<lır (Bursalıoglu, 1994: 29-31; Hoyve MIs~ 1991 : 28; Bursalıoglu 1991 : 57). Kuram ve uyguIamadakütüphaneclllk egıthnl genelolarak okul kütüphanecillglne yer Vermektedir. Kütüphanecilik egıtlmlnden geçenlerden bazılarının ok~ kütüphanelerinde çalışmasıyla. bu k0nunun uzmanlık aIanı oldugu görühnüştür. Okul kütüphanecillgi -egıtlınlnl genel kütüphaneclıik egıtlınlnden ayıran en ı>neınl1nokta : Egitim. öj!retlm teknikleri: ders programIan geliştirme; çocukların ve gençlerin gelişim özellıklerı; egıtim teknolojisi ve okul kütüphanesi hizmetleri gıbı ders1erln programa ..hnm..".dır. Kütüphanecl1lk egıtlıni almış kişiler kadar, ôgretınenler de _bu konuya özel ilgtgOır termektedirler (AASL ve AECr 1988 : 60; Herrıng 1988: 15; Carroll 1981 : 140; PoIette 1973 : 18). EgItIm örgütünün öneınl1-bır Unsuru olan okul kütüphanesinı geliştirme çabalan sürmektedir. Bu konuda: i- International_ Federation of'Llbrary AşşocIations and lnstitutions 1FLA (Ulus1artırası Kütüphane Dertıeklerı ve KuruluşIan , Fe_ _ -derasyonu); 2- United Nations Educatıonal, ScIentific ;md Organization - UNESCO (Birleşmiş MIlletler ~tim. Cultural Bilim ve Kültür Orgütü); 3- International AssocIatıon of School Librarlanshlp - IASL (Ulusl~ okul Kütüphanecıııgı Dernegı); Ile diger 4- Okul/tütt1phan,eleri. çoct.lk kütüphanelerı.. egıtlm ve araştırma konuIarıniİa ulusal ve ulJsIararası düzeyde çalışan derneklerin üyelerince yapılan araŞtıQDaIar 1986'da tamamlanarak uygulanmıştır (fIarmesdottır 1986: 3-5). _ 88 / Edebiyat FııId*oisi Dergisi Okul kütilphanec1lerinln görev. ve sorumluluklarının sürekli artması, egıtJm ve ogretim gerek:sınım1erJnln karşılanmasını zoruİ1Ju hale get1rınlştır. Ülkebırln beklenttIeı1: okunann türleı1, yönetımı, l\gretim programlan:okul kütüphaneıeıinin hızmet düzeyi ve te~loJIyI kuilainnı 1mkanIan farklı oldugundan, her ülkenin kendi şıırtianrui uygun olacak kuramı saptıımA'"' gerekmlştır. Uyg>ılAmAlannı ortaya çıkartnuştu'. Yukanda SaYıIan derneklerin üyelerince yapılan araştmnaIar ve 200'den fazla ülkenın okul kütüphanesı.personelinin egitim programlan ile uygulamalannın ıncelenmesi elde edilen bulgulara . temel dayaniık olmuştur. Okul kütüphanesi personelinin egitlmine yönelIk kuram ge-. . i1ştırillrken. uygulAmAlArbeklenirken: 1- AraştmnaIar geçmIŞten günümüze degın incelenmiş: 2- Elde edilen bilgiyi bütünleştirmek üzere çalışmalaryapıImış: 3- Kuraın hazırlanmıŞ: 4- Kuram gerekt1gtnce göZden geç1rilerek yapısal düzenlemeler . ..' sagIanmış; 5- İlgili gruplan yönlendinnek amacıyla kuraın taIutıImış: 6- Kuraından yararlanarak. uygulAmAcıların kendileı1nl olaylann rasgelellglnden kurtannalan tavsiye edilmiştır 1.1. . . BeItrtllen çalışmAlarla, 1995 yıImdaIFlA öncülügünde UNESCO ile IASL tarafından üyelerinin deste~le okul kütüphanesi . ve l\gIIidemek . personel1nin el!1tJm1ne yönelIk. kuram' geItştlı1lmJştır. UyguIamnak . üzere ülkelere sunulmuştur. Okul kütüphanesi perSoneli terlmlyle : Mesleki personel. teknik personel ve gönüllü kişiler Ifade edilmektedir. Görev a1anIan ve sorumluluk düzeylerıfarklı olan okul kütüphanesi personeli ıçın .geItştlı1leIı e/!1t1mprogramlamun temel . unsurlan Şekil 1'ıle gôsterilmeirtedir.. Kütüphanec1lik el!1tlminde "okul kütüphanecllig1", okul kütüphanelerinde tAhşmAkisteyenlere "mesleki egıtim" ve okul kütüphanelerinde çahşanlara "hızmetlçı egitim" düzeytndeki egitim prograınIiırında verilecekdersleriI!; bır başka deyjfle Şekıl 1'deki temel . unsurlann kapsanil aşa/tıda beilrtlimektedir: '. OKUL Kt1ı1)J>HANESt PER80NELtNiN . .. EÖİTiII.tROORAMI Okul kütüphanesi personelinln egitimi, görev alanı ve sori.tmlulugu farklı düzeylerde oldugundan "egitim prograI1)1''nm kapsamı : 'J'r<ıgramınJş\eyIŞ blçımını ve kullarumŞekl1nI göstermeyi hedeflemektedır. (') IFLA'nın okul KOIOphaneleri B61omO'OOn sorumlusuDr. !'aul8118. Bemherd ile 25 AOus"'s . 199&.1erihinde yapılan g&O,meyie bu bilgi elde ııdlimi}tir. 89 --_._- l ~. ,. i . incionaı e - oi~ i i tm ~..... ~~~ıj ~L ~r.ı ]p iii i~ ~e ~;1 ~i~ . .§ .. Illi ..!! ,,' .m~~ :;::ı::ı ıo:ı=~ ~] &!!'" 9 ~o::ı' :.:: ın ..... ,; B~~ ..ııIı cj~] ~L ~:;J .. E 1;1 ~E it) ~o) ~..... ~;? ~1 iii~ ~o::ı ~~ı::ı ~~, ~CQ' ~i' " ~j i a ~.11 ıi ~tl o ..o ı a 90 ~[ .lı ı:: ~o) ~~EI ı:: ~"ii ~'i <'~~ ~~C3 'E r.ı i~... ~CQ ı:: ~! Edebiyat FakQIt8Si Dergisi . Okul KütüphaneSi Persone1lnin Egitim programı : 1- Kütüphanecl ve öl!retmen yetiştiren yüksek ögretım kurum1a.nnın iLgIL1 ,pôlüm1ert ne hlzmetiÇi egitim progranüa.nnı düzenleyen kurumlar için "yeni egitim programını" kapsııT1'l"ktadır. Okul kütüphanesi personeli yettştiJmek istentrse; bu program. ders programı şeklinde kullanılaCaktır. . . 2- Okul kütüphanesi personeJtn1n görevlertni kapsamaktadır. )3e1trtilen görevler yerine getirilmek iSientrse. bu program okul kütüphanesi persone1lnin sahipobnasıgereken temel.ÖZel11k1ert gösteren rehber olarak kullanıJa('aJct.r. 3- Personelin bilgi gereksintm1ertni kapsamaktadır. Bilgi ge-o reksinim1ert karşılannıak tSlentrse. bu programın her bır. unsuruna gereksinim düzeyi (az orta yüksek şeklinde) ek. lenerek. anket veya liSIe olarak kullanlla.."khr. - - Her ülkenin egıtlm SISlerni. okul türü. ögı"etlmprogramı ne programı uyguhfuıa süreSi. bulundugu şai1lan. I)rgütı>e!yapısı. yönetim kurallan. madQi-manevi destek unsurlan ve teknollJj1k tmkan1an farklı oldugundan temel unsurlann kapsamı genelolarak belirt1lmektedlr. . Ayrınblı aç.kl"m"lann ülke şartlarına göre gelişUrthnesi bek.... lenmektedlr. A KtITt!J>!IANECiLbt A-L. Koieksıvan GelisUnne VE E1'QI'ORMASYON . a,- Saglama politikası hazırlama b- Bilgi kayııaklarım degerlendIrme c- sagışlar için kurallar geliştirme , . d- Bilgi kayııaklannın ürettnıi A-ll. v a- Saglama yöntemlertve teknik hizmetler b" Sınıflama c- Kata1og1ama d-Indeksleme e- Kütüphane !ıiiınet1eı1neyönelik otomasyon çal'şm"lau A-ID. a- Bilgi gerekslnim1ertnisaptarna b- Enformasyon hlzmet1ertniplanlama 91 -_.._- !nd onaı c- BiLgI kaynaklarının k1$ınmu d- Odfuıç venne Işlemleri e- Kütüphane türlerintn ve bilgi agJarınm hizmetleri f-Kütüphanelerarası , Işbırılgi g- Teknoloji kullaıumı vegenŞmeleri Izleme B. YÖJIIETiM B-I. a- Polıtıka ge1lşttnne ve program hazırlama b- Amaç ve görevleri belirleme . c- A~~çlara ulaşmak ıçın çahşma yÖntemıerisaptama .cı-Güncel gelişmeleri ve Istekleri Izleme e- DegeriendInDe f" Konuyla lIgi1Ikanuni yaptımn1aiı takıp etme B-U. Kavn"kh'ruıYönetlml' a- Yönetim süreçlerini uygulama . bcde- Denetleme Hızmet verilecek alanlann düzenlenmesi ve kullanımı BiLgIkaynaklannın ve ilgili araçlann korunması,ba1mnı '. HIzmetlerige1lşttnne B-III. Bütce Yönetımı a- Malı kaynaklan saglama b- Bütçe yönetımı c- Rapoı-hazırlama C. . C-I. EÖirlM. OORETiM, ÖÖREJIfiM öır,.tfm PrQiPJID1 H:l7.1r1~m::t , a- ögretlm.prograID1hazır1amadaIşbn:llgi b- ögremne ~e 6gretme kuramlan. uygulama1an . c- ögretimprogramını zeiıgln1eşttnne d- Gelışım psikolojisi, egıtim sosyolojlsl ve davranış b1lImleri C.U. Bııııı Erislın a- Bılgı gereksinimleriniinceleme: 92 -- . çOzüınleme Edebiyat FakOltesi Dergisi b- Bilgi . gerek..ıntmlel'lni karşı1aına c- Kullarucı egıtJmi d- Kütüphane hizmetlerini ge1işttrme C-III. a- Rehberltk b- ögtetınenlerle ve ögrenellerle Işblrugı. c- özeı ögretim yöntemıeri d- Bılgısayar destekli ôgretim programlan / e- Kütüphanelerin ve kurumların tamtınıı~ kulliınımı Günümüzde "Okul Kütüphanesı Persone1lnln EgItJm Programı" (Harınesdottlr 1995: 18-31) sadece bır kuram olmaktan çıkmıştır. Bu programı birçok ülke kapsam, işleyiş, konu ve yöntem açısından kendı şartlanna göre geliştirip; uygulamaya başlamıştır (Janleri 1996: 2021). ll. TthudYE'DE OKUL Jd)Ti)pJL\NESi , ÜZERiNE BİRARAŞTIRMA . PERSONELiNiN EÖiTiMİ Ankara ÜnlverslteSl, Hacettepe Ünlversltesl ve İstanbul Üniversltesi'nde' üniversite düzeyinde kütüphanecll1k egitimi yapılmaktadır. Sayılan üniversitelerin kütüphanecll1k bölümlerinde "okul kütüphaneleri" dersıeriverilmektedir. okul kütüphanelerinde çeşiti! düzeylerde egitim görmüş personel çalışniaktadır. Mesleki egıtimden geçenler çok az olmakla blrl1kte, genell1k1e mesleki egıtim görmemiş persOnel görev yapmaktadır. Egıtim düzeyıne bakılmaksızın okul kütftphanelerinde çalışan personel ıçın egı~ : Mllll Egıtim Bakanlıgı bftnyeslnde Yayımlar Dalresl'nili Okul Kütüphaneleri Şubesi lle HIzmetiçi Egıtim Dairesi tarafındari bırlıkle gerçekleştirilmekledlr. Her yıl bır llde "okul kütftphanecillgı kUrsu" açılmaktadır. Yılda 100 kişiYe kurs verllmesi dÜ$ooftlmdrtedlr. Kunt lar 2 veya 3 hafta düzenlenmektedlr. VaJlliklere genelge gönderilerek, okul müdürlerlrıln kütftphanc:lerde görev yapiınlan kursa göndermesi istenmektedir. Kurstaki dersler: MiLLİEgıtıın BakanIıgı'ndan ve Kftltür BakanIıgı'ndan görevlendirilen k1şllerce verllmekledlr. Kurs1arda ftniverslte kütüphanecilik bölftmlerinden görevlendirilen konu uzman1arı da ders vennlşlerdlr. ögreum üyesi olar~ katı1dıgımız okul kütüphanecillgı kurslarında ve sonrasında yaptıgım17. araştırmanuz şu aşamalar dahillnde gerçekleştirilmlştir: . 93 ------ . InciOnaJ ı. AŞAMA : Onceki kursJaı'dan: edJnllen deneytm1er ve kursa katıJacaklann bllgl.gereksinttnleri kursa başhl1't1Adı-rıOnce ortaya konmuştur. Beklentiler. gerekııtn1Qıler ve okul kiltillihaneleı1nJn bulundugt1 şartlar <if1ckAtealınarak program hazırlanmıştır. 2. AŞAMA : Araştırma yaptıgımız 11. Okul KiltilphanecılJgi Kursu 16.7.1990 - 3.8.1990 taıihleri arasında Bursa'dagerçekleşın1ş~. Katılan 129 kursiyere kurs baŞIangJCında \JIŞlsel özelliklertni ve bilgi dilzeylertni ortaya çıkartacak )'an'ket" uygulanmıştır. . Okul kil~phanecılJgi ktu'suna Ilkokul. ortaokul. LISe.meslek lisesi, teknik ve endilstri meslek lisesi. tıcaret ıısesl. askerilise. Imam hatıp lisesi. özel Lise ve Anadolu lisesi 01Iİ1ak üzere çeşitli- okul tilrlerinin kiltilphanelerindeçaJışaı:ııar kAtılmıştır. Araştırma kapsamındaki 129 kursiyerin 72 (%55.81 sı kadın. 57 (44.21 sı erkektlr. Kursiyerlerin 20 (%15.51 si 20-30 yaş grubunda; 63 (%48.81 il 31-40 yaş grubunda; 41 (%31.81141-50 yaş grubunda; 5 (%3.91151 ve yukarısı yaş grubunda . bulunmaktadır. Adana. Adıyaman. Afyon. Agn. Aınasya. Ankara. Antalya. Artvın. Aydın. Bahkeslr. Bolu. Burqur. Bursa, ÇarıAlcleAle. çorum. Çankırı. DenJzIi. Diyarbakır. Edırne. EIazıg. Es\JIŞehlr. Gaziantep. Gilmilşhane. Hatay. Isparta. İstanbul. fzıııır. Kastamonu. Kocaeıı. Kırklareıı. Kırşehir. Kayseri. Konya. Malatya. Kahrapummı-raş. Manısa, Mugla. Nevşehir. Ordu. Sa1nsun. Sivas. Tokat. Uşak. Zonguldak. Batman. Kınkkale olmak iizere 46 Ilden ve onların Ilçelerinden katılnn ol. . ~~~ Egitim dilzeylne ba1p1dıAmda, 2 si kiltüphanec1 01Iİ1akiizere 21 kışı yüksek ögrenlm görmilştür. Digerleri ortaögretlm' kurumlarından . . mezun ohnuşlarılır. . Okul kiltilphanecıııgı kursuna katı1anlann konuyla l1gILibilgi dilzey1ertni ortaya: çıkartmak ıçın "kütilphane Işlemleri ve hizmetleri hakkında bildiklerımn neler oldugu" soruhnuş!ur. Kiltüphanede hiç ça1ışmadıg\ halde kursa gönderilen 32 (%251 kışlgıbı. genelolarak . herkesin kurstan önceki bilgi diizeyl çok sınırlıdır. Hizmetvermeleri gerektlgtnden. Okul Kiltilphaneleri Yönetmeııgı (Milli Egitim Bakanlıgı 19771 dogruıtusunda ve kişisel gayretleriyle Çal.ışmaktadırlar. Sadece. 2 sı ilnlverslte kiltilphanecllik bOlilmil mezunu olan kilt.ilphanecl1erin konu Ile l1glli bilgi ve çahşm" 1:'1'1vardır. Ancak; egitim. ögretlm. Öj!renlm konularındayeterli bilgi ve tecriibeleri olm"<iigını Ifade etınIşlerdlr. Okul kiltilphanictııgı kursundan' önceki bllgidiizeyl Tablo . 1'de gösterilmektedir. 94 ---- Edebiyat F~ Deıgi&i Tablo 1 : Okul Kütüphaneclilgi Kursıuıdan Onceki BJlgI Düzeyi K1U'8taıL.Ooce BlJbaCDleı' ögrenct Işleri. sekreterlik. muhaseb~ kütüphaneclilk . YaaıtIanD SaJUI ('Mı) 103 (80) KişiSel degerlend!Ime1erle kütüphane ışlemlerının uygulanması 76 (59) Kütüphan~.dozenleIlD1esl 67 dışı işlerin yapılması . , . Okuyucu iSteklerinin karşılanması 65 (52) . (50) 3. AŞAMA: 1ı. Okul Kütüplıaneclllgt Kursunda gerçek1eşt1ı1len egıtim programında yeralan derslerin ve yapılan uygub.mAbnın kapsamı ~da ver1hnlştlr : : Okul Itütüphaneleri t:anımlanaı1ik aınaçlan. ve hızmetler!lnjn kapsanu. açıklanmıştır. Egit!me ve gençlerin yetişmesine katkılan tartJşı1mıştır. D"I""ler: Türk Milli Egltim1ne yOn veren kanun. yönetmelik ve geneJgeler1n okul kütüphanelerinin ça1ışınasUıa etkileri açıJclAmmştır. , Kat~lnd1ama ve Uvq11hnn::l~1: -Katal~ tanıınlanmıştır. Katalogun amaçları. Işlevleri, yararları ve türlerilncelenıİ11şttr. Kataloglama kurallan taıutılarak Anglo Amerikan Katalog!ama Kuralları 2'ye . . . göre uygulamAlAryapılmıştır. - S1n1t1::1m;1 ~ -ıjVO'111::1T11R~1_: Smıtlama tanıınlanmıştır. Snuflamanın amaçları. ışlevleri. yararları ve türleri Incelenmiştlr. Kullarulan en yaygın smıtlama sisteıİı1eri tanıtılarak, Dewey C5nIu Sınıflama SIs. temine göre uygulamaJar yapılImştır. Y""'!UT1" Usul Ve Teknlk),er1 : Yapı1acak y;ızı$malar pozisyon ve yazışma teknlgl hakkında bJlgl vet1lnıiştlr. v . Için kom- : ''De- Wirbaş Kitap Kayıt Defteri" tanıtı1arıik kayıt tut nım""", tutanak düzenlenmesi ve kayıttan düşüm Işlemleri konusundaki kUraDar açıklanmıştır. Kurallar ilg1li defter ozerlnde uygulanmıştır. Ödfinc Venne t..IPTT111,," : Okuyucuların üYe kaydı yapıldıktan sonra. ödünç ver1lecek eserlere yOnelik yapılaCak işlemler gösterilnıiştlr. Cılt yapımı ve öze1l1k1eriaçıklanmıştır. Cfide gönder1lecek eserler Için seçim yapılması ve tutanak hazırlanması ozertnde durulmuştur. 95 -- . InciOnaı - K- - ht. : Kütüphane koleksly()nu. okunan kitap sayısı. gOrsel-lşitsel materyaller. okuyucu hızmetlerı ve personelin Ozell1k1erlhakkında günljik. aylık ve yı11ıkolarak tutulması gereken istatistik çlzelgelerlnln nasıl hazırlanacagı beUrtilnılştlt. . Hızmetlerı : Müracaat I:!Olümünün gOrevieri ta-. Mürn"""t rumianmıştır. Başlıca müracaat eserleri tanıtıJarak bu eserlerin kullanımına ilişkin .temel bilgi kazandırılmıştır. Kltan SeC'tmt ve Saııanma5ı : Kitap seçlnılnde yararlanılacak başlılıa kaynaklannneler olacagı gOsterlhnlştır. Yayın sagıama yolları tatılmıŞtır. . . I Üç haftalık egitim pr<ıgramıve uygulamalar sonucunda "Okul Küt phaneClIIgI Kursu" tamamlanmıştır.. Kursbitiminde yapılan d~gerlendlrme sınavında başan]1!n yüksek oldugu gOrülmüştür. Kursiyerler başan Ue mezun olmuşlardır. i 'Verlleıiı dersler dogroltusunda kütüphane ışlemlerı ve hızmetlerı hakkında kurslyerlere tekrar anket uygulamnıştır. Okul kütüphanecUtgI kursunda.ogrenUen konular Tablo 2'de gOsterlhnlştlt. Tablo 2 : okıiı KütÜphanecillgı Kursunda ÖgrenUen Konular , Konular YlUUtiann Sayw ('iii) Okul kütüphanelerininamaçları ve hızmetlerı . Okul kütüphanelerlnll1gUendtren yOnetınellk ve genelgeler 126 (98) 124 (96) KatalqgIama veluygulaması Sınıflama ve uygulanıası 103 101 (80) (78) Yazışma usul ve t~erl 90 (70) Kitap kayıt. demirbaş ve demirbaştan düşüm ışlemlerı 85 (66) Ödünçverme ışlemıeri 77 Kütüphanede bulunan gazete. dergi ve kitaplann cl1t ışlemlerı 74 (57) Kütüphane Istatlst1klı;rlnlntutulması . 72 (56) Müracaat hızmetlerı 71 (55) Kitap seçımı ve saglanması 68 (53) - ~ kanun. . (60) 96 Üniversite mezunIarının başan düzeyi dıgerlerlnden fatkiı degildir. Üniversite kütüphanecl11k bölümü mezunu 2 kütüphanecl de diger Edebiyat FakOI\8oi Dergisi gıbı kursu ~ ve sınavlannda başarılı olmuşlardır. 2 KütüphanecJntıı başan düzeyi dlgerlertnden önelnıı derecede farlı:lı bulunmamıştır. Bu durum. üniversıte kütüphanecillk bölümü mezunlan ıçın kursun bilinenlerin tekran olmasından. bilgi düzeyine göre aymiı yapılmamasından ve gençlik. egıum. ögtetlm. ögrenım gibi okul kütüphaı'ıecilerlnln gereksınım duyacaklan bilginin verilmemesınden kaynaklanmaktadır. kursiyerler Bulunulan yaş grubu. görev yapılan okul. il.ilçe ve cinsiyet başarıyı etkileyen önemlı btr faktör olarak bulunmanuştır. KursiyerlerIIi hepsi kursa ilgi- göstermIŞ. derslere devam etmIŞ. başarılı olmuş ve araştırıruhnızı desteklemlŞlerdtr. , 4. AŞAMA : AraşbrrTU.m'!nT\bu son aşamıısında her kursiyerden ''bizimle sürekli baglanb kurarak. kursa ııışkln eleşttrilerlni ve ça" lışırui1anm bildirineleri" istemnlştir. Beklentileiımızc; yanıt veren 71 kütüphane personellyle Agustos 1990'dan Şubat 1996'yadegln görüşmelerve ya!nF"larla bag1anb sürdürülmüştür. - ' Okul- Kütüphanecillgl,Kursuna yönelik eleştiı1ler;olumlu yönde. özellikle "temel bilgi ve becerl1erln kazandırılmış olmasmda" topJanmaktadır. Bu kurstan geçip. okul kütüphanelerinde çalışanlar kütüphanecilik konusunda yeterince bilgili olduklannı Iddia etmemektedtr1er. Ancak görevlerini daha bilgili. daha bilinçli yapabilecek duruma gelmelerinde kursun önemlı katkılan oldugunu Ifade etmektedirler. Güncel ge1lşmeİer d<ıgrultusunda. egitimlerinin sürekli ('Im"...nı Istemektedtrler. ünWersıte kütüphanecillk bölümü mezunu 2 kütüphaneclsadece temel bilgi ve becerilerin kazandırılmasını degıı; kütüpbanecillk. egitim ve okul kütüphanelerine ilişkin en son geitşmelerın aktarılmasını _ _ gerekli görmektedtrler. " Elde ettlgtmtz bilgiye göre okulkütüppanelerine ayrı bütı;,1esaglanamamaktadır. 'Ko1ekslyonda yetersizlikler görülmektedtr. Her okulda hizmete elverişli ortamJar bulunamamaktadır. Daha Iyi okuyucu hizmeti vermek istenmektedir. Kurstan geçmış btr tek personelle hızmet verlldlgtnden; okul kütüphanelerinde çalışma yükü agır 01lTI"kt"dır. Bu zor şartlar altuıda; kütüphane personeli görevlerini severek, Isteyerek. ~lklik1erl olsa bile yararlı olma amacıy1a YaPın" kt"ı:j.r1ar. m. nEOpT R1'(DiıuıIE,SONUÇ VE Ö~ Kütüphanecillk mesiegındekl ve egıtlmdekl gelışmeler sonucunda "okul kütüphanelerinde peisonel" önemlı btr role sahıp olmuştur. Okul kütüphanelerinde çalışarılar ve çaJ>şa.."ldar ıçın ge)iştlrilen 97 -- , inciOnaı - programIann : "Egitim öAı.ıthu könuııunda bilgll1: ög1"etlm programlan l\Dnuııunda uzman: bılgı kaynaklarımn şeçtrni, orgsın17.al3)'onu,' kullammı ve ıçengı konusunda dertnlJgıne bılgı şahtbl; enformasyonbılgı hızmetlerı verebllen; kUllanıcı egitlİn1 programIannda d.m1ŞJTl,nı; ögfenc1. ve gençUk pş1koloj18inden anlayan; lletlş1m yöntemlerini uyguiayabllen: IIgfetmenlerln. ögrenenerin, yönettenerin, ve1llertn ve toplumun deııt~ ııagıayabllen: ııosyai ve !diltürel etk1nl1kleri planlayabilen ve' yönetim tekn1k1ertne glIre çahşan perliOneU yet1şt1nne!l1 beklenir. '.' TIlrklye'de iintWrStte kiltiiphanec1llk bölüntil mezunlanIun okul . kiit11phaneler1nde çsıl'ŞI""'" henüz yaygınJaŞmannşbr. {)yıla k! ':okul kilfl1phane!l1nde ça1~şııı"l(" ı1n1ve1."!l1te düzeyinde kiltl1phanecll1k egitIm1nden geçmekle\b1rl1kte. özel egıt1m1de gerektirmektedir. Hem me&lek! egttim. hem de h1zınet1çl egitim n1teUginde düzenlenen "Okul KI1tl1phanecnıgı Kıın!u" katılanlara temel bilgi ve beceriler kazıındusa da. okul kiltl1phaneler1n1n SOrUnIan ve yetenılzl1k1erı perşoneUn ça1ışmaJannda bl1y11ltzorluklara yol açmakt"rlır. Tiirk1ye'nfn çeşltU illerinden büyiik katıhm1a gerçeld«ıştlrllen ll. Okul KI1t11plıanec1!lgı Kurliu ve onu takıp eden 1990-1996 döneminde yaptıgımız araştırmanın liQnuç1an okul kilt11phanecillgı kurliunun önemini ve ge-' reklt11gını ortaya koymaktadır. Kıı>ada OlSa. aldıklan temel egitim kiltl1phane pen3OIlel1neçok yararlı o~"kt"d.r. Ü!kPmt7.dek! her düzeyden okul kiltl1phaneler1n111g11end1renbu sonuçlar, bır gerçegi görmem1z! sagIanıaktadır. Bu gerçek: KI1t11pJ1aneci!1kve egttIm b1l1ın1erlaÇJS1l1dan büyiik sorumluluklara şahtp olan oku! ki1t11phaneslpersonel1n1n!LorumlulukIanna eşde(!er egit1ınden geçn1P11V!Ier1d1r. Başan1ı çahŞınaIan kadar. başansız1ıklanda I!ÔZkonusudur. Meıılek! bilgi e~fkllkleri k!. Ş1ııeIgayretlerle kapatılmaya C"hşılm"ktııdır. Yardımcı perşortel olarak görev yapm"l"rı daha uygun olurken b1rÇOkJJ1~I(,1ôve yönetlmliel sorumluluklarla karşı karşıya bulunmaktadırlaF. . Okul kiltl1PhaIıoelerıDde çı>hş"<:'a1tve çalışmakta olan personeUn yettşt1r1lıneııllç1nönertm1z: IF'LA. UNESCO. lASL ve iIgIL1demek l1yeler1n1nu!uıılaraı"aııı deneytm1eri sonucu hazırlanan. "Okul KI1tl1phaneııt PerliOnel1n1n Egıt1ın Programı"nıian yararlanılarak hazırlanacak programlann uygulanmaliında toplanmaktadır. "Okul Kl1tl1phanelil PerlioneUnin EgttIm Programı"nda beUrtllen konular dl1nyanın neresinde, hangi egttim !I1!I1em1ndeolurlla omun, okul kl1tl1phanec181n1n sorumlulUk alanlaqnın ne kadar genIŞ ve önemU oldugunu gÖlitermekted1r. Bu durumda . güIıcelge1fşmelertn ızlenmesı ve . uygulsmm"". z'orunlu olmaktadır. Konuyla ilgill olarak 1lrıtver!l1tek11tüphanecll1k bölümleri ve MiIU Egitlm Bakanlıgı blrUkte ça1ışarak ıkı düzeyde prograıp dü. . 98 ---- . 99 -_..- InciOnaı ( 100
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ENGLISH BOOK-ILLUSTRATION OF TO-DAY
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54 54 I. SOME DECORATIVE ILLUSTRATORS. IF IF the famous' Poems by Alfred Tenny- son,' published in 1857 by Edward Moxon, Mr. Gleeson White wrote in 1897 • ' The whole modern school of decorative illustrators regard it rightly enough as the genesis of the modern movement.' The statement may need some modi- fication to touch exact truth, for the * modern movement' is no single file, straightforward move- ment. * Kelmscott,' 'Japan,' the * Yellow Book, black-and-white art in Germany, in France, in Spain, in America, the influence of Blake, the style of artists such as Walter Crane, have affected the form of decorative book-illustration in the nineties. Such perfect unanimity of opinion as is here ascribed to a large and rather indefinitely related body of men hardly exists, among even the smallest and most derided body of artists. Still, allowing for the impossibility of telling the whole truth about any modern and eclectic form of art in one sentence, there is here a statement of fact. What Rossetti and Millais and Holman Hunt achieved in the drawings to the illustrated ' Tennyson' was a vital change in the direction of English illustra- ENGLISH BOOK-ILLUSTRATION. 55 tion, and whatever form decorative illustration may assume, their ideal is effective while a personal interpretation of the spirit of the text is the creative impulse in illustrative art. The influence of tech- nical mastery is strong and enduring enough. It is constantly in sight and constantly in mind. But it is in discovering and making evident a principle in art that the influence of spirit on spirit becomes one of the illimitable powers. To Rossetti the illustration of literature meant giving beautiful form to the aspedt of delight, of penetration, that had kindled his imagination as he read. He illustrated the * Palace of Art' in the spirit that stirred him to rhythmic expression in words of the still music in Giorgione's * Pastoral,' or of the unpassing movement of Mantegna's * Parnassus.' Not the words of the text, nor those things precisely affirmed by the writer, but the meaning, the spell of beauty that held his mind to the exclusion of other images, gave him inspiration for his drawings. As Mr. I. SOME DECORATIVE ILLUSTRATORS. William Michael Ros- setti says: * He drew just what he chose, taking from his author's text nothing more than a hint and an opportunity.' It is said, indeed, that Tennyson could never see what the St. Cecily drawing had to do with his pqem. And that is strange enough to be true. It is clear that such an ideal of illustration is for the attainment of a few only. The ordinary illus- trator, making drawings for cheap reproduction in the ordinary book, can no more work in this mood than the journalist can model his style on the prose of Milton. But journalism is not literature, and ENGLISH BOOK-ILLUSTRATION. 56 pictured matter-of-fact is not illustration, though it is convenient and obligatory to call it so. How- ever, here one need not consider this, for the deco- rative illustrator has usually literature to illustrate, and a commission to be beautiful and imaginative in his work. He has the opportunity of Rossetti, the opportunity for significant art. at University of Arizona o http://library.oxfordjournals.org/ Downloaded from The ' Classics' and children's books divide the art of decorative illustrators. Those who have illustrated children's books chiefly, or whose best work has been for the playful classics of literature, it is convenient to consider in a separate article, though there are instances where the division is not maintainable. Walter Crane, for example, whose influence on a school of decorative design makes his position at the head of his following im- perative. Representing the * architectural' sense in the decoration of books, many years before the supreme achievements of William Morris added that ideal to generally recognized motives of book-decoration, Walter Crane is the precursor of a large and pro- lific school of decorative illustrators. Many factors, as he himself tells, have gone to the shaping of his art. Born in 1846 at Liverpool, he came to Lon- don in 1857, and there after two years was 'ap- prenticed ' to Mr. W. J. Linton, the famous wood- engraver. His work began with ' the sixties,' in contact with the enthusiasm and inspiration those years brought into English art. The illustrated * Tennyson,' and Ruskin's * Elements of Drawing, were in his thoughts before he entered Mr. Lin- ton's workshop, and the ' Once a Week' school had 'THE SIX-SWANS- I *HB* WlTH'IUiSHIfK"VVIM«5,"A I •»T»opc»"ttf>oNi>Mi»;3«rtHA I '(Hf'COIU-TmiW THC'kM FROM MR. I. SOME DECORATIVE ILLUSTRATORS. WALTER CRANE'S ' GRIMM'S HOUSEHOLD STORIES.' BY LEAVE OF MESSRS. MACMILLAN. at University of Arizo http://library.oxfordjournals.org/ Downloaded from FROM MR. WALTER CRANE'S ' GRIMM'S HOUSEHOLD STORIES.' BY LEAVE OF MESSRS. MACMILLAN. ENGLISH BOOK-ILLUSTRATION 58 a strong influence on his early contributions to * Good Words,'' Once a Week,' and other famous numbers. In 1865 Messrs. Warne published the first toy-book, and by 1869-70 the ' Walter Crane Toy-book ' was a fa£t in art. The sight of some Japanese colour-prints during these years suggested a finer decorative quality to be obtained with tint and outline, and in the use of black, as well as in a more delicate simplicity of colour, the later toy- books show the first influence of Japanese art on the decorative art of England. Italian art in Eng- land and Italy, the prints of Diirer, the Parthenon sculptures, these were influences that affe&ed him strongly. 'The Baby's Opera' (1877), and 'The Baby's Bouquet' (1879), are classics almost impos- sible to criticise, classics familiar from cover to cover before one was aware of any art but the art on their pages. So that if they seem less expres- sive of the supreme art of Greece, of Germany, of Italy, than of the countries by whose coasts ships 'from over the sea' go sailing by with strange cargoes and strange crews, of that land whence come broom-sellers and cradle-songs, it is not in their dispraise. As a decorative artist Mr. Crane is at his best when the use of colour gives clearness to the composition, but some of his most' serious' work is in the black-and-white pages of ' The Sirens Three,' ' Echoes iof Hellas,' ' The Shep- heardes Calendar/ and especially ' The Faerie Queene.' The number of books he has illustrated —upwards of seventy—makes a detailed account impossible. Nursery rhyme and fairy books, chil- dren's stories, the myths, of Greece, Spenser, Shake- at University of Arizona o http://library.oxfordjournals.org/ Downloaded from OF TO-DAY. 59 speare, ' pageant books' such as ' Flora's Feast' or 4 Queen Summer,' or the just published * Masque of Days,' his own writings, serious or gay, have given him subjects, as the great art of all times has touched the ideals of his art. But whatever the subject, how strong soever his artistic admirations, he is always Walter Crane, unmistakable at a glance. I. SOME DECORATIVE ILLUSTRATORS. Knights and ladies, fairies and fairy people, allegorical figures, nursery and school-room children, fulfil his decorative pur- pose without swerving, though not always without injury to their comfort and freedom and the life in their limbs. An individual apprehension that sees every situation as a decorative ' arrangement' is occasionally beside the purpose in rendering real life. But when his theme touches imagination, and is not a supreme expression of it—for then, as in the illustrations to ' The Faerie Queene,' an unusual sense of subservience appears to dull his spirit—his fancy knows no weariness nor sameness of device. The work of most of Mr. Crane's followers belongs to * the nineties,' when the 'Arts and Crafts' movement, the * Century Guild ' the Birmingham and other schools had attracted or produced artists working according to the canons of Kelmscott. Mr. Heywood Sumner was earlier in the field. The drawings to * Sintram ' (1883) and to * Undine' (1888) show his art as an illustrator. Undine— spirit of wind and water, flower-like in gladness— seeking to win an immortal soul by submission to the forms of life, is realized in the gracefully de- signed figures of frontispiece and title-page. Where ENGLISH BOOK-ILLUSTRATION 60 umner illustrates incident he is ' factual' t being matter-of-fact. The small drawing uced is hardly representative of his art, but f his work is adapted to a squarer page than that of 'The Library,' and has had to be re- jected on that account. Some of the 'most apt decorations in * The English Illustrated' were by Mr. Sumner, and during the time when art was represented in the magazine Mr. Ry- land and Mr. Louis Davis were frequent contributors. The grace- ful figures of Mr. Ry- land, uninterested in activity, a garden-world set with statues around them, and the carol-like grace of Mr. Davis's de- signs in that magazine, represent them better than the one or two books they have illus- trated. R. HEYWOOD SUMNER's ' UNDINE.' VE OF MESSRS. CHAPMAN AND HALL. ncident he is ' factual' ct. The small drawing sentative of his art, but d to a squarer page than that of 'The Library,' and has had to be re- jected on that account. Some of the 'most apt decorations in * The English Illustrated' were by Mr. Sumner, and during the time when art was represented in the magazine Mr. I. SOME DECORATIVE ILLUSTRATORS. Ry- land and Mr. Louis Davis were frequent contributors. The grace- ful figures of Mr. Ry- land, uninterested in activity, a garden-world set with statues around them, and the carol-like grace of Mr. Davis's de- signs in that magazine, represent them better than the one or two books they have illus- trated. g g reproduced is hardly representative of his art, but most of his work is adapted to a squarer page than that of 'The Library,' and has had to be re- jected on that account. Some of the 'most apt decorations in * The English Illustrated' were by Mr. Sumner, and during the time when art was represented in the magazine Mr. Ry- land and Mr. Louis Davis were frequent contributors. The grace- ful figures of Mr. Ry- land, uninterested in activity, a garden-world set with statues around them, and the carol-like grace of Mr. Davis's de- signs in that magazine, represent them better than the one or two books they have illus- trated. Among those associated with the' Arts and Crafts' who have given more of their art to book-decora- tion than these men, Mr. Anning Bell is first. He has gained the approval even of the most exigent of critics as an artist who understands drawing for FROM MR. HEYWOOD SUMNER's ' UNDINE.' Br LEAVE OF MESSRS. CHAPMAN AND HALL. FROM MR. HEYWOOD SUMNER's ' UNDINE.' Br LEAVE OF MESSRS. CHAPMAN AND HALL. at University of Arizona o http://library.oxfordjournals.org/ Downloaded from at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from FROM MR. HEYWOOD SUMNER's ' UNDINE.' Br LEAVE OF MESSRS. CHAPMAN AND HALL. FROM MR. HEYWOOD SUMNER's ' UNDINE.' Br LEAVE OF MESSRS. CHAPMAN AND HALL. Among those associated with the' Arts and Crafts' who have given more of their art to book-decora- tion than these men, Mr. Anning Bell is first. He has gained the approval even of the most exigent of critics as an artist who understands drawing for OF TO-DAY. 61 process. Since 1895, when the ' Midsummer Night's Dream' appeared, his winning art has been praised with discrimination and without dis- crimination, but always praised. Trained in an architect's office, widely known as the re-creator of coloured relief for architectural decoration, Mr. f " Wherein she passioned To see herself escaped from so sore ills," I. SOME DECORATIVE ILLUSTRATORS. Anning Bell's illustrative art shows constructive power no less than that fairy gift of seeming to improvise without labour and without hesitancy, which is one of its especial charms. In feeling, and in many of his decorative forms, he recalls the art of Florentine bas-relief, when Agostino di Duccio, or Rossellino or Mino da Fiesole, created forms of delicate sweetness, pure, graceful—so graceful that their power is hardly realized. The fairy by-play of the * Midsummer Night's Dream ' is exactly to Mr. Anning Bell's fancy. He knows better than to go about to expound this dream, and it is not likely that a more delightful edition will ever be put into the hands of children, or of anyone, than this in the white and gold cover devised by the artist. Of his illustrations to the ' Poems by John Keats' (1897), and to the 'English Lyrics from Spenser to Milton' of the following year—as illustrations—not quite so much can be said, dis- tinguished and felicitous as many of them are. The simple profile, the demure type of beauty that he affeds, hardly suits with Isabella when she hears that Lorenzo has gone from her, with Lamia by the clear pool f " Wherein she passioned To see herself escaped from so sore ills," f " Wherein she passioned To see herself escaped from so sore ills," f ENGLISH BOOK-ILLUSTRATION 62 or with Madeline,' St. Agnes' charmed maid.' Mr. Anning Bell's drawings to 'The Pilgrim's Pro- gress' (1898) reveal him in a different mood, as do those in ' The Christian Year' of three years earlier. His vision is hardly energetic enough, his energy of belief sufficient, to make him a strong illustrator of Bunyan, with his many moods, his great mood. A little these designs suggest Howard at University of http://library.oxfordjournals.org/ Downloaded from at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from FROM MR. ANNING BELL'S ' KEATS.' BY LEAVE OP MESSRS. CBORGB BELL. FROM MR. ANNING BELL'S ' KEATS.' BY LEAVE OP MESSRS. CBORGB BELL. Pyle, and Anning Bell is better in a way of beauty riot Gothic. So if Mr. I. SOME DECORATIVE ILLUSTRATORS. Anning Bell represents the 'Arts and Crafts' movement in the variety of decorative arts he has practised, and in the architectural sense underlying all his art, his work does not agree with the form in which the influence of William Morris on decorative illustration has chiefly shown itself. That form, of course, is Gothic, as the ideal of Kelmscott was Gothic. The work of the OF TO-DAY. 63 ' Century Guild' artists as decorative illustrators is chiefly in the pages of ' The Hobby Horse.' I do not think either Mr. Selwyn Image or Mr. Herbert Home has illustrated books, so in this connection one may not stop to consider the decorative strength of their ideal in art. The Birmingham school represents Gothic ideals with determination and rigidity. Morris addressed the students of the school and prefaced the edition of ' Good King Wenceslas,' decorated and engraved and printed by Mr. A. J. Gaskin c at the press of the Guild of Handicraft in the City of Birmingham,' with cordial words of appreciation for the pictures. These illus- trations are among the best Mr. Gaskin has done. The twelve full-page drawings to ' The Shepheardes Calendar,' printed at the Kelmscott Press in 1896, mark Morris's pleasure in Mr. Gaskin's work— this time seen in Andersen's * Stories and Fairy Tales.' If not quite in tune with Spenser's Eliza- bethan idyllism, these drawings are distinctive of the definite convictions of the artist. These convictions represent a splendid tradition. They are expressive, in their regard for the unity of the page, for harmony between type and de- coration, of the universal truth in all fine book- making. Only at times Birmingham work seems rather heavy in spirit, rather too rigid for develop- ment. Still, judging by results, a code that would appear to be against individual expression is in- spiring individual artists. Some of these—as Mr. E. H. New—have turned their attention to archi- tectural and " open air " illustration, in which con- nection their work will be considered, and many FROM MR. CASKJN'S * HANS ANDERSEN.' BY LEAVE OF Mft. GIOKOB ALLIN. FROM MR. CASKJN'S * HANS ANDERSEN.' BY LEAVE OF Mft. GIOKOB ALLIN. ENGLISH BOOK-ILLUSTRATION. 65 have illustrated children's books. Their quaint and naive fancy has there, at times produced a por- tentous embodiment of the * old-fashioned' child of fiction. Mr. in, F I. SOME DECORATIVE ILLUSTRATORS. Gere, though he has done little book-illustration, is one of the strongest artists ot the school. His original wood engravings show unmistakably his decorative power and his crafts- manship. With Mr. K. Fairfax Muckley he was responsible for 'The Quest' (1894.-96). Mr. Fairfax Muckley has illustrated and decorated a three- volume edition of * The Faerie Queene' (1897), wherein the forest branches and winding ways of woodland and of plain are more happily conven- tionalized than arc Spenser's figures. Some of the headpieces are especially successful. The artist uses the ' mixed convention' of solid black and line work with less confusion than many modern draughtsmen. Once its dangers must have been evident, but now the puzzle pattern, with solid blacks in the foreground, background, and mid- distance—only there is no distance in these draw- ings—is the most usual form of black and white. Miss Celia Levetus, Mr. Henry Payne, Mr. F. Mason, and Mr. Bernard Sleigh, are also to the credit of the school. Miss Levetus, in her later work, shows that an inclination towards a more flexible style is not incompatible with the training in mediaeval convention. Mr. Mason's illustrations to ancient romances of chivalry give evidence of conscientious craftsmanship, and of a spirit sym- pathetic to themes such as c Renaud of Montauban.' Mr. Bernard Sleigh's original wood-engravings are well known and justly appreciated. Strong in tra- i F F ENGLISH BOOK-ILLUSTRATION 66 dition and logic as is the work of these designers, it is, for many, too consistent with convention to be delightful. Perhaps the best result of the Birming- ham school will hardly be achieved until the formal effect of it is less patent.. p The " sixties" might have been void of art, so far as these designers are concerned, save that in those days Morris and Burne-Jones and Walter Crane, as well as Millais and Houghton and Sandys, were about their work. Far other is the case with artists such as Mr. Byam Shaw, or with the many draughtsmen, including Messrs. P. V. Woodroffe, Henry Ospovat, Philip Connard, and Herbert Cole, whose art derives its form and intention from the sixties. I. SOME DECORATIVE ILLUSTRATORS. Differing in technical power and fineness of invention, in all that distinguishes good from less good, they have this in common—that the form of their art would have been quite other if the illus- trated books of the sixties were among things unseen. Mr. Byam Shaw began his work as an illustrator in 1897 with a volume of * Browning's Poems,' edited by Dr. GarnetL He proved himself in these draw- ings, as in his pictures and later illustrations, an artist with a definite memory for the forms, and a genuine sympathy with the aims of pre-Raphaelitc art. Evidently, too, he admires the black-and- white of Mr. Abbey. He has the gift of dramatic conception, sees a situation at high pitch, and has a pleasant way of giving side-lights, pidtorial asides, in the form of decorative head and tailpieces. His illustrations to the little green and gold volumes of the * Chiswick Shakespeare ' are more emphatic than his earlier work, and in the decorations his at University of Arizona o http://library.oxfordjournals.org/ Downloaded from OF TO-DAY. 67 power of summarizing the chief motive is put to good use. There is no need of his signature to dis- tinguish the work of Byam Shaw, though he shows himself under the influence of various masters. Probably he is only an illustrator of books by the way, but in the meantime, as the * Boccaccio,' * Browning,' and * Shakespeare' drawings show, he works in black and white with vigorous intention. g Mr. Ospovat's illustrations to * Shakespeare's Sonnets' and to * Matthew Arnold's Poems' are interesting, if not very markedly his own. He illustrates the Sonnets as a celebration of a poet's passion for his mistress. In the Matthew Arnold drawings, as in these, he shows some genuine crea- tive power and an aptitude for illustrative decora- tion. Mr. Philip Connard has made spirited and well-realized illustrations in somewhat the same kind; Miss Amelia Bauerle, and Mr. Bulcock, who began by illustrating * The Blessed Damozel' in memory of Rossetti, have made appearance in the * Flowers of Parnassus' series, and Mr. Herbert Cole, with three of these little green volumes, pre- pared one for more important work in * Gulliver's Travels' (1900). The work of Mr. Woodroffe was, I think, first seen in the * Quarto'—the organ of the Slade School—where also Mr. A. Garth Jones, Mr. Cyril Goldie, and Mr. I. SOME DECORATIVE ILLUSTRATORS. Robert Spence, gave unmistakable evidence of individuality. Mr. Woodroffe's wood- engravings in the * Quarto' showed strength, which is apparent, too, in the delicately characterized figures to ' Songs from Shakespeare's Plays' (1898), with their borders of lightly-strung field flowers. ENGLISH BOOK-ILLUSTRATION 68 His drawings to * The Confessions of S. Augustine,' engraved by Miss Clemence Housman, are in keep- ing with the text, not impertinent. Mr. A. Garth Jones in the ' Quarto' seemed much influenced by Japanese grotesques; but in illustrations to Milton's ' Minor Poems' (1898) he has shown development towards the expression of beauty more austere, classical, controlled to the presentment of Milton's high thought. His recent * Essays of Elia' re- mind one of the forcible work of Mr. E. J. Sulli- van in * Sartor Resartus.' Mr. Sullivan's ' Sartor' and * Dream of Fair Women' must be mentioned. His mastery over an assertive use of line and solid black, the unity of his effects, the humour and imagination of his decorative designs, are not likely to be forgotten, though the balance of his work in books such as • White's Selborne' or ' The Com- pleat Angler,' obliges one to class him with ' Open- Air' illustrators, and so to leave a blank in this article. Mr. Laurence Housman stands alone among modern illustrators, though one may, if one will, speak of him as representing the succession of the sixties, or as connected with the group of artists whose noteworthy development dates from the publication of' The Dial' by Charles Ricketts and Charles Shannon in 1889. To look at Mr. Hous- man *s art in either connection, or to record the effect of Diirer, of Blake, of Eidward Calvert, on his technique, is only to come back to appreciation of all that is his own. As an illustrator he has hardly surpassed the spirit of the * forty-four de- signs, drawn and written by Laurence Housman,' OF TO-DAY. 69 that express his idea of George Meredith's * Jump to Glory Jane* (1890). These designs were the result of the appreciation which the editor, Mr. Harry Quilter, felt for Mr. I. SOME DECORATIVE ILLUSTRATORS. Housman's drawings to «The Green Gaffer' in * The Universal Review.* Jane the village woman with 'wistful eyes in a touching but bony face,' leaping with counten- ance composed, arms and feet 'like those who hang,' leaping in crude expression of the unity of soul and body, making her converts, failing to move the bishop, dying at last, though not in- gloriously, by the wayside — this most difficult conception has no ( burlesque outline ' in Mr. Housman's work, inexperienced and unacademic as is the drawing. at University of Arizona o http://library.oxfordjournals.org/ Downloaded from g 'Weird Tales from Northern Seas,' by Jonas Lie, was the next book illustrated by Mr. Housman. Christina Rossetti's 'Goblin Market' (1893), offered greater scope for freakish imagination than did 'Jane.' The goblins, pale-eyed, mole and rat and weasel-faced; the sisters, whose simple life they surround with hideous fantasy, are realized in har- mony with the unique effect of the poem—an effect of simplicity, 01 naive imagination, of power, of things stranger than are told in the cry of the goblin merchants, as at evening time they invade quiet places to traffic with their evil fruits for the souls of maidens. The frail-bodied elves of'The End of Elfin Town,' moving and sleeping among the white mushrooms and slender stalks of field flowers, are of another land than that of the goblin merchant-folk. Illustrations to ' The Imitation of Christ,' to ' The Sensitive Plant,' and drawings to ENGLISH BOOK-ILLUSTRATION 70 •The Were-Wolf,' by Miss Clemence Housman, complete the list of Mr. Housman's illustrations to writings not his own, with the exception of frontispiece drawings to several books. To explain Mr. Housman's reading of 'The Sensitive Plant' would be as superfluous as it would be ineffectual. In a note on the illustrations he has told how the formal beauty, the exquisite ministrations, the sounds and fragrance and sweet winds of the garden enclosed, seem to him as ' a form of beauty that springs out of modes and fashions,' too graceful to endure. I. SOME DECORATIVE ILLUSTRATORS. In his pictures he has realized the perfect ensemble of the garden, its sunny lawns and rose-trellises, its fountains, statues, and flower-sweet ways; the spirit of the Sensitive Plant, the lady of the garden, and Pan—Pan, the great god who never dies, who waits only with- out the garden, till in a little while he enters, 'effacing and replacing with his own image and superscription, the parenthetic grace . . . of the garden deity/ These drawings are very charming. Of a talent that treats always of enchanted places, where 'reality' is a long day's journey down a dusty road, it is difficult to speak without suggest- ing that it is all just a charming dalliance with pretty fancies, lacking strength. For the strength of Mr. Housman's imagination, however, his work speaks. His illustrations to his own writings, fairy tales, and poems, cannot with any force be dis- cussed by themselves. The words; belong to the pictures, the pictures to the words. The drawings to ' The Field of Clover' are seen to full advantage in the wood-engravings of Miss Housman. Only at University of Ariz http://library.oxfordjournals.org/ Downloaded from OF TO-DAY. 71 so, or in rcprodu&ion by photogravure, is the full intention of Mr. Housman's pen-drawings apparent. p g pp One may group the names of Charles Ricketts, C. H. Shannon, T. Sturge Moore, Lucien Pissarro, and Reginald Savage together in memory of * The Dial,' where the activity of five original artists first became evident, though, save in the case of Mr. Ricketts and Mr. Shannon, no continuance of the classification is possible. The first number of 'The Dial,' 1889, had a cover design cut on wood by Mr. C. H. Shannon—afterwards replaced by the design of Mr. Ricketts. Twelve designs by Mr. Ricketts may be said to represent the transi- tional—or a transitional—phase of his art, from the earlier work in magazines, which he disregards, to the reticent expression of * Vale Press' illustrations. In 1891 the first book decorated by these artists appeared, ' The House of Pomegranates,' by Oscar Wilde. There was, however, nothing in this book to suggest the form their joint talent was to take. Many delightful designs by Mr. Ricketts, somewhat marred by heaviness of line, and full-page illustra- tions by Mr. I. SOME DECORATIVE ILLUSTRATORS. Shannon, printed in an almost invisible, nondescript colour, contained no suggestion of ' Daphnis and Chloe.' at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from at University of Ari http://library.oxfordjournals.org/ Downloaded from The second' Dial '(1892) contained Mr. Ricketts' first work as his own wood-engraver, and in the following year the result of eleven months' joint work by Mr. Ricketts and Mr. Shannon was shown in the publication of * Daphnis and Chloe,' with thirty-seven woodcuts by the artists. Fifteen ot the pictures were sketched by Mr. Shannon and revised and drawn on the wood by Mr. Ricketts, RCHAND1SC FAVOURA BY LBATI OF MESIlt. KEGAM PAUL. at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ ded from BY LBATI OF MESIlt. KEGAM PAUL. invj uvoia 'tntion ao IAVII AB 3HJ. OJ.NI d33H5 dKLSdld 3HX OX AHQNIX 38 QlSLi 3H1 3HJ. OJ.NI d33H5 dKLSdld 3HX OX AHQNIX 38 ENGLISH BOOK-ILLUSTRATION. 74 who also engraved the initials. It is a complete achievement of individuality subordinated to an ideal. Here and there one can affirm that Mr. Shannon drew this figure, composed this scene, Mr. Ricketts that; but generally the hand is not to be known. The ideal of their inspiration—the im- mortal * Hypnerotomachia'—seems equally theirs, equally potent over their individuality. Speaking with diffidence, it would seem as though Mr. Shannon's idea of the idyll were more naive and humorous. Incidents beside the main theme of the pastoral loves of young Daphnis and Chloe— the household animals, other shepherds — are touched with humorous intent. Mr. Ricketts shows more suavity, and, as in the charming double- page design of the marriage feast, a more lyrical realization of delight and shepherd joys. at University of Arizona o http://library.oxfordjournals.org/ Downloaded from at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from The * Hero and Leander' of 1094 is a less elaborate, and, on the whole, a finer production. I must speak of the illustrations only, lest con- sideration of Vale Press achievements should fill the remaining space at my disposal. Obviously the attenuated type of these figures shows Mr. Ricketts' ideal of the human form as a decora- tion for a page of type. The severe reticence he imposes on himself is in order to maintain the balance between illustrations and text. I. SOME DECORATIVE ILLUSTRATORS. One has only to turn to illustrations to Lord de Tabley's * Poems/ published in 1893, to see with what eager imagination he realizes a subject, how strong a gift he has for dramatic expression. That a more per- suasive beauty of form was once his wont, much of his early and transitional work attests. But I do OF THE APPARITION OF THE THREE NYMPHS TO DAPHNE IN A DREAM. OF THE APPARITION OF THE THREE NYMPHS TO DAPHNE IN A DREAM. IN A DREAM. FROM MESSRS. RICKETTS AND SHANNON'S 'DAPHNJS AND CHLO1.' (MATHEWS AND LANE.) REPRODUCED BY THEIR LEAVE AND THE PUBLISHERS*. at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from FROM MESSRS. RICKETTS AND SHANNON'S 'DAPHNJS AND CHLO1.' (MATHEWS AND LANE.) REPRODUCED BY THEIR LEAVE AND THE PUBLISHERS*. ENGLISH BOOK-ILLUSTRATION 76 not think his power to achieve beauty need be de- fended. After the publication of ' Hero and Leander,' Mr. Shannon practically ceased wood- engraving for the illustration of books, though, as the series of roundel designs in the recent exhibi- at University of Arizona on http://library.oxfordjournals.org/ Downloaded from FROM MR. RICXBTTS' ' CUPID AND PSYCHE.' RIPfcODUCID BT HI1 PBfcMISUOM. FROM MR. RICXBTTS' ' CUPID AND PSYCHE.' RIPfcODUCID BT HI1 PBfcMISUOM. tion of his work proved, he has not abandoned nor ceased to go forward in the art. 4 tion of his work proved, he has not abandoned nor ceased to go forward in the art. 4 g 4 The Sphinx,' a poem by Oscar Wilde, * built, decorated and bound 'by Mr. Ricketts—but with- out woodcuts—was published in 1894, just after ' Hero and Leander,' and designs for a magnificent edition of 'The King's Quhair' were begun. 4 The Sphinx,' a poem by Oscar Wilde, * built, decorated and bound 'by Mr. Ricketts—but with- out woodcuts—was published in 1894, just after ' Hero and Leander,' and designs for a magnificent edition of 'The King's Quhair' were begun. OF TO-DAY. 77 Some of these are in ' The Dial,' as also designs for William Adlington's translation of ' Cupide and Psyches' in « The Pageant,' ' The Dial,' and ' The Magazine of Art.' The edition of the work pub- lished by the new Vale Press in 1897, is not that projected at this time. I. SOME DECORATIVE ILLUSTRATORS. It contains roundel designs in place of the square designs first intended. These roundels are, I think, the finest achievement of Mr. Ricketts as an original wood-engraver. The engraving reproduced shows of what quality are both line and form, how successful is the placing of the figure within the circle. On the page they are what the artist would have them be. With the beginning of the sequence of later Vale Press books—books printed from founts designed by Mr. Ricketts—a consecutive account is impossible, but the frontispiece to the ' Milton' and the borders and initials designed by Mr. Ricketts, must be mentioned. As a designer of book-covers only one failure is set down to Mr. Ricketts, and that was ten years ago, in the cover to 'The House of Pomegranates.' Mr. Reginald Savage's illustrations to some tales from Wagner by Mr. Farquharson Sharp lack the force of the c Rhinoceros and Peacock' in * The Pageant,' or of designs for * The Ancient Mariner' and * Sidonia the Sorceress.' Of M. Lucien Pissarro, in an article overcrowded with English illustrators, I cannot speak. His fame is in France as the forerunner of his art, and we in England know his coloured wood-engravings, his designs for * The Book of Ruth and Esther' and for * The Queen of the Fishes,' printed at his press at 6 Epping, but included among Vale Press books. ENGLISH BOOK-ILLUSTRATION 78 'The Centaur,' 'The Bacchant,' 'The Meta- morphoses of Pan,' ' Siegfried '—young Siegfried, wood-nurtured, untamed, setting his lusty strength against the strength of the brutes, hearing the bird- call then, and following the white bird to issues remote from savage life—these are subjects realized by the imagination of Mr. T. Sturge Moore. FROM MR. STURGE MOORE's ' THE CENTAUR.' REPRODUCED BY PERMISSION OF MR. RICKETTS. FROM MR. STURGE MOORE's ' THE CENTAUR.' REPRODUCED BY PERMISSION OF MR. RICKETTS. There is no artist illustrating books to-day whose work is more unified, imaginatively and technically. It is some years now since first Mr. Moore's wood- engravings attracted notice in * The Dial' and * The Pageant,' and the latest work from his graver —finer, more rhythmic in composition though it be—shows no change in ideals, in the direction of his powers. He has said, I think, that the easiest OF TO-DAY. 79 line for the artist is the true basis of that artist's work, and it would seem as though much delibera- tion in finding that line for himself had preceded any of the work by which he is known. The wood-engraving of Mr. Sturge Moore is of con- siderable importance. Always the true understand- ing of his material, the unhesitating realization of his subject, combine to produce the effect of inevitable line and form, of an inevitable setting down of forms in expression of the thought within. Only that gives the idea of formality, and Mr. Moore s art handles the strong impulse of the wild creatures of earth, of the solitary creatures, mighty and terrible, haunting the desert places and fearing the order men make for safety. Designs to Words- worth's ' Poems,' not yet published, represent with innate perception the earth-spirit as Wordsworth knew it, when the great mood of ' impassioned contemplation' came upon his careful spirit, when his heart leapt up, or when, wandering beneath the wind-driven clouds of March, at sight of daffodils, he lost his loneliness. 4 The Evergreen,' that ' Northern Seasonal,' re- presented the pictorial outlook of an interesting group of artists—Robert Burns, Andrew K. Worn- rath, John Duncan, and James Cadenhead, for example—and the racial element, as well as their own individuality, distinguishes the work of Mr. W. B. Macdougall and Mr. J. J. Guthrie of * The Elf.' Mr. ENGLISH BOOK-ILLUSTRATION Macdougall has been known as a book- illustrator since 1896, when 'The Book of Ruth,' with decorated borders showing the fertility of his designing power, and illustrations that were no less ENGLISH BOOK-ILLUSTRATION 80 representative of a unique use of material, appeared. The conventionalized landscape backgrounds, the long, straightly-draped women, seemed strange enough as a reading of the Hebrew pastoral, with its close kinship to the natural life of the free children of earth. Their unimpassioned faces, un- spontaneous gestures, the artificiality of the whole impression, were undoubtedly a new reading of the ancient charm of the story. Two books in 1897, and 'Isabella' and 'The Shadow of Love,' 1898, showed beyond doubt that the manner was not assumed, that it was the expression of Mr. Mac- dougall's sense of beauty. The decorations to ' Isabella' were more elaborate than to ' Ruth,' and inventive handling of natural forms was as marked. Again, the faces are dechara&erized in accordance with the desire to make the whole figure the symbol of passion, and that without emphasis. Mr. J. J. Guthrie is hardly among book-illustrators, since * Wedding Bells' of 1895 is not Mr. Guthrie, nor is the child's book of the following year, while the illustrations to Edgar Allan Poe's ' Poems' are still, I think, being issued from the Pear Tree Press in single numbers. His treatment of land- scape is inventive, but his rhythmic arrangements, his effects of white line on black, are based on a real sense of the beauty of earth, of tall trees and wooded hills, of mysterious moon—brightness and shade in the leafy depths of the woodlands. at University of Arizona o http://library.oxfordjournals.org/ Downloaded from Mr. Granville Fell made his name known in 1896 by his illustrations to 'The Book of Job.' In careful detail, drawn with fidelity, never ob- trusive, his art is pre-Raphaelite. He touches OF TO-DAY. 81 Japanese ideals in the rendering of flower-growth and animals, but the whole effedt of his decorative illustrations is far enough away from the art of Japan. In the 'Job' drawings he had a subject sufficient to dwarf a very vital imaginative sense by its grandeur. In the opinion of competent critics Mr. Granville Fell proved more than the technical distinction of his work by the manner in which he fulfilled his purpose. ENGLISH BOOK-ILLUSTRATION The solid black and white, the definite line of these drawings, were laid aside for the sympathetic medium of pencil in 'The Song of Solomon' (1897). Again, his con- ception is invariably dramatic, and never crudely dramatic, robust, with no trace of morbid or senti- mental thought about it. The garden, the wealth of vineyard and of royal pleasure ground, is used as a background to comely and gracious figures. His other work, illustrative of children's books and of legend, the cover and title-page to Mr. W. B. Yeats's * Poems,' show the same strong yet restrained imagination. Mr. Patten Wilson is somewhat akin to Mr. Granville Fell in the energy and soundness of his conceptions. Each of these artists is, as we know, a colourist, delighting in brilliant and iridescent colour-schemes, yet in black and white they do not seek to suggest colour. Mr. Patten Wilson's illus- trations to Coleridge's ' Poems' have the careful fulness of drawings well thought out, and worked upon with the whole idea definite in the imagina- tion. He has observed life carefully for the pur- poses of his art. But it is rather in rendering the circumstance of poems, such as ' The Ancient G ENGLISH BOOK-ILLUSTRATION 82 Mariner,' or, in his Chaucer illustrations—Con- stance on the lonely ship—that he shows his grasp of the subject, than by any expression of the spiritual terror or loneliness of the one living man among the dead, the solitary woman on strange seas. Mariner,' or, in his Chaucer illustrations—Con- stance on the lonely ship—that he shows his grasp of the subject, than by any expression of the spiritual terror or loneliness of the one living man among the dead, the solitary woman on strange seas. Few decorative artists express themselves habitu- ally in ' wash ' rather than by line. Among those who rarely use the pen Mr. Weguclin and Mr. W. E. F. Britten must be named. Mr. Weguelin has illustrated Anacreon in a manner to earn the appreciation of Greek scholars, and his illustrations to Hans Andersen have had a wider and not less appreciative reception. His drawings have move- ment and atmosphere. Mr. Britten illustrated poems on the months by Mr. ENGLISH BOOK-ILLUSTRATION Swinburne in the ' Magazine of Art' in 1892-3, and since that time his version of * Undine,' and illustrations to Tenny- son's ' Early Poems,' have shown the same power of graceful composition and sympathy with his subject. at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from at University of Arizona o http://library.oxfordjournals.org/ Downloaded from R. E. D. SKETCHLEY. BIBLIOGRAPHY. A I U U A BADIBLE. AUtgor'us. Frederic W. Funr. 8°. (Longmans, 1898.) 20 Ulust. Tbi Daj-Drtem. Alfred Tennyson. 8°. (Lane, 1901. ' Flowers of Parnassus.*) R. ANNDIO BEIJ.. Tbt Gddtn Trtantrj cf Art tmi Sng. Edited by R. E. Mack. 4°. (Nuter, 1890.; 18 illiat in colour. A I U U A BADIBLE. AUtgor'us. Frederic W. Funr. 8°. (Longmans, 1898.) 20 Ulust. Tbi Daj-Drtem. Alfred Tennyson. 8°. (Lane, 1901. ' Flowers of Parnassus.*) R. ANNDIO BEIJ.. Tbt Gddtn Trtantrj cf Art tmi Sng. Edited by R. E. Mack. 4°. (Nuter, 1890.; 18 illiat in colour. A I U U A BADIBLE. AUtgor'us. Frederic W. Funr. 8°. (Longmans, 1898.) 20 Ulust. Tbi Daj-Drtem. Alfred Tennyson. 8°. (Lane, 1901. ' Flowers of Parnassus.*) R. ANNDIO BEIJ.. Tbt Gddtn Trtantrj cf Art tmi Sng. Edited by R. E. Mack. 4°. (Nuter, 1890.; 18 illiat in colour. 0 Uust. Tbi Daj-Drtem. Alfred Tennyson. 8°. (Lane, 1901. ' Flowers of Parnassus.*) R. ANNDIO BEIJ.. R. ANNDIO BEIJ.. Tbt Gddtn Trtantrj cf Art tmi Sng. Edited by R. E. Mack. 4°. (Nuter, 1890.; 18 illiat in colour. OF TO-DAY. 83 Jack the Giant-Killer and Beauty and the Beast. Edited by Grace Rhys. 320. (Dent, 1894. Banbury Cross Series.) 35. The SUeping Beauty and Dick Whittingttn and bis Cat. Edited by Grace Rhys. 32°. (Dent, 1894. Banbury Cross Series.) 35- 35 Tht Christian Tear. 8°. (Methuen, 1895.) 5. 0 ( , ) A Midsummer Night's Dream. 40. (Dent, 1895.) 13, head and tailpieces, borders. and tailpieces, borders. Keats' Poems. Edited by Walter Raleigh. 8°. (Bell, 1897. Endymion Series.) 65. at University of Arizona on May 29, 2015 http://library.oxfordjournals.org/ Downloaded from at University of Ariz http://library.oxfordjournals.org/ Downloaded from y ) English Lyrics from Spenser U Milton. 8°. (Bell, 1898. En- dymion Series.) 20, head and tailpieces. y ) , p Pilgrim's Progress. 8°. (Methuen, 1898.) 39. Pilgrim's Progress. 8°. (Methuen, 1898.) 39. Lamb's Tales from Shakespeare. 8°. (Fremantle, 1899.) 15. Keats'Odes. 8". (Bell, 1901.) 15, from Endymion Series. Grimm's Fairy Tales. Edited by Marian Edwardes. (Dent, 1901.) 102. g g ( , ) Lamb's Tales from Shakespeare. 8°. (Fremantle, 1899.) 15. Keats'Odes. 8". (Bell, 1901.) 15, from Endymion Series. Grimm's Fairy Tales. Edited by Marian Edwardes. (Dent, g g , Lamb's Tales from Shakespeare. 8°. (Fremantle, 1899.) 15. Keats'Odes. 8". (Bell, 1901.) 15, from Endymion Series. Keats'Odes. 8". (Bell, 1901.) 15, from Endymion Series. Grimm's Fairy Tales. BIBLIOGRAPHY. Edited by Marian Edwardes. (Dent, 1901.) 102. ( , ) , y Grimm's Fairy Tales. Edited by Marian Edwardes. (Dent, 1901.) 102. ) W. E. F. BRITTEN. Undine. Translated from the German of Baron de la Motte Fouque by Edmund Gone. 4*. (Lawrence and Bullen, 1896.) 10 illust. The Early Poems if Alfred Lord Tennystn. Edited by John Churton-Collins. 8°. (Methuen, 1901.) 10. PERCY BULCOCK. The Blessed Damezel. Dante Gabriel Rossetti. 8°. (Lane, 1900. ' Flowers of Parnassus.*) 8 illust. The Blessed Damezel. Dante Gabriel Rossetti. 8°. (Lane, 1900. ' Flowers of Parnassus.*) 8 illust. A Dream of Fair Women. Alfred Tennyson. 8°. (Lone, 1902 'F of P") 9 ) A Dream of Fair Women. Alfred Tennyson. 8°. (Lone, 1902. 'F. of P.") 9. HERBERT COLE. Gulliver's Travels. 8°. (Lane, 190a) Profusely illustrated. The Rubaiyat. 8°. (Lane, 1901. 'Flowers of Parnassus.*) 9 illust 9 illust The Nut-Brtwn Maid. A new version by F. B. Money- Coutts. 8°. (Lane, 1901. *F. of P.*) 9. A Ballade upon a Wedding. Sir John Suckling. 8°. (Lane, 1901. «F. of P.') 9. 9 illust The Nut-Brtwn Maid. A new version by F. B. Money- Coutts. 8°. (Lane, 1901. *F. of P.*) 9. Coutts. 8 . (Lane, 1901. F. of P. ) 9. A Ballade upon a Wedding. Sir John Suckling. 8°. (Lane, 1901. «F. of P.') 9. ) The Rime of tin Auatnt Mariner. S. T. Coleridge. 8°. (Gay and Bird, 1900.) 6. PHILIP CONNARD. The Statue and the Bust. Robert Browning. 8*. (Lane, 1900. ' Flowers of Parnassus.1) 9 illust. Marpessa. Stephen Phillips. 8s. (Lane, 1900. «F. of P.") 7. PHILIP CONNARD. The Statue and the Bust. Robert Browning. 8*. (Lane, 1900. ' Flowers of Parnassus.1) 9 illust. The Statue and the Bust. Robert Browning. 8*. (Lane, 1900. ' Flowers of Parnassus.1) 9 illust. Marpessa. Stephen Phillips. 8s. (Lane, 1900. «F. of P.") 7. 1900. ' Flowers of Parnassus.1) 9 illust. Marpessa. Stephen Phillips. 8s. (Lane, 1900. «F. of P.") 7. f ) Marpessa. Stephen Phillips. 8s. (Lane, 1900. «F. of P.") 7. ENGLISH BOOK-ILLUSTRATION 84 WALTER CRANE. The New Fortst. J. R. Wise 40. (Smith, Elder, 1863.) 63 illust. engraved by W. J. Linton. (A new edition, pub- lished by Henry Sotheran, 1883, with the original illust. and 12 etchings by Hey wood Sumner.) g y y ) Storiesfrom Menu/. Mrs. De Haviland. 12°. (William Hunt, 1864.) 6. ) Walter Cram's Toy-Books. Issued in single numbers, from 1865- 1876. CtlltSti Editions, all published in 4% by George Routledgc, and printed throughout in colours. fralttr Crane's Piaurt Book. (1874.) 64 pp. Tbt Marquis of Carahas' Piaurt Book. (1874.) 64 pp. Tht Blut Btard Piaurt Book. (1876.) 32 pp. Song of Sixftnct Toy-Book. (1876.) 32 pp. Chattering Jack's Piaurt Book. (1876.) 32 pp. Tbt Thru Bears Piaurt Book. (1876.) 32 pp. Aladdin's Piaurt Book. (1876.) 24 pp. pp Tbt Magic of Kindntss. H. and A. Mayhew. 8°. (Cassell, Pctter and Galpin, 1869.) 8. p , Sunny Days, or a Month at tht Great Stewt. Author of ' Our White Violet.1 8°. (Griffith and Farran, 1871.) 4, in colours. Our Old Unclt's Homt. * Mother Carey.' 8°. (Griffith and Farran, 1871.) 4, Our Old Unclt's Homt. * Mother Carey.' 8°. (Griffith and Farran, 1871.) 4, Tbt Htadof tbt Family. Mrs. Craik. 8°. (Macmillan, 1875.) 6. Agatha's Husband. Mrs. Craik. 8°. (Macmillan, 1875.) 6. Tht Sluivtr of Lovt. A Collection of Valentines, Ancient and Mo3era. 40. (Marcus Ward, 1876.) Illust. in colour by Walter Crane and Kate Greenaway. Tbt Htadof tbt Family. Mrs. Craik. 8 . (Macmillan, 1875.) 6. Agatha's Husband. Mrs. Craik. 8°. (Macmillan, 1875.) 6. Tht Sluivtr of Lovt. A Collection of Valentines, Ancient and Mo3era. 40. (Marcus Ward, 1876.) Illust. in colour by Walter Crane and Kate Greenaway. g , Tht Sluivtr of Lovt. A Collection of Valentines, Ancient and Mo3era. 40. (Marcus Ward, 1876.) Illust. in colour by Walter Crane and Kate Greenaway. y Carrots. Mrs. Molesworth. 8°. (Macmillan, 1876.) 7. Songs of Many Seasons. Jemmett Browne. 4°. (Simpkin, Marshall, Pewtress, 1876.) Illust. by Du Maurier, Walter Crane, C. W. Morgan. 1 by Walter Crane. 0 , g y Tbt Baby's Optra. 40. (Routledge, 1877.) 11, and illustrative decorations on every page. In colours. Tbt Baby's Optra. 40. (Routledge, 1877.) 11, and illustrative decorations on every page. In colours. Tbt Cuckoo Clock. Mrs. Molesworth. 8°. (Macmillan, 1877.) 7. Grandmother Dear. Mrs. Moles worth. 8°. (Macmillan, 1878.^ 7. Tht Taptttry Room. Mrs. Molesworth. 8°. ) Tht Happy Princty and other tales. Oscar Wilde. 40. (Nutt, 18880 Illustrated by Walter Crane and Jacomb Hood. 3 by Walter Crane. ENGLISH BOOK-ILLUSTRATION ENGLISH BOOK-ILLUSTRATION 86 Tht Bo»k if Wedding Dayt. Quotations for every day in the Tht Bo»k if Wedding Dayt. Quotations for every day in the year, compiled by K. E. J. Reid, etc +°. (Longmans, 1889.) Tht Bo»k if Wedding Dayt. Quotations for every day in the year, compiled by K. E. J. Reid, etc +°. (Longmans, 1889.) Tht RtBtry Children. Mrs. Molesworth. 8°. (Macmillan, 1889.) 7. 0 year, compiled by K. E. J. Reid, etc + . (Longmans, 1889.) Tht RtBtry Children. Mrs. Molesworth. 8°. (Macmillan, 1889.) 7. ) Fltrcts Feast. A Masque of Flowers. Walter Crane. 40. (Catsell, 1889.) 40 pages of pictures, with text in artist's manuscript. In colours. p Tht TttrtU Dm/s Ntst. 8°. (Routledge, 1890.) 87 illust. by Walter Crane, W. McConncll, Harrison Weir and other artists. Renascence. A Book of Verse. Walter Crane. Including •The Sirens Three' and * Flora's Feast.' 40. (Elkin Mathews, 1891.) 39 designs, some engraved on wood by Arthur Leverett. A Wendtr Bttk fir Girls and Beys. Nathaniel Hawthorne. (Osgood, McHvaine, 1892.) 60 designs in colours. ( g , , ) g The Tempest. 8 illust to Shakespeare's ' Tempest.' Engraved and printed by Duncan C Dallas. (Dent, 1893.) p y Tin Gentlemen if Vtrma. 8 illust. to Shakespeare's *Two Gentlemen of Verona.' Engraved and printed by Duncan C. Dallas. (Dent, 1894.) 0 ( , ) Tht Sttrj if thi Glittering Plain. William Morris. 40. (Kelmscott Press. 1894.) 23 illust. by Walter Crane. Borders, titles and initials by William Morris. , y Tht His fry of Reynard tht Fix. English Verse by F. S. Ellis. 4°. (David Nutt, 1894.) Frontispiece and devices. Abo an edition in 1897. 8°. Merry Wives if Windsir. 8 illust. to Shakespeare's ' Merry Wives of Windsor.' Engraved and printed by Duncan C. Dallas. 40. (George Allen, 1894.) ( g , ) Tht Visim ifDanU. Miss Harrison. 8°. 1894. 4. Tht Fatrit Qmttue. In 3 volumes. Edited by Thomas J. Wise. 40. (George Allen, 1895.) 90, and headings and tailpieces to each Canto. p ^nttn Swnwury or the Tourney of the Lily and the Rose. Walter Crane. 40. (Cassell, 1896.) 40 pages of pictures with text in artist's manuscript. In colours. p Tht Shtphtarfs Calendar. Edmund Spenser. 4°. (Harper and Bros., 1898.) iSLand other devices. , ) Trtplttst comprising 'The Baby's Opera,' 'The Baby's Bouquet,' and 'The Baby's Own jEsop.' Walter Crane. ENGLISH BOOK-ILLUSTRATION (Macmillan, 1879.) 7. y p g Tbt Cuckoo Clock. Mrs. Molesworth. 8°. (Macmillan, 1877.) 7. Grandmother Dear. Mrs. Moles worth. 8°. (Macmillan, 1878.^ 7. Tht Taptttry Room. Mrs. Molesworth. 8°. (Macmillan, 1879.) 7. 0 ) The Baby's Bouquet. 40. (Routledge, 1879.) 11, and illustrative decorations on every page In colours. OF TO-DAY. 85 A Christmas Child. Mrs. Moles worth. 8°. (Macmillan, 1880.) 7. A Christmas Child. Mrs. Moles worth. 8°. (Macmillan, 1880.) 7. Tbi Necklace of Princtss Fiorimmdt. Mrs. De Morgan. 8°. (Macmillan, 188a) 8, and devices. ( , ) , Herr Baby. Mrs. Molesworth. 8°. (Macmillan, 1881.) 7. Also a 40 edition with 12. Tht First of May. A Fairy Masque. Walter Crane. Fol. (Henry Sotheran, 1881.) 5a designs. Text in artist's manuscript p Household Stories. Translated from the German of the Brothers Grimm by Lucy Crane. 8°. (Macmillan, 1882.) 11, and illustrative head and tailpieces and initials to each story. p y Rosy. Mrs. Molesworth. 8°. (Macmillan, 1882.) 7. y ( , ) Christmas Trtt Land. Mrs. Molesworth. 8°. (Macmillan, 1884.) 7. ) Walttr Crane's New Series of Pitlure Books. 40. (Marcus Ward, 1885-6.) ) Slate and Pendhania.—Little Quen Ann*.—Pothooks and Perseverance. 24 pages each, printed in colours. p g , p The Golden Primer. ]. M. £>. Meiklejohn. 8°. (Blackwood, 1885.) Part I. ana Part IL 14 decorative pages in colours in each part. p Folk and Fairy Tales. C. C. Harrison. 8°. (Ward and Downey, 1885.) 24. y, ) uUs." Mrs. Molesworth. 8°. (Macmillan, 1885.) 7. 0 , The Sirens Three. Walter Crane. 40. (Macmillan, 1886.) 41 pages of pictures, with text in artist's manuscript. 0 The Baby's Own Msop. 40. (Routledge, 1886.) 56 pages of pi&ures and text in artist's manuscript. In colours. Echoes of Hellas. The Tale of Troy and the Story of Orestes from Homer and Aeschylus. With introductory essay and sonnets by Prof. George C. Warr. Fol. (Marcus Ward, 1887.) 02 illustrative decorations. ) Four Winds Farm. Mrs. Molesworth. 8°. (Macmillan, 1887.) 7. ) Legends for Lionel 4°. (Cassell, 1887.) 40 pages of pictures, text in artist's manuscript In colours. p A Christmas Posy. Mrs. Molesworth. 8°. (Macmillan, 1888.) 7. 0 ) Tht Happy Princty and other tales. Oscar Wilde. 40. (Nutt, 18880 Illustrated by Walter Crane and Jacomb Hood. 3 by Walter Crane. ENGLISH BOOK-ILLUSTRATION (Routledge, 1899.) Printed by Edmund Evans. 40. OF TO-DAY. OF TO-DAY. 87 A Floral Fantasy, in an Old English Garden. Walter Crane. 8°. (Harper, 1899.) 40 pages of pictures with text in artist's manuscript. A Floral Fantasy, in an Old English Garden. Walter Crane. 8°. (Harper, 1899.) 40 pages of pictures with text in artist's manuscript. p H. GRAHVILLE FELL. Our Laifs Tumbltr. A Twelfth Century legend transcribed for Lady Day, 1894. 40. (Dent, 1894.) 4 Must. Wagners Heroes. Constance Maud. 8°. (Arnold, 1895.) 8. Alt Baba and tht Forty Tbievts. 32*. (Dent, 1895. Banbury Cross Series.) 36. y y, ( , ) Wagners Heroes. Constance Maud. 8°. (Arnold, 1895.) 8. Alt Baba and tht Forty Tbievts. 32*. (Dent, 1895. Banbury Cross Series.) 36. ) Tht Fairy Gifts and Tom Hickatbrift. 32*. (Dent, 1895. Banbury Cross Series.) 36. y ) The Book of Job. 40. (Dent, 1896.) 22. 0 f ( , ) The Song of Solomon. 40. (Chapman and Hall, 1897.) 12, and decorations. Wonder Stories from Herodotus. Re-told by C. H. Boden and W. Barrington D*Almeida. 8*. (Harper and Brothers, 1900.) 12, and decorations. A. T. GASKIN. Wonder Stories from Herodotus. Re-told by C. H. Boden and W. Barrington D*Almeida. 8*. (Harper and Brothers, 1900.) 12, and decorations. A. A A. T. GASKIN. A Book of PiOurtd Carols. Designed by members of the Birmingham Art School under the dire£bon of A. J. Gaskin. 4°. (George Allen, 1893.) IDust. by C. M. Gere. A. J. Gaskin, Henry Payne, Bernard Sleigh, Fred. Mason,and othen. Stwits and Fairy Tales. Hans Andersen. 8°. (George Allen, ) A Book of Fairy Tales. Re-told by S. Baring Gould. 8°. (Methuen, 1894/) 20. ( et ue , 89 /) 0. Good King Wenceslas. Dr. Neak. 4°. (Cornish Brother*, Birmingham, 1895.) 6. g a , ) The Shefhearis Calendar. 8°. (Kelmscott Press, 1896.) 12. C. M.GBSB. C. M.GBSB. Russian Fairy Tales. R. Nisbet Bain. 8*. (Lawrence and Bullen, 1893.) ° iamt- Bullen, 1893.) Th* Imitation of Christ. Thomas i Kempis. Introdudion by F. W. Farrar. 8°. (Methuen. 1894.) $. ( ) $ A Book ofPiOurtd Carols. SeeA.J. Gaskin. J. J.GuTHaa. J J tFtddmg BtUt. A new old Nursery Rhyme by A. F. S. and (Simpkin, Marshall, 1895.) E. de Passemore. 4*. (Simpkin, Marshall, 1895.) 7 iOust. ThtGardmofTtmt. Mrs. Davidson. 8°. (JarroU and Sons, 1896.) 38. ) An Album of Drawings. FoL (The White Cottage, Shorne, Kent, 1900.) 20 from various magazines. ENGLISH BOOK-ILLUSTRATION 88 LAURENCE HOUSMAN. Jump-to-Glory Jane. George Meredith. 8°. (Swan, Sonnen- schein, 1892.) 44 designs and text in artist's manuscript. Goblin Marktt. Christina Rossetti. 8°. (Macmillan, 1093.) Title-page, decorations and 12. p g , Weird Taus from Northern Sias. From the Danish of Jonas Lie. 8°. (Kegan Paul, 1893.) 12. ( g , ) Th* End of Elfin-town. Jane Barlow. 8°. (Macmillan, 1894.) Title-page, decorations and 8. p g , A Farm in Fairyland. Laurence Housman. 8°. (Kegan Paul, 1894.) Frontispiece, title-page and 12. , p , p g Th* House of Joy. Laurence Housman. 8°. (Kegan Paul, 1895.) Frontispiece, title-page and 8. ) p , p g Green Arras. Laurence Housman. 8°. (Lane, 1896.) Frontispiece, title-page and 4. p , p g All-Fellows. Laurence Housman. 8°. (Kegan Paul, 1896.) 7. The Were-Wolf. Clcmence Housman. 8°. (Lane, 1896.) 6. 0 f ( , ) Tht Sensitive Plant. P. B. Shelley. 40. (Aldine House, 1808.) 12, in photogravure. ) , p g Tht Field of Clover. Laurence Housman. 8°. (Kegan Paul, 1898.) Frontispiece, title-page and 10, engraved by Clcmence Housman. Of tht Imitation of Christ. Thomas a Kempis. 8°. (Kegan Paul, 1898.) 5. , ) Tht Little Land. Laurence Housman. 8°. (Grant Richards, 1899.) 4. A. GARTH JONES. Th* Minor Poems of John Milton. 8°. (Bell, 1898. Endymion Series.) 46 illust., and decorations. CELIA LKVETUS. Verse Fancies. Edward L. Levetus. 8°. (Chapman and Hall, 1898.) 7 illust. 1898.) 7 illust. Songs 0/ Innocence. William Blake. 320. (Welb, Gardner, and Darton, 1899.) 14. , W. B. MACDOUGALL. Chronicles of Strathearn. 8°. (David Philips, 1896.) 15 illust. Th* Fall of the Nibelungs. In Two Books. Translated by Margaret Armour. 8°. (Dent, 1897.) 8 in each book. Thames Sonnets and Semblances. Margaret Armour. 8°. (Elkin Mathews, 1897.) 12. Th* Book of Ruth. Introduction by Ernest Rhys. 40. (Dent, 1896.) 8. W. B. MACDOUGALL. Chronicles of Strathearn. 8°. (David Philips, 1896.) 15 illust. Th* Fall of the Nibelungs. In Two Books. Translated by Margaret Armour. 8°. (Dent, 1897.) 8 in each book. Thames Sonnets and Semblances. Margaret Armour. 8°. (Elkin Mathews, 1897.) 12. W. B. MACDOUGALL. Chronicles of Strathearn. 8°. (David Philips, 1896.) 15 illust. Th* Fall of the Nibelungs. In Two Books. Translated by Margaret Armour. 8°. (Dent, 1897.) 8 in each book. g ( , ) Thames Sonnets and Semblances. Margaret Armour. 8°. (Elkin Mathews, 1897.) 12. ( , ) Th* Book of Ruth. ENGLISH BOOK-ILLUSTRATION Introduction by Ernest Rhys. 40. (Dent, 1896.) 8. OF TO-DAY. 89 Isabella, or tht Pet of Basil. John Keats. 40. (Kegan Paul, 1898.) 8. The Shadow of Love and ether Pterns. Margaret Armour. 8°. (Duckworth, 1898.) 2. A Book of PiBurtd Carols. See A. J. Gas kin 0 A Book of PiBurtd Carols. See A. J. Gas kin. The Story of Alexander. Robert Steele. 40. (David Nutt, 1894.) 5 Must, and headings. The Story of Alexander. Robert Steele. 40. (David Nutt, 1894.) 5 Must, and headings. ) , g Hum of Bordeaux. Robert Steele. 8°. (George Allen, 1895.) 6, and headings. , g Renaud of Mentauban. Robert Steele. 8°. (George Allen, 1897.) 9- The Centaur. The Bacchant. Translated from the French of Maurice de GueYin by T. Sturge Moore. (Vale Press, 1899.) 40. 4 wood engravings. ) g g Same Fruits ef Solitude. William Penn. 8°. (Essex House Press, 1901.) Wood engraving on title-page. L. FAIRFAX MUCKLEY. The Faerie Queen*. Introduction by Prof. Hales. 40. (Dent, 1897.) 3 vols. Frontispiece, many decorations and 24. HENRY OSPOVAT. Shakespeare's Sonnets. 8°. (Lane, 1899.) 10 illust. Poems. Matthew Arnold. 8°. Edited by A. C. Benson. (Lane, 1900.) 16, and illustrative decorations. Shakespeare s Sonnets. 8 . (Lane, 1899.) 10 illust. Poems. Matthew Arnold. 8°. Edited by A. C. Benson. (Lane, 1900.) 16, and illustrative decorations. ( , ) , CHARLES RICKSTTS. A House ef Pomegranates. Oscar Wilde. 40. (Osgood, Mcllvaine, 1891.) 13 illust. by Charles Ricketts, 4 by C. H. Shannon. Poems, Dramatic and Lyrical. Lord de Tabley. 8°. (Mathews and Lane, 1893.) 5. Dapbnis and Cblot. Longus. Translated by Geo. Thornley. 4°. (Mathews and Lane, 1893.) 37 woodcuts drawn on die wood by Charles Ricketts from the designs of Charles Ricketts and Charles Shannon. Engraved by both artists. Ibe Sphinx. Oscar Wilde. 40. (Printed at the Ballantyne Press, 1894.) 9. Hero and Leandtr. Christopher Marlowe and George Chapman. 8°. (Vale Press, 1894.) 7, designed and engraved on wood by Charles Ricketts and Charles Shannon. Nymtbidia and the Musts Elixium. Michael Drayton. 8°. (Vale Press, 1896.) Frontispiece engraved on wood. CHARLES RICKSTTS. A House ef Pomegranates. Oscar Wilde. 40. (Osgood, Mcllvaine, 1891.) 13 illust. by Charles Ricketts, 4 by C. H. Shannon. A House ef Pomegranates. Oscar Wilde. 4 . (Osgood, Mcllvaine, 1891.) 13 illust. by Charles Ricketts, 4 by C. H. Shannon. Poems, Dramatic and Lyrical. Lord de Tabley. 8°. ENGLISH BOOK-ILLUSTRATION (Mathews and Lane, 1893.) 5. Poems, Dramatic and Lyrical. Lord de Tabley. 8°. (Mathews and Lane, 1893.) 5. Dapbnis and Cblot. Longus. Translated by Geo. Thornley. 4°. (Mathews and Lane, 1893.) 37 woodcuts drawn on die wood by Charles Ricketts from the designs of Charles Ricketts and Charles Shannon. Engraved by both artists. 0 g y Ibe Sphinx. Oscar Wilde. 40. (Printed at the Ballantyne Press, 1894.) 9. , Hero and Leandtr. Christopher Marlowe and George Chapman. 8°. (Vale Press, 1894.) 7, designed and engraved on wood by Charles Ricketts and Charles Shannon. y Nymtbidia and the Musts Elixium. Michael Drayton. 8°. (Vale Press, 1896.) Frontispiece engraved on wood. ENGLISH BOOK-ILLUSTRATION 9o Mlion's Early Poems. 8°. (Vale Press, 1896.) Frontispiece engraved on wood. Mlion's Early Poems. 8°. (Vale Press, 1896.) Frontispiece engraved on wood. g The Excellent Narration of the Marriage of Cupid* and Psyches. Translated from the Latin of Lucius Apuleius, by William Adlington. 8°. (Vale Press, 1897.) 6 wood engravings. g ( , ) g g The Book of Thel, Songs of Innocence and Songs of Experience. William Blake. 40. (Vale Pres3, 1897.) Frontispiece, en- graved on wood. 0 g Blake's Poetical Sketches. 40. (Vale Press, 1899.) Frontis- piece engraved on wood. HBNRY RYLAND. Holy Gladness. Words by Edward Oxenford and Music by Sir John Stainer. 40. (Griffith, Farran, 1889.) 8 illust. in colour by Henry Ryland, and illust. by Louis Davis and others. REGINALD SAVAGE. Der Ring des Nibelungen. Described by R. Farquharson Sharp. 40. (Marshall, Russell, 1898.) 5 illust. ( , , ) CHARLES SHANNON. See Charles Ricketts. 1 House of Pomegranates,' c Hero and Leander,' • Daphnis and Chloe.' See Charles Ricketts. 1 House of Pomegranates,' c Hero and Leander,' • Daphnis and Chloe.' BYAM SHAW. Poems by Robert Browning. 8°. (Bell, 1897.) 20 illust. Talis from Boccaccio. Joseph Jacobs. 40. (George Allen, 1899.) 20. ) The Predicled Plagut, etc Hippocrates Junior. 8°. (Simpkin, Marshall, 1900.) Many illust. (chiefly vignettes) by Byam Shaw and others. The Chiswick Shakespeare. 8°. (Bell, 1899, etc.) 6, and decorations in each volume. BERNARD SLEIGH. The Sea-King's Daughter, and other Poems. Amy Mark. Printed at the Press of the Birmingham Guild of Handicraft. (G. Napier, Birmingham. Tylston and Edwards, and A. P. Marston, London, 1895.) 4 illust and 35 decorated pages, engraved by the designer and L. A. Talbot. g y g A Book of PiQured Carols. See A. J. Gaskin. f Q HEY WOOD SUMNER. The Avon from Naxbj to Tewkesbury. FoL (Sceley, Jackson and Halb'day, 18820 2 I etchings. Cinderella : A Fairy Opera. John Farmer and Henry Leigh. f Q HEY WOOD SUMNER. The Avon from Naxbj to Tewkesbury. FoL (Sceley, Jackson and Halb'day, 18820 2 I etchings. and Halb day, 18820 etchings. Cinderella : A Fairy Opera. John Farmer and Henry Leigh. OF TO-DAY. 91 4°. (Harrow, J. C. Wilbee; London, Novcllo, Ewer, 1882.) 17 illust. Sintram and his Companions. Translated from the German of De la Motte Fouque". 40. (Seeley, Jackson and Halliday, 1883.) Frontispiece and 21. ) p The New Forest. J. R. Wise. ENGLISH BOOK-ILLUSTRATION See Walter Crane. 0 Undine. 40. (Chapman and Hall, 1888.) 16. The Besom Maker, and other country Folk Songs. Collected by Heywood Sumner. 40. (Longmans, 1888.) Frontb- piece and 25 decorated pages, with text in artist's manuscript. p p g p Jacob and the Raven. Frances M. Peard. 8°. (George Allen, 1896.) 15, and illustrative decorations. J. R. WEGUELIN. Lays of Ancient Rom*. Lord Macaulay. 8°. (Longmans, 1881.) 41 illust. Anacreon: with Thomas Stanley's translation. Edited by A. H. Bullen. 8°. (Lawrence and Bullen, 1892.) 11. The Little Mermaid and other Stories. Hans Andersen. Trans- lated by R. Nisbet Bain. 40. (Lawrence and Bullen, 1893.) 61. Catullus : with tht Pervigilium Veneris. Edited by S. G. Owen. 8°. (Lawrence and Bullen, 1893.) 8- The Wooing of Malkatoon. Commodus. Lewis Wallace. 8°. (Harper and Bros., 1898.) Illustrated by J. R. Weguelin and Du Mond. 6 to Commodus by J. R. Weguelin. J. R. WEGUELIN. Lays of Ancient Rom*. Lord Macaulay. 8°. (Longmans, 1881.) 41 illust. Anacreon: with Thomas Stanley's translation. Edited by A. H. Bullen. 8°. (Lawrence and Bullen, 1892.) 11. J Lays of Ancient Rom*. Lord Macaulay. 8°. (Longmans, 1881.) 41 illust. ) Anacreon: with Thomas Stanley's translation. Edited by A. H. Bullen. 8°. (Lawrence and Bullen, 1892.) 11. The Little Mermaid and other Stories. Hans Andersen. Trans- lated by R. Nisbet Bain. 40. (Lawrence and Bullen, 1893.) 61. Catullus : with tht Pervigilium Veneris. Edited by S. G. Owen. 8°. (Lawrence and Bullen, 1893.) 8- The Wooing of Malkatoon. Commodus. Lewis Wallace. 8°. (Harper and Bros., 1898.) Illustrated by J. R. Weguelin and Du Mond. 6 to Commodus by J. R. Weguelin. y g PATTEN WILSON. Miracle Plays. Our Lord's Coming and Childhood. {Cathe- rine Tynan Hinkson. 8°. (Lane, 1895.) 6 illust. A Houseful of Rebels. Walter C. Rhoades. 8°. (Archibald Constable, 1897.) I 0- SeUftions from Coleridge. Andrew Lang. 8°. (Longmans, 1898.) 18. King John Edited by J W Young 8° (Longmans 1899 PATTEN WILSON. Miracle Plays. Our Lord's Coming and Childhood. {Cathe- rine Tynan Hinkson. 8°. (Lane, 1895.) 6 illust. A Houseful of Rebels. Walter C. Rhoades. 8°. (Archibald C t bl 1897 ) I 0 PATTEN WILSON. Miracle Plays. Our Lord's Coming and Childhood. {Cathe- rine Tynan Hinkson. 8°. (Lane, 1895.) 6 illust. Miracle Plays. Our Lord's Coming and Childhood. {Cathe- rine Tynan Hinkson. 8°. (Lane, 1895.) 6 illust. A Houseful of Rebels. Walter C. Rhoades. ENGLISH BOOK-ILLUSTRATION 8°. (Archibald Constable, 1897.) I 0- SeUftions from Coleridge Andrew Lang 8° (Longmans y ( , ) A Houseful of Rebels. Walter C. Rhoades. 8°. (Archibald Constable, 1897.) I 0- A Houseful of Rebels. Walter C. Rhoades. 8 . (Archibald Constable, 1897.) I 0- SeUftions from Coleridge. Andrew Lang. 8°. (Longmans, 1898.) 18. , ) SeUftions from Coleridge. Andrew Lang. 8°. (Longmans, 1898.) 18. ) King John. Edited by J. W. Young. 8°. (Longmans, 1899. Swan Shakespeare.) 9. p ) PAUL V. WOODROFFE. PAUL V. WOODROFFE. Songs from Shakespeare1 s Plays. Edited by E. Rhys. 4° (Aldine House, 1898.) 12 illust. The Little Flowers of St. Francis of Assist. 8°. (Kegan Paul, 1899.) 8. The Confessions »/ St. Augustine. 8°. (Kegan Paul, 1900.) Title-page by Lawrence Housman, Ulua. by P. V. Woodxoffie. Engraved upon wood by Miss Housman. PAUL V. WOODROFFE. Songs from Shakespeare1 s Plays. Edited by E. Rhys. 4° (Aldine House, 1898.) 12 illust. Songs from Shakespeare1 s Plays. Edited by E. Rhys. 4° (Aldine House, 1898.) 12 illust. The Little Flowers of St. Francis of Assist. 8°. (Kegan Paul, 1899.) 8. ( , ) The Little Flowers of St. Francis of Assist. 8°. (Kegan Paul, 1899.) 8. ) The Confessions »/ St. Augustine. 8°. (Kegan Paul, 1900.) Title-page by Lawrence Housman, Ulua. by P. V. Woodxoffie. Engraved upon wood by Miss Housman.
https://openalex.org/W2060971566
https://europepmc.org/articles/pmc3848693?pdf=render
English
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Inconsistency in the items included in tools used in general health research and physical therapy to evaluate the methodological quality of randomized controlled trials: a descriptive analysis
BMC Medical research methodology
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11,605
* Correspondence: susanarmijo@gmail.com 1Postdoctoral Fellow, CLEAR (Connecting Leadership and Research) Outcomes Research Program, University of Alberta, 5-115A Edmonton Clinic Health Academy (ECHA), 11405 – 87 Avenue, Edmonton, Alberta T6G 1C9 Canada 2Faculty of Rehabilitation Medicine, Department of Physical Therapy, University of Alberta, 3-48 Corbett Hall, Edmonton T6G 2G4, Canada Full list of author information is available at the end of the article Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Open Access Inconsistency in the items included in tools used in general health research and physical therapy to evaluate the methodological quality of randomized controlled trials: a descriptive analysis Susan Armijo-Olivo1,2*, Jorge Fuentes2,3, Maria Ospina4, Humam Saltaji5 and Lisa Hartling6 Abstract Background: Assessing the risk of bias of randomized controlled trials (RCTs) is crucial to understand how biases affect treatment effect estimates. A number of tools have been developed to evaluate risk of bias of RCTs; however, it is unknown how these tools compare to each other in the items included. The main objective of this study was to describe which individual items are included in RCT quality tools used in general health and physical therapy (PT) research, and how these items compare to those of the Cochrane Risk of Bias (RoB) tool. Methods: We used comprehensive literature searches and a systematic approach to identify tools that evaluated the methodological quality or risk of bias of RCTs in general health and PT research. We extracted individual items from all quality tools. We calculated the frequency of quality items used across tools and compared them to those in the RoB tool. Comparisons were made between general health and PT quality tools using Chi-squared tests. Results: In addition to the RoB tool, 26 quality tools were identified, with 19 being used in general health and seven in PT research. The total number of quality items included in general health research tools was 130, Results: In addition to the RoB tool, 26 quality tools were identified, with 19 being used in general health and seven in PT research. The total number of quality items included in general health research tools was 130, compared with 48 items across PT tools and seven items in the RoB tool. The most frequently included items in general health research tools (14/19, 74%) were inclusion and exclusion criteria, and appropriate statistical analysis. In contrast, the most frequent items included in PT tools (86%, 6/7) were: baseline comparability, blinding of investigator/assessor, and use of intention-to-treat analysis. Key items of the RoB tool (sequence generation and allocation concealment) were included in 71% (5/7) of PT tools, and 63% (12/19) and 37% (7/19) of general health research tools, respectively. Conclusions: There is extensive item variation across tools that evaluate the risk of bias of RCTs in health research. Results call for an in-depth analysis of items that should be used to assess risk of bias of RCTs. Further empirical evidence on the use of individual items and the psychometric properties of risk of bias tools is needed. Background We conducted a previous systematic review that de- scribed which tools have been used to evaluate the methodological quality of RCTs in physical therapy (PT) research [5]. RCTs conducted in the area of PT have unique characteristics compared with pharmaco- logical trials. Because of the nature of PT treatments (e.g., manual therapy, exercises), RCTs assessing PT interventions are often complex [20], and diverse as- pects of their design (e.g., type and intensity of ther- apy, standardized or individually tailored approaches, therapists’ skills and experience) are likely to affect study results. It is unknown whether the tools to as- sess the quality of RCTs in PT differ from those used in general health research in terms of the items and type of bias they aim to address. The present study was designed to expand and update the analysis of our previous review [5] on quality tools for evidence synthesis. While the impact of trial bias on evidence synthesis has been largely recognized, the approaches to quality assessment have been inconsistent and controversial [5]. A wide variety of tools have been developed to evaluate RCT quality in different health areas.[5,6]; many of them have not been developed using scientifically rigorous methods nor have they been validated [5]. In addition, there is no agreement on the optimal tool to accurately assess trial quality. The use of different tools for evaluat- ing the quality of primary research in SRs can lead to discrepancies and skewed interpretations of SR results [7-9] and ultimately impact recommendations for clin- ical care. In 2008, The Cochrane Collaboration [10] intro- duced the Risk of Bias (RoB) tool as a way to address shortcomings associated with existing tools and methods for quality assessment in SRs. Individual RoB items were selected based on a growing body of empirical evidence quantifying the association be- tween certain characteristics related to the conduct of the trial and estimates of treatment effects [11-15]. For example, there is evidence that inadequate alloca- tion concealment or lack of double-blinding are likely to overestimate treatment effects by 18% and 9%, re- spectively [12,14,15]. The main objective of the present study was to de- scribe the frequency of individual items included in tools that assess RCT quality in general health and PT re- search, and how they compare to items included in the RoB tool [4]. Background tools and whether these items truly evaluate the likeli- hood of bias, as defined by The Cochrane Collaboration [10,16] and other criteria [17]. This would be an import- ant contribution for evidence synthesis. Randomized controlled trials (RCTs), and systematic re- views (SRs) and meta-analyses of these trials, are consid- ered the gold standard to evaluate the effectiveness of health care interventions. Results of these studies are crucial for informing the implementation of the best treatments to improve patient outcomes and the effi- ciency of the health care system. Evaluating the meth- odological quality of trials is an essential component of SRs as only the best available evidence should inform clinical and policy decisions. An accurate assessment of study quality is key for the synthesis and interpret- ation of results across studies to effectively guide health care [1]. Most of the studies that have evaluated the use of tools for quality assessment of RCTs [5,6,18,19] have not exhaustively assessed how these tools compare to each other in terms of their individual items and whether their use varies across different areas of health care research. For example, a recent study [19] examined the characteristics and methods of reviews assessing the quality of RCTs. While substantial vari- ation in the use of quality tools across reviews was identified, the study did not describe in detail which items were most frequently included in the tools. The present study was designed to refine the analysis of existing tools by conducting a more comprehensive search (i.e., no language restrictions, larger number of databases), describing the psychometric properties of the tools used in general health research, and com- paring the items included in these tools with the Cochrane RoB tool. The term “methodological quality” has evolved since its inception and involves the evaluation of the internal validity as well as the external validity of a given study [2,3]. Recently, The Cochrane Collaboration has lead a shift in the approach to quality assessment, in which the concept of trial quality is linked to the internal validity of the study, namely risk of bias [4]. However, there is still inconsistency among researchers on how study qual- ity is defined, and several terms have been used inter- changeably in the literature (i.e. quality assessment, methodological quality, risk of bias, critical appraisal, trial quality). Abstract Keywords: Bias, Methodological quality, Quality assessment, Critical appraisal, Risk of bias, Quality of reporting © 2013 Armijo-Olivo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Page 2 of 19 Design: observational, descriptive study Search strategy An update of a previous SR [5] on quality assessment tools was carried out to identify scales and their items used in the assessment of RCT quality in health and PT research. The updated search strategy incorporated key words identified by Dechartres et al. [19], with searches conducted from January 1st, 2007 to June 10, 2013 in the following bibliographic databases: Medline, Embase, Cinahl, ISI Web of Science, EMB Reviews-Cochrane Central Register of Controlled Trials and Cochrane Library and Best Evidence, All EBM Reviews -CDSR, ACP journal Club, DARE, CCTR, Global health, and HealthSTAR. Key words used in the search were: tool, critical appraisal, critical appraisal review, appraisal of methodology, appraisal of research methodology, re- search design review, quality assessment, methodological quality tool, RoB (tool), randomized (randomised) controlled trial, and RCT. Additionally, we manually searched the bibliographies of potentially relevant pa- pers. The search was not limited by language of publica- tion. For a sample search strategy, see Additional file 1. Second, two researchers independently extracted infor- mation on individual items used in the tools and the fre- quency of items across tools. Tools were categorized as relevant to PT if the authors specifically stated that the scale was developed for PT research, it was developed by a PT group, or if, according to Scopus searches, the tool was used in at least 5 PT reviews. Otherwise the tool was considered a general health research tool. One of the tools commonly used in both general health and PT research is the Jadad scale. This tool was included in both categories. Criteria for inclusion of studies in the review Studies were included if they described or used a newly developed tool to evaluate the methodological quality/ RoB of RCTs in any area of medical/health research and described any of its psychometric properties (i.e. validity, reliability, responsiveness). We excluded studies in which quality tools were developed for only one specific SR, studies that were not related to the development or psy- chometric testing of quality tools, and studies on generic tools that evaluated different types of research design (e.g., qualitative and quantitative studies). In addition, studies using modifications of existing tools were not considered for inclusion as they were likely not system- atically developed. The RoB tool [4,10] was known to be newly developed after our previous SR (2008), and was included prior to the updated search; however, we searched for manuscripts reporting psychometric prop- erties of the RoB tool. Items from the quality tools were grouped according to nine content categories that have been previously described [5]: 1) introduction, objectives, and design; 2) patient selection (inclusion and exclusion criteria, description of study participants); 3) assignment, ran- domization, and allocation concealment; 4) blinding; 5) interventions; 6) attrition, follow up and protocol deviations; 7) outcomes; 8) statistical analysis; and 9) miscellaneous. Background Secondary objectives were to 1) determine the nature of items included in general health and PT quality tools (i.e., evaluation of “conduct” versus “reporting”); 2) report on the psychometric properties of quality tools that have been formally evaluated; 3) deter- mine whether individual items in the tools relate to cer- tain threats to validity or precision [10,16,17] and 4) quantify the number of citations per tool, as a measure of usage since each tool’s inception and after inception of the RoB tool. In order to guide a proper assessment of study quality or risk of bias to inform decision-making, it is important to identify which items have been included in different Page 3 of 19 Page 3 of 19 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 items for inclusion, time to complete, scoring instruc- tions), and psychometric properties of the new tools. Information on face, content, construct, and concurrent validity, internal consistency, and reproducibility (intra and inter-rater reliability/agreement) was extracted. For this update, authors of original studies were not contacted to obtain additional information. The defini- tions of psychometric properties from Streiner and Norman [21-23] were used in the present study. Guide- lines developed by Terwee et al. [24] were used to define quality of measurement properties. Briefly, quality of measurement included internal (internal consistency, relevance of items and representativeness of items of the scale-content validity) as well as external components of validity (the relationship with other tests in a manner that is consistent with theoretically derived hypotheses- construct validity). Intra and inter-rater reliability (i.e. repeatability of measurements taken by the same tester at different times and repeatability of measurements taken by different testers, respectively) were also consid- ered. Definitions of psychometric properties for this review are provided in Additional file 2. Data screening Two reviewers independently screened abstracts and titles obtained from the database searches. The full text of potentially relevant articles was retrieved for further assessment. Disagreements were resolved by consensus. Methodological quality (conduct) and quality of repor- ting are two concepts that overlap to some degree; how- ever, they relate to different aspects of study quality. We defined methodological quality as “the confidence that the trial design, conduct, and analysis has minimized or avoided biases in its treatment comparisons” [6] (e.g., allocation concealment was appropriate). We defined quality of reporting as authors providing “information about the design, conduct and analysis of the trial,” [6] (e.g., method for concealing allocation was reported). Data extraction Data extraction was conducted in two phases. First, two researchers independently extracted information on content, construction, special features (e.g. area of development-clinical area-, number of items, selection of Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Page 4 of 19 Statistical Software: Version 12, 2012 (College Station, TX: StataCorp LP). Two researchers independently classified individual items based on whether they evaluated “reporting” and/ or “conduct” of the trial. Two researchers independently classified individual items based on whether they evaluated “reporting” and/ or “conduct” of the trial. Classifying quality items is a complex task due to un- clear description of items in the tools, lack of general agreement in bias definitions [25], and the need for em- pirical evidence linking these items to bias. Two re- searchers independently classified each item according to whether they potentially addressed threats to validity (i.e., selection bias, detection bias, performance bias, at- trition bias) or precision (Additional file 3). These cate- gorizations have been used in other relevant sentinel work [17,26-28]. Items that dealt with several threats to validity were classified as addressing multiple biases [29]. Reviewers considered each item by asking “What type of threats to validity or precision are addressed by a given item?” or “What do authors intend to capture with a given quality assessment item?” Thus, items were classi- fied into the threats to validity or precision that best repre- sented the concepts being addressed. We performed this task in duplicate and based on the guidelines established. The same type of analysis has been conducted previously for prognosis research [28]. Disagreements in item classifi- cation were resolved by consensus. Results The updated electronic searches identified 32,627 cita- tions. Manual searches identified four additional studies based on their titles and abstracts. After screening titles and abstracts, 154 articles were deemed potentially rele- vant. The application of the selection criteria resulted in 148 excluded studies. The main reasons for exclusion of studies were: 1) the study used a quality tool for which information on construction, development and/or psy- chometric properties was not available (n = 40); 2) the tool was already included in the original review (n = 39); 3) the study used a tool that was not specific for RCT quality assessment (n = 23); 4) the study used a modified tool already included in the review (n = 20); 5) the study used an instrument that was not a quality tool (n = 11); 6) the study used a tool developed for the purposes of a single review (n = 8); 7) the study focused on animal re- search (n = 4); 8) the study did not focus on a particular tool (n = 2); and 9) information on the name of the tool was not provided (n = 1). A list of excluded studies and reasons for exclusion is available in Additional file 4. The level of agreement for study selection between reviewers was excellent (kappa = 0.96). Tool citation h l Six manuscripts [31-36] reporting on four newly devel- oped tools met the eligibility criteria (Figure 1). These four new tools that evaluated the methodological qual- ity/RoB of RCTs in health research in addition to the RoB tool [4,10] were: the Cochrane Collaboration De- pression, Anxiety, And Neurosis (CCDAN) tool [34,35], the Randomized Controlled Trial Psychotherapy Quality Rating Scale (RCT-PQRS Tool) [32,33], 3) the Random- ized Controlled Trial -Natural Products Tool (RCT-NP) [31], and the CLEAR NPT (a checklist to evaluate the report of nonpharmacological trials) [36]. New PT- specific tools were not identified. The five tools were added to the 21 tools identified in our previous review [2] (i.e. Jadad [37], Maastricht [38], Delphi [39], PeDro [40,41], Maastricht-Amsterdam [42], Van Tulder [43], Bizzini [44], Chalmers [45], Reisch [46], Andrew [47], Imperiale [48], Detsky [49], Cho and Bero [50], Balas [51], Sindhu [52], Downs and Black [53], Nguyen [54], Oxford Pain Validity Scale (OPVS) [55], Arrive [56], CONSORT [57], and Yates [58]). Therefore, this update includes 26 quality tools. Details on the characteristics and psychometric properties of the new quality tools are presented in Tables 1 and 2. Each quality tool was tracked in the Scopus database to determine the number of times that the tool was cited since its original paper/citation. The number of citations per tool was tracked from January 1, 2007 to July 4, 2013 to describe recent uses of the tool and to ascertain whether the use of the tool declined after introduction of the RoB tool. The RoB tool was originally described in Chapter 8 of the Cochrane Handbook [10]. Since books and book chapters are not indexed in electronic databases, it was more challenging to track citations for the RoB tool. We tracked RoB citations using Google Scholar and the journal publication by Higgins et al. [4], that reported on the RoB tool. Analysis Data were summarized descriptively as the frequency of each item across quality tools, and within general health and PT research. Comparisons of items from PT and general health research tools with the RoB tool were also conducted. Comparisons between the proportion of in- dividual items used by PT tools and general health re- search tools were performed using Chi-squared or Fisher exact tests. The alpha level was set at α = 0.05. The level of agreement between reviewers for study selection and data extraction from quality tools was cal- culated using percentage agreement and the Kappa (κ) statistic [30]. Analyses were performed using Stata Most of the new tools have been tested for face and content validity (Table 2 and Additional file 5). Evalua- tions of other types of validity, such as criterion validity, have been conducted only for the RCT-NP and the RoB tool; however, the criterion used was a non-gold Page 5 of 19 Page 5 of 19 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Articles selected for full evaluation for inclusion in the review N = 154 Reasons for exclusions No info on construction, development and/or psychometric properties=40 -Tool included in original review=39 -Tool not specific for RCT assessment=23 -Modified existing tool= 20 -Not a quality assessment tool=11 -Tool developed for single review purposes=8 - Animal research= 4 -No focus on a particular tool=2 -Name of scale not reported=1 Duplicates removed = 5825 References selected for further examination of titles and abstracts N = 32,627 Tools identified in previous SR - N = 21 - PT = 7 - General = 14 New tools from update N = 5 Excluded N = 148 Total number of references identified from the updated electronic searches = 38,452 Included studies N = 6 Manual literature searches = 4 Total number of tools analyzed N = 26 Figure 1 PRISMA flow diagram for identification of studies. Excluded N = 148 Included studies N = 6 Total number of tools analyzed N = 26 gure 1 PRISMA flow diagram for identification of studies. standard tool (since to date, there is no accepted gold standard to evaluate the risk of bias or quality of RCTs in health research). Tool reproducibility has been evaluated for the CCDAN tool, the RCT-PQRS, and the RoB tool. Analysis The inter-rater reliability of the RoB tool was fair (k = 0.41- 0.60) in contrast to the CCDAN and RCT-PQRS tools which showed good inter-rater reliability (r = 0.75-0.86; intra class correlation coefficient [ICC] =0.76-0.79). quality items according to the nine content categories for general health versus PT tools were: introduction, objectives, and design: 8 versus 0 items; patient selec- tion: 18 versus 4 items; assignment, randomization, and allocation concealment: 8 versus 5 items; blinding: 12 versus 10 items; interventions: 17 versus 8 items; attri- tion, follow up and protocol deviation: 10 versus 9 items; outcomes: 15 versus 7 items; statistical analysis: 31 versus 5 items, and miscellaneous: 11 versus 0 items. Items from all 26 tools were summarized according to their frequency of use. The level of agreement between reviewers for item categorization in both PT (kappa = 0.92) and general health research tools (kappa = 0.98) was very good to excellent. Frequency of items: General health research tools, physical therapy tools and RoB tool Items addressing inclusion and exclusion criteria and the appropriateness of statistical analysis were the most fre- quently used among the general health research tools (74%, 14/19 tools). The second most commonly used items in general health research tools were description of withdrawals and drop outs, description and appropri- ateness of randomization process, blinding of investiga- tors/assessors, and description of treatment protocol for both intervention and control groups (63%, 12/19 tools). 4 item total and the mnibus item (item 25) was 88. he correlation between the 4-item total and study year as 0.51, significant at P < 001. he correlation of the mnibus item and study year as 0.47 (P < .0001). h ( T v o 1 r ols to measure methodological quality/risk of bias Of the 26 tools, 19 have been used in general health re- search and seven in PT research (including the Jadad scale, which is commonly used in both research areas). A total of 130 items have been used across general heath research tools compared with 48 items used in PT tools. The RoB tool has 6 domains with 7 items in total. Additional files 5 and 6 provide a detailed description of individual items contained in the tools. The numbers of In contrast, baseline comparability, blinding of investi- gator/assessor, and use of intention-to-treat analysis g uence ared with tion (k=0.79) allocation mpared with 0.73) [13] T m (m a tw T w o a s ow for the ween the f bias ome data t (t 0.02) were s r Time to complete Guidel for use availab hrough among was =0.75- 15-20 minutes No al items ll 23 n 0.51 s n 8, s % m % bility of the total o 24 items was 0.76 e of the individual individual ICCs be and 0.8 (items 2, 4, 14, 15, and 19). lve items had indivi between 0.3 and 0 ms 1, 3, 5, 9, 11, 12, 1 8 20 and 24) and bility of the total o 24 items was 0.76 e of the individual individual ICCs be and 0.8 (items 2, 4, 14, 15, and 19). lve items had indivi between 0.3 and 0 ms 1, 3, 5, 9, 11, 12, 1 8 20 and 24) and dity Re e, content and construct dity Re co 3 hi 0. res from raters correlated hly with year of lication (r=0.37-0.6) Re w Th ite to In m al to Cronbach α for all 25 ms as rated by the primary r was 0.87. Th re om of correlation between the l d h Th dity R e, content and construct dity Re co 3 h 0. res from raters correlated hly with year of lication (r=0.37-0.6) Re w Th it to In m al to Cronbach α for all 25 ms as rated by the primary r was 0.87. Th re o o correlation between the Th R nd construct R c 3 h 0 ers correlated r of p and mbled. n to s trials from ssential critical n item the study participants investigators was asse A systematic review regarding tools used i evaluate quality of NP was performed. Items all of these tools were compiled To be designated as e to include in the new appraisal instrument, a wal risk and and In m o m [6 en adad cation T a th 0 e No concealmen low [60]. tura p ated antial to 0.76) e of ement elective and 6) for [13]. monst o subst es 0.56 on thre s [59]. er agre 0) for s porting ect (0.8 eneration he RoB de oderate t mean valu greement welve item he interrat as fair (0.4 utcome re most perf quence g he No Content validity: items were included based on empirical evidence. In in ris sl re (κ Concurrent validity: A high degree of correlation was found between the domains wal risk and nd In m o m [6 en adad cation T a t 0 and the Jadad withdra item, risk of bias overa and total Jadad score, risk of bias overall risk Schulz allocation concealment [13] The correlations betwe overall risk of bias assessments and total score (t= 0.04) and allo l t (t 0 02) http://www.biomedcentral.com/1471 2288/13/116 CI 0.20–0.81)[61] There was very poor agreement between the Effective Public Health Practice Project Quality Assessment Tool (EPHPP) and the RoB tool in the final grade assigned to each study (kappa = 0.006)[61] The inter-rater reliability was substantial for sequence generation (k=0.79) and fair for the other 5 items (k=0.24- 0.37). Interrater reliability between consensus evaluations across rater pairs was fair for allocation concealment and “other sources of bias” (k=0.37-0.27), and moderate for sequence generation (k=0.60). [62] natural products, NPT natural products trials, RCT randomized controlled trial, atura Page 10 of 19 Page 10 of 19 Armijo-Olivo et al. p BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Table 2 Summary of the quality of measurement properties of quality tools from our previous systematic review and this update Scale Internal consistency Face validity Content validity Criterion Validity* Construct validity Reproducibility (agreement/reliability) TOOLS FOR PT Jadad Tool [37] - + + + + + Maastricht Tool [38] - + - + - + Delphi Tool [39] - + + + - + PEDro Tool [40,41] - + + - - + Maastricht-Amsterdam Tool [42] - + + - - + Van Tulder Tool [43] - + + + - + Bizzini tool [44] - + + - - + TOOLS FOR GENERAL HEALTH RESEARCH Tools from previous systematic review Chalmers tool [45] - + + + - + Reisch Tool [46] - + - + - - Andrew tool [47] - + - - - + Imperiale tool [48] - + - + - - Detsky tool [49] - + - + - + Cho Tool [50] - + - + - + Balas tool [51] - + - - - - Sindhu tool [52] - + + + - + Downs and Black tool [53] + + + + - + Nguyen tool [54] - + - - - - Oxford pain validity tool [55] - + - - - - Arrive tool [56] - + - - - + CONSORT tool [57] - + + - - + Yates Tool [58] - + + - + + New tools identified in search update Cochrane Collaboration Depression, Anxiety, and Neurosis (CCDAN) [34,35] + + + - + + The Randomized Controlled Trial Psychotherapy Quality Rating Tool (RCT-PQRS Tool) [32,33] + + + - + + RCT-Natural Products Tool (RCT-NP) [31] - + + + - - CLEAR NPT (Checklist to evaluate a report of a nonpharmacological trial [36] - + + - - - Risk of Bias Tool (RoB) [4,10] - + + + + + +Quality of measurements properties were based on guidelines established by Terwee et al. [13]. (+): criterion accomplished. (−): Criterion not accomplished. * In all cases, criterion validity was established with “no gold standard tools”. PT physical therapy, RCT randomized controlled trial.\ he quality of measurement properties of quality tools from our previous systematic review and New tools identified in search update Inclusion of the following items was significantly more frequent in PT tools compared with general health re- search tools: “intention to treat” (p = 0.014), “withdraws and drop outs acceptable” (p < 0.001), and “baseline comparability” (p = 0.027). were the most frequently used items among PT tools (86%, 6/7) (Additional files 5 and 6, Figures 2 and 3). The second most frequently used items in PT tools were: reporting of withdrawals and dropouts, method of randomization concealment, description of inclusion/ex- clusion, reporting of descriptive measures for point esti- mates, blinding of therapist, and blinding of participants (71%, 5/7 tools) (Figures 2 and 3). were the most frequently used items among PT tools (86%, 6/7) (Additional files 5 and 6, Figures 2 and 3). The second most frequently used items in PT tools were: reporting of withdrawals and dropouts, method of randomization concealment, description of inclusion/ex- clusion, reporting of descriptive measures for point esti- mates, blinding of therapist, and blinding of participants (71%, 5/7 tools) (Figures 2 and 3). When RoB items were individually examined, we found that sequence generation and allocation conceal- ment were included in 5 of the 7 PT tools (Figure 4). Page 11 of 19 Armijo-Olivo et al. 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BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Page 11 of 19 73.7 73.7 63.2 63.2 63.2 63.2 57.9 52.3 52.3 52.3 47.4 47.4 42.1 42.1 42.1 36.8 36.8 36.8 31.6 31.6 31.6 31.6 31.6 0 10 20 30 40 50 60 70 80 Inclusion and exclusion criteria clearly defined Appropriate statistical analysis Description of withdrawals and Drop outs Description of randomization process reported and appropiate Blinding of investigator/assessor Treatment protocol adequately described for the treatment and control groups Study described as randomized Study question/hypothesis described and well defined Description of sample characteristics Description of outcome measures/criteria for measuring outcomes Blinding of subjects Sample size calculation performed prior to initiation of the study Type of Statistical test used clearly stated Power calculation Testing of subject compliance to treatment protocol Baseline comparability Allocation concealment Blinding of therapist Clear reporting of number of subjects excluded from the trial Reliability reported for main outcome measures p value and/or confidence intervals reported Study conclusion supported by the findings of the study Intention to treat % Figure 2 Frequency of items used by tools used in general health research to measure methodological quality RCTs. New tools identified in search update RCT = randomized controlled trial. Figure 2 Frequency of items used by tools used in general health research to measure methodological quality RCTs. RCT = randomized controlled trial. Only four PT tools evaluated whether randomization was performed. Twelve (63%) general health research tools included randomization whereas seven (37%) in- cluded an item for allocation concealment. Further, fewer of the general health research tools included items related to blinding compared with the PT tools: blinding of participants (47% versus 71%) and blinding of out- come assessors (63% versus 86%). Intention to treat ana- lysis, a component of the incomplete outcome data domain in the RoB tool was more frequently used in PT tools (86%) compared with general health research tools (32%). Other items related to incomplete outcome data in the RoB tool are “description of withdrawals and drop outs” and “appropriateness of withdrawal/drop outs rate”. Compared with the general health research tools (63%), a larger proportion of PT tools (71%) included items for the description of withdrawals and drop outs. In contrast, none of the general health research tools in- cluded an item about whether the withdrawal/drop-out rate was acceptable compared with 57% of the PT tools. Another quality item used in the RoB tool is baseline comparability. This item was included in 86% of PT tools compared with 37% of the general health research tools. In general, PT tools appeared more similar in con- tent to the RoB tool than those used for general health research (Figure 4). Reporting versus conduct items and threats to validity and precision Of the 130 items included in the general health research tools, 62 (48%) evaluated trial “reporting” whereas 60 items (46%) evaluated “conduct” (i.e. methodological quality or risk of bias). Eight items (6%) were considered to evaluate both quality of reporting and conduct of tri- als (i.e. sample representativeness and description of par- ticipants source, description of randomization process reported and appropriate; testing of subject compliance to treatment protocol /report of compliance; therapist training and level of experience in the treatment(s) under investigation; validity, reliability and responsive- ness of the outcome measures reported; post-hoc power calculations and confidence intervals reported). Classification of items from general health research tools according to type of bias and threats to precision was as follows: selection bias (25 items, 19%); perform- ance bias (six items, 4.6%); performance and detection bias (six items, 4.6%); performance bias and contamin- ation (seven items, 5.4%); performance bias and compli- ance (two items, 1.6%); attrition bias (ten items, 7.8%); information bias (six items, 4.6%); detection bias (five items, 3.8%); reporting bias (17 items, 13%); threats to precision (four items, 3.1%); statistical bias (three items, 2.3%); threats to precision and statistical bias (two items, Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Page 12 of 19 Page 12 of 19 85.7 85.7 85.7 71.4 71.4 71.4 71.4 71.4 71.4 71.4 57.14 57.14 57.1 42.9 42.9 42.9 42.9 42.85 28.6 28.6 28.6 28.6 28.6 28.6 14.3 0 10 20 30 40 50 60 70 80 90 Intention to treat analysis Blinding Investigator/assessor Baseline comparability Report of Withdraws and dropouts Method of randomization concealed Inclusion/exclusion criteria defined Descriptive measures for point estimates Blinding Therapist Blinding participants Method of randomization appropriate Withdraws and dropouts rate acceptable Co-interventions avoided or comparable Randomization method performed Study described as randomized Treatment protocol described for treatment group Appropiate statistical analysis used Method of randomization described Reasons for withdrawal and drop outs The timing of outcome measure was comparable Relevant outcomes included Adverse effects described adherence/compliance acceptable in all groups Adequate sample size Treatment protocol described for control group Study described as double blinded % Figure 3 Frequency of items used from tools used in PT research to measure methodological quality of RCTs. PT = physical therapy; RCT = randomized controlled trial. Results expressed as percentages. Figure 3 Frequency of items used from tools used in PT research to measure methodological quality of RCTs. Reporting versus conduct items and threats to validity and precision PT = physical therapy; RCT = randomized controlled trial. Results expressed as percentages. scale was, by far, the most cited and used tool with 5,326 citations from inception (i.e., year 1996) to July 4, 2013. The second most cited tool was the Downs and Black tool, with 962 citations since its introduction in 1998. Other tools frequently cited were: PeDro, Delphi, and Chalmers tools (651, 625, and 584 citations from in- ception, respectively), followed closely by the Van Tulder (560 citations) and Maastricht-Amsterdam (360 citations) tools. Among the most frequently cited tools, a larger proportion (5/7) were PT tools com- pared with only two of the 19 general health research tools (i.e. Jadad and Chalmers). The relative number of citations for the tools after 2008 was similar to those of previous years. Particularly, the use of the Jadad tool (i.e. number of citations from 2007 to July 4, 2013: 3,672) did not show a decrease (in terms of absolute numbers of citations) after the inception of the RoB tool in 2008 (Table 3). Tracking of the RoB tool showed that it has been cited approximately 1230 times since inception. This number is likely an underestimate because of the challenges described above with respect to tracking the Cochrane Hand- book chapter that first described the tool. However, this information provides a reference point to track usage of the RoB tool over time. 1.6%); multiple biases (three items, 2.3%); and other (i.e., not classified as related to threats to validity or preci- sion) (27 items, 21%) (Additional file 5). Of the 48 items included in PT tools, 16 (33.3%) evalu- ated trial “reporting” whereas 28 (58.3%) evaluated “con- duct”. Four items (8.3%) were considered to evaluate both quality of trial reporting and conduct: testing/re- port subject compliance to treatment protocol, and validity, reliability and responsiveness of the outcome measures reported. The classification of items from PT tools according to type of bias and threats to precision was as follows: se- lection bias (10 items, 21%); performance and detection bias (five items, 10%); performance bias only (six items, 135%); performance and contamination bias (two items, 4.2%); performance and compliance bias (two items, 4.2%); information bias (five items, 10%); attrition bias (eight items, 17%); detection bias (three items, 6.3%); reporting bias (three items, 6.3%); threats to precision (two items, 4.2%); and statistical bias (1 item, 2.1%). (Additional file 6). Frequency of citations of quality tools The number of citations per quality tool since its incep- tion and after 2007 are detailed in Table 3. The Jadad Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Page 13 of 19 71.4 57.1 71.4 71.4 85.7 71.4 85.7 71.4 57.14 85.7 63.2 63.2 36.8 47.4 63.2 36.8 31.6 63.2 0 36.8 0 10 20 30 40 50 60 70 80 90 Appropriate Performed Participants Assessors/outcomes Therapists Intention to treat Withdraws and drop outs reported Withdraws and drop outs rate acceptable Baseline Comparability Randomization Allocation concealment Blinding Incomplete outcome data Other Bias % PT tools Health tools Figure 4 Comparison between RoB tool domains and items from PT and health sciences tools. PT = physical therapy; RoB = risk of bias. Results expressed as percentages. Figure 4 Comparison between RoB tool domains and items from PT and health sciences tools. PT = physical therapy; RoB = risk of bias. Results expressed as percentages. Discussion tools. According to Verhagen et al. [2], methodological quality assessment involves the evaluation of internal validity (the degree to which the study design, conduct and analysis have minimized biases), external validity (the extent to which study results are generalizable be- yond the experimental situation), and statistical analysis of primary research. According to The Cochrane Collab- oration [10], internal validity of a trial is linked to “risk of bias” and it should be the primary focus of quality as- sessment since external validity differs upon context. In addition, “quality of reporting” is commonly used as a proxy for trial quality, which has complicated the construct of “quality” even more. This study examined tools and individual items used in general health and PT research to assess the quality of RCTs. A variety of tools are still widely used despite crit- icisms raised regarding their limitations [8,63]. This find- ing is consistent with previous reviews on this topic that have identified inconsistencies in the use of quality tools [5,6,18]. There is extensive variation in individual items included across quality assessment tools. Many of these items may not be indicators of bias nor related to over- or under-estimations of treatment effects. Moreover, there is lack of empirical evidence supporting the associ- ation of many individual quality items with changes in the magnitude and direction of treatment effects. This finding raises important concerns in the field of quality assessment regarding the appropriateness of evaluating the evidence based on the use of these tools and items. A clear and consistent definition of “quality” across health research areas is necessary to advance the field of quality assessment. Furthermore, concepts such as internal validity, external validity, and quality of reporting should be explicitly and clearly defined for the constructs that the individual items are meant to address. Finally, items assessing the methodological quality (or internal validity) of RCTs should be based on empirical evidence of their association with treat- ment effects. Results of this study agree with those of Deschartres et al. [19] which found that a large number of tools have been used in reviews that assessed the quality and reporting of RCTs. According to Deschartres et al. Discussion [19], ambiguity and lack of a unique definition of trial “qual- ity” accounts for the heterogeneity of quality assessment ase ary 2007- 3 Year 2007 before RoB tool Year 2008 Year 2009 Year 2010 Year 2011 Year 2012 Year 2013 Until July 4, 2013 Subject area most used 2 393 468 616 514 634 706 341 Medicine 3 6 10 5 7 9 5 Medicine/health professions 4 39 65 71 77 87 75 40 Medicine/health professions 5 49 52 87 91 101 108 67 Health professions/ Medicine 8 34 36 23 21 21 19 4 Medicine/health professions 2 43 58 101 86 65 93 34 Medicine/health professions 4 15 7 6 9 4 5 Medicine/health professions 1 19 21 29 19 19 29 15 Medicine/Psychology 2 4 5 2 8 4 1 Medicine/ Nursing 0 0 0 0 0 0 0 Medicine 6 7 8 4 8 10 4 Medicine/ Pharmacology, Toxicology and Pharmaceutics 0 15 20 21 14 17 20 13 Medicine/ Biochemistry/ Nursing 4 4 5 3 5 5 4 Medicine/ Pharmacology, Toxicology and Pharmaceutics 0 3 1 0 0 0 1 Medicine 2 2 3 1 3 0 0 Medicine/Nursing 3 80 72 100 125 146 163 97 Medicine/Nursing 6 9 9 9 14 11 5 Dentistry/Medicine 11 14 7 10 2 3 2 Medicine/Neuroscience 2 2 2 0 0 0 0 Medicine 4 30 21 20 14 14 17 8 Medicine/Biochemistry 2 1 4 7 8 6 5 Medicine/Neuroscience p p 1 Medicine/psychology 4 Medicine/psychology 0 Pharmacology, Toxicology and Pharmaceutics 8 Medicine 75 Medicine 1155 Medicine Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Page 16 of 19 The number of items across quality tools is large; 130 and 48 items have been used by tools in general health and PT research, respectively. Some items are subjective, confusing, and lack a clear definition (e.g., subjects ap- propriate to study questions, discussion of bias resulting from non-blinding assessment). These factors make the evaluation of individual items challenging and likely con- tribute to low inter-rater agreement. Many quality items relate to “reporting” rather than “conduct” of trials; ap- proximately half of the items from these tools relate to reporting only. This finding is consistent with results de- scribed by Deschartres et al. PT and general health research tool items and threats to validity and precision f l h l h h d T l Empirical evidence has supported many items in the RoB tool. There is a substantial interest in investigating which methodological features of RCTs are associated with treatment effects. Evidence informing this associ- ation comes mainly from RCTs in the area of medicine and is based primarily on evaluations of dichotomous outcomes [12,14,15]. Therefore, empirical evidence on the relationship between trial quality and treatment effects may not be readily applicable to other health re- search areas such as PT and other areas of rehabilitation. Morever, information regarding the importance of in- cluding certain items in quality tools within different clinical areas is limited. As mentioned previously, RCTs in the area of PT have distinct characteristics compared with pharmacological trials conducted in medicine. PT interventions are complex interventions [20]; they com- prise certain characteristics such as the type of therapy and its intensity, a standardized or individually tailored approach, and the skills and experience of the therapists, that are likely to affect trial results. In addition, because of the nature of certain PT interventions (e.g., manual therapy, exercises), blinding of therapists and/or patients is not always possible. Appropriate blinding of study participants and all key study personnel is unlikely to be accomplished for most PT trials; however, blinding of outcome assessment has been commonly used as a Most items from general health research and PT tools were classified according to one or more categories of threats to validity or precision; however, some items could not be placed in any category. For example, the item “study question/hypothesis/purpose described and well defined” was not linked to any type of bias and was found irrelevant for study quality. Nevertheless, this item was included in 10 (53%) health research tools. This situation raises concerns about the usefulness of certain items to determine trial quality; therefore, these types of items should be carefully considered when deciding whether they should be part of these tools. Classifying quality items was a complex task due to unclear descriptions of the items and lack of empirical evidence linking these items to bias. The number of items that was linked to different types of bias varied by tool. For example, a high percentage of items dealt with selection bias (approximately 19% of general health and 21% of PT tools). In contrast, attrition bias was more frequently represented in items found in PT (17%) com- pared with general health research (7%) tools. Discussion [19], in which 25% of meth- odological reviews stated that RCTs reported details of sample size calculation, but only 6% reported on ad- equacy of the sample size. Although clear reporting is necessary to assess the quality of trial conduct, a focus on quality of reporting can hide differences in trial con- duct and lead to under- or over-estimation of the meth- odological quality [64]. proxy quality measure without validation. Therefore, more empirical evidence on trial bias is needed in the area of PT to determine which factors are likely to affect treatment effect estimates and thus provide accurate re- sults for the clinical community. Further research should examine the appropriateness of using certain items/ domains when evaluating the risk of bias of primary re- search in a variety of health areas. This information would provide clear benchmarks to assess the quality or risk of bias of primary research included in SRs and meta-analysis, and ultimately strengthen the evidence for decision-making in all areas of health care. The RoB tool is recommended by The Cochrane Col- laboration. Some groups within the Collaboration have developed their own tools and have not yet adopted the RoB approach (e.g. Cochrane Bone, Joint and Muscle Trauma Group). Other Cochrane groups have modified the RoB tool for their own purposes (i.e. Cochrane Back Review Group, Cochrane Renal Review Group). The RoB tool was developed more recently than many of the other tools; current research [9,13] recommends further testing of its psychometric properties and validation of the tool in a wide range of research fields. Additional guidelines will help users in applying and interpreting the results of the RoB tool. Comparison of items between PT and general health research tools with RoB tool We found that items frequently included in the PT tools were more closely linked to items/domains included in the RoB tool than those of general health research tools. This result suggests that PT tools are more closely linked to an examination of bias than the general health research tools. Tools most cited The Jadad scale [37] is the most frequently cited tool in health sciences research despite criticisms regarding its lack of responsiveness [8] and applicability to other health research areas such as PT and rehabilitation [5]. Herbison et al. [8], found that the Jadad scale might not be responsive enough to distinguish among different levels of trial quality. The use of the Jadad scale has been discouraged in many areas of health research. The dis- cordance between recommendations against using the Jadad scale and its ongoing use is a matter of concern and reasons for this discrepancy should be further ex- plored. It is likely that the Jadad tool is popular among SR authors because it is simple and requires little time to apply [13]. None of the other quality tools used in general health research and PT is as highly cited as the Jadad tool. Some tools are specific to certain areas (e.g., PT, nursing, psychology, pharmacology); most of them are long in- struments and require a greater amount of time to complete; and some lack clear guidelines for item assess- ment, which can discourage their use. PT and general health research tool items and threats to validity and precision f l h l h h d T l These re- sults call for an in-depth analysis of individual items of tools that evaluate trial quality or risk of bias of RCTs in health research in order to provide a more complete as- sessment of their internal validity. Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Page 17 of 19 between PT and general health research tools in the type of items that are included. There is a need for clarity and consistency of the constructs evaluated by items in quality assessment tools, particularly for aspects related to internal validity, external validity, precision, and qual- ity of reporting. The selection of items to assess internal validity, or risk of bias, should be based on empirical evi- dence of an association with distortions of treatment ef- fects. Finally, tools and items should undergo a thorough validation process to examine their psychometric prop- erties. Future studies in this area should investigate which items are linked to bias through empirical evi- dence or psychometric testing. This information will be valuable for the field of knowledge synthesis. Additional files Additional file 1: Search Strategy Example. Additional file 2: Definition of psychometric properties according to Terwee et al., [24]. Additional file 3: Bias definitions. Additional file 4: Excluded Studies. Strengths and limitations To the best of our knowledge, this study is the first to exhaustively explore the type and frequency of individual items included in tools that evaluate the quality or risk of bias of RCTs in health research. A comprehensive search was performed for all published research in this area, with no language restrictions, and using several strategies (i.e., manual search, Scopus) to identify rele- vant literature. However, because of indexing problems of research on the evaluation of quality assessment tools for RCTs [19], some studies may have been missed; this would not likely change our general findings. Data extraction and item classification was performed independently by two researchers with disagreements re- solved by consensus. The process of classifying items was somewhat subjective; therefore, classification of some items may be debated. Difficulties in classifying items as potentially linked to bias have been acknowl- edged in previous studies that analyzed bias in different types of research designs [25,28,65]. What is the implication, what should change now? Re- sults of this study call for an in-depth empirical analysis of the items that should be used to assess risk of bias of RCTs in health research. This information is urgently needed to develop guidelines for the design, conduct, and implementation of trials. In addition, this informa- tion is important for systematic reviewers and meta- analysts to evaluate the risk of bias of intervention trials in different areas of health research. We used Scopus database to track all original papers describing quality tools. We acknowledge that this ap- proach is only an indirect measure of the usage of qual- ity tools and should not be interpreted as absolute indicator of usage over time. What is new? Key findings h There is extensive item variation across tools that evalu- ate the risk of bias of RCTs. There is a lack of empirical evidence to support the association with bias for many items. There is extensive item variation across tools that evalu- ate the risk of bias of RCTs. There is a lack of empirical evidence to support the association with bias for many items. What this adds to what is known: Although some studies have previously addressed the use of tools for quality assessment of RCTs, this is the first study that exhaustively explores the type and frequency of items in- cluded in different tools that evaluate the risk of bias of RCTs in health research. The number of items included across quality tools is large: 130 and 48 different items have been used by general health research and physical therapy (PT) tools, respectively. Many items are used without a clear identification of their link to bias, or in- ternal validity. The frequency of use of these items varies according to health area (as demonstrated by our com- parison between PT and general health research), which suggests a lack of agreement regarding their relevance to trial quality or risk of bias. Author’s information Susan Armijo-Olivo has a Bsc in Physical therapy (PT) from the Pontifical University Catholic of Chile, a MSc PT and a PhD in Rehabilitation Sciences from the University of Alberta. Her major field of research is diagnosis, evaluation, and treatment of patients with musculoskeletal pain especially temporomandibular disorders and cervical spine disorders along with physical therapy evidence based practice. She currently is a postdoctoral fellow at the CLEAR (Connnecting Leaadership and Research Program in the Faculty of Nursing at the University of Alberta. Her post-doctoral project will focus on the methodological predictors of effect size estimates in PT trials. This research is critical to accurately provide conclusions for health care and decision making. This project will be an important contribution to the area of knowledge synthesis and translation in the PT field and allied health professions. 8. Herbison P, Hay-Smith J, Gillespie WJ: Adjustment of meta-analyses on the basis of quality scores should be abandoned. J Clin Epidemiol 2006, 59(12):1249–1256. 9. Armijo-Olivo S, Stiles C, Hagen N, Biondo P, Cummings G: Assessment of study quality for systematic reviews: a comparison of the Cochrane Collaboration Risk of Bias tool and the Effectve Public Health Practice Project Quality Assessment Tool: methodological research. J Eval Clin Pract 2012, 18(1):12–18. 10. Higgins J, Altman D: Chapter 8: Assessing risk of bias in included studies. In Cochrane Handbook for Systematic Reviews of Interventions version 50. Edited by Higgins J, Green S. Chichester, UK: John Wiley & Sons, Ltd; 2008. 11. 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Arch Phys Med Rehabil 2002, 83(12):1745–1752. Competing interests The authors declare that they have no competing interests. 5. Armijo-Olivo S, Macedo LG, Gadotti IC, Fuentes J, Stanton T, Magee DJ: Scales to assess the quality of randomized controlled trials: a systematic review. Phys Ther 2008, 88(2):156–175. Received: 26 December 2012 Accepted: 12 September 2013 Published: 17 September 2013 References Additional file 5: Heath Sciences Tools and items to Measure Methodological Quality of RCTs. 1. Khan K, Ter Riet G, Popay J, Nixon J, Kleijnen J: Satge II. Conducting the Review. Phase 5 Study Quality Assessment. In Undertaking Systematic Reviews of research Effectiveness CRD’s Guidance for those carrying out or commissioning reviews. York: Centre for Reviews and Dissemination; 2001:1–20. Additional file 6: Tools and Items to Assess Quality of RCTs in Physical Therapy. Additional file 6: Tools and Items to Assess Quality of RCTs in Physical Therapy. 2. Verhagen AP, de Vet HC, de Bie RA, Boers M, van den Brandt PA: The art of quality assessment of RCTs included in systematic reviews. J Clin Epidemiol 2001, 54(7):651–654. Conclusion h There is a considerable number of tools to evaluate the quality of RCTs in health research. There is extensive variation in the number of individual items across qual- ity assessment tools and an apparent lack of agreement Additional file 3: Bias definitions. Page 18 of 19 Page 18 of 19 Armijo-Olivo et al. BMC Medical Research Methodology 2013, 13:116 http://www.biomedcentral.com/1471-2288/13/116 Acknowledgements 12. Wood L, Egger M, Gluud LL, Schulz KF, Juni P, Altman DG, Gluud C, Martin RM, Wood AJG, Sterne JAC: Empirical evidence of bias in treatment effect estimates in controlled trials with different interventions and outcomes: Meta-epidemiological study. BMJ 2008, 336(7644):601–605. 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Author details 1Postdoctoral Fello 1Postdoctoral Fellow, CLEAR (Connecting Leadership and Research) Outcomes Research Program, University of Alberta, 5-115A Edmonton Clinic Health Academy (ECHA), 11405 – 87 Avenue, Edmonton, Alberta T6G 1C9 Canada. 2Faculty of Rehabilitation Medicine, Department of Physical Therapy, University of Alberta, 3-48 Corbett Hall, Edmonton T6G 2G4, Canada. 3Department of Physical Therapy, Catholic University of Maule, Talca, Chile. 4School of Public Health, University of Alberta, Institute of Health Economics, Edmonton, Alberta, Canada. 5School of Dentistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada. 6Alberta Research Centre for Health Evidence, Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada. 19. 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Shifting perspectives from “oncogenic” to oncofetal proteins; how these factors drive placental development
Reproductive biology and endocrinology
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West et al. Reproductive Biology and Endocrinology (2018) 16:101 https://doi.org/10.1186/s12958-018-0421-3 West et al. Reproductive Biology and Endocrinology (2018) 16:101 https://doi.org/10.1186/s12958-018-0421-3 REVIEW Open Access REVIEW © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shifting perspectives from “oncogenic” to oncofetal proteins; how these factors drive placental development Rachel C. West* , Gerrit J. Bouma and Quinton A. Winger Abstract Early human placental development strongly resembles carcinogenesis in otherwise healthy tissues. The progenitor cells of the placenta, the cytotrophoblast, rapidly proliferate to produce a sufficient number of cells to form an organ that will contribute to fetal development as early as the first trimester. The cytotrophoblast cells begin to differentiate, some towards the fused cells of the syncytiotrophoblast and some towards the highly invasive and migratory extravillous trophoblast. Invasion and migration of extravillous trophoblast cells mimics tumor metastasis. One key difference between cancer progression and placental development is the tight regulation of these oncogenes and oncogenic processes. Often, tumor suppressors and oncogenes work synergistically to regulate cell proliferation, differentiation, and invasion in a restrained manner compared to the uncontrollable growth in cancer. This review will compare and contrast the mechanisms that drive both cancer progression and placental development. Specifically, this review will focus on the molecular mechanisms that promote cell proliferation, evasion of apoptosis, cell invasion, and angiogenesis. Keywords: Cell proliferation, Migration, Invasion, Angiogenesis, Genomic instability, Placenta, Placental insufficiency * Correspondence: rachcwest2@gmail.com Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University, 10290 Ridgegate Circle, Lone Tree, Fort Collins, CO 80124, USA Background Currently, treatments for pathologies caused by placental insufficiency are lacking, with no known treat- ment for pre-eclampsia other than the immediate deliv- ery of the fetus. the histotroph, an endometrial secretion that facilitates implantation and conceptus development during the ini- tial weeks of pregnancy [18]. The histotroph also con- tains factors that regulate the invasion potential of the early trophoblast cells if an embryo implants [19]. While the understanding of the consequences of IUGR and preeclampsia has increased exponentially over the past few decades, there is still a need to elucidate the underlying cause behind placental insufficiency during development. Understanding what is driving placental insufficiency during early development will be essential in the development of better diagnostic and treatment tools for the prevention and treatment of both pathologies. The delicate interplay between cell proliferation and differ- entiation could be a key event that malfunctions early on in pregnancy, eventually leading to placental dysfunction. y p y p Once fertilization occurs, the zygote travels from the ampulla of the Fallopian tube to enter the endometrial cavity within 3 days [20]. During this journey, the zygote divides and undergoes a series of mitotic divisions to become the morula [21]. Approximately 5 days after fertilization, the morula transforms into a newly ex- panded blastocyst of 58-cells partitioned into a periph- eral layer called the trophectoderm, that will eventually become the placenta and the inner cell mass (ICM), which will become the fetus [22]. Approximately 9 days after fertilization, the blastocyst implants into the uterine wall in a three step process called apposition, adhesion, and invasion [23]. At this timepoint, a multinucleated, primitive syncytium has formed, penetrating the de- cidua, hollowing out areas of the stromal layer, and forming the lacunae that will eventually be filled with maternal blood [24]. Additionally, by day 9 the progeni- tor trophoblast cells, cytotrophoblast cells, have begun to form villous structures that will eventually differentiate into the two main cell types of the placenta; the weakly proliferative and fusional syncytiotrophoblast and the terminally differentiated, invasive extravillous trophoblast (EVT) [25]. At day 12 of gestation, cytotrophoblast cells begin to penetrate the primitive syncytium, forming the first primary chorionic villi of the placenta [26]. The cyto- trophoblast cells proliferate rapidly and accumulate in floating villi which will differentiate to form the syncyt- ium. This layer of cells will eventually come into contact with the maternal blood [27]. Background cerebral palsy, deafness, chronic lung disease, neurode- velopmental delays, and metabolic disorders [9–11], leading to substantial health care costs and emotional burdens on families. Both preeclampsia and IUGR appear to be heritable as they both are associated with an increased likelihood of IUGR and fetal death in subsequent pregnancies of the affected mothers [11]. Additionally, IUGR often occurs frequently in women suffering from placental morbidities such as preeclamp- sia, and gestational diabetes, putting the mother’s life in significant danger as well as the fetus [12]. During pregnancy, the female body undergoes incredible anatomic, metabolic, and physiological changes in the process of providing for the needs of a developing fetus. One of the most essential developments is the genesis of a placenta, which is critical for hormone production and gas and nutrient exchange between the mother and the fetus [1–3]. Any aberration in these physiological pro- cesses can cause devastating placental pathologies like preeclampsia and intrauterine growth restriction (IUGR) [4], leading to severe pregnancy complications [5]. Pre- eclampsia affects 4–8% of pregnancies in the United States and is attributed as the cause behind 500,000 fetal and 75,000 maternal deaths each year [6, 7]. IUGR also affects 7–9% of newborn infants and is thought to cause up to 50% of unexplained stillbirths [8]. These preg- nancy complications can also cause long-term develop- mental delays and health consequences including; The conditions affecting fetal growth can either be placental or fetal in origin. Fetal growth is dependent upon the overall health of the fetus, the ability of the mother to metabolize and provide sufficient amounts of substrates necessary for growth, and the competency of the placenta to transport these substrates from the mother to the fetus [13]. However, impaired placental function seems to drive the most severe cases of IUGR [14]. This placental insufficiency is a common phenotype associated with both IUGR and maternal placental co- morbidities including preeclampsia and hypertension * Correspondence: rachcwest2@gmail.com Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University, 10290 Ridgegate Circle, Lone Tree, Fort Collins, CO 80124, USA * Correspondence: rachcwest2@gmail.com Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University, 10290 Ridgegate Circle, Lone Tree, Fort Collins, CO 80124, USA West et al. Reproductive Biology and Endocrinology (2018) 16:101 Page 2 of 12 Page 2 of 12 [15]. Background Alternatively, cytotropho- blast cells will also form anchoring villi that will eventually attach to and invade into the mother’s decidualized endo- metrium, myometrium, and eventually her spiral arterioles [28] (Fig. 1). This balance between cytotrophoblast cell proliferation and subsequent differentiation into the inva- sive and migratory EVT has a marked similarity to how cancer cells form tumors and metastasize. Typically, when one considers oncogenes it’s hard to ignore the profound effects these proteins have during normal homeostasis in adult tissues. These genes pro- mote rampant cell proliferation in otherwise healthy tis- sues. Proliferative cells eventually begin to migrate towards other organ systems, invading into tissues to form metastatic tumors. However, to only consider on- cogenes as “bad” fails to consider the original purposes of these genes. These oncogenic processes are essential during early embryonic, fetal, and placental development and any aberrant signaling by these genes can cause dev- astating effects on fetal growth. These proteins are re- sponsible for the cancer-like processes that characterize early placental development. However, in direct contrast to carcinogenesis, the placenta uses these factors in a tightly controlled, highly regulated environment. This regulation exploits these factors so that they create a re- markably efficient organ in a short amount of time without the adverse consequences that often come with the expres- sion of oncogenic proteins. Therefore, we propose that on- cogenes instead be considered as oncofetal proteins. This review will focus on the similarities of oncogenic processes like proliferation, escape of apoptosis, cell in- vasion and migration, angiogenesis, and the signaling pathways that drive these mechanisms in both cancer and placental development. Understanding these paral- lels between placentation and tumorigenesis will provide insight into not only better ways to treat cancer but also understand how these processes can fail during develop- ment leading to placental insufficiency. Cell proliferation As the placenta begins forming 1 week after fertilization and must begin to facilitate nutrient and gas exchange by the end of the first trimester, rapid and substantial cell proliferation is essential. However, unlike cancer, this cell proliferation is tightly regulated and cells lose their proliferative capacity once they undergo differentiation into the invasive EVT lineage. One group of genes that are responsible for cytotrophoblast cell proliferation are growth factors and their receptors [29]. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and placental Human placental development Placentation begins with the uterine endometrium chan- ging its structure to prepare for implantation, a process known as decidualization [16]. The fibroblast-like cells of the endometrium transform into secretory decidual cells. These decidual cells comprise an immunoprivi- leged matrix that protects the implanting embryo from attack by maternal immune cells [17]. It also secretes West et al. Reproductive Biology and Endocrinology (2018) 16:101 Page 3 of 12 Fig. 1 Early Placental Development. The progenitor cells of the placenta, the cytotrophoblast proliferate rapidly during the first trimester of pregnancy. During this time they also differentiate to become part of the syncytiotrophoblast layer that fuses and becomes the layer of the placenta that comes into contact with the maternal blood. Additionally, cytotrophoblast cells differentiate to become part of the extravillous trophoblast, the cells that invade into the mother’s endometrium, seeking out her spiral arteries Fig. 1 Early Placental Development. The progenitor cells of the placenta, the cytotrophoblast proliferate rapidly during the first trimester of pregnancy. During this time they also differentiate to become part of the syncytiotrophoblast layer that fuses and becomes the layer of the placenta that comes into contact with the maternal blood. Additionally, cytotrophoblast cells differentiate to become part of the extravillous trophoblast, the cells that invade into the mother’s endometrium, seeking out her spiral arteries for cytotrophoblast cell proliferation while jun-B likely plays a role in terminal differentiation. This conclusion is at least partially supported by another finding using stimulation by epidermal growth factor (EGF) to induce differentiation of human primary cytotrophoblast cells towards the syncytio- trophoblast fate. Cells were treated with EGF for 40 min pulses and, while both c-jun and jun-B mRNA levels rapidly increased 2–4 h after exposure, EGF’s effects on jun-B were the most striking. Jun-B was significantly increased in cyto- trophoblast cells differentiating towards the syncytiotropho- blast lineage, indicating that EGF and its activation of jun-B is important in the terminal differentiation of cytotropho- blast cells [44]. Interestingly, the hormone adiponectin has also been implicated as an important regulator for the JUN kinase pathway, with a particular emphasis on c-jun regula- tion. In normal placentas, adiponectin has an antiprolifera- tive effect. However, in gestation diabetes mellitus (GDM) placentas, adiponectin levels are decreased with an increase in cell proliferation, potentially thought to be a contributor to the macrosomia seen in GDM babies. Human placental development Reproductive Biology and Endocrinology (2018) 16:101 West et al. Reproductive Biology and Endocrinology (2018) 16:101 regulates the transition from a pluripotent, highly prolif- erative state to a terminally differentiated cell [46]. One of the main targets of LIN28 is the let-7 family of miR- NAs. When cells are highly proliferative, LIN28 nega- tively regulates the let-7 family. However, as cells begin to differentiate the let-7 family of miRNAs is upregu- lated and can bind to the 3’ UTR of LIN28 to inhibit its translation into protein [47]. Because of this negative feedback loop, LIN28 and the let-7 s are often inversely expressed in many cancers [48]. In addition to this, in- creased LIN28 has been correlated with highly aggres- sive cancers and poor prognosis [49]. The let-7 s also regulate several other oncofetal proteins including HMGA2, c-Myc, RAS, and VEGF [49]. In placental cells, a knockdown of LIN28A led to spontaneous differenti- ation and syncytialization in human trophoblast cells [50]. Furthermore, knockdown of LIN28B and knockout of both LIN28A and LIN28B leads to trophoblast cells that are driven to differentiate towards only the syncytio- trophoblast lineage, but not extravillous trophoblast cells [51]. Collectively these data suggest that, as with pluri- potent cells, LIN28 is an essential gatekeeper in tropho- blast cell proliferation and differentiation. Additionally, the IGFs regulate caspase expression. Activa- tion of IGF1-R can prevent cleavage of caspases in both can- cer cells and fetal brain cells, preventing apoptosis [70, 71]. In accordance with the findings of Bcl-2 expression, there appears to be no caspase-mediated apoptosis in the syncy- tiotrophoblast of term villi of the placenta. There was also no response to stimulus-induced apoptosis in syncytiotro- phoblast of villous explants from term placental tissue [72]. These data suggest that the syncytiotrophoblast can protect itself against apoptotic signals to continue to function and contribute to fetal growth until the end of pregnancy. In most cell types, the transcription factor p53 antago- nizes IGF signaling to promote apoptosis and cell cycle arrest [73]. Several papers report that p53 closely monitors the IGF-1/Akt pathway and, upon sensing stress, nega- tively regulates IGF-1/Akt to halt cell proliferation and induce autophagy [74–76]. This negative regulation occurs by p53 transactivating IGF-BP3. The family of IGF-BPs regulates ligand availability to their IGF receptors [77]. Cell survival The ability to bypass apoptosis is another hallmark of can- cer and is essential during placentation. Again, the growth receptors and receptor tyrosine kinase pathways mentioned above play an important role in cell survival, specifically IGF-1 and IGF-2 binding to IGF-1R [38, 52].The relation- ship between IGF-1R and the PI3K/Akt and MAPK path- ways has been described as a crucial cell protectant in many different cancer cell types [53–56]. In immortalized human placental BeWo cells and in placental tissue ex- plants both IGF1 and IGF2 rescued serum-starved cells from apoptosis [57]. Additionally, mutated IGF1-R in preg- nant women leads to both intrauterine and post-natal growth restriction [58] and there is a direct correlation be- tween IGF levels and birth weight [59]. g p y g Finally, another important anti-apoptotic factor often found in cancer is survivin [82]. Belonging to the “in- hibitor of apoptosis” family, upregulation of survivin in cancers is directly correlated with apoptotic resistance, increased cell survival, and poor response to chemother- apy [83]. Survivin is yet another anti-apoptotic protein increased by IGF-1. In prostate cancer cells, stimulation with IGF-1 lead to increased survivin expression due to the increased stabilization and translation of survivin mRNA [84]. Alternatively, survivin has also been de- scribed as negatively regulated at the transcriptional level by p53 with the surivivin promoter having a p53 binding element although the exact mechanism of regu- lation by p53 is still poorly understood [82]. In the pla- centa, survivin is thought to play a crucial role in cell survival and proliferation of trophoblast cells [85, 86]. Messenger RNA levels of survivin were analyzed in first, second, and third trimester placentas of pre-eclamptic women, compared to normal placentas, survivin was sig- nificantly decreased. Additionally, survivin levels were dir- ectly correlated with severity of pre-eclampsia, with levels There are two distinct mechanisms the IGF system targets to promote cell survival; the Bcl-2 family and caspase proteins [60]. Increased Bcl-2 expression has been reported in several cancer cell lines and tumors [61–64] and leads to increased cell survival and resist- ance to chemotherapy treatment [65]. Bcl-2 immunolo- calization in the placenta has been described in several papers [66–68]; however its involvement in trophoblast cell apoptosis is still unclear. Soni et al. describe a grad- ual increase in Bcl-2 expression throughout pregnancy with maximal immunoreactivity occurring at term [69]. Ishihara et al. Human placental development It has been shown that a p53-induced accumulation of IGF-BP3 in the extracellular medium of cells can inhibit mitogenic function of IGF-1 in vitro [78]. Increased IGF-BP3 leads to increased complexing to IGF-1, reducing their ability to bind IGF-1R to promote cell survival and proliferation [79]. However, over 50% of human cancers have p53 mutations, preventing it’s pro-apoptotic function to promote spontaneous tumorigenesis [80]. In the pla- centa, increased p53 protein expression in placental villi is correlated with pre-eclampsia [81]. As excessive apoptosis in the villous trophoblast of placental villi is a characteris- tic of pre-eclampsia, these data suggest that upregulated p53 induces a disproportionate amount of apoptosis, lead- ing to placental insufficiency (Fig. 2). Human placental development To test whether adiponectin actually inhibits c-Jun in GDM placentas, the choriocarcinoma cell line, BeWo, was treated with high levels of glucose. These high glucose treated cells had signifi- cantly lower levels of adiponectin, leading to increased c-Jun protein and increased cell proliferation. Furthermore, addition of adiponectin to high glucose treated cells inhib- ited c-Jun activation, suppressing cell proliferation [45]. growth factor (PLGF), insulin like growth factor (IGF), trans- forming growth factor (TGF) and their subsequent receptors have all been identified in the cytotrophoblast and are specu- lated to act in a paracrine and autocrine manner on the dif- ferentiated cells of the placenta [30–36]. These growth factors bind to tyrosine kinase receptors on cytotrophoblast cell membranes inducing self-dimerization to activate the MEK/ERK proliferation pathway and the PI3K/Akt anti-apoptosis pathway [37]. These kinase signaling cascades are potent catalysts that influence cell proliferation and sur- vival in many cell types, including the placenta [38]. Gene editing experiments targeting the MAPK pathway in mice was embryonic lethal by E11.5 due to severe placental defects [39]. Additionally, gene disruption of the PI3K/Akt pathway led to depleted cells in the spongiotrophoblast layer (cells of the junctional zone of the mouse placenta, the specific func- tion is still unclear [40]) and decreased vascularization [41]. These data indicate a necessary role for growth factor driven activation of the MAPK/PI3K pathways during early placen- tal development. Interestingly, the phosphorylated forms of ERK1 and ERK2 were only detected in proliferative cytotro- phoblast cells until the end of the first trimester. This alludes to their importance in cell proliferation, losing expression once cells begin to terminally differentiate [42]. Additional oncogenic downstream target of the MAPK pathway, JUN has also been implicated in early placental cell proliferation and differentiation. However, different mem- bers of the JUN family are expressed at different time points. Messenger RNA for c-Jun was found at its highest levels in early gestational placental tissue whereas jun-B was at its highest levels between 35 and 40 weeks [43]. The authors of this study concluded that in the placenta c-jun is essential There are also several oncofetal proteins outside of the family of growth factors that promote cell proliferation. For example, our laboratory studies the LIN28-let7- HMGA2 molecular axis. LIN28 is an RNA binding protein considered to be a key molecular factor that Page 4 of 12 West et al. Cell survival also suggest that based on their findings that abundant expression of Bcl-2 in term syncytiotro- phoblast prevents cell death, allowing for the mainten- ance of placental mass near the end of pregnancy [66]. West et al. Reproductive Biology and Endocrinology (2018) 16:101 Page 5 of 12 Fig. 2 IGF signaling in the placenta. IGF regulates cell proliferation and survival in placenta cells through several mechanisms. Both IGF-1 and IGF-2 bind to the IGF-1R to stimulate the MEK/ERK pathway and the PI3K pathway to promote cell proliferation and evasion of apoptosis. Additionally, downregulation of p53 leads to higher levels of IGF’s allowing for more proliferation and cell survival Fig. 2 IGF signaling in the placenta. IGF regulates cell proliferation and survival in placenta cells through several mechanisms. Both IGF-1 and IGF-2 bind to the IGF-1R to stimulate the MEK/ERK pathway and the PI3K pathway to promote cell proliferation and evasion of apoptosis. Additionally, downregulation of p53 leads to higher levels of IGF’s allowing for more proliferation and cell survival promote invasion. This review will only focus on the integrins that regulate cell invasion in the placenta. decreasing as pre-eclampsia became more severe [87]. Due to the upregulated levels of p53 in pre-eclampsia it has been suggested that the negative regulation of survivin by p53 is a potential cause of the low levels of survivin mRNA found in pre-eclamptic placentas [86]. During placental development, there is a delicate balance between adhesion-promoting integrin expression and invasion-promoting integrins. This balance in early cytotrophoblast cells is regulated in large part by α5β1 and α1β1. In contrast to cancer, cytotrophoblast cells use the invasion-restraining role of α5β1 to balance the invasion-promoting role of α1β1 to tightly regulate the depth of invasion into the mother’s decidua [91]. During early gestation, the proliferating cytotrophoblast cells begin to upregulate α1β1 as they differentiate to become more invasive. However, as gestation continues and invasion becomes less of a priority, expression of the α1β1 integrin complex declines [91]. Additionally in pre-eclamptic placental tissue, α1β1 immunostaining is almost nonexistent while the invasion-restraining α5β1 is still detectable at levels similar to normotensive placentas [92]. This suggests that the shallow invasion of uterine vasculature, a hallmark of pre-eclampsia, is at least in part caused by altered integrin expression. Angiogenesis Angiogenesis is a mandatory process driving tumor pathogenesis leading to tumor metastasis and poor can- cer prognosis. Alternatively, the ability to not only join existing vessels but also to create vessels in avascular tis- sue is an essential component of placental development. Any aberration in the signaling pathways that drive angiogenesis and vasculogenesis can lead to shallow in- vasion into the maternal spiral arteries, a known cause of placental insufficiency. The angiopoietin (ANG) and vascular endothelial growth factor (VEGF) families of growth factors are two critical families for vessel devel- opment in the placenta [112]. Similar to the balancing and counterbalancing effects of integrins regulating cell invasion, VEGF and placenta growth factor (PlGF) work in a synergistic fashion to promote angiogenesis in a controlled environment [113]. Both growth factors are key components that control two different types of angiogenesis, branching and non-branching. (Fig. 3). Vasculogenesis begins approximately at 21 days post- conception when mesenchymal stem cells inside the mesenchymal villi of the placenta differentiate to be- come hemangiogenic progenitor cells [114]. These pro- genitor cells eventually migrate towards the periphery of the villous columns and coalesce to form hemangiogenic cords, the primitive original vessels of the villous [115]. Eventually these cords will mature into a more sophisti- cated network of vessels, differentiating into intermedi- ate villi with capillary networks of branched vessels [116]. This process is almost totally driven by paracrine signaling of VEGF-A from the cytotrophoblast [114]. VEGF-A works through receptor tyrosine kinase recep- tors, VEGFR-1 and VEGFR-2, to stimulate branched angiogenesis [117]. Branching angiogenesis requires a series of steps including permeabilization of vascular tissue, degradation of the basement membrane, and increased proliferation and migration of endothelial cells. This leads to the formation of endothelial cell tubes and recruitment of pericytes to the exterior of the capillary, forming a stable vessel [115, 118]. These mechanisms lead to the creation of a network of immature intermedi- ate villi containing superficially located capillaries lying directly beneath the trophoblast layer of the villous p [ ] Finally, the metalloproteinase (MMP) family of pro- teins is a critical group of enzymes that facilitate inva- sion. In addition to degrading the ECM, MMPs also can modify cell adhesion molecules like integrins and acti- vate cytokines to stimulate epithelial-mesenchymal tran- sition and drive cell invasion [104]. Cell invasion Human placentation is unique in that the EVT cells of the placenta invade fully into the maternal decidua to encapsu- late and erode the spiral arteries, exposing the placenta to maternal blood [88]. The similarities between cell invasion of EVT cells and cancer cells are striking. However, one key difference is that trophoblast cells adhere to a tightly regu- lated pattern of proliferation then differentiation and inva- sion without metastasis into new tissues. Cancer cells proliferate rapidly, eventually seeking out other tissues to metastasize towards. Not surprisingly, many of the same factors are required for both neoplastic cells and tropho- blast cells. Some of these requirements for invasion include altered expression of cell adhesion molecules, secretion of proteinases, and epithelial-mesenchymal transition. The integrin family is inextricably linked with the TGF- β signaling pathway. TGF- β is both a regulator and regulated by several integrins in many different cell types [93]. Both α1β1 and α5β1 expression is stimulated by TGF- β in fibroblast cells. Additionally, α5β1 has been found to be upregulated by TGF- β in both carcin- oma cells and lung cancer cells [93, 94]. As TGF- β is known to have an important role in both the inhibition and promotion of trophoblast cell invasion [95, 96], these data imply that there is a delicate interplay between TGF- β signaling and the regulation of the integ- rins α5β1 and α1β1 during early placental development. In non-invasive cells, there is a network of proteins that harness cells to the extracellular matrix (ECM) and to each other. However, in invasive or metastatic cells, this network is downregulated [89] which allows cells to seek out new tissues. One group of altered proteins is the integrin family. Integrins are a heterodimeric family of cell membrane proteins that are made up of at least 18 α subunits and 8 β subunits [90]. These subunits dimerize to form at least 24 different receptors, allowing them to bind to a variety of different ECM ligands. Because of this diversity, some integrins promote adhesion and some Page 6 of 12 Page 6 of 12 West et al. Reproductive Biology and Endocrinology (2018) 16:101 West et al. Reproductive Biology and Endocrinology (2018) 16:101 West et al. Reproductive Biology and Endocrinology (2018) 16:101 Another driver of cell invasion shared between cancer and placentation is the loss of expression of the cell adhesion molecule E-cadherin. Cell invasion Found at the adherens junctions of epi- thelial cells, E-cadherin is a potent promoter of cell-cell adhe- sion [97]. Known as a suppressor of invasion, decreased function of E-cadherin is directly correlated with invasion and tumor metastasis [98, 99]. E-cadherin also plays a critical role in the maintenance of the epithelial cell phenotype, with a loss of E-cadherin being the final step to trigger the epithelial-mesenchymal transition (EMT) [100], a process that is not only important during early embryonic develop- ment but also cancer. E-cadherin is predominantly expressed in anchored placental villi of first and second trimester pla- centas, gradually becoming down-regulated as cells differen- tiate to become EVT [101]. The transcription factor Snail, transcriptionally regulates E-cadherin, by binding to the E-box elements found on Snail’s promoter region to trigger EMT and has also been suggested to regulate E-cadherin ex- pression in EVT [102]. There is a layer of proliferative, non-invasive EVTcells found in the proximal and distal parts of anchored villi and as these cells undergo EMT to become invasive and migratory, there is a change in E-cadherin expression. However, term placentas from women with HELLP syndrome and pre-eclampsia found a reduction of E-cadherin in EVT cells with an apparent increase in Snail expression [102]. Snail appears to be the main regulator of decreased E-cadherin in most tumor expression and it now appears to be an important regulator of E-cadherin in EVT cells as well. Addtionally, E-cadherin is known to be essential for early embryonic and placental development as E-cadherin −/−mice have severe epithelial trophoblast defects and die at the time of implantation [103]. recognition of pregnancy during the first trimester, the developing embryo secretes proteins to decidualized endometrial stromal cells, allowing for upregulation of MMPs [109]. In immortalized JEG-3 cells and in villous tissue explants, addition of hCG to culture medium in- creased invasion in a dose dependent manner [110, 111]. Interestingly, these data suggest that the uterus has the ability to influence invasion, keeping this process regu- lated and local. This is in direct contrast to the unregu- lated and rampant invasion seen in metastatic cancer. Angiogenesis Several MMPs, including MMP-2, MMP-3, and MMP-9 have been de- scribed in different locations in the placenta; however there is evidence to suggest that MMP-9 is the most in- fluential proteinase during placental invasion [105, 106]. MMP-2 and MMP-9 are found at their highest levels in the extravillous cytotrophoblast between 6 and 8 weeks of pregnancy, appearing to facilitate trophoblast invasion into the decidua [107]. Interestingly, MMP expression isn’t restricted to the invasive trophoblast cells as MMPs have been described in the endometrial stromal and nat- ural killer cells of the decidua [108]. Furthermore, per- missiveness to invasion by the decidua seems to be influenced by the presence of cytotrophoblast cells. This interaction between uterine and trophoblast MMPs could be regulated by the pregnancy hormone, human chorionic gonadotropin (hCG). To stimulate maternal West et al. Reproductive Biology and Endocrinology (2018) 16:101 Page 7 of 12 Fig. 3 Angiogenesis in the placenta. During the first trimester FGF promotes vasculogenesis by promoting the differentiation of mesenchymal stem cells into hemangiogenic progenitor cells. These cells aggregate to form hemangiogenic cords and eventually primitive capillaries. VEGF-A promotes the angiogenesis of these capillaries through branching angiogenesis. As pregnancy progresses, PlGF is upregulated leading to non-branching angiogenesis and elongated capillaries Fig. 3 Angiogenesis in the placenta. During the first trimester FGF promotes vasculogenesis by promoting the differentiation of mesenchymal stem cells into hemangiogenic progenitor cells. These cells aggregate to form hemangiogenic cords and eventually primitive capillaries. VEGF-A promotes the angiogenesis of these capillaries through branching angiogenesis. As pregnancy progresses, PlGF is upregulated leading to non-branching angiogenesis and elongated capillaries upregulate both VEGF and VEGFR expression [126– 128]. As with tumorigenesis, hypoxia is necessary in early placental development. During the first trimester, placental development occurs in a low-oxygen environ- ment due to the absence of access to maternal circula- tion [129]. These conditions are considered key to stimulating placental vasculogenesis. In placental fibro- blasts, hypoxia upregulates both VEGF mRNA and pro- tein [130]. One mechanism working to regulate VEGF through hypoxia is the glycoprotein Fibronectin. Fibro- nectin works through its high affinity integrin receptor, α5β1 to stimulate VEGF during angiogenesis of embryos as well as several tumors [131, 132]. Bovine aortic endo- thelial cells grown in a low pH environment to mimic hypoxia, had increased interactions between fibronectin and VEGF [133]. Genomic instability Genomic instability is widely acknowledged as a hallmark of cancer. Ranging widely from nucleotide mutations to al- ternations in chromosome number or structure (known as chromosome instability), genomic instability can have major deleterious effects on normal cells [139]. However, some degree of instability appears to be tolerated by cells and has been documented in human embryos. One study analyzed blastomeres from women under 35 years of age that had undergone in vitro fertilization (IVF). Upon ana- lysis, researchers found that 70% of all embryos had some chromosomal genomic abnormality. Additionally, only 9% of the embryos analyzed had a 100% occurrence of diploid blastomeres [140]. This suggests that genomic instability is prevalent in human embryos and potentially explains the low levels of fertility in women compared to other species. Another study analyzed levels of aneuploidy in fertilized oocytes, cleavage stage embryos, and blastocyst stage em- bryos. There was a large increase in aneuploidy between the fertilized oocyte stage and cleavage stage embryos. As embryos developed to the blastocyst stage, there was a sig- nificant decrease in the aneuploidy rate (83% aneuploidy in cleavage stage versus 58% in blastocyst stage). However, while there was a decrease in rates of aneuploidy, there were still high levels of overall chromosomal abnormality [141]. These data suggest that, as with tumors, for rapid placental development to occur a lapse in the cell-cycle checkpoint machinery must occur. Additionally, it has been suggested that this genomic instability actually provides an advantage for embryo implantation [142]. Finally, even with less priority attributed to cell-cycle checkpoints and DNA repair, there must be some regula- tion of DNA repair in the placenta for it to develop into a proper functioning organ. Our laboratory is currently fo- cused on the regulation of DNA repair and genome stabil- ity in trophoblast cells by the tumor suppressor BRCA1. BRCA1 is a multifunctional protein involved in many dif- ferent aspects of cell cycle regulation including; regulation of transcription of several proliferation factors, homologous recombination of double-stranded breaks (DSBs), cell-cycle checkpoint regulation, and chromatin remodeling [148]. BRCA1 works to repair DNA damage by acting as a scaf- folding protein for other DNA repair proteins and also pro- motes strand-invasion by interacting with the recombinase protein, Rad51 [149, 150]. Additionally, BRCA1 forms a re- pressor complex with CtIP and ZNF350. This repressor complex binds to promoter regions of several oncofetal proteins to prevent transcription [151]. Angiogenesis In tumors, hypoxia has been shown to Page 8 of 12 Page 8 of 12 West et al. Reproductive Biology and Endocrinology (2018) 16:101 proposed that endoreduplication occurs during times of genomic instability to increase tissue mass while cell proliferation is decreased to prevent propagation of cells with damaged chromosomes [146]. In the placenta, extravillous trophoblast cells invade into the decidua as two different cell types, interstitial cytotrophoblast cells (iCTBs) and endovascular cytotrophoblast cells (eCTBs). The iCTBs are the cells that invade into the decidua, moving as deep as the first third of the myometrium. Once at the myometrium, these cells undergo a final step of differentiation where they undergo endoredupli- cation to become multinucleated [147]. Similarly to how damaged cells undergo endoreduplication to increase size, it is thought that iCTBs undergo endoreduplication to further penetrate into the myometrium of the uterus. phenomenon is also seen in uteroplacental hypoxia, where maternal oxygen levels are normal but there is impaired oxygen circulation throughout the placenta and fetus [137]. However, in instances of post-placental hypoxia where the mother has normal oxygen levels but the fetus is hypoxic, the placenta may become hyperoxic leading to inappropriate levels of oxygen during early development, causing increased levels of PlGF and in- creased non-branching angiogenesis [138]. This early onset placental hyperoxia often leads to the most severe form of pre-eclampsia, with increased adverse outcomes and fetal mortality [138]. Angiogenesis Additionally, low pH conditions stimu- lated the secretion of fibronectin into culture medium in human trophoblast cells [134]. Finally, in differentiated placental multipotent mesenchymal stromal cells (PMSCs), α5β1 has been show to interact with fibronec- tin to promote VEGF-A induced differentiation and mi- gration [135]. surface [119]. These branched vessels are responsible for the dramatic increase in villous blood vessels facilitating enhanced fetoplacental blood flow to accommodate the rapidly developing fetus [120]. Branching angiogenesis and VEGF-A expression continues to dominate placental vascularization quickly producing a multitude of vessels until approximately the 26th week of gestation [121]. At this point, villous vascularization undergoes a switch from branching to non-branching angiogenesis. At this point, the focus moves from producing more vessels to increasing the length of the existing vessels [122]. Non-branching angiogenesis is driven by another mem- ber of the VEGF family of proteins, PlGF. Whereas VEGF-A and VEGFR-2 are expressed at high levels during early pregnancy, waning as pregnancy advances [122]; PlGF is expressed at relatively low levels during the first trimester of pregnancy but increases at 11–12 weeks, reaching peak levels at week 30 of pregnancy [123]. PlGF is thought to have an antagonistic effect on VEGF-A, forming a heterodi- mer that prevents VEGF-A from activating either VEGF1-R or VEGF2-R [124]. At peak PlGF expression, the immature intermediate villi begin to form the mature intermediate villi. Non-branching angiogenesis leads to the formation of long, thin vessels found at the tips of the villous. These ves- sels continue to grow in length, eventually surpassing the boundaries of the mature intermediate villi to form ter- minal villi. Each terminal villous has a thin trophoblast layer covering only one or two capillary coils [125]. These villous structures are critical for diffusional gas exchange from mother to fetus [121] (Fig. 2). Additionally, PlGF is also regulated by low oxygen conditions, albeit in an opposite fashion to VEGF. Hu- man placental cells exposed to low oxygen conditions had decreased PlGF mRNA and protein [121]. Abnormal oxygen levels during early placental development are thought to lead to altered VEGF/PlGF expression lead- ing to pre-eclampsia. For example, in the instances of pre-placental hypoxia where mother, placenta, and fetus are hypoxic (due to high altitude or anemia) there is an increase of VEGF and branched angiogenesis [136]. This Similarly to cancer, both VEGF and PlGF are regulated by hypoxia. Genomic instability Additionally, this genomic in- stability of immortalized cells leads to a higher propen- sity for these cells to behave as cancer cells, no longer regulated in the controlled manner that characterizes trophoblast cells. This creates a need for a better model Genomic instability One oncofetal pro- teins target already discussed in the “cell proliferation” sec- tion is HMGA2. In addition to promoting cell proliferation, increased levels of HMGA2 causes genomic instability by preventing non-homologous end-joining as well as delaying clearance of γ-H2AX, a marker for DSBs, [152]. BRCA1−/− knockout mice are embryonic lethal before gestational day 7.5 due to dramatic decreases in cell proliferation and poor differentiation of the extraembryonic tissue. These knock- out embryos have a complete loss of diploid trophoblast cells with an overabundance of trophoblast giant cells [153]. Interestingly, mouse trophoblast giant cells are poly- ploid and are potentially accustomed to levels of genomic instability through endoreduplication, which is necessary for trophoblast giant cell function. In addition to aneuploidy, extravillous trophoblast cells of the placenta are also polyploid [143]. These cells are analogous to murine trophoblast giant cells that are also invasive. However, rodent trophoblast giant cells have ploidy levels that can reach up to 1024 N compared to the 4–8 N recorded in extravillous trophoblast cells [144]. These cells become polyploid through a process known as endoreduplication, where cells undergo mitosis but fail to divide after DNA replication. Endore- duplication is another phenomenon that occurs in can- cer to promote genomic instability [145]. It has been Unfortunately, this question will be hard to prove using today’s current models of trophoblast cell development. Trophoblast cells derived from first trimester placentas are very difficult to obtain. Additionally these cells are hard to culture, making alternative model systems to study tropho- blast development essential. Immortalized cell lines are ex- tensively used as a model for trophoblast development and West et al. Reproductive Biology and Endocrinology (2018) 16:101 Page 9 of 12 Page 9 of 12 differentiation. However, these cells present their own shortcomings that make them less than ideal candidates for use. These shortcomings are especially apparent when it comes to studying DNA damage and genomic instability. For example, cytogenetic analysis of the extravillous first trimester Swan71 cell line immortal- ized with hTert revealed that these cells were near pentaploid in karyotype [136]. This is almost certainly due to chromosomal missegregation during mitosis, leading to a heterogeneous population of aneuploid cells. Additionally, when our lab began using this cell line to investigate BRCA1 in human trophoblast cells we found high levels of markers for DNA damage. Genomic instability We created a BRCA1 knockout trophoblast cell line using CRISPR-Cas9 genome editing to investigate levels of DNA damage by immunostaining for markers of double and single-stranded breaks. Surprisingly, the level of DSBs, as evidenced by immunostaining for γ-H2AX, was indistinguishable between BRCA1 knockout cells (BrKO) and wild-type Swan71 cells (Fig. 4). This high level of double-stranded breakage was confirmed using another marker for DSBs, 53BP1 (data not shown). These data corroborate the idea that immortalized cells suffer from cellular crises when cultured in vitro, resulting in microsatellite and chromosomal instability. Due to this propensity towards genomic instability in culture, immortalized cells are unlikely to provide insight into the role of genomic instability during early placental development. Additionally, this genomic in- stability of immortalized cells leads to a higher propen- sity for these cells to behave as cancer cells, no longer regulated in the controlled manner that characterizes trophoblast cells. This creates a need for a better model system to investigate the regulation of oncogenic pro- cesses during trophoblast development. differentiation. However, these cells present their own shortcomings that make them less than ideal candidates for use. These shortcomings are especially apparent when it comes to studying DNA damage and genomic instability. For example, cytogenetic analysis of the extravillous first trimester Swan71 cell line immortal- ized with hTert revealed that these cells were near pentaploid in karyotype [136]. This is almost certainly due to chromosomal missegregation during mitosis, leading to a heterogeneous population of aneuploid cells. Additionally, when our lab began using this cell line to investigate BRCA1 in human trophoblast cells we found high levels of markers for DNA damage. We created a BRCA1 knockout trophoblast cell line using CRISPR-Cas9 genome editing to investigate levels of DNA damage by immunostaining for markers of double and single-stranded breaks. Surprisingly, the level of DSBs, as evidenced by immunostaining for γ-H2AX, was indistinguishable between BRCA1 knockout cells (BrKO) and wild-type Swan71 cells (Fig. 4). This high level of double-stranded breakage was confirmed using another marker for DSBs, 53BP1 (data not shown). These data corroborate the idea that immortalized cells suffer from cellular crises when cultured in vitro, resulting in microsatellite and chromosomal instability. Due to this propensity towards genomic instability in culture, immortalized cells are unlikely to provide insight into the role of genomic instability during early placental development. References 30. Ladines-Llave CA, et al. Cytologic localization of epidermal growth factor and its receptor in developing human placenta varies over the course of pregnancy. 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Medical record validation of mate of preeclampsia. J Clin Epidemiol. 2010;63(8):932–7. 35. Maglione D, et al. Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc Natl Acad Sci U S A. 1991; 88(20):9267–71. 8. Ergaz Z, Avgil M, Ornoy A. Intrauterine growth restriction-etiology and consequences: what do we know about the human situation and experimental animal models? Reprod Toxicol. 2005;20(3):301–22. 8. Ergaz Z, Avgil M, Ornoy A. Intrauterine growth restriction-etiology and consequences: what do we know about the human situation and experimental animal models? Reprod Toxicol. 2005;20(3):301–22. 36. Conclusion While understanding the consequences of fetal growth re- striction has increased exponentially over the past few de- cades, there is still a need to elucidate the underlying cause behind placental insufficiency during placental organogen- esis. Understanding what is driving placental insufficiency during early fetal development will be essential in the devel- opment of better diagnostic and treatment tools for the prevention and treatment of IUGR. The ability of placental cells to divide rapidly, differentiate, invade and migrate into tissues, and eventually create their own vascular network makes these cells an ideal system to gain insight into cancer biology and tumor metastasis. Alternatively, as placental pathologies like intrauterine growth restriction (IUGR) and pre-eclampsia are multi-faceted disorders with no known cause, better understanding the molecular mechanisms that drive oncogenic processes will provide better insight into how the early placenta develops. Pre-eclampsia and IUGR are rarely diagnosed until after 20 weeks of gestation, significantly later than pathogenesis begins. Therefore it is critical to start thinking of oncofetal proteins in their original roles, namely as drivers of cell proliferation, differentiation, invasion, and cell survival during early embryogenesis and placental development. Studying how oncofetal proteins drive placentation is essential to facilitate the process of providing better diagnostics for earlier screenings as well as treatment, ensuring the proper care for healthier babies and happier mothers. Fig. 4 γ-H2AX in BrKO and WT Swan71 cells. Immunostaining for γ-H2AX (green) and merged with DAPI (blue) in BRCA1 knockout cells and wild-type Swan71 cells imaged at 20x magnification Fig. 4 γ-H2AX in BrKO and WT Swan71 cells. Immunostaining for γ-H2AX (green) and merged with DAPI (blue) in BRCA1 knockout cells and wild-type Swan71 cells imaged at 20x magnification Page 10 of 12 Page 10 of 12 West et al. Reproductive Biology and Endocrinology (2018) 16:101 West et al. 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Anti-CD20 as the B-Cell Targeting Agent in a Combined Therapy to Modulate Anti-Factor VIII Immune Responses in Hemophilia a Inhibitor Mice
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ORIGINAL RESEARCH ARTICLE published: 06 January 2014 doi: 10.3389/fimmu.2013.00502 Anti-CD20 as the B-cell targeting agent in a combined therapy to modulate anti-factor VIII immune responses in hemophilia A inhibitor mice ORIGINAL RESEARCH ARTICLE bli h d 06 J 2014 Chao Lien Liu1,2, PeiqingYe1, Jacqueline Lin1, Chérie L. Butts 3 and Carol H. Miao1,4* 1 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA 2 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 3 Biogen Idec, Inc., Weston, MA, USA 4 Department of Pediatrics University of Washington Seattle WA USA Chao Lien Liu1,2, PeiqingYe1, Jacqueline Lin1, Chérie L. Butts 3 and Carol H. Miao1,4* 1 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA 2 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 3 Biogen Idec, Inc., Weston, MA, USA 4 Department of Pediatrics University of Washington Seattle WA USA 4 Department of Pediatrics, University of Washington, Seattle, WA, USA Neutralizing antibody formation against transgene products can represent a major compli- cation following gene therapy with treatment of genetic diseases, such as hemophilia A. Although successful approaches have been developed to prevent the formation of anti-factor VIII (FVIII) antibodies, innovative strategies to overcome pre-existing anti-FVIII immune responses in FVIII-primed subjects are still lacking. Anti-FVIII neutralizing anti- bodies circulate for long periods in part due to persistence of memory B-cells. Anti-CD20 targets a variety of B-cells (pre-B-cells to mature/memory cells); therefore, we investigated the impact of B-cell depletion on anti-FVIII immune responses in hemophilia A mice using anti-CD20 combined with regulatoryT (Treg) cell expansion using IL-2/IL-2mAb complexes plus rapamycin. We found that anti-CD20 alone can partially modulate anti-FVIII immune responses in both unprimed and FVIII-primed hemophilia A mice. Moreover, in mice treated with anti-CD20+IL-2/IL-2mAb complexes+rapamycin+FVIII, anti-FVIII antibody titers were significantly reduced in comparison to mice treated with regimens targeting only B or T cells. In addition, titers remained low after a second challenge with FVIII plasmid. Treg cells and activation markers were transiently and significantly increased in the groups treated with IL-2/IL-2mAb complexes; however, significant B-cell depletion was obtained in anti-CD20-treated groups. Importantly, both FVIII-specific antibody-secreting cells and memory B-cells were significantly reduced in mice treated with combination therapy.This study demonstrates that a combination regimen is highly promising as a treatment option for modulating anti-FVIII antibodies and facilitating induction of long-term tolerance to FVIII in hemophilia A mice. Reviewed by: Reviewed by: David William Scott, Uniformed Services University of the Health Sciences, USA Federico Mingozzi, UPMC Paris 6 and Genethon, France Reviewed by: David William Scott, Uniformed Services University of the Health Sciences, USA Federico Mingozzi, UPMC Paris 6 and Genethon, France *Correspondence: Carol H. Miao, Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, 1900 Ninth Avenue, C9S-7, Seattle, WA 98101, USA Keywords: anti-CD20, factor VIII, hemophilia, tolerance induction, immunomodulation, B-cell depletion Edited by: y Federico Mingozzi, UPMC Paris 6 and Genethon, France Federico Mingozzi, UPMC Paris 6 and Genethon, France Keywords: anti-CD20, factor VIII, hemophilia, tolerance induction, immunomodulation, B-cell depletion INTRODUCTION Control mice were treated with rat IgG2a (250 µg/mouse) on days 0 and 14. Anti-CD20 significantly reduced total B220+/CD19+ B-cells (80–90% reduction) both in blood (Figures S1A,B in Supplementary Material) and spleen (Figure S1C in Supplementary Material). B-cell depletion was sustained over 4– 6 weeks with gradual return to normal levels at 8 weeks following treatment. No reduction in B-cell levels were observed in IgG2a isotype-treated control and naive mice. pp y g In our previous studies, administration of IL-2/IL-2mAb com- plexes prevented anti-FVIII immune responses in hemophilia A mice following gene or protein replacement therapy (24, 25). Nev- ertheless, overcoming pre-existing antibody responses in primed subjects remains challenging. Anti-FVIII neutralizing antibodies persist in part due to memory B-cells (26). Moreover, molec- ular studies have shown that long-lived plasma cells (LLPCs) can support chronic inflammatory processes by secreting path- ogenic antibodies for long periods (27, 28). It is hypothesized that LLPCs may also play an important role in prolonged pro- duction of anti-FVIII antibodies in hemophilia A patients. In this study, we developed a treatment strategy of single or combina- tion therapy using agents targeting B-cells (to eliminate memory responses) and those inducing Treg cell expansion (to suppress T helper cell function). By using a combination of anti-CD20+IL- 2/IL-2mAb complexes+rapamycin, anti-FVIII immune responses were significantly reduced. Hemophilia A mice treated with com- bination therapy showed little or no anti-FVIII antibodies titers, and this was also evident after a second challenge with FVIII plas- mid. This study sought to identify strategies toward induction of immune tolerance to FVIII transgene product following gene ther- apy and to demonstrate that combination therapy targeting B and T lymphocytes can be a viable option. yp In order to investigate the best treatment schedule and thera- peutic effects of anti-CD20 treatment, three groups of hemophilia A mice were injected with three different dosages: 100 µg/mouse (on days −2, 0, 3, 6, and 9), 250 µg/mouse (on days 0 and 14), and 500 µg/mouse (on day 0). Control mice were treated with rat IgG2a 250 µg/mouse on days 0 and 14. All mice were injected with FVIII plasmid (50 µg/mouse) at day 0. Following treatment, FVIII activities and neutralizing antibody titers were assessed by aPTT and Bethesda assays at different time points. INTRODUCTION lysis (CDC) (13), which are considered to be immediate and com- paratively short-acting. Nevertheless, the clinical response to a single course of the anti-CD20 mAb can be late acting and pro- longed. This has led to the suggestion that anti-CD20 could also have an immunization effect (14); however,it is unknown whether this correlates with clinical outcome. Recently anti-CD20 IgG1 (15) and IgG2a (16) molecules have been used successfully to prevent the production of anti-FVIII antibodies. Hemophilia A is an X-linked, congenital bleeding disorder result- ing from a deficiency of factor VIII (FVIII). Approximately 35% of patients with hemophilia A develop complications of anti- FVIII neutralizing antibodies following FVIII protein replace- ment therapy (1, 2). In order to overcome anti-FVIII immune responses, we sought transient immunosuppressive strategies that can reduce pre-existing antibodies and induce long-term tolerance to FVIII. CD20 is a 35-kDa transmembrane protein expressed on B-cells from the pre-B-cell stage to mature B lymphocytes, but not plasma cells (3). Monoclonal antibodies (mAbs) against human CD20 (Rituximab) induce rapid B-cell depletion (4) and is currently approved by the Food and Drug Administra- tion (FDA) for treatment of non-Hodgkin B-cell lymphomas (5, 6) and several autoimmune disorders including type 1 diabetes (T1D) (7), rheumatoid arthritis (8), and Sjögren’s syndrome (SS) (9). Anti-CD20 depletes B-cells via several mechanisms (10, 11), such as direct induction of apoptosis, antibody-dependent cell- mediated cytotoxicity (ADCC) (12), and complement-dependent Webster et al. (17) defined a strategy in which complexes of IL-2/IL-2-specific mAbs (JES6-1A12) can be used to selectively expand CD4+CD25+Foxp3+ regulatory T (Treg) cells in vivo with little or no change in other cell populations. This approach has been used to successfully treat asthma (18) and experimen- tal myasthenia gravis (MG) (19) in mouse models. In addition, rapamycin is currently used as an immunosuppressive agent to prevent acute graft rejection in humans (20). Rapamycin com- bines with the intracellular immunophilin FK506-binding protein (FKBP12) to form FKBP12-rapamycin complexes that inhibit the activity of mammalian target of rapamycin (mTOR) and result January 2014 | Volume 4 | Article 502 | 1 www.frontiersin.org Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. Liu et al. in inhibiting effector T-cell (Teff) proliferation (21). Rapamycin not only increased Treg:Teff cell ratios but also improved the suppressive activity of Treg cells (22, 23). B domain-deleted hFVIII under the control of the liver-specific hAAT promoter (HP) and the hepatic control region (HCR) on day 0. ANTI-CD20 TREATMENT CAN REGULATE ANTI-FVIII PRODUCTION IN A NON-VIRAL GENE THERAPY MODEL To test if B-cell depletion can regulate anti-FVIII immune responses, we utilized anti-CD20 IgG2a antibody (anti-CD20) in a murine model. Hemophilia A mice were divided into two treatment groups (Figure 1 and Figure S1 in Supplemen- tary Material): FVIII plasmid-treated mice were given anti- CD20 (250 µg/mouse) on days 0 and 14 combined with a FVIII plasmid (pBS-HCRHPI-FVIIIA; 50 µg/mouse) expressing A similar treatment was given to FVIII plasmid-primed hemo- philia A mice with pre-existing neutralizing antibodies. These mice were developed by hydrodynamic injection of 50 µg of FVIII FIGURE 1 | Factor VIII gene expression and anti-FVIII antibody formation after FVIII plasmid+anti-CD20 treatment in hemophilia A mice. Four groups of hemophilia A mice were treated with FVIII plasmid (50 µg/ treatment/mouse) at day 0 and i.v. injection of anti-CD20 at various doses and schedules as listed in the following: group 1: 100 µg/treatment/mouse, injected at days −2, 0, 3, 6, and 9. Group 2: 250 µg/treatment/mouse, injected at days 0 and 14. Group 3: 500 µg/treatment/mouse, injected at day 0. Group 4: control rat IgG, 250 µg/treatment/mouse, injected at days 0 and 14. Peripheral blood samples were collected at different time points to examine FVIII activities (A) and inhibitor titers (B). Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments. Frontiers in Immunology | Microbial Immunology January 2014 | Volume 4 | Article 502 | 2 mation ur es and injected at days 0 and 14. Group 3: 500 µg/treatment/mouse, injected at day 0. Group 4: control rat IgG, 250 µg/treatment/mouse, injected at days 0 and 14. Peripheral blood samples were collected at different time points to examine FVIII activities (A) and inhibitor titers (B). Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments. injected at days 0 and 14. Group 3: 500 µg/treatment/mouse, injected at day 0. Group 4: control rat IgG, 250 µg/treatment/mouse, injected at days 0 and 14. Peripheral blood samples were collected at different time points to examine FVIII activities (A) and inhibitor titers (B). Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments. injected at days 0 and 14. Group 3: 500 µg/treatment/mouse, injected at day 0. Group 4: control rat IgG, 250 µg/treatment/mouse, injected at days 0 and 14. INTRODUCTION In the rat IgG2a control group, anti-FVIII antibody appeared within 2 weeks post plasmid injection, increased to high-titers at 3–4 weeks, and main- tained high-titer levels through 24 weeks. In addition, initially high levels of FVIII activity decreased to low-undetectable levels within 4 weeks (Figure 1). In the anti-CD20-treated groups, one mouse from each group of mice had persistent FVIII activity with- out detectable inhibitory anti-FVIII antibodies (Figure 1). The remaining mice displayed delayed immune responses; however, all mice generated moderate to high-titers of neutralizing antibodies with FVIII activity decreasing to undetectable levels at 6–15 weeks. While antibody titers were clearly reduced following anti-CD20 treatment, these titers increased over time. Although mice treated with anti-CD20 were not completely resistant to FVIII immune responses, they all exhibited partial modulatory effects compared to the rat IgG2a treated control mice. EFFECTS ON T/B-CELL RESPONSES IN PERIPHERAL BLOOD AND SPLEEN WERE SIGNIFICANT IN TREATED HEMOPHILIA A MICE / SPLEEN WERE SIGNIFICANT IN TREATED HEMOPHILIA A MICE Next, we evaluated changes in T- and B-cell popula- tions of hemophilia A mice following treatment. We ana- lyzed peripheral blood in mice treated with IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (n = 4). Mice with neutralizing antibodies and those treated with anti-CD20+FVIII; IL-2/IL-2mAb complexes+rapamycin+FVIII; and IL-2/IL-2mAb complexes+anti-CD20+FVIII were used as control groups. Mice were treated weekly with the indicated regimen for 4 weeks. Flow cytometry analysis showed that the CD4+ T cells in total T-cell populations did not significantly change over time (Figure 4A). Interestingly, there was a slight decrease in the percentage and numbers of CD4+ T cells in anti-CD20 treated groups. However, the percentage of CD4+CD25+Foxp3+ T cells within the CD4+ T- cell compartment was significantly increased in the IL-2/IL-2mAb complexes treated groups compared to other groups for 4 weeks during treatment period (Figure 4B; P < 0.05). The expanded Treg cells declined rapidly to baseline levels within 2 weeks post treatment. Similar to our previous studies (24, 25), IL-2/IL-2mAb complex-expanded Treg cells showed considerably higher expres- sion of molecules crucial for the suppressive function of Treg cells, including CD25, glucocorticoid-induced tumor necrosis factor receptor (GITR), and cytotoxic T-lymphocyte antigen 4 (CTLA- 4) (Figure 4C). The substantial increase in Treg cells following injection of IL-2/IL-2mAb complexes occurred not only in blood but also appeared as a fivefold increase in spleen (Figure S2A in COMBINATION TREATMENT WITH IL-2/IL-2mAb COMPLEXES, RAPAMYCIN, AND ANTI-CD20 ENHANCED FVIII PLASMID-MEDIATED GENE THERAPY IN HEMOPHILIA A MICE Since anti-CD20 treatment can partially modulate anti-FVIII immune responses in hemophilia A mice, we investigated whether a combination therapy using anti-CD20 to deplete B-cells and IL-2/IL-2mAb complexes to expand Treg cells (24) can more consistently reduce anti-FVIII responses. Hemophilia A mice were treated with: IL-2/IL-2mAb complexes+rapamycin+anti- CD20+FVIII (n = 4, group 1; Figure 3A); IL-2/IL-2mAb complexes+anti-CD20+FVIII (n = 4, group 2; Figure 3B); IL-2/IL-2mAb complexes+rapamycin+FVIII (n = 3, group 3; Figure 3C); anti-CD20+FVIII (n = 4, group 4; Figure 3D); and mock agents (control inhibitor mice; n = 2, group 5; Figure 3E) weekly for 4 weeks. FVIII protein (1 U/mouse) was given weekly for 4 weeks for induction of FVIII-specific tolerance during the treatment period. FVIII plasmid second challenge was applied at 5 weeks following 4 weeks of treatment (week 9). Except for con- trol mice, all treated groups showed decreased antibody titers. The most significant and prolonged reduction of neutralizing antibody titers was observed using the combination treatment of IL-2/IL- 2mAb complexes+anti-CD20+rapamycin+FVIII (Figure 3A). FIGURE 2 | Factor VIII gene expression and anti-FVIII antibody titers following anti-CD20 treatment in FVIII plasmid-primed hemophilia A mice with pre-existing inhibitors. Mice were primed with FVIII plasmid to induce high-titer inhibitory antibodies at 8 weeks before anti-CD20 treatment. The inhibitor mice were then treated with anti-CD20. Group 1: control rat IgG, 250 µg/treatment/mouse, injected at days −7, −4, and 0. Group 2: anti-CD20, 250 µg/treatment/mouse, injected at days −7, −4, and 0. Peripheral blood samples were collected at different time points to evaluate FVIII activities (A) and inhibitor titers (B). Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments. www frontiersin org Jan ar 2014 | Vol me 4 | Article 502 | 3 h d at days −7, −4, and 0. Group 2: anti-CD20, 250 µg/treatment/mouse, injected at days −7, −4, and 0. Peripheral blood samples were collected at different time points to evaluate FVIII activities (A) and inhibitor titers (B). Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments URE 2 | Factor VIII gene expression and anti-FVIII antibody FIGURE 2 | Factor VIII gene expression and anti-FVIII antibody titers following anti-CD20 treatment in FVIII plasmid-primed at days −7, −4, and 0. Group 2: anti-CD20, 250 µg/treatment/mouse, injected at days −7, −4, and 0. ANTI-CD20 TREATMENT CAN REGULATE ANTI-FVIII PRODUCTION IN A NON-VIRAL GENE THERAPY MODEL Peripheral blood samples were collected at different time points to examine FVIII activities (A) and inhibitor titers (B). Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments. FIGURE 1 | Factor VIII gene expression and anti-FVIII antibody formation after FVIII plasmid+anti-CD20 treatment in hemophilia A mice. Four groups of hemophilia A mice were treated with FVIII plasmid (50 µg/ treatment/mouse) at day 0 and i.v. injection of anti-CD20 at various doses and schedules as listed in the following: group 1: 100 µg/treatment/mouse, injected at days −2, 0, 3, 6, and 9. Group 2: 250 µg/treatment/mouse, FIGURE 1 | Factor VIII gene expression and anti-FVIII antibody formation ft FVIII l id+ ti CD20 t t t i h hili A i F FIGURE 1 | Factor VIII gene expression and anti-FVIII antibody formation after FVIII plasmid+anti-CD20 treatment in hemophilia A mice. Four Frontiers in Immunology | Microbial Immunology Frontiers in Immunology | Microbial Immunology January 2014 | Volume 4 | Article 502 | 2 Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. plasmid via tail vein, and only mice with neutralizing antibody titers >30 Bethesda units (BU) were used. Mice were then treated with anti-CD20 (n = 5; 250 µg/mouse) on days −7, −4, and 0. Control mice were treated with IgG2a (n = 2; 250 µg/mouse) on days −7,−4,and 0. Plasma samples were collected on day 1 follow- ing treatment. As shown in Figure 2, neutralizing antibody titers were maintained at high levels in rat IgG2a control-treated mice. In contrast, neutralizing antibody titers were significantly reduced following anti-CD20 treatment in 80% (four of five) treated mice. In particular, 20% (one of five) of anti-CD20-treated mice showed therapeutic FVIII activities levels for 14 weeks (Figure2B). These results indicate that anti-CD20 can partially modulate anti- FVIII immune responses both in the FVIII unprimed and primed hemophilia A mice. Neutralizing antibody titers were reduced to 0 in 50% (two of four) treated mice, and a reversion of 8% FVIII gene expres- sion was observed. Additional animals (n = 3–5/group) have been treated in repeated experiments with similar results to those shown (Figures 3A–E). In addition, treated mice were chal- lenged with non-specific antigen, TNP-ficoll (24), at 16 weeks following treatment (week 20). These animals responded simi- larly to control/naive mice, demonstrating the tolerance effect was FVIII-antigen-specific. COMBINATION TREATMENT WITH IL-2/IL-2mAb COMPLEXES, RAPAMYCIN, AND ANTI-CD20 ENHANCED FVIII PLASMID-MEDIATED GENE THERAPY IN HEMOPHILIA A MICE Peripheral blood samples were collected at different time points to evaluate FVIII activities (A) and inhibitor titers (B). Each symbol represents data obtained from an individual mouse. Data shown is representative of two independent experiments. FIGURE 2 | Factor VIII gene expression and anti-FVIII antibody tit f ll i ti CD20 t t t i FVIII l id i d | ers following anti-CD20 treatment in FVIII plasmid-primed g hemophilia A mice with pre-existing inhibitors. Mice were primed with FVIII plasmid to induce high-titer inhibitory antibodies at 8 weeks before anti-CD20 treatment. The inhibitor mice were then treated with anti-CD20. Group 1: control rat IgG, 250 µg/treatment/mouse, injected January 2014 | Volume 4 | Article 502 | 3 www.frontiersin.org Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. FIGURE 3 | Immunomodulation with separate or combined therapy by IL-2/IL-2mAb complexes, rapamycin, and anti-CD20 in FVIII plasmid-primed hemophilia A mice with pre-existing inhibitors. Four groups of hemophilia A inhibitor mice were treated separately with different combined regimens: (A) IL-2/IL-2mAb complexes+rapamycin+ anti-CD20+FVIII injection (n = 4, group 1), (B) IL-2/IL-2mAb complexes+anti-CD20+FVIII (n = 4, group 2), (C) IL-2/IL-2mAb complexes+rapamycin+FVIII injection (n = 3, group 3), (D) anti-CD20+FVIII injection (n = 4, group 4), and (E) Control inhibitor mice (n = 2, group 5). Each experimental group was treated with indicated immunomodulation regimen weekly for 4 weeks. Anti-FVIII antibody titers were assessed by Bethesda assay over time. Each symbol represents data obtained from an individual mouse. Data shown is representative of three independent experiments. complexes+rapamycin+FVIII injection (n = 3, group 3), (D) anti-CD20+FVIII injection (n = 4, group 4), and (E) Control inhibitor mice (n = 2, group 5). Each experimental group was treated with indicated immunomodulation regimen weekly for 4 weeks. Anti-FVIII antibody titers were assessed by Bethesda assay over time. Each symbol represents data obtained from an individual mouse. Data shown is representative of three independent experiments. FIGURE 3 | Immunomodulation with separate or combined therapy by IL-2/IL-2mAb complexes, rapamycin, and anti-CD20 in FVIII plasmid-primed hemophilia A mice with pre-existing inhibitors. Four groups of hemophilia A inhibitor mice were treated separately with different combined regimens: (A) IL-2/IL-2mAb complexes+rapamycin+ anti-CD20+FVIII injection (n = 4, group 1), (B) IL-2/IL-2mAb complexes+anti-CD20+FVIII (n = 4, group 2), (C) IL-2/IL-2mAb in Supplementary Material); and plasma B-cells (B220−CD138+) (Figure 5D). in Supplementary Material); and plasma B-cells (B220−CD138+) (Figure 5D). COMBINATION TREATMENT WITH IL-2/IL-2mAb COMPLEXES, RAPAMYCIN, AND ANTI-CD20 ENHANCED FVIII PLASMID-MEDIATED GENE THERAPY IN HEMOPHILIA A MICE Supplementary Material; middle panel and Figure S2B in Supple- mentary Material; upper panel). Almost all expanded Treg cells were Helios+ (Figure S2A in Supplementary Material; right panel and Figure S2B in Supplementary Material; upper panel) nat- ural Treg cells derived from thymus. In contrast, no significant change in the CD4+ T-cell population was observed in the spleen (Figure S2A in Supplementary Material; left panel and Figure S2B in Supplementary Material; upper panel). The expression levels of the activation markers of Treg cells including CD25, GITR, and CTLA-4 after IL-2/IL-2mAb complex treatment also reached high levels in the spleen (Figure S2B in Supplementary Material; lower panel). Frontiers in Immunology | Microbial Immunology COMBINATION THERAPY DEPLETES ANTI-FVIII-SPECIFIC ANTIBODY-SECRETING CELLS (ASCS) AND FVIII-SPECIFIC MEMORY B-CELLS IN MICE (C) Blood cells were also stained and analyzed for Treg cells markers: CD25, GITR, and CTLA-4. Data shown are median fluorescence intensity (MFI) values of the three activation markers. Data shown is representative of two independent experiments. FIGURE 4 | Effects of immunomodulation on CD4 T cells, CD4+CD25+Foxp3+ Treg cells andTregs activation markers in peripheral blood of treated hemophilia A inhibitor mice over time. Lymphocytes were isolated from the blood of naive (light grid), inhibitor only (light slant), anti-CD20+FVIII (white), IL-2/IL-2mAb complexes+rapamycin+FVIII (light gray), IL-2/IL-2mAb complexes+anti-CD20+FVIII (dark gray), and IL-2/IL-2mAb FVIII-specificASCs and memory B-cells at later time points (4 and 6 weeks) after treatment showed similar results as those obtained at 2 weeks. B and plasma cells. In a preliminary experiment, we found that either use of bortezomib (an inhibitor of 26S proteasome that can reduce/eliminate plasma cells) alone or in combination with T-cell-regulating agents such as IL-2/IL-2mAb complexes did not help reduce pre-existing neutralizing antibody titers (data not shown). Meslier et al. (29) reported similar results that borte- zomib only delayed the onset of FVIII neutralizing antibodies in hemophilia A mice but failed to eliminate established anti- FVIII IgG-producing cells. Anti-CD20 mAbs can deplete pan B lymphocytes, from pre-B-cells to memory B-cells, ranging from 50 to 90%. Rituximab (anti-human CD20) is beneficial in treat- ing patients with acquired hemophilia (30). Limited data have also been described in case reports with respect to the use of Rituximab in children (31, 32) and adults (31, 33) with con- genital hemophilia A and neutralizing antibodies. It has been hypothesized that concurrent administration of anti-CD20 and high-dose FVIII might be beneficial to treat hemophilia A patients with neutralizing antibodies. Emerging data suggests that repop- ulating transitional murine and human B-cells (which increase markedly in numbers following anti-CD20 depletion therapy) exhibited potent regulatory activity (34). Therefore, use of anti- CD20 may promote effects distinct from merely reducing the mature B-cell pool. IL-10 expressing immature B-cells may pro- mote a regulatory environment to aid in tolerance induction (35, 36). As anti-CD20 treatment exhibits a distinct mechanism of action and relatively few side effects, it is an excellent can- didate agent for combinational approaches. Thus, we set out We also assessed whether depletion of FVIII-specific memory B-cells occurred following treatment. COMBINATION THERAPY DEPLETES ANTI-FVIII-SPECIFIC ANTIBODY-SECRETING CELLS (ASCS) AND FVIII-SPECIFIC MEMORY B-CELLS IN MICE Hemophilia A mice were treated with IL-2/IL-2mAb complexes+ rapamycin+anti-CD20+FVIII; IL-2/IL-2mAb complexes+ rapamycin+FVIII; anti-CD20+FVIII; and FVIII alone as described previously. CD138+ cells were obtained from spleens of treated mice 2 weeks following treatment. Plasma cells were incubated with FVIII and analyzed for the formation of spots in an ELISPOT assay. Anti-FVIII ASCs correlated with the number of cells plated. Total anti-FVIII ASCs were reduced in mice treated with IL-2/IL-2mAb complexes+rapamycin+anti- CD20+FVIII and IL-2/IL-2mAb complexes+rapamycin+FVIII compared to other groups (Figure 6A). To confirm specificity of the assay, naive mice that had not been treated with FVIII were included. No FVIII-specific ASCs were detected in these mice (Figure 6A). Furthermore, no background staining in plates without FVIII immobilization was observed using cells obtained from FVIII-treated mice (data not shown). Evaluation of Effects of anti-CD20 treatment were also evaluated on B-cell populations in the hemophilia A mice treated with combination therapy. After two treatments of anti-CD20, proportions of B- cell populations were measured by flow cytometry. We observed a significant decrease in proportions in the anti-CD20-treated mouse groups of total B-cells (B220+ cells) (Figure 5A and Figure S2C in Supplementary Material); mature B-cells (IgD+IgMlow) (Figure 5B and Figure S2C in Supplementary Material); transi- tional B-cells (IgM+IgDlow) (Figure 5C and Figure S2C in Sup- plementary Material); memory B-cells (IgM−IgD−) (Figure S2C Frontiers in Immunology | Microbial Immunology January 2014 | Volume 4 | Article 502 | 4 Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. Liu et al. FIGURE 4 | Effects of immunomodulation on CD4+ T cells, CD4+CD25+Foxp3+ Treg cells andTregs activation markers in peripheral blood of treated hemophilia A inhibitor mice over time. Lymphocytes were isolated from the blood of naive (light grid), inhibitor only (light slant), anti-CD20+FVIII (white), IL-2/IL-2mAb complexes+rapamycin+FVIII (light gray), IL-2/IL-2mAb complexes+anti-CD20+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (black) treated mice. (A) CD4+ in total T cells and (B) CD4+CD25+Foxp3+ in CD4+ T cells were stained and analyzed by flow cytometry during the treatment. (C) Blood cells were also stained and analyzed for Treg cells markers: CD25, GITR, and CTLA-4. Data shown are median fluorescence intensity (MFI) values of the three activation markers. Data shown is representative of two independent experiments. FIGURE 4 | Effects of immunomodulation on CD4+ T cells, complexes+rapamycin+anti-CD20+FVIII (black) treated mice. (A) CD4+ in total T cells and (B) CD4+CD25+Foxp3+ in CD4+ T cells were stained and analyzed by flow cytometry during the treatment. COMBINATION THERAPY DEPLETES ANTI-FVIII-SPECIFIC ANTIBODY-SECRETING CELLS (ASCS) AND FVIII-SPECIFIC MEMORY B-CELLS IN MICE We isolated CD138− spleen cells (presumably containing FVIII-specific memory B- cells) from hemophilia A mice treated with different sin- gle or combined regimens and re-stimulated these cells with high-dose FVIII (2 U/well). FVIII-specific memory B- cells were detected by ELISPOT. Interestingly, FVIII-specific memory B-cells were significantly reduced only in the IL- 2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII and anti- CD20+FVIII-treated mouse groups (Figure 6B). DISCUSSION Treatment of hemophilia A patients with inhibitors is very challenging and costly. In addition, probability of morbidity is increased in these patients. Although treatments with inhibitors has been successful in hemophilia mice, it is very challenging to decrease pre-existing anti-FVIII neutralizing antibodies in FVIII- primed hemophilia A mice. In vivo expansion of activated Tregs had profound suppressive effects and was able to prevent forma- tion of anti-FVIII antibodies in both gene therapy and protein replacement therapy treated mice; however, this regimen can only transiently modulate pre-existing anti-FVIII immune responses. Anti-FVIII neutralizing antibodies can circulate for long periods and is thought to be partly due to the persistence of memory January 2014 | Volume 4 | Article 502 | 5 www.frontiersin.org Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. Liu et al. FIGURE 5 | Effects of immunomodulation on total B, mature B, transitional B, and plasma B-cells in peripheral blood of each mouse group. Lymphocytes were isolated from the blood of naive (light grid), Inhibitor only (light slant), anti-CD20+FVIII (white), IL-2/IL-2mAb complexes+rapamycin+FVIII (light gray) IL-2/IL-2mAb complexes+anti-CD20+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (black) treated mice. (A) B220+ B-cells, (B) IgM+IgDhi B-cells, (C) transitional B-cells, and (D) plasma cells were stained and analyzed by flow cytometry during the treatment period (week 2, 4, and 5). Data shown are cell percentages (A–C) and real numbers (D) of the B-cell populations. Data shown is representative of two independent experiments. FIGURE 6 | Depletion of FVIII-specific ASCs (Antibody-Secreting Cells) and memory B-cells in the inhibitor mice treated with IL-2/IL-2mAb complexes plus rapamycin and anti-CD20. Cells were isolated by MACS from spleens of naive (light slant), FVIII plasmid only (white), anti-CD20+FVIII (light gray), IL-2/IL-2mAb complexes+rapamycin+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (black) treated mice (n = 2, each group) 2 weeks after treatment. (A) 3 × 106 cells were used to detect FVIII-specific ASC cells by ELISPOT assay. (B) 5 × 105 non-plasma cells were cultured and stimulated with FVIII at 10 U/ml for 6-days, and memory B-cells were detected by ELISPOT assay. Data shown are mean of spot numbers for each treated group (n = 2). to study whether anti-CD20 treatment alone or in combina- tion with other immune tolerance therapies can be more ben- eficial to treat hemophilia subjects with pre-existing inhibitory ib di Withanti-CD20treatmentalone,wefoundthatanti-FVIIIneu- tralizing antibody titers were reduced in both FVIII unprimed and primed hemophilia A mice. DISCUSSION However, antibodies were not l l li i d d FVIII i i did i i h FIGURE 5 | Effects of immunomodulation on total B, mature B, transitional B, and plasma B-cells in peripheral blood of each mouse group. Lymphocytes were isolated from the blood of naive (light grid), Inhibitor only (light slant), anti-CD20+FVIII (white), IL-2/IL-2mAb complexes+rapamycin+FVIII (light gray) IL-2/IL-2mAb complexes+anti-CD20+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (black) treated mice. (A) B220+ B-cells, (B) IgM+IgDhi B-cells, (C) transitional B-cells, and (D) plasma cells were stained and analyzed by flow cytometry during the treatment period (week 2, 4, and 5). Data shown are cell percentages (A–C) and real numbers (D) of the B-cell populations. Data shown is representative of two independent experiments. complexes+rapamycin+anti-CD20+FVIII (black) treated mice. (A) B220+ B-cells, (B) IgM+IgDhi B-cells, (C) transitional B-cells, and (D) plasma cells were stained and analyzed by flow cytometry during the treatment period (week 2, 4, and 5). Data shown are cell percentages (A–C) and real numbers (D) of the B-cell populations. Data shown is representative of two independent experiments. FIGURE 6 | Depletion of FVIII-specific ASCs (Antibody-Secreting Cells) and memory B-cells in the inhibitor mice treated with IL-2/IL-2mAb complexes plus rapamycin and anti-CD20. Cells were isolated by MACS from spleens of naive (light slant), FVIII plasmid only (white), ti CD20+FVIII (li ht ) IL 2/IL 2 Ab l + i +FVIII (black) treated mice (n = 2, each group) 2 weeks after treatment. (A) 3 × 106 cells were used to detect FVIII-specific ASC cells by ELISPOT assay. (B) 5 × 105 non-plasma cells were cultured and stimulated with FVIII at 10 U/ml for 6-days, and memory B-cells were detected by ELISPOT D t h f t b f h t t d (black) treated mice (n = 2, each group) 2 weeks after treatment. (A) 3 × 106 cells were used to detect FVIII-specific ASC cells by ELISPOT assay. (B) 5 × 105 non-plasma cells were cultured and stimulated with FVIII at 10 U/ml for 6-days, and memory B-cells were detected by ELISPOT assay. Data shown are mean of spot numbers for each treated group (n = 2). (black) treated mice (n = 2, each group) 2 weeks after treatment. (A) 3 × 106 cells were used to detect FVIII-specific ASC cells by ELISPOT assay. DISCUSSION Addi- tionally, rapamycin enriched antigen-specific Foxp3+ Treg cells to promote organ transplant tolerance (40) and inhibited relapsing experimental autoimmune encephalomyelitis (EAE) by modula- tion of both effector/regulatory T cells (41). Moghimi et al. (42) reported that transient oral delivery of rapamycin combined with repeated injections of low doses of FVIII prevented induction of neutralizing antibody responses in hemophilia A mice. Thus, we adopted a treatment strategy that included rapamycin with IL- 2/IL-2mAb complexes to enhance Treg cell function in vivo for targeting T-cell-mediated immune responses and induction of FVIII-specific tolerance in hemophilia A mice with pre-existing antibodies. attributed to impaired TH cell responses or directly mediated by the immune complexes is unclear. Upon second challenge with FVIII plasmid,theantibodytiterremainedlowwithoutanincrease inFVIIIactivity,indicatingthatonlypartialtoleranceagainstFVIII was achieved with combination therapy. Furthermore, treated mice were challenged with non-specific antigen, TNP-ficoll (24) at 16 weeks following treatment and responded similarly to control mice, demonstrating that this tolerance effect is FVIII-antigen- specific. Based on these results, we hypothesize that residual, long-lived FVIII-specific plasma cells may contribute to sustained low-titer neutralizing antibodies that limit the therapeutic benefits of immunomodulatory regimens. y g Zhang et al. showed that a single dose of anti-CD20 IgG1 pre- treatment prevented the increase of neutralizing antibodies in hemophilia A mice receiving high-dose protein replacement ther- apy (15). These antibodies can selectively deplete follicular B-cells while sparing marginal zone (MZ) B-cells as potential tolerogenic antigen-presenting cells. Interestingly, this treatment also led to an increase of Treg cells. In a similar case, Sarikonda et al. showed that transient B-cell depletion with anti-CD20 IgG2a in combi- nation with proinsulin resulted in modest increases in Treg cells and offered limited efficacy in type 1 diabetes (T1D) prevention in NOD mice (7). In rhesus macaques, Mingozzi et al. (43) success- fully used Rituximab in combination with cyclosporine to erad- icate anti-human factor IX antibody following AAV8-mediated gene therapy. In addition, transient B-cell depletion by anti-CD20 IgG2a prevented FVIII inhibitor formation in hemophilia A mice receiving protein therapy but failed to induce long-term toler- ance (16). In our study, administration of anti-murine CD20 IgG2a significantly reduced CD19+ B-cells in blood, spleen, and lymph nodes, as well as neutralizing antibody titers in FVIII plasmid-treated mice. Furthermore, our results showed that com- bination therapy targeting both B and T cells had better results to more significantly reduce anti-FVIII immune responses. DISCUSSION As shown in our previous experiments, FVIII expression persisted in the liver for very long periods following hydrodynamic deliv- ery of FVIII plasmids. With reversion of FVIII expression, FVIII antigen is present continuously during follow-up. Thus, it is con- cluded that long lasting FVIII-specific partial tolerance has been achieved with the combination therapy. In addition, viral gene transfer with adenoid-associated viral (AAV) vectors has been pro- posed as a therapeutic strategy for hemophilia A (44, 45). The immunomodulation regimens developed for transgene-specific immune responses may also be helpful in the design of modu- latory protocols for immune responses to gene transfer vectors, in particular pre-existing immunity against viral vectors such as AAV. In addition, liver-directed gene transfers with several vec- tors have been associated with the induction of tolerance to the expressed transgene (46, 47). Combination of the immunomod- ulatory regimen developed in this study with liver gene transfer should increase the efficacy of tolerization against FVIII trans- gene. In summary,we demonstrated that IL-2/IL-2mAb complexes plus rapamycin acted synergistically with anti-CD20 to promote induction of immune tolerance to FVIII by increasing the num- ber and function of CD4+CD25+Foxp3+ Treg cells as well as eliminating both FVIII-specific ASCs and memory B-cells. These findings provide important preclinical evidence for the safety and enhanced therapeutic efficacy of the combined treatment Our results demonstrate that treatment with IL-2/IL-2mAb complexes+rapamycin had a synergistic effect when combined with anti-CD20 antibody to reduce neutralizing antibody titers. In contrast, mice treated with anti-CD20 only or different com- binations only transiently reduced neutralizing antibody titers. In ourimmunomodulationstudies,wealsoincludedweeklyinjection of FVIII protein (1 U/mouse/treatment) for induction of FVIII- specific tolerance. Furthermore, no increase in neutralizing anti- body titers was observed following a second FVIII plasmid chal- lenge, indicating induction of tolerance to FVIII. The mechanistic studies showed that the combined therapy promoted immune tolerance by increasing CD4+CD25+Foxp3+ Treg cells and their activation markers CD25, GITR, and CTLA-4. Furthermore, com- bined treatment using anti-CD20 showed 98% depletion of total B, mature B, transitional B, and plasma cells. Changes in T/B-cell populations were not only detectable in the peripheral blood but also found in splenocytes isolated from treated mice. Most impor- tantly, FVIII-specific ASCs and memory B-cells were reduced fol- lowing treatment with combination therapy. It was demonstrated that anti-CD20 predominantly depleted FVIII-specific memory B-cells, and IL-2/IL-2mAb complexes+rapamycin helped reduce FVIII-specific ASCs. DISCUSSION (B) 5 × 105 non-plasma cells were cultured and stimulated with FVIII at 10 U/ml for 6-days, and memory B-cells were detected by ELISPOT assay. Data shown are mean of spot numbers for each treated group (n = 2). FIGURE 6 | Depletion of FVIII-specific ASCs (Antibody-Secreting Cells) and memory B-cells in the inhibitor mice treated with IL-2/IL-2mAb complexes plus rapamycin and anti-CD20. Cells were isolated by MACS from spleens of naive (light slant), FVIII plasmid only (white), anti-CD20+FVIII (light gray), IL-2/IL-2mAb complexes+rapamycin+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII FIGURE 6 | Depletion of FVIII-specific ASCs (Antibody-Secreting Cells) and memory B-cells in the inhibitor mice treated with IL-2/IL-2mAb complexes plus rapamycin and anti-CD20. Cells were isolated by MACS from spleens of naive (light slant), FVIII plasmid only (white), anti-CD20+FVIII (light gray), IL-2/IL-2mAb complexes+rapamycin+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII to study whether anti-CD20 treatment alone or in combina- tion with other immune tolerance therapies can be more ben- eficial to treat hemophilia subjects with pre-existing inhibitory antibodies. Withanti-CD20treatmentalone,wefoundthatanti-FVIIIneu- tralizing antibody titers were reduced in both FVIII unprimed and primed hemophilia A mice. However, antibodies were not completely eliminated and FVIII activity did not improve in these Frontiers in Immunology | Microbial Immunology Frontiers in Immunology | Microbial Immunology January 2014 | Volume 4 | Article 502 | 6 Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. animals. To further improve the therapeutic efficacy, we com- bined B-cell depletion using anti-CD20 with in vivo Treg cell expansion using IL-2/IL-2mAb complexes+rapamycin to treat hemophilia A inhibitor mice. Our previous studies using IL-2/IL- 2mAb complexes alone showed five to sevenfold expansion of highly suppressive Treg cells in vivo, which induced long-term tolerance to FVIII in unprimed hemophilia A mice following gene therapy (24). IL-2/IL-2mAb complexes that can selectively promote human Treg expansion are currently under develop- ment. Although no clinical trials have been initiated so far, IL- 2/IL-2mAb complexes have high potential as a clinically feasible strategy; however, IL-2/IL-2mAb complex single treatment in the hemophilia A “inhibitor” mice only transiently reduced neutral- izing antibody titers during treatment. It has been shown that rapamycin blocked T-cell-cycle progression from G1 to S phase after activation (37) and promoted TCR-induced T-cell anergy (38), achieving induction of operational tolerance (39). www.frontiersin.org MICE Peripheral blood samples were taken from the experimental mice and collected in a 3.8% sodium citrate solution. FVIII activities were measured by a modified clotting assay using FVIII deficient plasma and reagents to measure activated partial thromboplastin time (APTT) and FVIII deficient plasma (49, 50). FVIII activities were calculated from a standard curve generated by using serially diluted normal human pooled plasma. Anti-FVIII activities were measured by Bethesda assay as previously described (51). All mice were kept according to the National Institutes of Health guidelines for animal care and the guidelines of Seattle Children’s Research Institute, and maintained at a specific pathogen-free (SPF) facility. Hemophilia A mice in a 129/SV × C57BL/6 mixed genetic background were generated by targeted disruption of exon 16 of FVIII gene (48) and were used at the age of 6–8 weeks. ACKNOWLEDGMENTS This work is supported by a R01 grant (R01 HL69049) from NIH-NHLBI. ELISPOT ASSAY i Hemophilia A mice were intravenously (i.v.) injected with 50 µg of FVIII plasmid [pBS-HCRHPI-FVIIIA (49)] in 2 ml phosphate- buffered saline (PBS) via tail vein in 8–10 s. IL-2/IL-2mAb com- plexes were prepared as previously described (17). One microgram recombinant mouse IL-2 (PeproTech, Rocky Hill, NJ, USA) was mixed with 5 µg anti-IL-2mAb (JES6-1A12) (eBioscience, San Diego, CA, USA), incubated at 37°C for 30 min, and then injected intraperitoneally (i.p.) into mice according to schedules specified in Results. Groups of IL-2/IL-2mAb complexes only treated mice, FVIII plasmid only treated mice, and naive mice were included as controls. Selected mice treated with immunomodulation received a second plasmid challenge at 9 weeks after the first treatment with immunomodulation therapy. Blood samples were taken from the retro-orbital plexus at serial time points and assessed for FVIII activity and anti-FVIII antibody levels. In preparation for the enzyme-linked immunospot (ELISPOT) assay, spleen cells from the treated mice were prepared to isolate the CD138+ (ASCs plasma) cells, 96-well filter plates (Millipore, MAHA N4510) were coated with the ASC cells (1 × 106/well) and incubated overnight at 4°C. To detect the ASC cells, plates were washed and blocked with RPMI-1640 supplemented with 10% preselected fetal calf serum (Hyclone, Logan, Utah), 2 mM l- glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Life Technologies), and 5 × 105 M β-mercaptoethanol (Sigma- Aldrich) for 1 h at 37°C prior to detection. The restimulation of memory B-cells in vitro was achieved as described. (26) Briefly, spleen cells were isolated and depleted of CD138+ ASCs. CD138− spleen cells were cultured at 3 × 105 cells/well in RPMI-1640 medium at 37°C for 6-days. About 2 U/well of FVIII was added to the cells on day 0. After 6-days culture, newly formed ASCs were detected by ELISPOT assays. B-CELLS DEPLETION BY ANTI-CD20 TREATMENT Mice were depleted of B-cells using anti-murine CD20 IgG2a anti- body (clone 18B12; Biogen Idec, Weston, MA, USA) at indicated schedules and dosages. To assess B-cell depletion, mice were given two i.v. doses 14 days apart, and peripheral blood and spleen tissues were collected at different time points after the first and second doses. Peripheral blood was collected in microcapillary tubes with 3.8% sodium citrate solution via retro-orbital plexus, centrifuged to remove plasma, and remaining cells suspended in PBS for staining and flow cytometry. AUTHOR CONTRIBUTIONS Chao Lien Liu designed and performed research, analyzed data, and wrote the paper. Peiqing Ye performed research. Jacqueline Lin performed research and helped revise the manuscript. Chérie L. Butts provided anti-CD20 antibody and helped revise the man- uscript. Carol H. Miao designed the project and research, analyzed data, and wrote the paper. FLOW CYTOMETRY AND ANTIBODIES Cell suspensions of peripheral blood, lymph nodes (LNs from superficial cervical),and spleens of each treated mouse group were prepared according to standard protocols. Cell suspensions were stained for FACS analysis using the following antibodies (obtained from eBioscience unless otherwise stated): PE-Cy5-anti-mouse CD25; FITC-anti-mouse CD62L (L-selectin); Alexa Fluor®647- anti-mouse/rat Foxp3; PE-anti-mouse CD152 (CTLA-4); Alexa Fluor®700-anti-mouse CD4 (BD Pharmingen™; San Jose, CA, USA); PE-Cy7-anti-mouse GITR (BD Pharmingen™); Alexa Fluor®700-anti-mouseB220;FITC-anti-mouseIgD;PE-Cy7-anti- mouse IgM and PE-anti-mouse CD138. Cells were first stained for T-cell surface markers CD4, CD25, CD62L, and GITR, and subsequently stained intracellularly with T-cell markers for Foxp3 and CTLA-4 following the company protocol (eBioscience). For B-cell populations, cells were stained with surface markers B220, IgD, IgM, and CD138. Samples were analyzed on an LSRII flow DISCUSSION In Figure S2C in Supplementary Material,the percentage of total memory T cells (gated as IgM−IgD−popula- tion) increased due to the more significant depletion (98%) of total B, mature B, and transitional B-cells. However, we found that FVIII-specific memory B-cells were significantly reduced after anti-CD20 treatment (shown in Figure 6B) in both mouse groupstreatedwithanti-CD20combinationregimen.Whetherthe effects of IL-2/IL-2mAb complexes+rapamycin on B-cells were January 2014 | Volume 4 | Article 502 | 7 www.frontiersin.org Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. cytometer (Becton Dickinson, Palo Alto, CA, USA) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). cytometer (Becton Dickinson, Palo Alto, CA, USA) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). for antibody responses in hemophilia A patients with pre-existing inhibitors. for antibody responses in hemophilia A patients with pre-existing inhibitors. MATERIALS AND METHODS MICE REFERENCES Beers SA, French RR, Chan HT, Lim SH, Jarrett TC, Vidal RM, et al. Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for anti- body selection. Blood (2010) 115(25):5191–201. doi:10.1182/blood-2010-01- 263533 32. Carcao M, St Louis J, Poon MC, Grunebaum E, Lacroix S, Stain AM, et al. Rit- uximab for congenital haemophiliacs with inhibitors: a Canadian experience. Haemophilia (2006) 12(1):7–18. doi:10.1111/j.1365-2516.2005.01170.x 13. Cheson BD. Ofatumumab, a novel anti-CD20 monoclonal antibody for the treatment of B-cell malignancies. J Clin Oncol (2010) 28(21):3525–30. doi:10. 1200/JCO.2010.27.9836 33. Fox RA, Neufeld EJ, Bennett CM. Rituximab for adolescents with haemophilia and high titre inhibitors. Haemophilia (2006) 12(3):218–22. doi:10.1111/j.1365- 2516.2006.01215.x 14. Cartron G, Watier H, Golay J, Solal-Celigny P. From the bench to the bedside: ways to improve rituximab efficacy. Blood (2004) 104(9):2635–42. doi:10.1182/ blood-2004-03-1110 34. Sabahi R, Anolik JH. B-cell-targeted therapy for systemic lupus erythematosus. Drugs (2006) 66(15):1933–48. doi:10.2165/00003495-200666150-00004 15. Zhang AH,Skupsky J,Scott DW. Effect of B-cell depletion using anti-CD20 ther- apy on inhibitory antibody formation to human FVIII in hemophilia A mice. Blood (2011) 117(7):2223–6. doi:10.1182/blood-2010-06-293324 35. Blair PA, Noreña LY, Flores-Borja F, Rawlings DJ, Isenberg DA, Ehrenstein MR, et al. CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity (2010) 32(1):129–40. doi:10.1016/j.immuni.2009.11.009 16. Sack BK, Merchant S, Markusic DM, Nathwani AC, Davidoff AM, Byrne BJ, et al. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy. PLoS One (2012) 7(5):e37671. doi:10.1371/journal.pone.0037671 36. Evans JG, Chavez-Rueda KA, Eddaoudi A, Meyer-Bahlburg A, Rawlings DJ, Ehrenstein MR, et al. Novel suppressive function of transitional 2 B cells in experimental arthritis. 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Failure of rituximab to induce immune tolerance in a boy with severe haemophilia A and an alloimmune factor VIII antibody: a case report and review of the literature. Haemophilia (2006) 12(3):280–4. doi:10.1111/j.1365-2516.2006.01212.x 12. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fimmu. 2013.00502/abstract Figure S1 | B-cell depletion following anti-CD20 treatment in hemophilia A mice. Mice were treated with i.v. injection of FVIII plasmid (50 µg at day 0) and anti-CD20 (gray) or IgG2a isotype control (white) at a dose of 250 µg/injection at days 0 and 14. PBMCs and spleen cells isolated from anti-CD20 treated hemophilia A mice were stained with FITC-CD19, and APC-B220 at 0.5, 2, 4, 8, 12, and 16 weeks following plasmid treatment and analyzed by flow cytometry. Naïve (black) and IgG control-treated hemophilia A mice were used as controls. (A) Representative plot for blood cells at different time points, (B) Total B-cell (CD19+B220+) depletion in blood over time, (C) Total B-cell (CD19+B220+) depletion in spleen over time. Data shown is representative of two independent experiments. Frontiers in Immunology | Microbial Immunology January 2014 | Volume 4 | Article 502 | 8 Anti-CD20 modulates pre-existing anti-FVIII response Liu et al. Liu et al. 19. Liu R, Zhou Q, La Cava A, Campagnolo DI, Van Kaer L, Shi FD. Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia. Eur J Immunol (2010) 40(6):1577–89. doi:10.1002/eji.200939792 Figure S2 | Effects of immunomodulation on bothT and B-cells isolated from spleens of each treated mouse group. Spleen (A–C) cells were collected and isolated at serial time points from naive (light slant), FVIII plasmid only (white), anti-CD20+FVIII (light gray), IL-2/IL-2mAb complexes+rapamycin+FVIII (dark gray), and IL-2/IL-2mAb Figure S2 | Effects of immunomodulation on bothT and B-cells isolated from spleens of each treated mouse group. Spleen (A–C) cells were collected and isolated at serial time points from naive (light slant), FVIII plasmid only (white), anti-CD20+FVIII (light gray), IL-2/IL-2mAb complexes+rapamycin+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (black) treated mice (n = 2, each group). Cells were stained and analyzed for T-cell populations (A,B) and B-cell populations (C). Data shown is representative of two independent experiments. Figure S2 | Effects of immunomodulation on bothT and B-cells isolated from spleens of each treated mouse group. Spleen (A–C) cells were collected and isolated at serial time points from naive (light slant), FVIII plasmid only (white), anti-CD20+FVIII (light gray), IL-2/IL-2mAb complexes+rapamycin+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (black) treated mice (n = 2, each group). Cells were stained and analyzed for T-cell populations (A,B) and B-cell populations (C). Data shown is representative of two independent experiments. 20. SUPPLEMENTARY MATERIAL Kahan BD, Camardo JS. Rapamycin: clinical results and future opportuni- ties. Transplantation (2001) 72(7):1181–93. doi:10.1097/00007890-200110150- 00001 complexes+rapamycin+FVIII (dark gray), and IL-2/IL-2mAb complexes+rapamycin+anti-CD20+FVIII (black) treated mice (n = 2, each 21. Zhang C,Shan J,Lu J,HuangY,Feng L,Long D,et al. Rapamycin in combination with donor-specific CD4+CD25+Treg cells amplified in vitro might be realize the immune tolerance in clinical organ transplantation. 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Citation: Liu CL, Ye P, Lin J, Butts CL and Miao CH (2014) Anti-CD20 as the B-cell targetingagentinacombinedtherapytomodulateanti-factorVIIIimmuneresponsesin hemophilia A inhibitor mice. Front. Immunol. 4:502. doi: 10.3389/fimmu.2013.00502 This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology. Copyright © 2014 Liu, Ye, Lin, Butts and Miao. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Citation: Liu CL, Ye P, Lin J, Butts CL and Miao CH (2014) Anti-CD20 as the B-cell targetingagentinacombinedtherapytomodulateanti-factorVIIIimmuneresponsesin hemophilia A inhibitor mice. Front. Immunol. 4:502. doi: 10.3389/fimmu.2013.00502 This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology. Citation: Liu CL, Ye P, Lin J, Butts CL and Miao CH (2014) Anti-CD20 as the B-cell targetingagentinacombinedtherapytomodulateanti-factorVIIIimmuneresponsesin hemophilia A inhibitor mice. Front. Immunol. 4:502. doi: 10.3389/fimmu.2013.00502 This article was submitted to Microbial Immunology a section of the journal Frontiers 45. Zhong L, Jayandharan GR, Aslanidi GV, Zolotukhin S, Herzog RW, Srivas- tava A. Development of novel recombinant AAV vectors and strategies for the potential gene therapy of hemophilia. J Genet Syndr Gene Ther (2012) S1:008. doi:10.4172/2157-7412.S1-008 This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology. Copyright © 2014 Liu, Ye, Lin, Butts and Miao. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2014 Liu, Ye, Lin, Butts and Miao. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Immunology | Microbial Immunology REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 46. Markusic DM, Herzog RW. Liver-directed adeno-associated viral gene therapy for hemophilia. J Genet Syndr Gene Ther (2012) 1:1–9. doi:10.4172/2157-7412. S1-009 47. Mátrai J, Cantore A, Bartholomae CC, Annoni A, Wang W, Acosta-Sanchez A, et al. Hepatocyte-targeted expression by integrase-defective lentiviral vectors Frontiers in Immunology | Microbial Immunology Frontiers in Immunology | Microbial Immunology January 2014 | Volume 4 | Article 502 | 10
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https://nutritionandmetabolism.biomedcentral.com/counter/pdf/10.1186/1743-7075-8-37
English
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Health aspects, nutrition and physical characteristics in matched samples of institutionalized vegetarian and non-vegetarian elderly (&gt; 65yrs)
Nutrition & metabolism
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Abstract Background: Epidemiological studies indicate that a well balanced vegetarian diet offers several health benefits including a lower prevalence of prosperity diseases in vegetarians compared to omnivores. It was the purpose of the present study to compare nutritional and physical characteristics in matched samples of institutionalized vegetarian (V) and non-vegetarian (NV) elderly. Methods: Twenty-two female and 7 male V (females: 84.1 ± 5.1yrs, males: 80.5 ± 7.5yrs) and 23 female and 7 male NV (females: 84.3 ± 5.0yrs, males: 80.6 ± 7.3yrs) participated. All subjects were over 65 years of age, and free of major disease or physical handicap. Dietary intake, blood profile, anthropometrics, and handgrip strength were determined. Results: Mean daily energy intake was 6.8 ± 2.0MJ in V females, and 8.0 ± 1.4MJ in the NV females, only the V did not reach the recommended value of 7.8 MJ. Male V and NV had a mean daily energy intake of 8.7 ± 1.6MJ and 8.7 ± 1.2MJ respectively (RDI: 8.8 MJ). Mean carbohydrate intake was significantly below the RDI in NV only (female V: 47.8 ± 7.5E%, female NV: 43.3 ± 4.6E%, male V: 48.1 ± 6.4E%, male NV: 42.3 ± 3.6E%), while protein (female V: 17.3 ± 3.4E%, female NV: 19.5 ± 3.5E%, male V: 17.8 ± 3.4E%, male NV: 21.0 ± 2.0E%), and saturated fat intake (female V: 25.4 ± 8.2 g/day, female NV: 32.2 ± 6.9 g/day, male V: 31.4 ± 12.9 g/day, male NV: 33.4 ± 4.7 g/day) were too high in both V and NV. Mean micronutrient intakes met the RDI’s in all 4 groups. Mean blood concentrations for vitamin B12, folic acid, iron, and calcium were normal in all 4 groups. Mean zinc blood serum was below the reference value in all groups, whereas estimated zinc intake was in agreement with the RDI. The mean blood cholesterol concentration was above the 200 mg/dl upper limit in the V group (213 ± 40 mg/dl) and below that limit in the NV (188 ± 33 mg/dl) group. Mean BMI was 26.1 ± 4.7 kg/m2 in the female V, 26.8 ± 3.7 kg/m2 in the female NV, 23.5 ± 3.7 kg/m2 in the male V, and 25.2 ± 4.2 kg/m2 in the male NV. V and NV scored below the reference values for the handgrip strength test. * Correspondence: peter.deriemaeker@vub.ac.be † Contributed equally Department of Human Biometry and Biomechanics, Faculty of Physical Education and Physiotherapy, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium © 2011 Deriemaeker et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH Open Access Health aspects, nutrition and physical characteristics in matched samples of institutionalized vegetarian and non-vegetarian elderly (> 65yrs) Peter Deriemaeker*, Dirk Aerenhouts†, Dolf De Ridder†, Marcel Hebbelinck† and Peter Clarys† Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Abstract Conclusions: Generally, our results show a similar profile for V and NV concerning dietary intake, blood values, and physical characteristics. Attention should be paid to the intake of mono- and disaccharides and saturated fats in the diet of both V and NV. This study indicates that a vegetarian lifestyle has no negative impact on the health status at older age. Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Page 2 of 8 Page 2 of 8 Participants Twenty-two female and 7 male V elderly living in a vege- tarian senior citizens home in the Netherlands (female: 84.1 ± 5.1yrs, male: 80.5 ± 7.5yrs) and 23 female and 7 male NV elderly living in a regular senior citizens home in the Dutch speaking part of Belgium (female: 84.3 ± 5.0yrs, male: 80.6 ± 7.3yrs) volunteered to participate in this study. All subjects were “apparently healthy”, which was defined as free of major disease or physical handicap. In agreement with the university ethics committee, all participants received explanation about the purpose and procedures of the study and signed an informed consent statement before participating. The national nutritional surveys in Belgium and the Netherlands revealed a poor eating pattern and lack of physical activity in the Belgian and Dutch population [15-17]. The reported macronutrient distribution in the elderly (65-75yrs) (17% protein, 39% fat, 44% carbohy- drate) was similar in both countries and not in accor- dance with the reference values proposed by their respective Health Councils [17,18]. The surveys indi- cated a too high intake of saturated fat and protein with meat, fish, eggs and dairy products as the main protein sources, a negligible consumption of plant-based pro- ducts (seeds, nuts, pulses, tofu, quorn and tempeh), and an intake of fruit and vegetables far below the recom- mendations [15-17]. Background vegetarian nutrition, particularly in the elderly [33-35], a matched samples study on institutionalized elderly vege- tarians compared with institutionalized non-vegetarians may contribute to the present knowledge of the dietary pattern in the elderly. Epidemiological studies on vegetarians show that appro- priately planned vegetarian diets are healthy and nutri- tionally adequate [1-5]. Compared to omnivorous diets, vegetarian diets can provide several health benefits [6-9]. However, these positive health-related outcomes in vegetarians may be influenced by factors other than dietary practices. Regular physical activity and the avoid- ance of harmful practices such as smoking and excessive drinking, and being more “health conscious” in general are influencing factors [10]. Most studies compared a self-selected vegetarian group with standard population references [1]. In order to truly attribute the health advantages associated with a vegetarian lifestyle, it is necessary to compare the vegetarian subjects with an adequate reference sample [11-14]. In such a design the vegetarians are compared with a comparable non-vege- tarian sample to exclude possible confounding factors. It is the purpose of the present study to compare the nutritional, health and physical status in comparable groups of vegetarian (V) and non-vegetarian (NV) elderly in order to gain more insight into the adequacy of a vegetarian diet at older age in particular. Measures and procedure Measurements were carried out accord- ing to standardized techniques [37,38]. 7.8MJ, in contrast to the 8.0 ± 1.4MJ in the NV females. Male V and NV met the RDI of 8.8MJ with a mean daily energy intake of 8.7 ± 1.6MJ and 8.7 ± 1.2MJ respectively. Mean protein intake (female V: 17.3 ± 3.4E %, female NV: 19.5 ± 3.5E%, male V: 17.8 ± 3.4E%, male NV: 21.0 ± 2.0E%) was too high in the female V and female and male NV. The female V had a lower protein intake compared to the female NV. Mean carbohydrate intake was significantly lower than the RDI in the NV only (female V: 47.8 ± 7.5E%, female NV: 43.3 ± 4.6E%, male V: 48.1 ± 6.4E%, male NV: 42.3 ± 3.6E%). The female V had a higher carbohydrate intake compared to their NV counterparts. Mono- and disaccharide intake was too high in female NV and male V. Polysaccharide intake was too low in all 4 groups compared to the RDI. Dietary fibre intake was sufficient in all 4 groups. Total fat intake (female V: 34.7 ± 7.2E%, female NV: 35.2 ± 4.5E%, male V: 32.8 ± 7.9E%, male NV: 32.6 ± 3.0E%) was in accordance with the RDI in all 4 groups. Satu- rated fat intake was significantly above the maximum Measures and procedure Dietary intake (Food Frequency Questionnaire, FFQ), blood profile, anthropometrics, and handgrip strength were registered. During our visit at their respective senior citizens homes, all V and NV participants completed a validated semi-quantitative 104 items FFQ [36] to estimate their dietary intakes over the last 6 months. A standard por- tion size and 9 possible food-frequency categories, ran- ging from never or less than 1 time per month to 6 or more times per day, were given for each food items. The FFQ was completed in the presence of the researcher, allowing clarification and help when necessary. In general, energy intake decreases throughout adult- hood, leading to a reduced macro- and micronutrient intake at older age [19,20]. Knowledge of the nutritional status of elderly people is of particular importance, since the elderly population has a higher prevalence of chronic medical conditions [21,22], malnutrition [23], and at the same time an increased prevalence of many chronic diseases which are associated with nutritional status [24-26]. Nonetheless, there are few studies on the nutritional status of elderly vegetarians, and dietary stu- dies on vegetarians usually include only a few elderly, if any [1-5]. Although the dietary intake in vegetarians appears favorable with respect to chronic disease risk factor profile, deficiencies in certain nutrients have been found [27-32]. Of these latter studies very few have been carried out in elderly subjects [32]. Although the number of vegetarians is relatively small in the Belgian and Dutch population [15], it is important to study their nutritional status, as in certain day care centers and senior citizens homes vegetarian meals are served. Since there is a growing interest in healthy diets and Blood samples were collected after an overnight fast and analysed for haemoglobin, red cell count, serum iron, transferrine, ferritine, white cells, ureum, albumin, serum calcium, serum zinc, vitamin B12, folic acid, tri- glycerides, total cholesterol, HDL-cholesterol and LDL- cholesterol in the clinical biology laboratory of the uni- versity hospital of the Vrije Universiteit Brussel, follow- ing validated standard procedures. Anthropometric variables were registered in order to determine the body mass index (BMI, kg/m2) and the waist-hip ratio. Additionally, triceps and subscapular skinfolds and upperarm girth were taken. The handgrip strength was measured using a hand dynamometer grip Page 3 of 8 Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 strength meter. Statistical analysis In case of different reference values [17,18] for men and women, males and females were analysed separately. After testing for normality (Kolmogorov Smirnov Good- ness of Fit test) the data at scale level were compared using an independent sample t-test for comparisons between the female V and NV. Because of the small number of male subjects, the Mann-Whitney test was used for the comparisons between the male V and NV groups. One sample t-tests were used for comparisons with the recommended daily intakes (RDI). Statistical analyses were performed using SPSS, and the signifi- cance level was set at 0.05. *Significantly different with NV (p < 0.05) †Significantly different from Recommended Daily Intake (RDI) (p < 0.05) Discussion A major problem is the difficulty to compare with refer- ence values as no references for very old people (> 75yrs) exist. Therefore this study compares two matched samples of elderly citizen home people with a mean age of 84.1 ± 5.1yrs in female V, 84.3 ± 5.0yrs in female NV, 80.5 ± 7.5yrs in male V, and 80.6 ± 7.3yrs male NV, which is far above the cut-offs of most reference values. Discussion Table 2 Daily micronutrient intake in vegetarian (V) and non-vegetarian (NV) elderly males (m) and females (f) (mean ± SD) Micronutrients Gender V NV RDI Calcium (mg) m 1096 ± 371 969 ± 399 800-1000 f 894 ± 306 858 ± 265 800-1000 Iron (mg) m 15.7 ± 3.4† 13.2 ± 1.5† 9 f 12.5 ± 5.6† 12.8 ± 3.0† 8 Zinc (mg) m 13.3 ± 3.8 11.9 ± 1.8† 10 f 13.0 ± 8.3 12.0 ± 4.3† 9 Sodium (mg) m 4083 ± 1231 4911 ± 831† 575-3500 f 2861 ± 907* 4944 ± 971† 575-3500 Potassium (mg) m 3976 ± 794*,† 3127 ± 395 1600-3100 f 3217 ± 963 3203 ± 580 1600-3100 Phosphorus (mg) m 1672 ± 429† 1395 ± 443 1000 f 1396 ± 658† 1322 ± 307† 1000 Magnesium (mg) m 388 ± 129* 248 ± 46† 325 f 340 ± 190* 252 ± 74 275 Vit A (mg) m 0.70 ± 0.21* 1.13 ± 0.10† 0.7 f 0.55 ± 0.21* 1.06 ± 0.21† 0.6 Beta-carotene (mg) m 1.30 ± 0.92 1.07 ± 0.49 - f 1.01 ± 0.41 1.04 ± 0.42 - Vit B1 (mg) m 1.60 ± 0.61*,† 0.99 ± 0.16 1 f 1.41 ± 1.22 1.08 ± 0.45 1 Vit B2 (mg) m 1.82 ± 0.41* 1.20 ± 0.20† 1.5 f 1.51 ± 0.62 1.24 ± 0.36 1.3 Vit B6 (mg) m 1.50 ± 0.42† 1.56 ± 0.18† 1.1 f 1.21 ± 0.42*,† 1.47 ± 0.26† 1.0 Vit D (μg) m 0.31 ± 0.01 0.27 ± 0.05 - f 0.11 ± 0.02* 0.23 ± 0.08 - Vit C (mg) m 162 ± 47*,† 96 ± 35 70 f 149 ± 82† 123 ± 46† 70 *Significantly different with NV (p < 0.05) †Significantly different from Recommended Daily Intake (RDI) (p < 0.05) Table 2 Daily micronutrient intake in vegetarian (V) and non-vegetarian (NV) elderly males (m) and females (f) (mean ± SD) The macronutrient intake was in agreement with the Belgian and Dutch food consumption surveys [17,18]. Only in male V, protein intake was within the recom- mendations, while for the other groups protein intake was too high. Mean carbohydrate intake was too low in the male and female NV compared to the RDI. Total fat intake met the RDI in all groups. Results Table 1 shows that the mean daily energy intake of 6.8 ± 2.0MJ in V females was not according the RDI of Table 1 Daily energy, macronutrient and water intake in vegetarian (V) and non-vegetarian (NV) elderly males (m) and females (f) (mean ± SD) Variable Gender V NV RDI Energy (MJ) m 8.7 ± 1.6 8.7 ± 1.2 8.8 f 6.8 ± 2.0*,† 8.0 ± 1.4 7.8 Protein (E%) m 17.8 ± 3.4 21.0 ± 2.0† 10-15 f 17.3 ± 3.4*,† 19.5 ± 3.5† 10-15 Total carbohydrate (E%) m 48.1 ± 6.4 42.3 ± 3.6† 50-55 f 47.8 ± 7.5* 43.3 ± 4.6† 50-55 Mono- and disaccharides (g) m 170 ± 37† 137 ± 35 131 f 137 ± 50 133 ± 30† 117 Polysaccharides (g) m 66 ± 22† 56 ± 3† 158 f 50 ± 24† 59 ± 14† 140 Fibre (g) m 28 ± 9 36 ± 10 27 f 28 ± 18 32 ± 11 27 Total fat (E%) m 32.8 ± 7.9 32.6 ± 3.0 30-35 f 34.7 ± 7.2 35.2 ± 4.5 30-35 Saturated fat (g) m 31.4 ± 12.9 33.4 ± 4.7† 23 f 25.4 ± 8.2*,† 32.2 ± 6.9† 21 MUFA (g) m 28.8 ± 11.7 39.1 ± 4.2† 28 f 22.8 ± 10.1* 37.1 ± 6.1† 25 PUFA (g) m 14.3 ± 2.8† 13.8 ± 1.3† 28 f 13.4 ± 7.0† 12.2 ± 3.4† 25 Cholesterol (mg) m 181 ± 56 208 ± 21 < 300 f 161 ± 69 205 ± 46 < 300 Alcohol (E%) m 1.7 ± 3.1 13.2 ± 13.1 < 4 f 0.3 ± 0.9* 5.6 ± 7.7 < 4 Water (ml) m 2050 ± 277 2224 ± 214 1600-2400 f 1663 ± 452* 2166 ± 538 1600-2400 *Significantly different with NV (p < 0.05) macronutrient and water intake in vegetarian (V) and non-vegetarian (NV) elderly males (m) SD) nergy, macronutrient and water intake in vegetarian (V) and non-vegetarian (NV) elderly males (m (mean ± SD) Table 1 Daily energy, macronutrient and water intake in vegetarian (V) and non-vegetarian (NV) and females (f) (mean ± SD) Deriemaeker et al. Results Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Page 4 of 8 Mean BMI did not differ between V and NV with respective BMI values of 26.1 ± 4.7 kg/m2 in the female V,26.8 ± 3.7 kg/m2 in the female NV, 23.5 ± 3.7 kg/m2 in the male V and 25.2 ± 4.2 kg/m2 in the male NV (Table 4). The waist-hip ratio was comparable between V and NV groups, and was below the upper reference value of 0.99 for males and 0.90 for females [39]. As to the strength test both groups scored below the reference values of 40 kg for males and 25 kg for females [40]. There were no differences between the V and NV groups. RDI (females: 21 g/day, males: 23 g/day) for both V and NV females and male NV. Comparable results in the V and the NV samples were found for the mono- and poly-unsaturated fatty acid intake. Alcohol intake was significantly lower in the female V compared to the NV, while no difference was found between the male V and NV. Mean daily water intake was sufficient in all groups. Mean vitamin and mineral intakes met the RDI in both groups (Table 2). Blood concentrations for vitamin B12, folic acid, iron, and calcium were within the recommended values in both groups (Table 3). Zinc blood serum was below the reference value in both groups. The mean blood choles- terol concentration was above the 200 mg/dl upper limit in the V group (213 ± 40 mg/dl) and below that limit in the NV (188 ± 33 mg/dl) group. As to the blood bio- chemistry, a comparable number of subjects in both groups did not reach the reference values. *Significantly different with NV (p < 0.05) y †Significantly different from Recommended Daily Intake (RDI) (p < 0.05) Discussion The saturated fat intake was comparable and, except in the male V, signif- icantly above the RDI in all groups. Mean daily energy intake was lower in V females compared to their NV counterparts and did not reach the recommended value of 7.8 MJ. Most studies found no differences in energy intake between V and NV [1-5]. The difference found between the female groups in this study can be due to several reasons. Female V had a lower protein and satu- rated fat intake and a higher carbohydrate intake com- pared to the female NV. The macronutrient profile in V females was closer to the recommendations compared to the NV females. However both groups had protein, saturated fat and carbohydrate intakes not in accordance with the RDI. Although only significant in the females, the V had a lower alcohol intake compared to the NV. The better macronutrient profile and a lower alcohol consumption may be attributed to the often cited “health consciousness” in vegetarians [1,10]. Mean vita- min and mineral intakes met the RDI in both groups, which indicates a varied choice of micronutrient dense foods in both groups. A study of Brants et al. [24] indi- cated a favourable food choice of independently living elderly vegetarians preparing their own meals compared to institutionalized elderly vegetarians. Compared to the institutionalized subjects studied by Brants et al. [24] our subjects (V and NV) show similar dietary shortcom- ings. Although the fact that vitamin B12, folic acid, iron, Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Page 5 of 8 Page 5 of 8 Table 3 Blood profile in vegetarian (V) and non-vegetarian (NV) elderly males (m) and females (f) (mean ± SD) Parameter Gender V (n, n a-typ.)(a) NV (n, n a-typ.)(a) Ref. Discussion Haemoglobin (mmol/l) m 13.4 ± 0.9 (4, 2) 13.6 ± 1.2 (6, 2) 13-16.5 f 12.8 ± 1.1 (18, 3) 13.1 ± 1.6 (20, 3) 11.8-14.5 Red cell count (106/mm3) m 4.6 ± 0.3 (4, 0) 4.5 ± 0.5 (6, 0) 4.2-5.7 f 4.3 ± 0.5 (21, 2) 4.4 ± 0.5 (20, 2) 3.9-5.0 Serum iron (mg/dl) m+f 76 ± 25 (24, 4) 82 ± 36 (26, 1) 50-170 Transferrine (mg/dl) m+f 243 ± 33* (25, 1) 217 ± 37 (26, 1) 181-332 Ferritine (mg/l) m 47 ± 26 (4, 1) 161 ± 165 (6, 0) 30-400 f 70 ± 46 (21, 1) 158 ± 180 (20, 1) 13-150 White cells (103/mm3) m+f 6.4 ± 1.5* (22, 0) 7.8 ± 2.5 (26, 5) 3.6-9.6 Ureum (mg/dl) m+f 36.9 ± 11.0 (24, 8) 42.5 ± 23.5 (26, 14) 15-40 Albumin (g/dl) m+f 3.9 ± 0.3* (24, 3) 3.7 ± 0.3 (26, 3) 3.7-5.0 Serum calcium (103/mm3) m+f 8.8 ± 0.4 (24, 5(b)) 9.0 ± 0.4 (26, 1) 8.6-9.8 Serum zinc (mg/dl) m+f 62.0 ± 11.1† (24, 20) 64.5 ± 12.2† (26, 18) 70-150 Total cholesterol (mg/dl) m+f 213 ± 40*,† (24, 18) 188 ± 33 (25, 12) < 200 LDL (mg/dl) m+f 121 ± 30 (24, 15) 111 ± 26 (24, 11) < 115 HDL (mg/dl) m+f 54 ± 15 (24, 4) 51 ± 12 (26, 2) > 40 Triglycerides (mg/dl) m+f 180 ± 106 (24, 10) 149 ± 103 (26, 6) < 180 Vitamin B12 (mg/l) m+f 0.58 ± 0.60 (22, 7) 0.46 ± 0.46 (26, 7) 0.22-0.94 Folic acid (mg/l) m+f 9.8 ± 5.7 (22, 6) 7.3 ± 6.0 (26, 6) 2.0-14.0 (a)(n: number of subjects studied, n a-typ.: number of subjects with a-typical values) (b)The values were close to the cut-off point, i.e. 8.1, 8.3, 8.4, 8.4, 8.4 *Significantly different with NV (p < 0.05) †Significantly different from reference values (Ref.) (p < 0.05) value in both groups. This indicates that the RDI may be underestimated for elderly as a consequence of their poor absorption due to aging. The mean blood choles- terol concentration was below the 200 mg/dl upper limit in the NV group and above that limit in the V group. These higher blood cholesterol levels are and calcium are often described as critical in a vegetar- ian diet, the mean blood concentrations of these para- meters were normal in both groups. *Significantly different with NV (p < 0.05) g y (p ) †Significantly different from reference values (Ref.) (p < 0.05) g y (p ) †Significantly different from reference values (Ref.) (p < 0.05) Discussion The main explanation can be found in the fact that our subjects were lacto-ovo vegetarians. While estimated zinc intake was sufficient, zinc blood serum was below the reference Table 4 Anthropometry and strength of vegetarian (V) and non-vegetarian (NV) elderly males (m) and females (f) (mean ± SD) Variable Gender V NV Ref. Body height (cm) m 174.6 ± 7.6 169.0 ± 7.0 f 154.5 ± 8.1* 158.8 ± 4.1 Body weight (kg) m 72.2 ± 14.9 68.7 ± 8.7 f 62.2 ± 10.8 67.6 ± 10.2 BMI (kg/m2) m 23.5 ± 3.7 25.2 ± 4.2 19-25 f 26.1 ± 4.7 26.8 ± 3.7 19-25 Waist-Hip ratio m 0.91 ± 0.10 0.94 ± 0.04 0.99 f 0.90 ± 0.10 0.90 ± 0.06 0.90 Triceps skinfold (mm) m 10.6 ± 4.3† 13.0 ± 2.8 15.0 f 12.7 ± 3.5† 11.2 ± 4.0† 21.6 Subscapular skinfold (mm) m 12.2 ± 3.0† 11.9 ± 6.0 16.2 f 9.2 ± 2.6*,† 13.5 ± 3.5† 19.0 Upperarm girth (cm) m 27.9 ± 4.0 27.4 ± 3.0 28.3 f 27.7 ± 2.9† 29.0 ± 3.9 30.3 Handgrip (kg) m 29.0 ± 9.1† 24.7 ± 7.2† 40.3 ± 7.9 f 17.5 ± 5.5† 16.1 ± 6.0† 25.4 ± 5.4 *Significantly different with NV (p < 0.05) †Significantly different from reference values (Ref.) (p < 0.05) able 4 Anthropometry and strength of vegetarian (V) and non-vegetarian (NV) elderly males (m) and females (f) mean ± SD) strength of vegetarian (V) and non-vegetarian (NV) elderly males (m) and females (f) Table 4 Anthropometry and strength of vegetarian (V) and non-vegetarian (NV) elderly males (m ( ) Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Page 6 of 8 comparable with those found in elderly in Flanders where 86% of the males and 91% of the females between 65 and 69 yrs have blood cholesterol levels above 200 mg/dl [41]. Since the subjects in this study have a mean age over 80 years the values in the vegetarian subjects can be considered as normal. Moreover, several studies suggest that slightly higher cholesterol levels may even protect against infections and atherosclerosis [42-44]. According to these authors, values from 200 to 239 mg/ dl are more appropriate in elderly (> 65 yrs) [42-44]. Comparable numbers of subjects not reaching blood reference values were found in both groups. Discussion Of note is that vitamin B12 deficiency is not always unique to vegetarians, although it is generally more difficult for vegetarians (especially vegans) to meet vitamin B12 requirements than it is for omnivores. The present results show a mean vitamin B12 status according to the reference values while a comparable number of subjects with atypical values were detected for the V and NV. Therefore a low vitamin B12 status is not likely to be related to neither the vegetarian nor the omnivorous diet. The present results confirm the advice of Elmadfa and Singer [45] that a regular monitoring of the vitamin B12 status in order to facilitate early detection of low vitamin B12 status and timely treatment before clinical manifestations can develop is required. These concerns should also be taken into account for the other blood parameters. compared to the reference values (65 - 75 yrs) may be due to the higher mean age (> 80 yrs) of the studied V and NV groups. Since in this study V and NV did not substantially dif- fer with respect to dietary intake and nutritional status, one should be careful with the conclusion that only dietary factors of a vegetarian diet cause different mor- bidity and mortality risks among V and NV. A limitation of the present study may be the fact that the compared senior citizens homes in this study are located in two different regions (Belgium and the Neth- erlands). This is mainly due to the fact that, to our knowledge, there is but one exclusively vegetarian senior citizens home in the Netherlands and none in the Dutch speaking region of Belgium. Nonetheless, the national food consumption surveys in both regions showed similar shortcomings typical for European diets regarding the macro- and micronutrient intake. Another drawback is the non-randomized sampling of the two populations studied. More extensive studies have to be undertaken in order to gain more knowledge about the value of vegetarian diets in elderly people. Acknowledgements Th h f Acknowledgements The authors are grateful to physician Kris Hebbelinck, and Tom Van Gyseghem, Sofie Rousseeuw, Vauke Synquintyn and Dries Claeys for their assistance in collecting the data. The authors would also like to thank all the elderly participants and the staff of the participating senior citizens homes for their cooperation. Conclusion h The macronutrient profile in V females was closer to the recommendations compared to the NV females. However, both groups had macronutrient intakes not in accordance with the RDI. Blood values, anthropometrics and handgrip strength were comparable for V and NV. These diet-related health risk factors found in both V and NV can be corrected by additional nutritional inter- ventions. Such interventions should be encouraged and monitored by the responsible authorities and caretakers of the senior citizens homes and institutions. The pre- sent results suggest that the shortcomings of health related characteristics and dietary intake are not likely to be specifically related neither to the vegetarian nor to the omnivorous diet. Finally, this study corroborates the assertion of many professional instances that a balanced, and well planned vegetarian diet can be a responsible choice also at older age [1]. p Mean BMI was around 25 and comparable between V and NV in both genders. This is in contrast with previous studies which showed vegetarians to have a lower BMI [7,46,47]. Appleby et al. [46] found a significant inverse association between dietary fibre and BMI, but this was not confirmed by our results. The lower reported satu- rated fat intake in female V as compared to the NV did not result in a significantly lower BMI or waist-hip ratio in female V. Except lower subscapular skinfolds in V females no anthropometric differences were found between the V and NV. This can be a consequence of working with small matched samples. The findings concerning the anthropo- metric characteristics indicate an adequate energy balance, also in the female V who reported an energy intake below the reference value. The waist-hip ratio was comparable between V and NV groups, and was below the upper reference value [39]. Price et al. [39] and Seidell [48] showed that for persons aged > 75 yrs, waist-hip ratio should be used instead of BMI because of the positive rela- tionship between mortality and abdominal adiposity. Therefore we can consider that the elderly V and NV in the present study are not at increased risk. Regarding the handgrip strength both groups scored below the reference value and no differences were found between the V and NV. The anthropometric and physical differences MH, DDR and PC conceived the study. PC and MH were responsible for the data collection. PD participated in its design and coordination, did the statistical analyses and wrote the manuscript. DA, MH, DDR and PC critically reviewed the manuscript for writing and intellectual content. All authors read and approved the final manuscript. References Deriemaeker P, Alewaeters K, Hebbelinck M, Lefevre J, Philippaerts R, Clarys P: Nutritional status of Flemish vegetarians compared with non- vegetarians: a matched sample study. Nutrients 2010, 2:770-780. 15. Debacker N, Temme L, Cox B, Huybrechts I, Van Oyen H: De Belgische voedsel consumptie peiling 2004. Voedingsgewoonten van de Belgische bevolking ouder dan 15 jaar. Wetenschappelijk Instituut voor Volksgezondheid, Afdeling Epidemiologie, Brussel; 2007, (Belgian food consumption survey 2004. Nutritional habits of the Belgian population over 15 years of age). 36. Deriemaeker P, Aerenhouts D, Hebbelinck M, Clarys P: Validation of a 3-Day Diet Diary: Comparison with a 7-Day Diet Diary and a FFQ. Medicine & Science in Sports & Exercise 2006, 38(5):S328. 16. Vandevijvere S, De Vriese S, Huybrechts I, Moreau M, Temme E, De Henauw S, De Backer G, Kornitzer M, Leveque A, Van Oyen H: The gap between food-based dietary guidelines and usual food consumption in Belgium, 2004. Public Health Nutrition 2008, 12(3):423-431. 37. Adam C, Klissouras V, Ravazzolo M, Renson R, Tuxworth W: EUROFIT: European Test of Physical Fitness. Rome, Italy: Council of Europe, Committee for Development of Sport; 1988. g 17. Health Council of the Netherlands: Committee on Trends in food consumption: Significant trends in food consumption in the Netherlands. The Hague: Health Council of the Netherlands; 2002 [http://www. gezondheidsraad.nl/sites/default/files/summary%20200212.pdf], publication no. 2002/12. ISBN 90-5549-437-2. 38. Eston R, Reilly T, Eds: Kinanthropometry and Exercise Physiology Laboratory Manual: Tests, procedures and data. London, UK: E & FN Spon;, 1 1996. 39. Price GM, Uauy R, Breeze E, Bulpitt CJ, Fletcher AE: Weight, shape, and mortality risk in older persons: elevated waist-hip ratio, not high body mass index, is associated with a greater risk of death. Am J Clin Nutr 2006, 84(2):449-60. 18. Hoge Gezondheidsraad (Higher Health Council): Voedingsaanbevelingen voor België. Brussel 2006, (Nutritional recommendations for Belgium) nr. 7145-2.. 40. van Heuvelen MJ, Kempen GI, Brouwer WH, de Greef MH: Physical fitness related to disability in older persons. Gerontology 2000, 46(6):333-41. 19. Roberts SB: Impaired regulation of energy intake in old age. In Nutrition and aging Nestlé nutrition workshop series clinical & performance program Edited by: Rosenberg IH, Sastre A 2002, 6:49-61. 41. Steens G, ed: Moet er nog sport zijn? Sport, beweging en gezondheid in Vlaanderen 2002-2006. Antwerpen: F&G Partners 2006, 1:244. 20. Lowik MR, Westenbrink S, Hulsof KFAM, Kistemaker C, Hermus RJJ: Nutrition and aging: dietary intake in “apparently healthy” elderly (Dutch nutrition surveillance system). References Reddy S, Sanders T: Haematological studies on premenopausal Indian and Caucasian vegetarians compared with Caucasian omnivores. Br J Nutr 1990, 64:331-8. 8. Key TJ, Appleby PN, Spencer EA, Travis RC, Allen NE, Thorogood M, Mann JI: Cancer incidence in British vegetarians. Br J Cancer 2009, 101:192-197. 32. Hunt IF, Murphy NJ, Henderson C: Food and nutrient intake of Seventh- day Adventist women. Am J Clin Nutr 1988, 48:850-1. 9. Craig WJ: Health effects of vegan diets. Am J Clin Nutr 2009, 89:1627S-33S. 10. Bedford JL, Barr SI: Diets and selected lifestyle practices of self-defined adult vegetarians from a population-based sample suggest they are more ‘health conscious’. Int J Behav Nutr Phys Act 2005, 2:4. 33. Knoops KT, de Groot LC, Kromhout D, Perrin A-E, Moreiras-Verela O, Menotti A, van Staveren WA: Mediterranean diet, lifestyle factors, and 10-year mortality in elderly European men and women. The HALE project. JAMA 2004, 292(12):1433-1439. 11. Nathan I, Hackett AF, Kirby S: The dietary intake of a group of vegetarian children aged 7-11 years compared with matched omnivores. Brit J Nutr 1996, 75:533-544. 34. Trichopoulou A, Orfanos P, Norat T, Bueno-de-Mesquita B, Ocké MC, Peeters PH, van der Schouw YT, Boeing H, Hoffmann K, Boffetta P, Nagel G, Masala G, Krogh V, Panico S, Tumino R, Vineis P, Bamia C, Naska A, Benetou V, Ferrari P, Slimani N, Pera G, Martinez-Garcia C, Navarro C, Rodriguez-Barranco M, Dorronsoro M, Spencer EA, Key TJ, Bingham S, et al: Modified Mediterranean diet and survival: EPIC-elderly prospective cohort study. BMJ 2005, 330:991. 12. Clarys P, Deriemaeker P, Hebbelinck M: Study of physical fitness and health related parameters in vegetarian and non-vegetarian students. Nutr Food Sc 2000, 30:243-249. 13. Deriemaeker P, Aerenhouts D, Hebbelinck M, Clarys P: Nutrient Based Estimation of Acid-Base Balance in Vegetarians and Non-vegetarians. Plant Foods Hum Nutr 2010, 65:77-82. 35. Bamia C, Trichopoulos D, Ferrari P, Overvad K, Bjerregaard L, Tjønneland A, Halkjær J, Clavel-Chapelon F, Kesse E, Boutron-Ruault M-C, Boffetta P, Nagel G, Linseisen J, Boeing H, Hoffmann K, Kasapa C, Orfanou A, Travezea C, Slimani N, Norat T, Palli D, Pala V, Panico S, Tumino R, Sacerdote C, Bueno-de-Mesquita HB, Waijers PMCM, Peeters PHM, van der Schouw YT, Berenguer A, et al: Dietary patterns and survival of older Europeans: the EPIC-elderly study (European Prospective Investigation into Cancer and Nutrition). Publ Hlth Nutr 2007, 10(6):590-598. 14. References g 24. Brants HA, Lowik MR, Westenbrink S, Hulshof KF, Kistemaker C: Adequacy 24. Brants HA, Lowik MR, Westenbrink S, Hulshof KF, Kistemaker C: Adequacy of a vegetarian diet at old age (Dutch Nutrition Surveillance System). J Am Coll Nutr 1990, 4:292-302. 1. Craig WJ, Mangels AR: Position of the American Dietetic Association: Vegetarian Diets. J Am Diet Assoc 2009, 109:1266-1282. of a vegetarian diet at old age (Dutch Nutrition Surveillance System). J Am Coll Nutr 1990, 4:292-302. 2. Chang-Claude J, Frentzel-Beyme R: Dietary and lifestyle determinants of mortality among German vegetarians. Int J Epidem 1993, 22:228-236. 25. Lowik MR, Schrijver J, Odink J, van den Berg H, Wedel M: Long-term effects of a vegetarian diet on the nutritional status of elderly people (Dutch Nutrition Surveillance System). J Am Coll Nutr 1990, 6:600-9. 3. Key TJ, Fraser GE, Thorogood M, Applby PN, Beral V, Reeves G, Burr ML, Chang-Claude J, Frentzel-Beyme R, Kuzma JW, Mann J, McPherson K: Mortality in vegetarians and nonvegetarians: detailed findings from a collaborative analysis of 5 prospective studies. Am J Clin Nutr 1999, 70:516S-524S. 26. Woo J, Kwok T, Ho SC, Sham A, Lau E: Nutritional status of elderly Chinese vegetarians. Age Ageing 1998, 4:455-61. 27. Alexander D, Ball MJ, Mann J: Nutrient intake and haematological status of vegetarians and age-sex matched omnivores. Eur J Clin Nutr 1994, 48:538-46. 4. Appleby PN, Thorogood M, Mann JI, Key TJ: The Oxford vegetarian study: an overview. Am J Clin Nutr 1999, 70:525S-531S. 28. Helman A, Darnton-Hill J: Vitamin and iron status in new vegetarians. Am J Clin Nutr 1987, 45:785-9. 5. Leblanc JCh, Yoon H, Kombadjian A, Verger Ph: Nutritional intakes of vegetarian populations in France. Eur J Clin Nutr 2000, 54:443-449. 29. Lewis N, Kies C, Fox H: Iron intake and nutritional status of lacto-ovo vegetarian and omnivorc students eating in a lacto-ovo vegetarian food service. Nutr Rep Int 1983, 27:199-206. 6. Davey GK, Spencer EA, Appleby PN, Allen NE, Knox KH, Key TJ: EPIC-Oxford: lifestyle characteristics and nutrient intakes in a cohort of 33883 meat- eaters and 31546 non meat-eaters in the UK. Publ Hlth Nutr 2003, 6:259-268. 30. Miller D, Specker B, Ho M, Norman E: Vitamin B12 status in a macrobiotic community. Am J Clin Nutr 1991, 53:524-9. 7. Key TJ, Appleby PN, Russell MS: Health effects of vegetarian and vegan diets. Proc Nutr Soc 2006, 65:35-41. 31. Received: 15 February 2011 Accepted: 14 June 2011 Published: 14 June 2011 23. Exton-Smith AN: Nutritional status: diagnosis and prevention of malnutrition. In Metabolic and Nutritional Disorders in the Elderly. Edited by: Exton-Smith AN, Caird FI. Bristol: John Wright; 1980:66-76. Authors’ contributions MH, DDR and PC conceived the study. PC and MH were responsible for the data collection. PD participated in its design and coordination, did the statistical analyses and wrote the manuscript. DA, MH, DDR and PC critically reviewed the manuscript for writing and intellectual content. All authors read and approved the final manuscript. Page 7 of 8 Page 7 of 8 Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Competing interests h h d l h 22. Woo J, Ho SC, Lau J, Yuen YK, Chan SG, Masarei J: Cardiovascular symptoms, electrocardiographic abnormalities and associated risk factors in an elderly Chinese population aged 70 years and above. Int J Cardiol 1993, 42:249-55. Received: 15 February 2011 Accepted: 14 June 2011 Published: 14 June 2011 Received: 15 February 2011 Accepted: 14 June 2011 Published: 14 June 2011 References J Am Coll Nutr 1989, 8:347-356. 42. Staessen J, Amery A, Birkenhäger W, Bulpitt C, Clement D, de Leeuw P, Deruyttere M, De Schaepdryver A, Dollery C, Fagard R, Fletcher A, Forette F, Forte J, Henry J-F, Koistinen A, Leonetti G, Nissinen A, O’Brien E, O’Malley K, Pelemans W, Petrie JC, Strasser Thomas K, Terzoli L, Thijs L, Tuomilehto J, 21. Ho SC, Woo J: Social and health profile of the Hong Kong old-old population. A report. The Chinese University of Hong Kong, Hong Kong; 1994. Page 8 of 8 Page 8 of 8 Deriemaeker et al. Nutrition & Metabolism 2011, 8:37 http://www.nutritionandmetabolism.com/content/8/1/37 Webster J, Williams BO: Is a high serum cholesterol level associated with longer survival in elderly hypertensives? J Hypertens 1990, 8:755-61. Webster J, Williams BO: Is a high serum cholesterol level associated with longer survival in elderly hypertensives? J Hypertens 1990, 8:755-61. y y y 43. Onder G, Landi F, Volpato S, Fellin R, Carbonin P, Gambassi G, Bernabei R: Serum cholesterol levels and in-hospital mortality in the elderly. 0 Am J Med 2003, 115(4):4265-71. 44. Ravnskov U: High cholesterol may protect against infections and atherosclerosis. Q J Med 2003, 96:927-934. 45. Elmadfa I, Singer I: Vitamin B-12 and homocysteine status among vegetarians: a global perspective. Am J Clin Nutr 2009, 89(suppl):1693S-8S. 46. Appleby PN, Thorogood M, Mann JI, Key TJ: Low body mass index in non- meat eaters: the possible roles of animal fat, dietary fibre and alcohol. Int J Obes 1998, 22:454-460. 47. Alewaeters K, Clarys P, Hebbelinck M, Deriemaeker P, Clarijs JP: Cross- sectional analysis of BMI and some lifestyle variables in Flemish vegetarians compared with non-vegetarians. Ergonomics 2005, 48:1433-1444. 48. Seidell JC: Waist circumference and waist/hip ratio in relation to all-cause mortality, cancer and sleep apnea. Eur J Clin Nutr 2010, 64:35-41. 48. Seidell JC: Waist circumference and waist/hip ratio in relation to all-cause mortality, cancer and sleep apnea. Eur J Clin Nutr 2010, 64:35-41. doi:10.1186/1743-7075-8-37 Cite this article as: Deriemaeker et al.: Health aspects, nutrition and physical characteristics in matched samples of institutionalized vegetarian and non-vegetarian elderly (> 65yrs). 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Vasculogenic mimicry as a poor diagnostic and prognostic indicator in patients with malignant‎ melanoma: A ‎systematic review and meta-analysis
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Vasculogenic mimicry as a poor diagnostic and prognostic indicator in patients with malignant‎ melanoma: A ‎systematic review and meta-analysis Zhenhua Zhang Research article Version of Record: A version of this preprint was published on November 21st, 2019. See the published version at https://doi.org/10.1186/s12885-019-6350-5. Page 1/20 Page 1/20 Abstract Background: Vasculogenic mimicry (VM), a brand-new tumor microvascular model of non-endothelial cells, has been proposed as an important therapeutic target in malignant melanoma (MM). We performed a systematic review to evaluate the diagnostic and prognostic accuracy of VM for overall survival of MM patients. Methods: Using QUADAS-2 tool, the quality of the included studies was evaluated. Diagnostic capacity of VM variables were pooled by Meta-Disc software in term of sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and the area under summary receiver operating characteristic (SROC). Results: A retrospective observational study was conducted based on ‎ten studies including 978 clinically melanoma patients with proportion (P). VM+ melanoma cells were associated with poor prognosis in 38% of MM group (P = 0.35, 95% confidence intervals (CI): 0.27-0.42,  p-value < 0.001 ). The pooled sensitivity and specificity were 0.82 (95% CI: 0.79- 0.84) and 0.69 (95% CI: 0.66-0.71), respectively. Furthermore, the pooled PLR, NLR, and DOR were 2.56 (95% CI: 1.94-3.93), 0.17 (95% CI: 0.07-0.42), and 17.75 (95% CI: 5.30-59.44), respectively. Also, the AUC of SROC was 0.63, indicating high conserving of VM as a biomarker‎. Importantly, subgroup results suggested that VM+ tumor was a significantly accurate prognostic biomarker when diagnosed by CD31-/PAS+ staining methods in Asian MM samples ( p-value  < 0.001 ). Conclusions: Our findings support the potential of VM+ tumor as a promising prognostic biomarker and emphasize on an effective adjuvant therapeutic strategy in prognosis of Asian MM patients.. Background Malignant melanoma (MM) is the most ‎aggressive skin cancer and the most common skin disorder in Caucasians with an estimated global incidence of about 200,000 new cases per year and 50,000 ‎cancer- related deaths in 2018 [1, 2]. Moreover, the incidence of MM has been rapidly increased over the last 10 years in Asian and Mediterranean population, and it is diagnosed as the seventh and the eighth most common cancer among men and women in Singapore, respectively [3-6]. Although the Asian population has a notably lower risk of MM than Caucasians due to ethnic differences, anatomic distribution, histologic subtypes, and stage at diagnosis [6, 7]. Interestingly, the Caucasians melanoma patients are characterized by aggressive and progressive disease state, leading to major cancer-related morbidity and mortality of skin disorder [3]. Ultraviolet (UV) radiation, race, lifestyle, and genetic differences are the most important reasons for the higher mortality of melanoma [8-12], which can be decreased via early-stage detection and prevention [12, 13]. Dermoscopy and intrinsic molecular subtyping of melanoma have been widely accepted as an accurate diagnostic method with more than 50% accuracy compared to the clinical diagnosis in patients from Asian-Pacific and Central European countries [14, 15]. Recent investigations introduced a new non-angiogenesis dependent pathway entitled vasculogenic mimicry (VM), which refers to the extremely aggressive tumor cells that imitate endothelial cells and form a vessel-like structure [16, 17]. Eventually, VM has been considered as a cancer hallmark that can independently facilitate tumor neovascularization by formation of fluid-conducting and vascular endothelial cells [18-20]. VM was dedifferentiated into numerous cellular phenotypes and obtained Page 2/20 Page 2/20 Page 2/20 endothelial-like features, resulting in the formation of the de novo matrix-rich vascular-like network, such as plasma and red blood cells [21, 22]. The co-generation of endothelial cells, channels, laminar structures, and heparin sulfate proteoglycans are the main pathophysiological characteristics of VM in human melanoma patients [23-25]. Aggressive VM+ tumor cells are characterized by a higher expression of the basement membrane extracellular matrix (ECM) component laminin5γ2 and metalloproteinases (MMPs)-1, -2, -9, and -14 [21, 22, 26]. In highly aggressive melanoma cells, downregulation of vascular endothelial cadherin and upregulation of ECM components promotes the perfusion of the VM pathway [19, 21]. Ultimately, the VM+ melanoma cells are associated with more aggressive and metastatic tumor biology. Background Accumulated evidence suggests that VM is related to a poor prognosis in various malignant human tumors, including breast [27], colorectal [28], prostate [29], hepatocellular carcinoma [30], lung [29], ovarian [31], gastric [32], and bladder cancers [33]. Despite numerous experimental studies, the prognostic value of VM for survival in MM patients is still controversial and inconclusive. Certainly, understanding the role of VM in MM pathogenesis can help to develop effective treatments for tumor invasion and drug resistance in MM [34]. Hence, we conducted a quantitative ‎systematic review along with a comprehensive meta-analysis investigation based on ‎eligible studies to resolve inconsistent and often ambiguous findings. Furthermore, we planned to identify the prognostic accuracy of VM+ cancer patients to predict other clinical pathological feature outcomes of MM. Methods This systematic review and meta-analysis were performed according to recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement guidelines [35]. Data extraction and quality assessment All selected articles were reviewed independently by three researchers (ZZ, SI, and MDS) according to PICO (population, intervention, control, and outcomes) principle [36] and any disagreements or inconsistencies in a search process were addressed through consultations and debate. If an acceptable consensus was not reached, a third partner (QW) would resolve these disagreements based on the original data. The key demographics and clinicopathological information of all the qualified data collections ‎were summarized ‎in Table 1 and Table 2; including the first author’s name, publication year, total cases, ‎gender, country of origin population, age, follow up time, VM+ or VM- rate, analyzing methods of VM, Clark level, and location of sampling. Besides, we e-mailed corresponding authors to obtain any missing and ‎additional information, as well as original data needed for the meta-analysis. If the above data were not cited in the original study or no reply was received by email, the item was reported as “not reported (NR)". All eligible studies were assessed based on the Newcastle-Ottawa scale (NOS) [37] and Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) [38] protocols. Also, the probability of bias was calculated based on the criteria from the Cochrane Collaboration’s tool (Cochrane handbook for systematic reviews of interventions version 5.1.0.). Inclusion/exclusion criteria The current meta-analysis covered all prospective and randomized controlled trials (RCTs) studies that were considered eligible if they met the following criteria: (i) Melanoma patients were confirmed by immunohistochemical or histochemical tests. (ii). VM+ tumor tissues samples were assessed by classical staining in the tissue specimens, positive Periodic Acid-Schiff (PAS) and/or negative endothelial cell markers, CD34 or CD31; (iii) No previously received systemic treatment for metastatic disease. Likewise, we excluded all non-comparative, review, case-control, ‎conference abstracts, meeting, comments, and unrelated articles, as well as family-based, in vitro, and animal studies. Moreover, we excluded duplicate studies, continued work of previous ‎publications, and poor quality studies, as well as those with incomplete and/or missing data such as sample size and VM frequency. Search strategy and study selection MEDLINE electronic databases of Pubmed, Embase, Wiley Online Library, Web of Science, Science Direct, Cochrane library, and VIP-Google Scholar were searched without using language restrictions to assess the prognostic value of VM in melanoma patients prior to April 18, 2019. Definitely, different spelling and synonyms were combined applying Boolean “OR” and main terms were linked applying Boolean “AND” to identify all the relevant studies. The search string was conducted using MeSH terms and following main headline terms or free word based on the research question (both the UK and US spellings), such as: “vascular mimicry OR vasculogenic mimicry OR tumor cell-lined vessels ‎OR ‎tumor-derived endothelial cells” AND “prognosis OR survival OR outcome” AND “melanoma OR basal cell carcinoma OR squamous cell carcinoma OR cancer OR neoplasms OR malignant melanoma OR basal-cell skin OR squamous-cell skin OR skin”. The comprehensive literature search strategies were detailed in Table S1 (Additional file 1: Table S1), which were retrieved and screened by three researchers separately (ZZ, SI, HH, and MDS). Page 3/20 Page 3/20 Description of studies A detailed PRISMA flowchart of the study identification, screening, and exclusion process was shown in Fig. 1. The primary manual search yielded ‎426 potentially eligible literatures ‎through searching of electronic databases and 1 record by manual search. ‎After excluding duplicate studies (198 studies), 229 publications were kept for screening, of which 102 records were excluded according to the inclusion and exclusion criteria from database searching. Then, the remaining 127 articles were further assessed by abstract reviewing, and 67 studies were discarded either due to cell or animal studies data. Following careful review of titles and abstracts, 60 studies were considered in full-text articles and assessed for suitability. 20 studies were excluded for obvious irrelevance, 16 studies were precluded for other cancer studies, and 12 studies dismissed due to no related essay (Also see Additional file 1: Table S2). Finally, 12 studies were presented ‎in this meta-analysis [43-53]. Statistical analysis Page 4/20 The current systematic meta-analysis was carried out applying Comprehensive Meta-Analysis (CMA) software (the USA, version 2.2.064). The diagnostic accuracy and ROC curves were conducted on MetaDiSc (version 1.4). Besides, the quality of study was calculated by RevMan version 5.2 [39, 40]. Pooled specificity, pooled sensitivity, negative likelihood ratio (NLR), positive likelihood ratio (PLR), and diagnostic odds ratio (DOR) were calculated by corresponding 95% CIs to evaluate the diagnostic value of VM. Furthermore, the summary receiver operating characteristic (SROC) curve was calculated for the Page 4/20 involved studies with an overall area under the curve (AUC). Results of the meta-analysis were reported as a proportion (P) with 95% confidence intervals (CIs). All data were reported as means ± STD, standard deviation (SD) or median (range), as well as a description of ‎qualitative variables as number and percentage. The chi square-based Q-test was applied to testify between-study heterogeneity. Subgroup analysis was performed to identify the source of existing heterogeneity between the VM+ and available sub analyses such as sample size, race, and VM detestation methods. Publication bias was assessed using Begg’s funnel plots [41] and Egger’s regression test [42]. A value of “Pr > |z|” less than 0.05 was considered to be potential publication bias. All reported p values were two-sided and p-value < 0.05 was considered statistically significant. Quality assessment All 12 papers were methodologically essayed according to NOS and QUADAS-2 quality evaluation standards of the Cochrane Reviewer handbook. Both systems’ tools focused on the study dependent on the ‎methodology. Overall, the average NOS score was approximately 7.4 out of 12‎, which could be classified nearly in the high quality group. For each study, the NOS score is sorted in Table 1. Furthermore QUADAS-2 results confirmed that significant bias was not detected in the present meta-analyses. Details of the quality evaluation of eligible studies according to the NOS score were summarized in the Additional file 1: Table S3. The reviewers' decisions about each risk of bias and applicability concerns graph were presented as ‎percentages across selected studies. Figure S1 shows all parameters of QUADAS-2 assessment individually (Additional file 2: Figure S1). In this study, no significant bias and applicability concerns were found in all the selected studies. Outcome of the meta-analysis The relationship between VM+ and overall survival of MM patients was ‎identified applying the pooled proportions test method. We used a random effect approach because the heterogeneity of the overall prognosis was relatively high, which is shown across the study (I2 = 79.8, p-value < 0.001). Based on heterogeneous cross of 12 studies, VM was associated with poor prognosis in 38% of MM group compared to the VM-‎group (P = 0.35, 95% confidence intervals (95% CIs): 0.27-0.42, p-value < 0.001). Therefore, these results suggested that VM+ indicated a poorer prognosis for MM patients (Fig. 2). Characteristics of studies The demographic information of all relevant studies was detailed in Table 1. According to this table, a total of 12 studies with 978 MM patients were included in this systematic review and meta-analysis, between 1999 and 2017. Most of the studies were conducted in people of the Asian race, tracked by 7 studies (58.4%) [47, 48, 50-54] and 4 studies (33.4%) in European countries [44, 46, 49, 55], one study in USA (8.2%) [43], and no study from African populations. Gender subgroups among 978 patients included 377 male and 307 female patients. The major clinicopathological features of the included studies are shown in Table 2. More than 80% of the MM patients were diagnosed by histopathological tests. PAS combined-staining with endothelial markers (CD31 or CD34) is a commonly used method for identification of tumor VM in paraffin-embedded tissue specimens (66.7%) in 8 studies [44, 48, 50-55] as well as PAS staining in 4 studies [43, 46, 47, 49]. Moreover, significant predictors of VM+ in both adjusted and unadjusted analyses were Clark level IV/V (84.4%). Finally, eleven studies reported the association Page 5/20 between VM and clinicopathological parameters regarding OS [43-54], with the follow-up period ranged from 39-480 months. Subgroup analysis Associations between VM+ and the possible demographic and clinicopathological features of MM patients are sorted in Table 3. According to the results, none of the above covariates contributed to the heterogeneity (all p-values > 0.05). Therefore, according to those covariates, the pooled sensitivity, specificity, PLR, NLR, DOR, and AUC were measured for significant sub-analysis parameters. We detected statistically significant relationships between VM and sample size, VM and race, as well as between VM expression and staining method (Fig. 5). As shown in Fig. 5a and Table 3, VM+ is a potentially accurate prognostic biomarker in CD31-/PAS+ (P = 0.24, 95% CI: 0.15-0.35) compared to CD34-/PAS+ (P = 0.39, 95% CI: 0.27–0.42) and PAS+ staining subgroups (P = 0.40, 95% CI: 0.30–0.52). As a result, the CD31-/PAS+ staining methods are relatively accurate diagnostic methods for detection of the VM, with 75% sensitivity and 70% specificity. The subgroups analysis was performed based on sample size (≤100 vs. >100; Fig. 5b). The proportion of population with a high sample size (3 studies with more than 100 MM cases) was 0.41 (95% CI: 0.28–0.56; p-value = 0.12); while that of a sample size with less than 100 MM patients (9 studies) was 0.31 (95% CI: 0.23-0.41; p-value < 0.001). Meanwhile, the highest specificity, NLR, and AUC in sample sizes less than 100 suggested that VM is more accurate in diagnosis of smaller sample sizes. Interestingly, our results show that the overexpression of the VM was a high risk prognosis factor in Asia populations (7 studies with 503 cases; P = 0.32; 95% CI: 0.23–0.42; p-value < 0.001; Fig. 5c). As seen in Table 3 and Fig. 5C, the pooled sensitivity and specificity were higher in the Asian patients compared to Caucasian patients (85% vs. 69% and 78% vs. 68%, respectively). Moreover, we could not find any significant correlation between the VM+ melanoma samples with gender, age, Clark level, and location of sampling (Data not shown). Publication bias and sensitivity analysis The publication bias and sensitivity were analyzed using Funnel plots and empirically utilizing regression tests according to Begg’s test. The analysis was conducted by excluding a single study at a time. A symmetric inverted funnel shape in this study implies a ‘well-behaved’ data set, in which publication bias is improbable. Following exclusion of the ten studies, there was no obvious statistical evidence for publication bias in our meta-analysis (t = 1.41; p-value = 0.19) (Fig. 6). Hence, the results of the current meta-analysis were credible and stable, due to no noticeable publication bias influencing overall results. Diagnostic accuracy The effect of heterogeneity on the diagnostic threshold was evaluated based on the Spearman correlation coefficient. Fig. 3 presents the forest plots of pooled sensitivity and specificity, with their 95% CIs for individual studies. According to the results, the overall pooled sensitivity of VM+ tumor was 0.82 (95% CI: 0.79-0.84, Fig. 4a), while the specificity of VM+ tumor was 0.69 (95% CI: 0.66-0.71; Fig. 4b), among the 12 included studies. Furthermore, the overall pooled results for PLR, NLR, and DOR were 2.56 (95% CI: 1.94-3.93), 0.17 (95% CI: 0.07-0.42), and 17.75 (95% CI: 5.30-59.44), respectively. Page 6/20 Discussion Page 7/20 Page 7/20 To the best of our knowledge, this is the first meta-analysis study to identify prognostic value of VM+ in advanced melanoma patients. Our results indicate that 38% MM patients with VM+ have a poor prognosis (P = 0.35, 95% CI: 0.27-0.42, p-value < 0.001).‎ Moreover, significant association was identified in the pathologic features of the VM+ melanoma samples by race, sample size, and VM detection methods, which adversely influenced cancer survival. In current study, the AUC of SROC was 0.63, indicating the high accuracy of VM as a biomarker for MM‎. In addition, our pooled results provided convincing evidence for a significant positive relationship between VM and less sample size. To the best of our knowledge, this is the first meta-analysis study to identify prognostic value of VM+ in advanced melanoma patients. Our results indicate that 38% MM patients with VM+ have a poor prognosis (P = 0.35, 95% CI: 0.27-0.42, p-value < 0.001).‎ Moreover, significant association was identified in the pathologic features of the VM+ melanoma samples by race, sample size, and VM detection methods, which adversely influenced cancer survival. In current study, the AUC of SROC was 0.63, indicating the high accuracy of VM as a biomarker for MM‎. In addition, our pooled results provided convincing evidence for a significant positive relationship between VM and less sample size. Accumulating evidences indicated that VM is a new model of tumor microcirculation in highly aggressive malignant tumor cells [16, 17]. Recently, in vivo and in vitro studies showed that twist-related protein 1 (Twist1), neurogenic locus notch homolog protein 4 (Notch4), hypoxia inducible factor (HIF)-1a‎, EPH receptor A2 (EphA2), matrix metalloproteinase (MMP)-1, 2, -9, -14, and vascular endothelial (VE)-cadherin are potential therapeutic targets and prognostic indicators in VM+ tumor samples [22, 56]. Moreover, these studies suggest that VM+ tumor samples were resistant to common antiangiogenic drugs, such as apatinib, bevacizumab, and sunitinib [23, 34, 57]. The high ratio of neovascularization in VM+ tumor promotes angiogenesis, metastasis, and tumor growth along with extensive hypoxia and necrosis as well as induced recruitment of various pro-angiogenic factors, such as bone marrow-derived CD45+ myeloid cells, pericyte progenitor cells, and mature F4/80+ tumor-associated macrophages [58, 59]. Location variety and heterogenic morphology of MM tumors have a close relationship with the VM formation, which represented a noteworthy challenge for dermatologists [16, 60]. Discussion Thus, we estimated OS in the meta-analysis, taking into account that the great majority of the studies do not report the information [62]. Several published meta-analyses have concerned to evaluate the dissimilarity of tumor VM relevant to the prognosis of cancers [27, 28, 30, 53, 63]. For example, Cao Z. et al. suggested that VM+ cancer patients have a poor 5-year overall survival rate compared to VM- cancer patients, particularly in metastatic diseases of sarcomas and lung, colon, liver, and melanoma cancers [19]. By contrast, Shen Y. et al. addressed the tumor VM formation as an unfavorable prognostic indicator in breast cancer patients (P = 0.23, 95% CI: 0.08-0.38, p-value = 0.003) [64]. In line with our results, Yang JP. et al. findings showed that tumor VM is significantly associated with cancer differentiation, lymph node metastasis and distant metastasis, (P = 2.16; 95% CI: 1.98-2.38; p-value < 0.001) [65]. With such foreground and assumptions, this current study allows us to reach a better understanding of the clinical role of VM formation in MM patients using the statistical approaches. Conversely, the correlation between VM and survival of cancer patients are controversial or inconclusive. We would like to point out that there are some significant limitations in the current work: First, we only include papers published in English, but papers published in other languages, especially Chinese and Russian, were excluded, which certainly causes selection bias. Also, we did not consider the sensitivity analysis when reflecting on the significant difference among individual articles. Importantly, in most selected studies, the comments detection methods were IHC technique. The different primary antibodies with a wide range of the antibody dilution might also affect the IHC sensibility. Furthermore, the small sample size, short follow-up times, and no homogeneous distribution of the population (no studies upon the African publication) might also affect the precision of the estimate. Finally, the publication bias results showed that these limitations were not sufficiently enough to influence of the analysis of late- stage and fatal complications. It is clear that future clinical studies with larger sample sizes, standardized protocols, and more homogenizes populations would be‎ required to fully understand the prognostics potential of tumor VM in melanoma patients. Conclusions The results of the present meta-analysis for the first time suggested that VM+ tumor is associated with a poor OS of MM patients, as well as it is a more accurate prognostic biomarker in less sample ‎size groups of Asian patients. Therefore, the tumor VM status could be a promising prognostic biomarker for surgical and effective adjuvant therapy of MM patients‎. Discussion Our results clearly showed that VM is considered to have a negative effect on the overall survival of MM patients with a risk ratio of 0.35 (95% CI: 0.27-0.42, p-value < 0.001). Furthermore, our findings from sub- analyses underlined the status of VM formation in MM patients. We showed a stronger association between VM+ and sample size, VM+ and race, as well as VM+ and detection method of VM (p-value < 0.001). Our findings suggested that VM+ can be a significantly accurate prognostic biomarker when diagnosed by CD31-/PAS+ staining methods, with relatively accurate diagnostic value for VM detection (75% sensitivity and 70% specificity). Also, results of subgroup analyses implied a better diagnosis of VM in less sample sizes compared to that in higher than 100 cases (P: 0.31, 95% CI: 0.23-0.41; p-value < 0.001), with a pooled sensitivity of 85% and specificity of 78%. Interestingly, our results proposed VM as a promising accurate biomarker and target for MM diagnosis and therapeutics in Asian patients than that in Caucasian patients, with a pooled sensitivity of 91% and specificity of 70.5%. Lifestyle factors such as UV radiation exposure and nutrition are synergistically effective on the prevalence of MM [61, 62]. Compared to Caucasians, Asian MM patients are diagnosed at older ages; hence, we face a large population of old MM patients [4, 12]. But considering that our study was limited to a small sample size of cases in the Caucasian group (475 cases), more large-size studies among Caucasian MM population should be performed to obtain a comprehensive result [61]. It is known that VM+ tumor samples profiling could be more accurate in the Asian population compared to the Caucasian population [62]. The meta- analysis showed that the CD31-/PAS+ staining is a more accurate detection method for VM+ tumor samples than CD34-/PAS+ and PAS+ staining. Meanwhile‎, this meta-analysis suggested that Page 8/20 Page 8/20 postoperative detection with CD34- and/or CD31- of VM+ tumor samples in MM would be useful in finding critical therapy targets as well as making better follow-up plans. Thus, we estimated OS in the meta-analysis, taking into account that the great majority of the studies do not report the information [62]. postoperative detection with CD34- and/or CD31- of VM+ tumor samples in MM would be useful in finding critical therapy targets as well as making better follow-up plans. Funding This work was supported in part by ‎Southwest Medical University Grant and the Research Foundation of the Science ‎and Technology Department of Sichuan Province (No, 18080). Availability of data and material Not applicable. Abbreviations MM: Malignant melanoma; UV: Ultraviolet; VM: Vasculogenic mimicry; ECM: Extracellular matrix; PRISMA: Preferred reporting items for systematic reviews and meta-analysis; PAS: Periodic acid schiff's; NOS: Page 9/20 Newcastle-Ottawa scale; QUADAS-2: Quality assessment of diagnostic accuracy studies 2; CAM: Comprehensive meta-analysis; PLR: Positive likelihood ratio; NLR: Negative likelihood ratio; DOR: Diagnostic odds ratio; SROC: Summary receiver operating characteristic; AUC: Under the curve; P: Proportion; CI: Confidence interval; Twist1: Twist-related protein 1; Notch4: Neurogenic locus notch homolog protein 4; HIF-1a: hypoxia inducible factor 1a; EphA2: EPH receptor A2; MMP: Matrix metalloproteinase; VE: Vascular endothelial; EKR: Extracellular signal-regulated kinas. Ethics approval and consent to participate This study was approved by an independent ethics committee/institutional review board at Department of Oncology, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China. Consent for publication Not applicable. Competing interests The authors declare that they no competing interests. Acknowledgements We thankfully acknowledge all authors for their continuous collaborations with us in sharing information. We also gratefully acknowledge all authors and coworkers for their contributions. We also gratefully acknowledge all authors and coworkers for their contributions. Authors' contributions Page 10/20 ZZ, SI, MDS participated in the design of the study. HH performed the statistical analysis. ZL, SI, and YF carried out the data extraction. SI and QW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All Authors read and approved the final manuscript. 1. 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Qin L, Ren Y, Chen AM, Guo FJ, Xu F, Gong C, Cheng P, Du Y, Liao H: Peroxisome proliferator-activated receptor gamma ligands inhibit VEGF-mediated vasculogenic mimicry of prostate cancer through the AKT signaling pathway. Molecular medicine reports 2014, 10(1):276-282. 64. Shen Y, Quan J, Wang M, Li S, Yang J, Lv M, Chen Z, Zhang L, Zhao X, Yang J: Tumor vasculogenic mimicry formation as an unfavorable prognostic indicator in patients with breast cancer. Oncotarget 2017, 8(34):56408-56416. 65. Yang JP, Liao YD, Mai DM, Xie P, Qiang YY, Zheng LS, Wang MY, Mei Y, Meng DF, Xu L et al: Tumor vasculogenic mimicry predicts poor prognosis in cancer patients: a meta-analysis. Angiogenesis 2016, 19(2):191-200. Additional Files Additional file 1: Table S1. The detailed search strategy. Table S2. The excluded full-text articles. Table S3. Quality assessment of the included studies according to the Newcastle-Ottawa Scale (NOS) Additional file 1: Table S1. The detailed search strategy. Table S2. The excluded full-text articles. Table S3. Quality assessment of the included studies according to the Newcastle-Ottawa Scale (NOS) Additional file 2: Figure S1. Risk of bias graph. The overall risk of bias was regarded as low in all qualified studies, in term of the QUADAS-2 assessment Additional file 2: Figure S1. Risk of bias graph. The overall risk of bias was regarded as low in all qualified studies, in term of the QUADAS-2 assessment Figures Page 15/20 Figure 1 Figure 1 Flow diagram of included studies (following PRISMA guidelines, n = number of studies). Flow diagram of included studies (following PRISMA guidelines, n = number of studies). Flow diagram of included studies (following PRISMA guidelines, n = number of studies). Flow diagram of included studies (following PRISMA guidelines, n = number of studies). Page 16/20 Figure 2 Forest plot of proportion ratios (P) in the random effect model. These plots show the prognostics accuracy for all objective response analyses. Forest plot of proportion ratios (P) in the random effect model. These plots show the prognostics accuracy for all objective response analyses. Figure 2 Forest plot of proportion ratios (P) in the random effect model. These plots show the prognostics accuracy for all objective response analyses. Page 17/20 Figure 3 Summary receiver operating characteristic (SROC) curve for VM in the diagnosis of MM cancer. Figure 3 Summary receiver operating characteristic (SROC) curve for VM in the diagnosis of MM cancer. Figure 3 Figure 3 Summary receiver operating characteristic (SROC) curve for VM in the diagnosis of MM cancer. Page 18/20 Figure 4 Figure 4 Page 18/20 orest plot of pooled sensitivity (a) and specificity (b) for VM in the diagnosis of MM cancer. Figure 5 Funnel plot of the sub-analysis parameters. Forest plots showed that MM cancer was associated with detection methods of VM (a), sample size (b), and race (c). CIs, confidence intervals. Weights are from random effects analysis. orest plot of pooled sensitivity (a) and specificity (b) for VM in the diagnosis of MM cancer. ensitivity (a) and specificity (b) for VM in the diagnosis of MM cancer. orest plot of pooled sensitivity (a) and specificity (b) for VM in the diagnosis of MM cancer. orest plot of pooled sensitivity (a) and specificity (b) for VM in the diagnosis of MM cancer. orest plot of pooled sensitivity (a) and specificity (b) for VM in the diagnosis of MM cancer. Figure 6 Funnel plots for the detection of a publication bias‎. All enrolled 12 studies represent by each point for the specified association, individually. The vertical and horizontal axes represent the standard error of a logarithmic ‎proportion and the proportion limits, respectively. Funnel plots for the detection of a publication bias‎. All enrolled 12 studies represent by each point for the specified association, individually. The vertical and horizontal axes represent the standard error of a logarithmic ‎proportion and the proportion limits, respectively. Figure 5 Funnel plot of the sub-analysis parameters. Forest plots showed that MM cancer was associated with detection methods of VM (a), sample size (b), and race (c). CIs, confidence intervals. Weights are from random effects analysis. Funnel plot of the sub-analysis parameters. Forest plots showed that MM cancer was associated with detection methods of VM (a), sample size (b), and race (c). CIs, confidence intervals. Weights are from random effects analysis. Page 19/20 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile2.doc Additionalfile1.pdf Page 20/20
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Binding interaction of a novel fluorophore with serum albumins: steady state fluorescence perturbation and molecular modeling analysis
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© 2015 Pal et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Uttam Pal1†, Sumit Kumar Pramanik2†, Baisali Bhattacharya1, Biswadip Banerji2 and Nakul Chandra Maiti1* *Correspondence: ncmaiti@ iicb.res.in †Uttam Pal and Sumit Kumar Pramanik contributed equally to this work 1 Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata, West Bengal, India Full list of author information is available at the end of the article Abstract Fluorescence emission and anisotropy are widely used to measure the binding param- eters and kinetic behavior of reactions that cause a change in the rotational time of a fluorescent molecule. We report here fluorescence emission and anisotropy behavior of a newly synthesized novel naphthalene base fluorophore (methyl 3-[(6-{[2-(tert- butoxy)-2-oxoethyl] (4-methoxyphenyl)amino}naphthalen-2-yl)formamido]propanoate) in several solution conditions including its binding to human and bovine serum albumin proteins both in their native and denatured states. The fluorescence yield of the compound substantially increased inside hydrophobic protein surface and ~30 nm decrease in Stokes’ shift, compared to aqueous solution, was observed. Shift in fluores- cence excitation peak position from the absorption peak of the molecule was ~8 nm in protein solution. This indicated possible alteration of excited state geometry of the compound by the globular fold of albumins. In addition, we measured the steady state fluorescence anisotropy of the molecule to evaluate several thermodynamic parameters and the results suggested the binding was energetically favorable. The measured ΔG° was ~−30 kJ mol−1 and the derived dissociation constant was ~10−6 M. The molecu- lar docking analysis further highlighted the nonspecific association of the compound with the proteins and hydrophobic forces may have a significant role in the binding processes. Under the denatured condition of the protein, the compound lost its bind- ing efficacy and reduction in fluorescence intensity was observed. Thus, the molecule appears as a new fluorescence probe to report the nature of its binding site in terms of increased fluorescence quantum yield and decreased Stokes’ shift. It can also report the changes in the binding site due to global change in protein structure such as unfolding/ misfolding often linked to several human disorder. Further it could be useful to detect and study the drug binding site of specific protein of interest. Keywords:  Serum albumin, New chemical entity, Binding site, Microenvironment, Fluorescence probe *Correspondence: ncmaiti@ iicb.res.in †Uttam Pal and Sumit Kumar Pramanik contributed equally to this work 1 Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata, West Bengal, India Full list of author information is available at the end of the article Pal et al. SpringerPlus (2015) 4:548 DOI 10.1186/s40064-015-1333-8 Pal et al. SpringerPlus (2015) 4:548 DOI 10.1186/s40064-015-1333-8 Open Access Binding interaction of a novel fluorophore with serum albumins: steady state fluorescence perturbation and molecular modeling analysis Uttam Pal1†, Sumit Kumar Pramanik2†, Baisali Bhattacharya1, Biswadip Banerji2 and Nakul Chandra Maiti1* Background The interaction and the energetics of small molecule binding to a protein largely depend on the molecular architecture and microenvironment provided due to folding/unfold- ing or even transformation of the protein structure. The observable properties of a small Page 2 of 13 Pal et al. SpringerPlus (2015) 4:548 molecule in such microenvironment, in turn, carry information about the binding site, which is crucial for drug development (Royer 2006; Cohen et al. 2002; Abou-Zied and Al-Shini 2008; Er et al. 2013) and many other investigations. Our attention was focused on fluorescence emission and binding aspects of the fluorophore in the hydrophobic milieu inside a globular fold of a protein under physiological and denaturing conditions. We have synthesized a naphthalene based fluorophore, methyl 3-[(6-{[2-(tert-butoxy)- 2-oxoethyl](4-methoxyphenyl)amino}naphthalen-2-yl) formamido]propanoate (com- pound 5) to study the immediate surroundings of the molecule inside the proteins. Serum albumin was chosen as model protein with at least seven hydrophobic grooves on its surface. It provides a unique microenvironment and acts as a universal receptor for many drug molecules (Er et al. 2013; Reichenwallner and Hinderberger 2013; Simard et  al. 2006; Curry et  al. 1998). This protein increases the solubility of hydrophobic ligands in plasma and modulates their delivery to cells. The precise architecture of the binding pockets is known from several crystallographic and NMR spectroscopic studies (Reichenwallner and Hinderberger 2013; Simard et al. 2006; Curry et al. 1998). Thus, the interaction pattern and the spectroscopic signature of a small molecule housed in the well defined environment of serum albumin could provide significant insight into the interaction pattern and its binding efficacy (Yamasaki et al. 2013). fi Intrinsic protein fluorescence originating from tryptophan and tyrosine residues or the fluorescence of the drug molecule itself provides ample information about the local environment, the changes in protein conformation and the interaction of a protein with a drug molecule (Royer 2006; Cohen et al. 2002; Abou-Zied and Al-Shini 2008). How- ever, the current investigation explored both the quantitative and qualitative aspect of the interaction and incorporation of compound 5 into the binding pockets of serum albumin, at the molecular level using fluorescence methodologies. The thermodynamic parameters were obtained by measuring the effect of temperature on binding constant. In addition to access the interaction site and binding specificity of the drug molecule computational modeling analysis was carried out. Absorption spectroscopy Ground-state absorption spectra were recorded with a Shimadzu UV-2401PC Spec- trometer. 1 cm path-length quartz cuvette was used and 250–450 nm wavelength range was scanned. Compound 5 absorption spectra as a function of BSA and HSA concentra- tion were recorded by titrating (Banerjee et al. 2012; Ray et al. 2012) compound 5 solu- tion with concentrated protein solutions. Small dilution error in the titration experiment was ignored. Procedure to synthesize compound 5 Synthesis was carried out following the Scheme 1. Detailed procedure and characteristic data are given in the Additional file 1. Methods Bovine and human serum albumins were purchased from Sigma–Aldrich Corporation (St. Louis, MO, USA). Tris–HCl and Urea were also purchased from Sigma-Aldrich. All the samples were prepared in 20 mM Tris–HCl buffer of pH 7.0. Deionized and triple distilled water was used for preparing buffer solution that was passed through 0.22 µm pore size Millipore filters (Millipore India Pvt. Ltd., Bangalore, India). i All air and water sensitive reactions were carried out in oven dried glassware under nitrogen atmosphere using standard manifold techniques. All the chemicals were pur- chased from Acros organics and Sigma-Aldrich, and used without further purification unless otherwise stated. Compounds that are not described in the experimental part were synthesized according to the literature procedures. Solvents were freshly distilled by standard procedures prior to use. Flash chromatography was performed on silica gel (Merck, 100–200 mesh) with the indicated elutant. All 1H and 13C-NMR spectra were recorded on a Bruker 600 MHz spectrometer. For 1H NMR, tetramethylsilane (TMS) served as internal standard (δ  =  0) and data are reported as follows: chemical shift, Pal et al. SpringerPlus (2015) 4:548 Page 3 of 13 integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and coupling constant(s) in Hz. For 13C NMR, TMS (δ = 0) or CDCl3 (δ = 77.26) was used as internal standard and spectra were obtained with complete proton decoupling. Mass spectra were obtained on a Jeol MS station 700 and ESI-TOF mass spectrometer. Determination of binding constants Kd for compound 5 binding with BSA and HSA were determined from the compound 5 fluorescence anisotropy perturbation with proteins. Compound 5 concentration was kept at 0.5 µM and the protein concentration was varied from 0 to 5.5 µM. Small dilu- tion error due to the titration was ignored. Anisotropy values as a function of protein concentration were recorded. To derive the binding parameters, data were analyzed using the non-linear Langmuir isotherm (Banerji et al. 2013): (2) r = rmax ∗[P]/(Kd + [P]) r = rmax ∗[P]/(Kd + [P]) (2) where Δr is the difference in fluorescence anisotropy in the absence and presence of the protein at concentration [P], Δrmax is the maximum possible change in the fluorescence anisotropy, Kd is the binding dissociation constant. The non-linear equation was fitted to the data using Wolfram Mathematica 10. Stern–Volmer quenching constant or the binding affinity constant, Ka was determined as a reciprocal of Kd (Banerji et al. 2013). Fluorescence emission and excitation spectroscopyhl The steady-state fluorescence emission and excitation spectra were recorded with a Cary Eclipse Fluorescence Spectrophotometer. The emission spectra of serum albumins and compound 5 were obtained by exciting the samples at the wavelengths 295 and 330 nm, respectively. The excitation spectra of compound 5 was obtained by recording the emis- sion at wavelength 450  nm. In all the cases, the excitation and emission slit widths were kept at 5 nm each. Compound 5 fluorescence emission or excitation spectra as a Scheme 1  Reagent and conditions: a Conc. H2SO4, MeOH, 0 °C to r.t., 6 h. b Palladium (II) acetate (0.05 equiv.), xantphos (0.1 equiv.) and cesium carbonate (3 equiv.), 80 °C, 4 h. c Potassium tert-butoxide (1.2 equiv.), DMF, 0 °C to r.t., 12 h. d Lithium hydroxide (3 equiv.), MeOH–water (5:1), r.t., 2 h. e EDC.HCl (3.0 equiv.), HOBT (2.5 equiv.), TEA (6 equiv.), 0 °C to r.t., 1.5 h Scheme 1  Reagent and conditions: a Conc. H2SO4, MeOH, 0 °C to r.t., 6 h. b Palladium (II) acetate (0.05 equiv.), xantphos (0.1 equiv.) and cesium carbonate (3 equiv.), 80 °C, 4 h. c Potassium tert-butoxide (1.2 equiv.), DMF, 0 °C to r.t., 12 h. d Lithium hydroxide (3 equiv.), MeOH–water (5:1), r.t., 2 h. e EDC.HCl (3.0 equiv.), HOBT (2.5 equiv.), TEA (6 equiv.), 0 °C to r.t., 1.5 h Scheme 1  Reagent and conditions: a Conc. H2SO4, MeOH, 0 °C to r.t., 6 h. b Palladium (II) acetate (0.05 equiv.), xantphos (0.1 equiv.) and cesium carbonate (3 equiv.), 80 °C, 4 h. c Potassium tert-butoxide (1.2 equiv.), DMF, 0 °C to r.t., 12 h. d Lithium hydroxide (3 equiv.), MeOH–water (5:1), r.t., 2 h. e EDC.HCl (3.0 equiv.), HOBT (2.5 equiv.), TEA (6 equiv.), 0 °C to r.t., 1.5 h Page 4 of 13 Pal et al. SpringerPlus (2015) 4:548 function of protein concentration were recorded by simple titration method (Banerjee et al. 2012, 2013; Ray et al. 2012). Fluorescence anisotropy Fluorescence anisotropy experiments were performed in the Cary Eclipse Fluorescence Spectrophotometer and a manual polarizer accessory was used. The excitation and emission wavelengths were set to 330 and 450 nm, respectively, with slit widths of 5 nm for each monochromator. Anisotropy (r) was determined using the following equation (Banerjee et al. 2012): (1) r = (IVV−G ∗IVH)/(IVV + 2G ∗IVH); G = IHV/IHH (1) where I is the fluorescence emission intensity when excitation and emission polarizes are aligned in a particular way denoted by the suffix. For example, VV indicates both the excitation and emission polarizers are vertically aligned whereas VH indicates excitation polarizer is vertical and the emission polarizer is horizontally aligned and so on. G is a correction factor. The changes in compound 5 fluorescence anisotropy as a function of protein concen- tration were recorded by simple titration method as mentioned earlier. Binding thermodynamics Kd values were determined as a function of temperature and the thermodynamic param- eters of binding was obtained by fitting van’t Hoff equation (Banerjee et al. 2012; Ray et al. 2012) to the data: (3) ln Keq = −H◦/RT + S◦/R ln Keq = −H◦/RT + S◦/R (3) where Keq is the equilibrium constant (here the Stern–Volmer quenching constant) of binding at corresponding temperature T, and R is the gas constant. The equation gives the standard enthalpy change (ΔH°) and standard entropy change (ΔS°) on binding. The Page 5 of 13 Pal et al. SpringerPlus (2015) 4:548 free energy change (ΔG°) has been estimated from the following relationship (Banerjee et al. 2012; Ray et al. 2012): (4) G◦= H◦−TS◦ G◦= H◦−TS◦ (4) Absorbance and fluorescence of compound 5 In aqueous buffer at pH 7.4, Compound 5 shows a strong absorption band with a peak at 330 nm. Compound 5 is also fluorescence active and the fluorescence band appeared at ~450 nm (Fig. 1). Figure 1 also shows the excitation spectrum of compound 5, which largely overlapped with the absorption spectrum suggesting that excited state conforma- tion of compound 5 in solution is homogeneous and close to ground state structure. Molecular docking Molecular docking experiments were performed using AutoDock 4.2 (Morris et  al. 2009) and AutoDock Vina (Trott and Olson 2010) of The Scripps Research Institute and the SwissDock server (http://www.swissdock.ch/). AutoDock 4.2, Vina and SwissDock uses different algorithm and scoring function for docking calculations. AutoDockTools (Morris et al. 2009) was used for the preparation of ligands and proteins for docking. BSA (PDB: 3V03) (Majorek et al. 2012) and HSA (PDB: 1E78) (Bhattacharya et al. 2000) structural information were obtained from Protein Data Bank (Berman et al. 2000). The ligand structures were drawn in Avogadro (Hanwell et  al. 2012) and geometry opti- mized in vaccuo using the steepest descent followed by conjugate gradient algorithms. Genetic algorithm was used in AutoDock 4.2 and it was run (ga_run) 100 times to gen- erate a statistically significant number of docked poses (Alam et al. 2012). AutoDock 4.2 results were clustered using binding free energy and standard deviation cut offs of 0.5 kcal mol−1 and 2Å, respectively. Lipophilicity and solubility calculations Lipophilicity in terms of calculated (clogP) and solubility in terms of calculated logS (clogS) were determined at Virtual Computational Chemistry Laboratory server (http:// www.vcclab.org/lab/alogps/) (Tetko et al. 2005). Polar surface area was calculated with a 1.4 Å radius probe size. Thermal and chemical denaturationh Thermal denaturation of protein was performed by increasing the temperature of the compound 5-protein solution from 10 to 80 °C in 8 steps (Banerjee et al. 2012). The sample was kept under continuous stirring condition and the changes in compound 5 fluorescence spectra were recorded. Chemical denaturation was achieved by increas- ing the concentration of urea (Banerjee et al. 2012) while recording the compound 5 fluorescence. Results and discussion Absorbance and fluorescence of compound 5 Fluorescence quantum yield of compound 5 in protein environment Compound 5 absorbs UV–visible light strongly at the wavelengths where protein absorbs. It also has a strong absorption band in the fluorescence emission range of protein. Therefore, protein intrinsic fluorescence perturbation with compound 5 or Page 6 of 13 Pal et al. SpringerPlus (2015) 4:548 300 400 500 600 0.0 0.2 0.4 0.6 0.8 Wavelength (nm) OD Fluorescence intensity Fig. 1  Absorption and fluorescence behavior of compound 5 in the UV–Visible range. Absorption spectrum of compound 5 in 20 mM Tris–HCl buffer of pH 7.4 is shown in blue. Fluorescence emission and excitation spectra of compound 5 are shown in green and yellow, respectively. Compound 5 fluorescence spectra were recorded in the same buffer and normalized against its absorption spectrum 300 400 500 600 0.0 0.2 0.4 0.6 0.8 Wavelength (nm) OD Fluorescence intensity Fig. 1  Absorption and fluorescence behavior of compound 5 in the UV–Visible range. Absorption spectrum of compound 5 in 20 mM Tris–HCl buffer of pH 7.4 is shown in blue. Fluorescence emission and excitation spectra of compound 5 are shown in green and yellow, respectively. Compound 5 fluorescence spectra were recorded in the same buffer and normalized against its absorption spectrum the energy transfer from protein to compound 5 is not an eminent choice to probe the binding interactions of compound 5 with proteins. However, the changes in com- pound 5 fluorescence may be monitored as a parameter of binding. With the increasing concentration of serum albumins, we have found that the fluorescence intensity of the compound 5 increases (Fig. 2a, Additional file 1: Figure S1A). A blue shift in the emis- sion maximum was also observed. It indicated compound 5 binding to the hydrophobic grooves of serum albumins. Such binding causes solvent exclusion of compound 5 and the energy that is otherwise spent in solvent relaxation, is gained by the emitting pho- tons. Here, about 30 nm decrease in the Stokes’ shift in compound 5 fluorescence cor- responds to ~0.2 eV energy gain by each emitted photon. Interaction of compound 5 with serum albumins increases its fluorescence quantum yield and compound 5 gets a hydrophobic environments while bound to the serum albu- mins. Thus, the changes in compound 5 fluorescence in the presence of proteins carry the information not only about the interaction but the microenvironment of the binding site on its target protein as well. Fluorescence anisotropy of compound 5 in presence of proteins Fluorescence anisotropy of compound 5 in presence of proteins Small molecules tumble faster in less viscous solvents. But when it binds to a large mol- ecule such as protein, its movement gets restricted. Fluorescence anisotropy is, there- fore, widely used to measure the binding constants and kinetics of reactions that cause a change in the rotational time of the fluorescent molecules (Heyduk et al. 1996). Fluo- rescence anisotropy measurements can also elucidate the microenvironment of a small molecule in terms of its rotational diffusion, interactions, and proximity to proteins. Compound 5 in buffer solution shows very low anisotropy. With the increasing con- centration of serum albumins, fluorescence anisotropy of compound 5 increases and gradually reaches the saturation (Fig. 2b, Additional file 1: Figure S1B). Binding disso- ciation constants for compound 5 binding with the two serum albumins were deter- mined from this experiment and were found to be in the low micromolar concentration range (Table 1). The molecule showed increased quantum yield along with a blue shift in presence of protein. Anisotropy experiment confirmed that the molecule goes inside the binding cavity of the protein, thus, restricting its free rotation. Therefore, the anisot- ropy suggests that the observed change in the fluorescence property of the molecule is a direct effect of the binding site environment. Thermodynamics of compound 5 binding to serum albumins Equilibrium constant of a reaction changes with the temperature (Fig. 3). Such a change can be explained by van’t Hoff’s equation, which in turn, gives the standard enthalpy and standard entropy changes for the reaction. The associations of the compound with serum albumins are thermodynamically favorable, which is evident from the decrease in Gibbs free energy (Table 2). Moreover, the binding with HSA is enthalpy driven (negative Table 1  The Kd and  Ka values for  the binding of  the compound 5 to  serum albumins as determined by the fluorescence and anisotropy perturbation experiments at room tem- perature Protein Kd (M) Ka (M−1) BSA 1.93 × 10−6 5.18 × 105 HSA 2.05 × 10−6 4.88 × 105 a ln K a = - 2582.95 T + 21.44 R 2 = 0.992 1/ 293 1/ 298 1/ 303 12.65 12.70 12.75 12.80 12.85 12.90 1/ T ( K - 1) ln K a b ln K a = 2920.62 T + 3.15 R 2 = 0.9994 1/ 293 1/ 298 1/ 303 12.80 12.85 12.90 12.95 13.00 13.05 13.10 1/ T ( K - 1) ln K a Fig. 3  Determination of thermodynamic parameters of compound 5 binding from van’t Hoff’s plot. a Decrease in the binding equilibrium constant with the decreasing temperature for BSA-compound 5 interac- tion and the fitted van’t Hoff equation. b Increase in the binding equilibrium constant with the decreasing temperature for HSA-compound 5 interaction and the fitted van’t Hoff equation Table 1  The Kd and  Ka values for  the binding of  the compound 5 to  serum albumins as determined by the fluorescence and anisotropy perturbation experiments at room tem- perature Protein Kd (M) Ka (M−1) BSA 1.93 × 10−6 5.18 × 105 HSA 2.05 × 10−6 4.88 × 105 a ln K a = - 2582.95 T + 21.44 R 2 = 0.992 1/ 293 1/ 298 1/ 303 12.65 12.70 12.75 12.80 12.85 12.90 1/ T ( K - 1) ln K a b ln K a = 2920.62 T + 3.15 R 2 = 0.9994 1/ 293 1/ 298 1/ 303 12.80 12.85 12.90 12.95 13.00 13.05 13.10 1/ T ( K - 1) ln K a Fig. 3  Determination of thermodynamic parameters of compound 5 binding from van’t Hoff’s plot. a Decrease in the binding equilibrium constant with the decreasing temperature for BSA-compound 5 interac- tion and the fitted van’t Hoff equation. Fluorescence quantum yield of compound 5 in protein environment 0 µM [BSA ] 5.5 µM a 350 400 450 500 550 600 0 2 4 6 8 Wavelength ( nm) Fluorescence intensity ( a.u.) b ∆r = 0.18 [ BSA ] 1.93 + [ BSA ] ,R 2 =0.9995 0 1 2 3 4 5 0.00 0.05 0.10 0.15 [BSA ] ( µM ) ∆r 453 nm Fig. 2  Fluorescence emission and anisotropy change of compound 5 in presence of serum albumin. Compound 5 concentration was kept constant at 0.5 μM and the protein concentration was varied from 0 through 5.5 μM. a The changes in fluorescence emission spectrum of compound 5 as a function of BSA concentration. b The changes in compound 5 fluorescence anisotropy with increasing concentration of BSA and the fitted Langmuir isotherm 0 µM [BSA ] 5.5 µM a 350 400 450 500 550 600 0 2 4 6 8 Wavelength ( nm) Fluorescence intensity ( a.u.) b ∆r = 0.18 [ BSA ] 1.93 + [ BSA ] ,R 2 =0.9995 0 1 2 3 4 5 0.00 0.05 0.10 0.15 [BSA ] ( µM ) ∆r 453 nm Fig. 2  Fluorescence emission and anisotropy change of compound 5 in presence of serum albumin. Compound 5 concentration was kept constant at 0.5 μM and the protein concentration was varied from 0 through 5.5 μM. a The changes in fluorescence emission spectrum of compound 5 as a function of BSA concentration. b The changes in compound 5 fluorescence anisotropy with increasing concentration of BS and the fitted Langmuir isotherm Page 7 of 13 Pal et al. SpringerPlus (2015) 4:548 Fluorescence anisotropy of compound 5 in presence of proteins Excited state geometry of compound 5 in protein environment Fluorophore binding to a protein often results in an altered ground state electronic property, which can be visualized by a change in the absorption spectrum. However, the absorption spectrum of compound 5 does not change due to its binding with the serum albumins (Additional file 1: Figure S2). It indicates that the ground state geometry of compound 5 inside the hydrophobic groove of serum albumins remains the same as in the solution. Interestingly, when we recorded the excitation spectrum of compound 5 in presence of serum albumins (Fig. 4, Additional file 1: Figure S3), we observed ~8 nm red shift in the wavelength of excitation maximum (330–338 nm). It indicates that the excited state geometry of compound 5 within the binding site of serum albumin gets altered with the possible formation of an exciplex. Thermodynamics of compound 5 binding to serum albumins b Increase in the binding equilibrium constant with the decreasing temperature for HSA-compound 5 interaction and the fitted van’t Hoff equation Page 8 of 13 Pal et al. SpringerPlus (2015) 4:548 Table 2  Thermodynamics of compound 5 binding to serum albumins Serum albumins ΔG° (kJ mol−1) at 25 °C ΔH° (kJ mol−1) ΔS° (J mol−1 K−1) BSA −31.64 21.47 178.25 HSA −32.09 −24.28 26.18 Table 2  Thermodynamics of compound 5 binding to serum albumins ΔH°) whereas the binding with BSA is entropy driven (positive ΔS°). It suggests that, despite the structural similarity in the two proteins, the interactions with HSA are ther- modynamically different from that of BSA. Denaturation of proteins bound to compound 5 b The changes in fluorescence of BSA compound 5 complex with the increasing concentration of Urea 0 M [Urea ] 8 M b 350 400 450 500 550 600 0.0 0.2 0.4 0.6 0.8 1.0 Wavelength ( nm) Fluorescence intensity ( a.u.) 10 °C 80 °C a 350 400 450 500 550 600 0.0 0.2 0.4 0.6 0.8 1.0 Wavelength ( nm) Fluorescence intensity ( a.u.) Fig. 5  Compound 5 interaction with denaturing proteins. a The changes in fluorescence of BSA compound 5 complex with the increasing temperature. b The changes in fluorescence of BSA compound 5 complex with the increasing concentration of Urea Drug like properties of compound 5h The molecular properties of the compound, such as clogP, clogS, polar surface area etc. (Bickerton et al. 2012) are listed in the Table 3. The clogP value of a compound is the logarithm of its partition coefficient between n-octanol and water. It is a well established measure of the compound’s lipophilicity, which influences its behaviour in a range of biological processes such as solubility, membrane permeability, lack of selectivity and non-specific toxicity (Alam et al. 2011). It has been shown for compounds to have a rea- sonable probability of being well absorbed, their logP value must not be greater than 5.0 (Lipinski et al. 1997). Besides, the aqueous solubility of a compound is also defined by logS, which significantly affects its absorption and distribution characteristics. Typically, a low solubility goes along with a bad absorption. Most of the drugs on the market have an estimated logS value of about −4. Table 3 lists the polar surface area of the compound as well, which should be less than 140 Å2 for a drug molecule (Lipinski et al. 1997). Apart from lipophilicity/solubility and the polar surface area, the molecular weight and the number of hydrogen bond acceptor/donor in compound 5 also follow the Lipinski’s rule of five (Lipinski et al. 1997) to be a candidate drug molecule. Denaturation of proteins bound to compound 5 Serum albumin-compound 5 complexes were denatured chemically and thermally. When compound 5 is bound to the protein, fluorescence is blue shifted. With the grad- ual increase in the temperature or the urea concentration, the fluorescence intensity of compound 5 gradually decreases and the Stokes’ shift increases until the fluorescence returns to its solution state nature (Fig. 5, Additional file 1: Figure S4). This experiment demonstrated that compound 5 binds to the structured protein and not to a denatured protein. In other words, it reports the progressive loss of binding sites on its receptor when the receptor is undergoing a massive structural change. b 250 300 350 400 450 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Wavelength ( nm) Normalized intensity ( a.u.) 0 µM [BSA ] 5.5 µM a 250 300 350 400 450 0 2 4 6 8 Wavelength ( nm) Fluorescence intensity ( a.u.) b 250 300 350 400 450 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Wavelength ( nm) Normalized intensity ( a.u.) Fig. 4  Compound 5 binding is an excited state phenomenon. a The changes in compound 5 fluorescence excitation spectra upon addition of BSA. b Normalized absorption (blue) and fluorescence excitation (yellow) spectra of compound 5 overlapped with fluorescence excitation spectrum (green) in presence of BSA 0 µM [BSA ] 5.5 µM a 250 300 350 400 450 0 2 4 6 8 Wavelength ( nm) Fluorescence intensity ( a.u.) Fig. 4  Compound 5 binding is an excited state phenomenon. a The changes in compound 5 fluorescence excitation spectra upon addition of BSA. b Normalized absorption (blue) and fluorescence excitation (yellow) spectra of compound 5 overlapped with fluorescence excitation spectrum (green) in presence of BSA Page 9 of 13 Pal et al. SpringerPlus (2015) 4:548 10 °C 80 °C a 350 400 450 500 550 600 0.0 0.2 0.4 0.6 0.8 1.0 Wavelength ( nm) Fluorescence intensity ( a.u.) 0 M [Urea ] 8 M b 350 400 450 500 550 600 0.0 0.2 0.4 0.6 0.8 1.0 Wavelength ( nm) Fluorescence intensity ( a.u.) Fig. 5  Compound 5 interaction with denaturing proteins. a The changes in fluorescence of BSA compound 5 complex with the increasing temperature. a  The data represent mean ± SD Molecular modeling In silico molecular docking calculation shows that the interactions of the compound with serum albumins are thermodynamically favorable (Table 4). The binding free ener- gies computed by AutoDock Vina and SwissDock are very similar to that of the experi- mentally obtained values (Table 2). Molecular docking also provides the insight into the most favorable binding site for these compounds on the serum albumins. The lowest energy complexes obtained by the three different algorithms consistently showed that Table 3  Molecular properties of compound 5 a  The data represent mean ± SD Molecular properties Values logPa 4.56 ± 0.62 logS −5.97 Polar surface area 94.17 Å2 Lipinski’s rule of five Yes Table 3  Molecular properties of compound 5 Molecular properties Page 10 of 13 Pal et al. SpringerPlus (2015) 4:548 Table 4  Theoretical binding free energies as obtained by molecular docking experiments using three different algorithms, AutoDock 4.2, AutoDock Vina and SwissDock a  The data represent mean ± SEM Protein AutoDock 4.2 (kJ mol−1)a AutoDock Vina (kJ mol−1) SwissDock (kJ mol−1) BSA −17.08 ± 0.35 −33.05 −36.15 HSA −20.78 ± 0.44 −35.56 −35.52 Table 4  Theoretical binding free energies as obtained by molecular docking experiments using three different algorithms, AutoDock 4.2, AutoDock Vina and SwissDock the binding sites for compound 5 lay in the groove between domain I and domain III of BSA, whereas it was within the domain I in case of HSA (Fig. 6, Additional file 1: Figure S5). This may, in part, explain the enthalpy driven nature of binding with HSA and the entropy driven binding with BSA (Table 2). Moreover, the non-specific nature of the binding is apparent from the lack of clustering in the AutoDock 4.2 results (Additional file 1: Figure S6). We have shown in earlier works that the low energy high frequency clusters in the AutoDock 4.2 output signifies specificity in the binding inter- actions (Alam et al. 2012; Rudra et al. 2012; Bhowmik et al. 2013). Serum albumin with its many hydrophobic binding pockets acts like a universal receptor for almost all drug molecules. Binding to serum albumin is generally non-specific in nature and driven by mainly hydrophobic interactions, which is evident in the molecular docking results as well (Additional file 1: Figures S7, S8). i Detailed interaction diagrams of the protein–ligand complexes showing the interact- ing residues and the types of interactions obtained by three different docking programs (AutoDock 4.2, AutoDock Vina and SwissDock) are given in Additional file 1 (Figures S7, S8). The consensus of the interacting residues of BSA and HSA with compound 5 are produced from those interacting diagrams depicted in Additional file 1: Figures S7, S8 and is shown in Fig. 7. We have found that Lys114 and His145 of BSA forms H-Bond Fig. 6  Interaction of compound 5 with serum albumins as obtained from molecular docking experiments. a Best binding conformation of compound 5 with BSA and the close up view. b Best binding conformations of compound 5 with HSA; it is also shown in close up. AutoDock Vina generated complexes are depicted here. Proteins are shown in ribbon diagram and the ligands in stick model. Molecular properties The three domains of serum albumin are marked with I–III. Standard color representation is used to denote the elements, H, N and O in the ligand Fig. 6  Interaction of compound 5 with serum albumins as obtained from molecular docking experiments. a Best binding conformation of compound 5 with BSA and the close up view. b Best binding conformations of compound 5 with HSA; it is also shown in close up. AutoDock Vina generated complexes are depicted here. Proteins are shown in ribbon diagram and the ligands in stick model. The three domains of serum albumin are marked with I–III. Standard color representation is used to denote the elements, H, N and O in the ligand Fig. 6  Interaction of compound 5 with serum albumins as obtained from molecular docking experiments. a Best binding conformation of compound 5 with BSA and the close up view. b Best binding conformations of compound 5 with HSA; it is also shown in close up. AutoDock Vina generated complexes are depicted here. Proteins are shown in ribbon diagram and the ligands in stick model. The three domains of serum albumin are marked with I–III. Standard color representation is used to denote the elements, H, N and O in the ligand Page 11 of 13 Pal et al. SpringerPlus (2015) 4:548 a b Fig. 7  Detailed interaction diagram of compound 5 with serum albumins as obtained by molecular docking experiments. Only the residues common in all three lowest energy complexes generated by AutoDock 4.2, AutoDock Vina and SwissDock are shown. a Interacting residues of BSA and the types of interactions with compound 5. b Interacting residues of HSA and the types of interactions with compound 5 a a b Fig. 7  Detailed interaction diagram of compound 5 with serum albumins as obtained by molecular docking experiments. Only the residues common in all three lowest energy complexes generated by AutoDock 4.2, AutoDock Vina and SwissDock are shown. a Interacting residues of BSA and the types of interactions with compound 5. b Interacting residues of HSA and the types of interactions with compound 5 b Fig. 7  Detailed interaction diagram of compound 5 with serum albumins as obtained by molecular docking experiments. Only the residues common in all three lowest energy complexes generated by AutoDock 4.2, AutoDock Vina and SwissDock are shown. a Interacting residues of BSA and the types of interactions with compound 5. Conclusion We have reported here, the spectroscopic behavior and binding parameters of a novel synthetic fluorophore in aqueous buffer and in the presence of albumin proteins. The compound showed drug like properties and bound to serum albumin with the binding constants in low micromolar concentration range. Moreover, the compound showed some interesting properties that could be used to probe the microenvironment, which reflect the immediate surroundings of the molecule inside the target proteins. The com- pound binding to the hydrophobic sites of serum albumin significantly increased its fluorescence quantum yield, caused significant decrease in the Stokes’ shift indicating changes of excited state geometry of the molecule inside protein binding pocket. We observed an overall effect of the microenvironment of the binding site on the fluoro- phore. Whether it was hydrophobicity alone or some other factor such as polarity or charge or a combination of all and how those environmental variables correlate with the fluorescence property of the molecule requires further elaborate experiment to understand. In the future experiment we plan to study the fluorescence properties of this molecule in different solution condition, micellar and liposomal or membranous environments. Acknowledgements Uttam Pal thanks INSPIRE Fellowship Programme, Department of Science and Technology, Government of India, India for financial support. Sumit Kumar Pramanik thanks CSIR-IICB, India for financial support. We also thank CSIR for financial support through Genesis (BSC 0121) and miND (BSC 0115) projects. Additional file Additional file 1. Detailed procedure of compound 5 synthesis and the characteristic data. 1H and 13C NMR spect of compound 5 are given in Additional file. It also contains Figures S1–S8. Author details 1 1 Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata, West Bengal, India. 2 Chemistry Division, Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata, West Bengal, India. Authors’ contributions C i d d d i d Conceived and designed the experiments: UP, SKP and NCM. Performed the experiments: UP, SKP and Baisali Bhattacha- rya. Analyzed the data: UP, SKP, Baisali Bhattacharya, Biswadip Banerji and NCM. Contributed reagents/materials/analysis tools: Biswadip Banerji and NCM. Wrote the paper: UP, SKP, Baisali Bhattacharya, Biswadip Banerji and NCM. All authors read and approved the final manuscript. Molecular properties b Interacting residues of HSA and the types of interactions with compound 5 with compound 5, whereas, Arg458 forms pi-cation interaction. Other important inter- acting residues of BSA are Arg144, Ser192, Pro110 and Leu189. In case of HSA, it is found that Arg117 and Tyr138 forms H-bond, whereas, Arg186 and Tyr161 forms pi- stacking with compound 5. Other important interacting residues of HSA are Leu115, Phe157, Leu182, Leu185 and Gly189. Page 12 of 13 Pal et al. SpringerPlus (2015) 4:548 Competing interests g The authors declare that they have no competing interests. Received: 3 June 2015 Accepted: 10 September 2015 Additional file 1. Detailed procedure of compound 5 synthesis and the characteristic data. 1H and 13C NMR spectra of compound 5 are given in Additional file. It also contains Figures S1–S8. Alam A, Haldar S, Thulasiram HV, Kumar R, Goyal M, Iqbal MS, Pal C, Dey S, Bindu S, Sarkar S et al (2012) Novel anti-inflam- matory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor. J Biol Chem 287:24844–24861 References Abou-Zied OK, Al-Shini OIK (2008) Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes. J Am Chem Soc 130(32):10793–10801 Alam A, Pal C, Goyal M, Kundu MK, Kumar R, Iqbal MS, Dey S, Bindu S, Sarkar S, Pal U et al (2011) Synthesis and bio-evalu- ation of human macrophage migration inhibitory factor inhibitor to develop anti-inflammatory agent. Bioorg Med Chem 19:7365–7373 Alam A, Haldar S, Thulasiram HV, Kumar R, Goyal M, Iqbal MS, Pal C, Dey S, Bindu S, Sarkar S et al (2012) Novel anti-inflam- matory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor. J Biol Chem 287:24844–24861 References Abou-Zied OK, Al-Shini OIK (2008) Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes. J Am Chem Soc 130(32):10793–10801 Alam A, Pal C, Goyal M, Kundu MK, Kumar R, Iqbal MS, Dey S, Bindu S, Sarkar S, Pal U et al (2011) Synthesis and bio-evalu- ation of human macrophage migration inhibitory factor inhibitor to develop anti-inflammatory agent. Bioorg Med Chem 19:7365–7373 Alam A, Haldar S, Thulasiram HV, Kumar R, Goyal M, Iqbal MS, Pal C, Dey S, Bindu S, Sarkar S et al (2012) Novel anti-inflam- matory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor. J Biol Chem 287:24844–24861 Page 13 of 13 Pal et al. SpringerPlus (2015) 4:548 Banerjee M, Pal U, Subudhhi A, Chakrabarti A, Basu S (2012) Interaction of Merocyanine 540 with serum albumins: photo- physical and binding studies. J Photochem Photobiol B 108:23–33 Banerjee M, Pal U, Subudhhi A, Chakrabarti A, Basu S (2012) Interac physical and binding studies. J Photochem Photobiol B 108:2 Banerji B, Pramanik SK, Pal U, Maiti NC (2013) Potent anticancer activity of cystine-based dipeptides and their interaction with serum albumins. Chem Cent J 7:91 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242 Bhattacharya AA, Curry S, Franks NP (2000) Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures. J Biol Chem 275:38731–38738 Bhowmik A, Das N, Pal U, Mandal M, Bhattacharya S, Sarkar M, Jaisankar P, Maiti NC, Ghosh MK (2013) 2,2′-Diphenyl-3,3′- diindolylmethane: a potent compound induces apoptosis in breast cancer cells by inhibiting EGFR pathway. PLoS One 8:e59798 Bickerton GR, Paolini GV, Besnard J, Muresan S, Hopkins AL (2012) Quantifying the chemical beauty of drugs. Nat Chem 4:90–98 Cohen BE, McAnaney TB, Park ES, Jan YN, Boxer SG, Jan LY (2002) Probing protein electrostatics with a synthetic fluores- cent amino acid. Science 296:1700–1703 Curry S, Mandelkow H, Brick P, Franks N (1998) Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Nat Struct Biol 5:827–835 asymmetric distribution of binding sites. Nat Struct Biol 5:827–835 reveals an asymmetric distribution of binding sites. References Nat Struct B Er JC, Vendrell M, Tang MK, Zhai D, Chang YT (2013) Fluorescent dye cocktail for multip serum albumin. ACS Comb Sci 15(9):452–457 Er JC, Vendrell M, Tang MK, Zhai D, Chang YT (2013) Fluorescent dye cocktail for multiplex drug-site mapping on human serum albumin. ACS Comb Sci 15(9):452–457 Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR (20 chemical editor, visualization, and analysis platform. J Cheminform 4:17 Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR (2012) Avogadro: An advanced semantic chemical editor, visualization, and analysis platform. 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Academic Press, San Diego, pp 492–503. doi:10.1016/S0076-6879(96)74039-9 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and computational approaches to estimate solubil- ity and permeability in drug discovery and development settings Adv Drug Deliv Rev 23:3 25 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and computational app y y y Majorek KA, Porebski PJ, Dayal A, Zimmerman MD, Jablonska K, Stewart AJ, Chruszcz M, Minor W (2012) Structural and immunologic characterization of bovine horse and rabbit serum albumins Mol Immunol 52:174 182 y y y Majorek KA, Porebski PJ, Dayal A, Zimmerman MD, Jablonska K, Stewart AJ, Chruszcz M, Minor W (2012) Structural and Majorek KA, Porebski PJ, Dayal A, Zimmerman MD, Jablonska K, Stewart AJ, Chruszcz M, Minor W (2012) Structural and immunologic characterization of bovine, horse, and rabbit serum albumins. Mol Immunol 52:174–182 immunologic characterization of bovine, horse, and rabbit serum albumins. 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Biochim Biophys Acta BBA Gen Subj 1830:5382–5393 Royer CA (2006) Probing protein folding and conformational transitions with fluorescence. Chem Rev 106(5):1769–1784 Rudra DS, Pal U, Maiti NC, Reiter RJ, Swarnakar S (2012) Melatonin inhibits matrix metalloproteinase-9 activity by binding to its active site. J Pineal Res 54(4):398–405. doi:10.1111/jpi.12034 Si d JR Z i PA H il JA C S (2006) L i f hi h d l ffi i f id bi di i h albumin: spectroscopic and docking studies. Spectrochim Acta A Mol Biomol Spectrosc 92:164–174 Reichenwallner J, Hinderberger D (2013) Using bound fatty acids to disclose the functional structure of serum albumin. Biochim Biophys Acta BBA Gen Subj 1830:5382–5393l Reichenwallner J, Hinderberger D (2013) Using bound fatty acids to disclose the functional structure of serum albumin. Biochim Biophys Acta BBA Gen Subj 1830:5382–5393 Biochim Biophys Acta BBA Gen Subj 1830:5382–5393 Royer CA (2006) Probing protein folding and conformational transitions with fluorescence. Chem Rev 106(5):1769–1784 Rudra DS, Pal U, Maiti NC, Reiter RJ, Swarnakar S (2012) Melatonin inhibits matrix metalloproteinase-9 activity by binding to its active site. J Pineal Res 54(4):398–405. doi:10.1111/jpi.12034 Simard JR, Zunszain PA, Hamilton JA, Curry S (2006) Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis. J Mol Biol 361:336–351 Tetko IV, Gasteiger J, Todeschini R, Mauri A, Livingstone D, Ertl P, Palyulin VA, Radchenko EV, Zefirov NS, Makarenko AS et al (2005) Virtual computational chemistry laboratory-design and description. J Comput Aided Mol Des 19:453–463 Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. References J Comput Chem 31:455–461 Yamasaki K, Chuang VTG, Maruyama T, Otagiri M (2013) Albumin–drug interaction and its clinical implication. Biochim Biophys Acta BBA Gen Subj 1830:5435–5443 Royer CA (2006) Probing protein folding and conformational transitions with fluorescence. Chem Rev 106(5):1769–1784 Rudra DS, Pal U, Maiti NC, Reiter RJ, Swarnakar S (2012) Melatonin inhibits matrix metalloproteinase-9 activity by binding to its active site. J Pineal Res 54(4):398–405. doi:10.1111/jpi.12034 fi Yamasaki K, Chuang VTG, Maruyama T, Otagiri M (2013) Albumin–drug interaction and its clinical implication. Biochim Biophys Acta BBA Gen Subj 1830:5435–5443
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Place cell maps slowly develop via competitive learning and conjunctive coding in the dentate gyrus
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ARTICLE 1 Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea. 2 Center for Neuroscience Imaging Research, Institute for Basic Science (IBS), Suwon 16419, Republic of Korea. 3 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea. 4 Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea. ✉email: royers@kist.re.kr Place cell maps slowly develop via competitive learning and conjunctive coding in the dentate gyrus Soyoun Kim1,2, Dajung Jung 1,3 & Sébastien Royer1,4✉ Place cells exhibit spatially selective firing fields that collectively map the continuum of positions in environments; how such activity pattern develops with experience is largely unknown. Here, we record putative granule cells (GCs) and mossy cells (MCs) from the dentate gyrus (DG) over 27 days as mice repetitively run through a sequence of objects fixed onto a treadmill belt. We observe a progressive transformation of GC spatial representations, from a sparse encoding of object locations and spatial patterns to increasingly more single, evenly dispersed place fields, while MCs show little transformation and preferentially encode object locations. A competitive learning model of the DG reproduces GC transfor- mations via the progressive integration of landmark-vector cells and spatial inputs and requires MC-mediated feedforward inhibition to evenly distribute GC representations, suggesting that GCs slowly encode conjunctions of objects and spatial information via competitive learning, while MCs help homogenize GC spatial representations. 1 Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea. 2 Center for Neuroscience Imaging Research, Institute for Basic Science (IBS), Suwon 16419, Republic of Korea. 3 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea. 4 Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea. ✉email: royers@kist.re.kr 1 NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications TURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 P r fi c competitive learning model of the DG that received inputs from EC object-vector cells, grid cells and non-grid spatial cells, and explored the mechanisms and parameters critical to reproduce the experimental data. Our findings suggest that a slow integra- tion of spatial cell and landmark-vector cell inputs, achieved via competitive learning, is the mechanism underlying both the emergence of single place field representations and the con- tinuous mapping of the space by GCs, while an increase in MC feedforward inhibition in the cue locations is required to ensure a uniform distribution of GC representations. P rincipal cells in the hippocampus exhibit spatially selective firing fields called “place fields” that collectively map the continuum of positions in environments1. How such activity patterns emerge during learning is a fundamental ques- tion. Results d fi Consistent with anatomical figures, putative GCs were located closer to the reversal point of LFP type-2 dentate spikes (DS2) above the granule cell layer, while putative MCs were relatively deeper in the positive phase of DS2, i.e., in the hilus41 (Fig. 1e); accordingly, on shanks where both putative GCs and MCs were detected, MCs were located on average 31.5 ± 1.5 µm below GCs (t824 = 20.6, p = 2e−76, two-tailed paired t test, n = 47 shanks, average over all GC-MC combinations; Fig. 1f, top). Furthermore, a group of unclassified cells showing short ACG refractory gaps similar to those of GCs and similar gamma relationships to those of MCs (n = 168; Supplementary Fig. 3b) were found on average 43.8 ± 2.7 µm below GCs (t300 = 16.2, p = 5e−43, two-tailed paired t test, n = 13 shanks; Fig. 1f, bottom), suggesting these cells might be pyramidal cells from CA3. Finally, a number of cells (n = 164) were not included in this classification because the number of spikes discharged was too A theory postulates that the place fields of GCs are generated through competitive learning18,19, that is, through the combina- tion of competition between GCs, mediated by feedback inhibi- tion, and Hebbian synaptic plasticity at the level of EC-to-GC inputs. In support of this theory, single place field representations emerge automatically in network models featuring GC competi- tion when EC synaptic weights are recursively updated by Heb- bian synaptic plasticity mechanisms18,19. Furthermore, object and spatial information is hypothesized to be integrated at the level of the GCs8,20. Inputs from both the medial (MEC) and lateral (LEC) divisions of the EC converge onto GCs5, and are largely believed to supply diverse types of spatial and non-spatial information. Given that landmark-vector cells (or object-vector cells) in the MEC21 and LEC22 encode animal spatial relation- ships with objects, that most cells in the MEC show spatial activity23, and that grid cells in MEC exhibit periodic firing fields that convey spatial information related to path integration24,25, GCs might be able to bind object and spatial information via the integration of inputs from landmark-vector cells, grid cells and non-grid spatial cells in the EC. Finally, MCs receive direct inputs from the CA3, semilunar GCs and possibly the EC26,27 and are particularly well positioned to shape GC activity via both direct and indirect connections. Results d fi Identification of putative GCs and MCs. We recorded neuronal activity during treadmill running every day for 27 days using a 6- shank silicon probe (64 channels) implanted in the DG of the right brain hemisphere (Fig. 1a, b; Supplementary Fig. 1). A total of 4003 cells were isolated (from 4 mice, 16 sessions per mouse) following standard criteria for unit detection and clustering37,38. In addition, to help identify putative GCs and MCs, we used a previous data set in which a subset of GCs and MCs expressed the Chronos opsin (via AAV/hSyn-Flex-Chronos-GFP injections in POMC-Cre and DRD2-Cre mice, respectively) and showed excitatory responses to light stimuli (18 POMC light-excited cells from 3 mice and 33 DRD2 light-excited cells from 2 mice)17. g ) To identify putative GCs and MCs, we assessed differences in cells’ spike autocorrelogram (ACG) and spike relationship with hilar local field potential gamma (30–80 Hz) oscillations (Sup- plementary Fig. 2a–d), as previously17. We measured an ‘ACG refractory gap’ (defined as the duration for the ACG to reach 75% of its peak value; Supplementary Fig. 2a), a gamma coupling index (defined as the difference in gamma power between window periods within [−10 to +10 ms] and outside [+40 to +100 ms] epochs of maximal firing activity; Supplementary Fig. 2d) and the mean spike gamma phase for each cell and examined the cell clustering and overlap with POMC/DRD2 light-excited cells and putative excitatory neurons (detected from short-latency peaks in cell-pair cross-correlograms)39. First, some cells were categorized as putative interneurons (n = 248) based on their high firing rate, short ACG refractory gap and lack of overlap with putative excitatory neurons (Supplementary Fig. 3a) and were excluded from the next analysis. Then, putative GCs (n = 2323) were characterized by short ACG refractory gaps, high gamma coupling indexes, a preference to discharge before the troughs of gamma oscillations and overlap with POMC light- excited cells (Fig. 1c, d; Supplementary Fig. 3b–e). In contrast, putative MCs (n = 408) were characterized by large ACG refractory gaps (consistent with in vivo intracellular recording data)40, low gamma coupling indexes, a preference to discharge before the peaks of gamma oscillations, and overlap with DRD2 light-excited cells. Place cell maps slowly develop via competitive learning and conjunctive coding in the dentate gyrus The granule cell (GC) population of the dentate gyrus (DG) is the first processing stage of the hippocampal trisynaptic loop; accounts for nearly half of the neurons in the mammalian hip- pocampus2; locally interacts with GABAergic interneurons and mossy cells (MCs), a small population of excitatory neurons located in the hilus (104 MCs versus 106 GCs in rodents)3–5; and is largely believed to perform pattern separation6–10 on inputs from the entorhinal cortex (EC) via the generation of sparse-orthogonal output patterns11–15 and to assist memory formation in the CA3 via powerful GC-to-CA3 synapses4,5,7,8,16. Recently, several methods were developed to segregate and assess the spatial representations of specific DG cell types, leading to reports of clear differences between GCs and MCs in terms of the scale, sparseness, stability and remapping of spatial representa- tions11–15,17. An emerging picture is that the GCs generate spatial representations that are relatively stable over time and consist predominantly of a small and unique place field, whereas the MCs generate several large place fields that are strongly altered by small changes in the environments; moreover, the GCs efficiently differentiate the environments via the recruitment of small context-specific cell ensembles, whereas the MCs engage large cell ensembles that largely overlap across contexts11–15,17. However, the fundamental question on how these diverse spatial repre- sentations develop during the learning of an environment remain largely untested. NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications Results d fi f Depth difference (µm) ( GC – MC ) % of cell pairs –110 0 110 0 10 20 P=2×10 – 76 Depth difference (µm) ( GC – Unclassifed ) –110 0 110 P=5×10 – 43 0 10 20 10 ms % of cell pairs f Depth difference (µm) ( GC MC ) % of cell pairs –110 0 110 0 10 20 P=2×10 – 76 f d e 50 100 MC GC 5 10 ms 1 0 ( GC MC ) Depth difference (µm) ( GC – Unclassifed ) –110 0 110 P=5×10 – 43 0 10 20 % of cell pairs Fig. 1 Recording of putative GCs and MCs. a Left, 3D representation of the mouse brain (Allen Mouse Brain Institute; www.alleninstitute.org) showing recording electrode configuration in the dentate gyrus (light green). Dark green, cornus ammonis (CA). Right, the electrode positions (ellipsoid) relative to the lateral/medial edges of the granule cell layer (dashed lines) for all mice. b Left, scheme showing the location of the hilus and granule cell layer on a coronal section of the hippocampus. Right, layout of recording sites for a shank of the silicon probe and profile of local field potential dentate spike 2 (LFP DS2). Red arrow, position of DS2 reversal. c 3D scatter plot for cells’ spike gamma phase, ACG refractory gap and gamma coupling index. Putative MCs (orange dots) and GCs (light blue dots) are identified by overlap with DRD2 (red filled circle) and POMC (blue circle) light-excited cells. d Spike ACGs (upper, color-coded representation of individual cell; lower, population average) for putative GCs (blue), MCs (red) and inhibitory cells (green). e Layout of LFP DS2 (upper) and putative GCs and MCs (lower) along the silicon probe shanks for one session. f Top, distribution of depth differences for all possible pairs of putative GCs and MCs recorded concurrently on the same shanks (n = 825 pairs). Bottom, same analysis between GCs and the population of unclassified cells in panel (c) (n = 301 pairs). Arrows, the means. P-values, two-tailed paired t test. low (<50 spikes) to reliably measure ACG refractory gaps and gamma phases. low (<50 spikes) to reliably measure ACG refractory gaps and gamma phases. through a lick port on every trial (belt cycle) at the same belt position (position 0 cm). We distinguished four types of place field activity among GCs (Fig. Results d fi In particular, MC-to-GC feedforward inhibition27–33 is expected to affect GC competition and thus competitive learning in the GC network. To investigate the development of DG spatial representations during the learning of an environment, we recorded putative GCs and MCs over 27 days as mice ran head-fixed on a long treadmill enriched with visual-tactile landmarks34. Such an apparatus is particularly suited for differentiating spatial mechanisms35,36 and assessing learning effects since the animal trajectory is con- sistent across trials and the spatial information is reduced to a sequence of individual landmarks fixed on the belt and path integration. We analyzed how spatial representations evolved over days as mice learned the belt layout and, subsequently, how cells encoded other belt layouts. To test the contributions of competitive learning, EC inputs and MCs, we implemented a URE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 ACG peak time (ms) Gamma coupling index –100 –50 50 100 Autocorrelograms Lag (ms) GC MC Inh c d Gamma phase (degree ) e MC GC 5 mV 10 ms 10 20 30 40 50 0 1 0 -1 0 1 0 1 2323 408 1 248 GC/POMC MC/DRD2 LFP DS2 0 a.u. f Depth difference (µm) ( GC – MC ) % of cell pairs –110 0 110 0 10 20 P=2×10 – 76 Depth difference (µm) ( GC – Unclassifed ) Unclassifed –110 0 110 P=5×10 – 43 0 10 20 1 2 Shank position for mouse # 3 4 DG edges a 6-shank silicon probe DG CA 5 mV 10 ms LFP DS2 Mol Hilus Hilus Mol Granule cell layer CA1 CA3 b % of cell pairs 180 360 540 0 720 Recording of putative GCs and MCs. a Left, 3D representation of the mouse brain (Allen Mouse Brain Institute; www.alleninstitute.org) showing ding electrode configuration in the dentate gyrus (light green). Dark green, cornus ammonis (CA). Right, the electrode positions (ellipsoid) relative to ateral/medial edges of the granule cell layer (dashed lines) for all mice. b Left, scheme showing the location of the hilus and granule cell layer on a nal section of the hippocampus. Right, layout of recording sites for a shank of the silicon probe and profile of local field potential dentate spike 2 (LFP . Red arrow, position of DS2 reversal. c 3D scatter plot for cells’ spike gamma phase, ACG refractory gap and gamma coupling index. Results d fi Putative MCs ge dots) and GCs (light blue dots) are identified by overlap with DRD2 (red filled circle) and POMC (blue circle) light-excited cells. d Spike ACGs er, color-coded representation of individual cell; lower, population average) for putative GCs (blue), MCs (red) and inhibitory cells (green). e Layout of DS2 (upper) and putative GCs and MCs (lower) along the silicon probe shanks for one session. f Top, distribution of depth differences for all possible of putative GCs and MCs recorded concurrently on the same shanks (n = 825 pairs). Bottom, same analysis between GCs and the population of ssified cells in panel (c) (n = 301 pairs). Arrows, the means. P-values, two-tailed paired t test. ACG peak time (ms) Gamma coupling index c Gamma phase (degree ) 10 20 30 40 50 0 1 0 -1 GC/POMC MC/DRD2 Unclassifed 1 2 Shank position for mouse # 3 4 DG edges a 6-shank silicon probe DG CA 5 mV 10 ms LFP DS2 Mol Hilus Hilus Mol Granule cell layer CA1 CA3 b 180 360 540 0 720 ACG peak time (ms) Gamma coupling index c Gamma phase (degree ) 10 20 30 40 50 0 1 0 -1 GC/POMC MC/DRD2 Unclassifed 180 360 540 0 720 c 1 2 Shank position for mouse # 3 4 DG edges a 6-shank silicon probe DG CA 5 mV 10 ms LFP DS2 Mol Hilus Hilus Mol Granule cell layer CA1 CA3 b 1 2 Shank position for mouse # 3 4 DG edges a a 6-shank silicon probe DG CA 5 mV 10 ms LFP DS2 Mol Hilus Hilus Mol Granule cell layer CA1 CA3 b b –100 –50 50 100 Autocorrelograms Lag (ms) GC MC Inh d 0 1 0 1 2323 408 1 248 0 a.u. e MC GC 5 mV 10 ms LFP DS2 f Depth difference (µm) ( GC – MC ) % of cell pairs –110 0 110 0 10 20 P=2×10 – 76 Depth difference (µm) ( GC – Unclassifed ) –110 0 110 P=5×10 – 43 0 10 20 10 ms % of cell pairs –100 –50 50 100 Autocorrelograms Lag (ms) GC MC Inh d e MC GC 5 mV 10 ms 0 1 0 1 2323 408 1 248 LFP DS2 0 a.u. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 a Treadmill b Single Multiple LV Periodic Unspecific 20 50 30 40 0 Spines Tubes Velcro 0 201 Position ( cm ) 25 25 1 1 Trials Trials Hz Single Multiple LV Periodic Unspecific c 0 201 Position (cm) 0.6 0 1 Cell number a.u. 100 Mean 50 150 0 Hz Max Fig. 2 Types of place field activity among GCs. a Scheme of the treadmill showing the 3 pairs of landmarks fixed on the belt. b Individual cell examples for various types of GC representations: a single-field cell; a landmark-vector (LV) cell (showing two fields matching a type of landmark); a periodic cell (with three periodic fields); and an unspecific cell (with more than one field that are neither LV nor periodic). Top, scheme of the belt; middle, spike raster and color-coded firing rate map; bottom, mean firing rate. c Color-coded, firing rate maps of GCs from all sessions, sorted according to field positions and grouped by type of representation. Line plots, average of firing rate maps for each type of representation (lines, the mean; shadows, s.e.m; n = 178 single, 105 unspecific, 53 LV and 11 periodic cells). The color scale is the same as that used in (b). Single Multiple LV Periodic Unspecific c 0 201 Position (cm) 0.6 0 1 Cell number a.u. 100 Mean 50 150 z c a a Treadmill b Single Multiple LV Periodic Unspecific 20 50 30 40 0 Spines Tubes Velcro 0 201 Position ( cm ) 25 25 1 1 Trials Trials Hz 0 Hz Max b Position (cm) Fig. 2 Types of place field activity among GCs. a Scheme of the treadmill showing the 3 pairs of landmarks fixed on the belt. b Individual cell examples for various types of GC representations: a single-field cell; a landmark-vector (LV) cell (showing two fields matching a type of landmark); a periodic cell (with three periodic fields); and an unspecific cell (with more than one field that are neither LV nor periodic). Top, scheme of the belt; middle, spike raster and color-coded firing rate map; bottom, mean firing rate. c Color-coded, firing rate maps of GCs from all sessions, sorted according to field positions and grouped by type of representation. Results d fi 2b, c): (1) single place field cells; (2) unspecific cells, which exhibited several firing fields with no apparent periodicity or relationship with landmarks; (3) landmark-vector (LV) cells, which, similar to LV cells in the EC and CA1 (refs. 21,35), encoded spatial relationships with landmarks; and (4) periodic cells, which exhibited three periodic firing fields with similar periodicity but various offsets. Progressive transformation of GC firing fields over days. To investigate place cell activity during familiarization with the belt layout, mice were trained to run head-fixed for a water reward on an empty 150-cm-long belt for a week and then were introduced to a 201-cm-long belt displaying visual-tactile landmarks (Fig. 2a; Supplementary Fig. 4). The landmarks consisted of 5-cm-long arrays of small erect objects that lined both edges of the belt and provided visual-tactile stimulation to both sides of the mice. We used three types of landmarks: an array of shrink tubes, an array of Velcro pieces, and an array of glue spines. To detect cell activity associated with a given landmark, each landmark was fixed to two locations of the belt. A water reward was delivered GC representations gradually transformed across days (Fig. 3a–c). On day 1, a few GCs exhibited place fields (1.6 ± 0.5% single and 5.8 ± 2.2% multiple-field cells (n = 14 GCs)) and were mostly LV cells and periodic cells (Fig. 3b, c; among multiple-field cells, 43.3 ± 19.0% LV, 44.4 ± 22.2% periodic and 12.2 ± 6.2% unspecific cells). Then, the fraction of GCs with place TURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 Line plots, average of firing rate maps for each type of representation (lines, the mean; shadows, s.e.m; n = 178 single, 105 unspecific, 53 LV and 11 periodic cells). The color scale is the same as that used in (b). c Day 1 2 3 5 7 10 13 16 20 10 13 16 20 % of cells Day Multiple Single 1 2 3 5 7 a GCs with place fields 0 201 Position ( cm ) 0 10 20 0 201 cm LV Periodic Unspecific 10 20 1 0 1 Day LV peak ratio (a/b) 0 40 80 r = –0.5 % of multiple d 0 15 Hz 0 201 cm a b P = 10–4 Day 1 Day 13–20 b Single Unspecific LV Periodic 0 Hz Max Fig. 3 Progressive transformation of GC firing fields across days. a Color-coded, firing rate maps of GCs across days. Only the GCs showing firing fields are displayed for clarity. The rows of the matrices correspond to individual GCs and are sorted according to firing field positions. Top, scheme of the belt. b Color-coded, firing rate maps of GCs on day 1 (left) and on days 13, 16, and 20 combined (right), for each type of GC firing field. The color scale is the same as that used in (a). c Upper, proportion of GCs with a single field (black) and multiple fields (gray) across days. Lower, proportion of LV (light blue), periodic (dark blue) and unspecific GCs (red), among multiple-field GCs, across days. d Upper, definition of LV peak ratio as the ratio between LV fields’ peak firing rates (smaller peak over larger peak). Lower, distribution of LV peak ratios across days. Each dot is the LV peak ratio of one LV cell. Red line, linear fit. n = 53, r = −0.5, p = 1e−4, Pearson’s correlation, two-tailed t test. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 4c; change in firing rate from trial −50 to −1 before extinction, r = −0.75, p = 5e−10, Pearson’s correlation) and mostly occurred late in the sessions (Fig. 4d; percent of events before versus after trial 30, 20.8 ± 8.9% versus 79.2 ± 8.9%, t15 = −3.3, p = 5e−3, two- tailed paired t test). fields progressively increased via an increase in unspecific and single-field GCs, reaching a plateau after 5 and 10 days, respectively, while the fraction of LV cells and periodic cells decreased (Fig. 3b, c; for days 13–20, 20.2 ± 3.3% single and 11.8 ± 1.6 multiple-field cells (n = 41 GCs); among multiple- field cells, 21.0 ± 5.3% LV, 1.7 ± 1.7% periodic and 77.4 ± 5.6% unspecific cells). Moreover, the peak firing rates of the two fields of LV cells became increasingly uneven (Fig. 3d; n = 53, r = −0.5, p = 1e−4, Pearson’s correlation; peak rate ratio on day 1 versus days 13–20, 81.8 ± 5.8% (n = 6 LV cells) versus 36.8 ± 5.3% (n = 10 LV cells), t14 = 5.5, p = 8e−5, two-tailed unpaired t test). Importantly, emergence and extinction rates changed across days, which was consistent with the gradual transformation of GC representations. While both emergence and extinction rates decreased across days (Fig. 4e; F8,42 = 3.7, p = 2e−3, two-way ANOVA, emergence rate across days 1–7 versus days 10–20, 43.1 ± 6.6% versus 19.9 ± 5.1%, t24 = 2.8, p = 0.01, two-tailed unpaired t test; extinction rate, 34.6 ± 5.6% versus 21.5 ± 3.5%, t24 = 2.0, p = 0.06, two-tailed unpaired t test), the emergence rate was initially higher than the extinction rate and reached an equivalent level after 7 days (emergence versus extinction, days 1–7, t4 = 5.0, p = 7e−3; days 10–20, t3 = −0.26, p = 0.81, two- tailed paired t test), matching the increase in and stabilization of Emergence and extinction of firing fields within sessions. The increase of single-field representations and the reduced propor- tion of LV and periodic cells implies that new place fields emerged and that existing place fields became extinct. Both place field emergence and extinction events could be observed within sessions (see Methods, Fig. 4a) and produced preferentially incremental changes in the number of firing fields in each cell (Fig. 4b, F1,48 = 4.5, p = 0.04, two-way ANOVA). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 Place field emergences were characterized by a steep rise of the in-field rate 100 0 201 0 30 10 20 20 100 1 1 Trials Trials Hz 40 30 90 0 90 1 1 none–>single multi–>single none–>multi single–>multi a b single–>none multi–>none f Position ( cm ) 0 5 15 % of cells 10 none–>single single–>none none–>multi multi–>none multi–>single single–>multi Field emergence Field extinction Day 1–7 1 60 Trials Emerging fields Extinguishing fields 1 60 Trials 0.3 0.6 60 40 20 0 Trial number Firing rate (a.u.) Day 1–7 Day 10–20 Day Rate (%) 10 7 2 1 3 5 131620 Emergence Extinction 0 100 50 241 161 Place field number 1 1 0 4 40–45 30–35 10–15 Trial number 20–25 Rate (%) e c d 0 0.6 50 0 –50 Trials Emergence Firing rate (a.u.) Extinction g Average in-field rate Day 10–20 In-field rate Emergence Extinction Emergence Extinction 0 Hz Max 4 Emergence and extinction of firing fields within sessions. a Individual cell examples for field emergences and field extinctions within a session for converting between no field, single-field and multiple-field conditions. Top, scheme of the belt; middle, spike raster plot and color-coded firing rate ; bottom, mean firing rate. b Proportion of GC conversion types for field emergences (black, n = 41 cells) and field extinctions (salmon, n = 21 cells). ortions are relative to the number of GCs with place fields. c Average in-field firing rate (lines, the mean; shadow, s.e.m) for field emergences (black, 25 cells) and field extinctions (salmon, n = 88 cells), after aligning to emergence (or extinction) trials of in-field firing rate vectors. Note that field rgence is relatively instantaneous, as the in-field firing rate reaches an immediate plateau, while field extinction is preceded by a gradual decrease in n-field firing rate. d Proportion of field emergence (black, n = 53 cells) and field extinction (salmon, n = 30 cells) events as a function of session trials. oportion of field emergence (black, n = 125 cells) and field extinction (salmon, n = 88 cells) events as a function of days. f Color-coded representation -field firing rate vectors for all place fields on days 1–7 (left) and days 10–20 (right). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 URE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 5 100 0 201 0 30 10 20 20 100 1 1 Trials Trials Hz 40 30 90 0 90 1 1 none–>single multi–>single none–>multi single–>multi a single–>none multi–>none Position ( cm ) Field emergence Field extinction 0 Hz Max b 0 5 15 % of cells 10 none–>single single–>none none–>multi multi–>none multi–>single single–>multi Emergence Extinction b a % of cells c 0 0.6 50 0 –50 Trials Emergence Firing rate (a.u.) Extinction c Firing rate (a.u.) c 100 0 201 0 30 10 20 20 Hz 40 30 90 0 f Position ( cm ) Day 1–7 1 60 Trials Emerging fields Extinguishing fields 1 60 Trials 0.3 0.6 60 40 20 0 Trial number Firing rate (a.u.) Day 1–7 Day 10–2 Day Rate (%) 10 7 2 1 3 5 131620 0 100 50 241 161 Place field number 1 1 0 4 40–45 30–35 10–15 Trial number 20–25 Rate (%) e d 0 50 0 –50 Trials Firing rate (a g Average in-field rate Day 10–20 In-field rate Emergence Extinction Emergence Extinction 0 Hz f Day 1–7 1 60 Trials Emerging fields Extinguishing fields 1 60 Trials Fi i ( ) Day Rate (%) 10 7 2 1 3 5 131620 0 100 50 241 161 Place field number 1 1 0 4 40–45 30–35 10–15 Trial number 20–25 Rate (%) e d g Day 10–20 In-field rate Emergence Extinction Emergence Extinction f Day 1–7 1 60 Trials Emerging fields Extinguishing fields 1 60 Trials 241 161 Place field number 1 1 Day 10–20 In-field rate 0 4 40–45 30–35 10–15 Trial number 20–25 Rate (%) d Emergence Extinction f d 0.3 0.6 60 40 20 0 Trial number Firing rate (a.u.) Day 1–7 Day 10–20 g Average in-field rate g Extinguishing fields 1 60 Trials Firing rate (a Day Rate (%) 10 7 2 1 3 5 131620 0 100 50 241 161 Place field number 1 1 Trial number e Emergence Extinction Day Rate (%) 10 7 2 1 3 5 131620 0 100 50 Trial number e Emergence Extinction Fig. 4 Emergence and extinction of firing fields within sessions. a Individual cell examples for field emergences and field extinctions within a session for GCs converting between no field, single-field and multiple-field conditions. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 Day 1 2 3 5 7 10 13 16 20 a GCs with place fields 0 201 cm 0 Hz Max a 5 3 c % of cells Multiple Single 0 10 20 d d 0 15 Hz 0 201 cm a b 0 201 Position ( cm ) Day 1 Day 13–20 b Single Unspecific LV Periodic d b 10 13 16 20 Day 1 2 3 5 7 0 LV Periodic Unspecific 10 20 1 0 1 Day LV peak ratio (a/b) 0 40 80 r = –0.5 % of multiple P = 10–4 10 20 1 0 1 Day LV peak ratio (a/b) r = –0.5 P = 10–4 10 13 16 20 Day 1 2 3 5 7 LV Periodic Unspecific 0 40 80 % of multiple Fig. 3 Progressive transformation of GC firing fields across days. a Color-coded, firing rate maps of GCs across days. Only the GCs showing firing fields are displayed for clarity. The rows of the matrices correspond to individual GCs and are sorted according to firing field positions. Top, scheme of the belt. b Color-coded, firing rate maps of GCs on day 1 (left) and on days 13, 16, and 20 combined (right), for each type of GC firing field. The color scale is the same as that used in (a). c Upper, proportion of GCs with a single field (black) and multiple fields (gray) across days. Lower, proportion of LV (light blue), periodic (dark blue) and unspecific GCs (red), among multiple-field GCs, across days. d Upper, definition of LV peak ratio as the ratio between LV fields’ peak firing rates (smaller peak over larger peak). Lower, distribution of LV peak ratios across days. Each dot is the LV peak ratio of one LV cell. Red line, linear fit. n = 53, r = −0.5, p = 1e−4, Pearson’s correlation, two-tailed t test. NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 to a plateau value (Fig. 4c) and occurred preferentially at the beginning of the sessions (Fig. 4d; percent of events before versus after trial 30, 75.6 ± 10.2% versus 24.4 ± 10.2%, t15 = 2.5, p = 0.02, two-tailed paired t test), while place field extinctions were pre- ceded by gradual decreases in the in-field firing rate (Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 In-field firing rate vectors are sorted to emphasize field rgences (upper, sorted by trial number for which cumulative sums >20% of vector integrals) and field extinctions (lower, using the same sorting hod in the reversed direction). The color scale is the same as that used in (a). g Average in-field firing rate (lines, the mean; shadow, s.e.m) over trials days 1–7 (blue, n = 241 place fields) and days 10–20 (yellow, n = 161 place fields). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 To test the dependence of place cell activity on belt features, after day 21, we recorded the same neurons across three consecutive sessions using three distinct belt layouts: the original belt layout; a reor- dered belt, presenting the same landmarks as the original belt but in a rearranged order; and a novel belt, which was a different length (211 cm) and presented a new set of landmarks (Fig. 5a). Furthermore, a relationship was apparent between the number of place fields and the number of belts represented by each cell (Fig. 5d). Cells exhibiting multiple place fields tended to show place fields for several belts, while cells exhibiting single place fields tended to be active in only one belt (average number of belts represented by multiple-field cells, 2.06 ± 0.11 (n = 50, days 22–24), 2.02 ± 0.12 (n = 41, days 25–27), and by single-field cells, 1.38 ± 0.09 (n = 48, day 22–24), 1.30 ± 0.10 (n = 44, day 25–27); t96 = 4.7, p = 9e−6, t83 = 4.8, p = 6e−6, for days 22–24 and 25–27 respectively, two-tailed unpaired t test). Consistent with the idea that an engram specific to the layout of the original belt was created, GCs exhibiting single place fields were less frequent in sessions using the reordered and novel belts than in sessions using the original belt (Fig. 5b; original belt, 15.4 ± 2.3%, 15.8 ± 1.6%; reordered belt, 10.6 ± 2.3%, 12.7 ± 3.3%; novel belt, 8.1 ± 2.5%, 6.7 ± 2.2% for days 22–24 and 25–27 respectively; F2,68 = 6.0, p = 4e−3, two-way ANOVA; original vs. reordered, t23 = 1.7, p = 0.10; original vs. novel, t23 = 5.0, p = 4e−5, ad hoc two-tailed paired t test; across days 22–27); for LV cells, the magnitude of the two firing fields was more similar in sessions using the reordered and novel belts than in sessions using the original belt (Fig. 5c; peak rate ratio, original belt, 0.50 ± 0.11 (n = 8, days 22–24), 0.53 ± 0.13 (n = 3, days 25–27); reordered belt, 0.74 ± 0.08 (n = 6, days 22–24), 0.75 ± 0.07 (n = 8, Representations of MCs and CA3 cells. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 We distinguished three types of firing activity patterns among MCs: (1) cells with a single place field, (2) cells with multiple place fields, and (3) cells with relatively high firing rates (i.e., a mean firing rate >3 Hz) but low spatial modulation (Fig. 6a, see Methods). In contrast to GCs, the fraction of MCs showing firing activity on the belt was initially high (70.6 ± 10.7% of MCs compared to 7.3 ± 2.7% of GCs on day 1; t3 = 6.4, p = 7e−3, two-tailed paired t test) and did not change Number of active belts 1 2 3 % of cells 0 Multiple Single Original Reordered Novel a b c d LV peak ratio 5 3 1 Number of fields 40 80 Cell number 1 202 0 201 0 201 0 211 Position ( cm ) Original Reordered Novel 1 0.5 0 % of cells 0 10 20 5 3 1 Original Reordered Novel Number of fields Original Reordered Novel Original Reordered Novel Day 22–24 Day 25–27 0 Hz Max Fig. 5 GC encoding of other belt layouts. a Firing rate maps of GCs for the original (left), reordered (center) and novel (right) belts. Top, scheme of the belt layouts. Color-coded, each row represents the firing rate maps of a GC for the 3 belts, normalized by the peak firing rate of the cell across all belts. Only GCs exhibiting place fields in at least one of the belts are displayed. GCs are sorted according to the type of belt and the belt position of the largest firing fields. b Fraction of GCs with single (black) and multiple (gray) place fields for each belt, during early (days 22–24, left; n = 67 single and 50 multiple- field cells) and late (days 25–27, right; n = 62 single and 41 multiple-field cells) periods on the new belt layouts. c LV peak ratio (circle, individual cell; bar, the mean; error bar, s.e.m.) of LV cells for the 3 belts, during early (left, n = 18 LV cells) and late (right, n = 13 LV cells) periods on the new belt layouts. Note that the magnitude of the two firing fields is more similar in the reordered and novel belts than in the original belt. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 Top, scheme of the belt; middle, spike raster plot and color-coded firing rate map; bottom, mean firing rate. b Proportion of GC conversion types for field emergences (black, n = 41 cells) and field extinctions (salmon, n = 21 cells). Proportions are relative to the number of GCs with place fields. c Average in-field firing rate (lines, the mean; shadow, s.e.m) for field emergences (black, n = 125 cells) and field extinctions (salmon, n = 88 cells), after aligning to emergence (or extinction) trials of in-field firing rate vectors. Note that field emergence is relatively instantaneous, as the in-field firing rate reaches an immediate plateau, while field extinction is preceded by a gradual decrease in the in-field firing rate. d Proportion of field emergence (black, n = 53 cells) and field extinction (salmon, n = 30 cells) events as a function of session trials. e Proportion of field emergence (black, n = 125 cells) and field extinction (salmon, n = 88 cells) events as a function of days. f Color-coded representation of in-field firing rate vectors for all place fields on days 1–7 (left) and days 10–20 (right). In-field firing rate vectors are sorted to emphasize field emergences (upper, sorted by trial number for which cumulative sums >20% of vector integrals) and field extinctions (lower, using the same sorting method in the reversed direction). The color scale is the same as that used in (a). g Average in-field firing rate (lines, the mean; shadow, s.e.m) over trials for days 1–7 (blue, n = 241 place fields) and days 10–20 (yellow, n = 161 place fields). 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 place cells observed in Fig. 3c. This effect was also observable in the matrix concatenation of in-field firing rates for all GC place fields, sorted by time of field emergence or extinction (Fig. 4f) and was also revealed by distinct profiles of average in-field rate for days 1–7 and 10–20 (Fig. 4g). days 25–27); novel belt, 0.69 ± 0.07 (n = 6, days 22–24), 0.74 ± 0.11 (n = 3, days 25–27); F2,30 = 3.5, p = 0.04, two-way ANOVA; original vs. reordered, t23 = 2.5, p = 0.02, original vs. novel, t18 = 1.8, p = 0.08, ad hoc two-tailed unpaired t test; average across days 22–27). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 However, the fraction of single-field cells was higher in sessions using the reordered belt than in those using the new belt (t23 = 2.1, p = 0.04, ad hoc two-tailed paired t test), suggesting that the engram may have helped place field generation for other belts according to the degree of belt similarity. Changing the belt. The gradual transformation of GC repre- sentations might be associated with the development of an engram specific to the particular features of the belt. To test the dependence of place cell activity on belt features, after day 21, we recorded the same neurons across three consecutive sessions using three distinct belt layouts: the original belt layout; a reor- dered belt, presenting the same landmarks as the original belt but in a rearranged order; and a novel belt, which was a different length (211 cm) and presented a new set of landmarks (Fig. 5a). Consistent with the idea that an engram specific to the layout of the original belt was created, GCs exhibiting single place fields were less frequent in sessions using the reordered and novel belts than in sessions using the original belt (Fig. 5b; original belt, 15.4 ± 2.3%, 15.8 ± 1.6%; reordered belt, 10.6 ± 2.3%, 12.7 ± 3.3%; novel belt, 8.1 ± 2.5%, 6.7 ± 2.2% for days 22–24 and 25–27 respectively; F2,68 = 6.0, p = 4e−3, two-way ANOVA; original vs. reordered, t23 = 1.7, p = 0.10; original vs. novel, t23 = 5.0, p = 4e−5, ad hoc two-tailed paired t test; across days 22–27); for LV cells, the magnitude of the two firing fields was more similar in sessions using the reordered and novel belts than in sessions using the original belt (Fig. 5c; peak rate ratio, original belt, 0.50 ± 0.11 (n = 8, days 22–24), 0.53 ± 0.13 (n = 3, days 25–27); reordered belt, 0.74 ± 0.08 (n = 6, days 22–24), 0.75 ± 0.07 (n = 8, Changing the belt. The gradual transformation of GC repre- sentations might be associated with the development of an engram specific to the particular features of the belt. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 d For the groups of GCs that are active in 1 (black), 2 (green), and 3 (purple) belts, the distribution of the number of fields per cell (in the belt with the largest number of fields), during early (left) and late (right) periods on the new belt layouts. Note that cells exhibiting multiple place fields tend to represent several belts, while cells exhibiting a single place field tend to represent only one belt, t96 = 4.7, p = 9e−6 (days 22–24), t83 = 4.8, p = 6e−6 (days 25–27), two-tailed unpaired t test). Multiple Single b % of cells 0 10 20 Original Reordered Novel Original Reordered Novel Day 22–24 Day 25–27 b Original Reordered Novel a Cell number 1 202 0 201 0 201 0 211 Position ( cm ) 0 Hz Max Number of active belts 1 2 3 % of cells 0 Multiple Single Original Reordered Novel a b c d LV peak ratio 5 3 1 Number of fields 40 80 Cell number 1 202 0 201 0 201 0 211 Position ( cm ) Original Reordered Novel 1 0.5 0 % of cells 0 10 20 5 3 1 Original Reordered Novel Number of fields Original Reordered Novel Original Reordered Novel Day 22–24 Day 25–27 0 Hz Max Day 25–27 a c LV peak ratio Original Reordered Novel 1 0.5 0 Original Reordered Novel c d Fig. 5 GC encoding of other belt layouts. a Firing rate maps of GCs for the original (left), reordered (center) and novel (right) belts. Top, scheme of the belt layouts. Color-coded, each row represents the firing rate maps of a GC for the 3 belts, normalized by the peak firing rate of the cell across all belts. Only GCs exhibiting place fields in at least one of the belts are displayed. GCs are sorted according to the type of belt and the belt position of the largest firing fields. b Fraction of GCs with single (black) and multiple (gray) place fields for each belt, during early (days 22–24, left; n = 67 single and 50 multiple- field cells) and late (days 25–27, right; n = 62 single and 41 multiple-field cells) periods on the new belt layouts. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 Note the increase in MC (but no C) activity in landmark positions. Day 1 2 3 5 7 10 13 16 20 0 201 Cells d d 10 16 10 c 0 50 –20 20 0 Field-to-cue distance ( cm ) e # of fields Day 10–20 Day 1–7 Cue MC GC 0.8 0 201 Position ( cm ) Day 10–20 f 0.3 a.u. MC GC Day 1–7 f e Fig. 6 MC overrepresentation of landmarks. a Individual cell examples for various types of MC representations. From left to right, a single-field cell, three multiple-field cells, and a cell with low spatial modulation. Top, scheme of the belt; middle, spike raster plot and color-coded firing rate map; bottom, the mean firing rate. b Proportion of MCs with single fields (black), multiple fields (gray) and low spatial modulation (blue), across days. c Distribution of the number of fields per cell for days 1–7 and days 10–20 among MCs exhibiting place fields. The number of place fields per cell was reduced across days (t89 = 2.8, p = 7e−3, two-tailed unpaired t test). d Color-coded rate maps for MCs exhibiting firing fields across days. The rows of the matrices correspond to individual MCs and are sorted according to firing field positions. The color scale is the same as that used in (a). e Distribution of field-to-landmark distances for GC (blue) and MC (red) place fields for days 1–7 (left) and days 10–20 (right). f Average of GC (blue) and MC (red) rate maps (line, the mean; shadow, s.e.m.) for days 1–7 (left, n = 1159 GCs and 181 MCs) and days 10–20 (right, n = 540 GCs and 112 MCs). Note the increase in MC (but not GC) activity in landmark positions. of fields with peak <5 cm from landmarks, days 1–7, MCs, 70.2 ± 5.0% (n = 19), GCs, 50.0 ± 5.4% (n = 19), t36 = 2.8, p = 9e−3; days 10–20, MCs, 49.6 ± 5.8% (n = 12), GCs, 43.4 ± 4.3% (n = 16), t26 = 0.9, p = 0.39; days 1–7 versus days 10–20, MCs, t29 = 2.6, p = 0.01, GCs, t33 = 0.9, p = 0.40, two-tailed unpaired t test). Given such difference between MC and GC spatial activities, MC spatial activity was likely not predomi- nantly generated by GC inputs (consistent with ref. 14). significantly across days (Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 c LV peak ratio (circle, individual cell; bar, the mean; error bar, s.e.m.) of LV cells for the 3 belts, during early (left, n = 18 LV cells) and late (right, n = 13 LV cells) periods on the new belt layouts. Note that the magnitude of the two firing fields is more similar in the reordered and novel belts than in the original belt. d For the groups of GCs that are active in 1 (black), 2 (green), and 3 (purple) belts, the distribution of the number of fields per cell (in the belt with the largest number of fields), during early (left) and late (right) periods on the new belt layouts. Note that cells exhibiting multiple place fields tend to represent several belts, while cells exhibiting a single place field tend to represent only one belt, t96 = 4.7, p = 9e−6 (days 22–24), t83 = 4.8, p = 6e−6 (days 25–27), two-tailed unpaired t test). NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 0 40 Multiple Day 20 1 % of cells 20 Day 1 2 3 5 7 10 13 16 20 0 201 cm Cells d a 60 0 30 30 10 20 Single Multiple 201 Position ( cm ) Low spatial modulation Trials Trials 60 1 0.8 0 201 Position ( cm ) Day 10–20 0 b c f 0 50 0 30 Single Low spatial 3 5 7 Day 1–7 Day 10–20 # of fields –20 20 0 Field-to-cue distance ( cm ) e 0.3 Hz a.u. MC GC 1 Day 1–7 % of cells 2 3 5 7 10 13 16 2 4 6 # of fields Day 10–20 Day 1–7 Cue MC GC 0 Max 6 MC overrepresentation of landmarks. a Individual cell examples for various types of MC representations. From left to right, a single-field cell, three tiple-field cells, and a cell with low spatial modulation. Top, scheme of the belt; middle, spike raster plot and color-coded firing rate map; bottom, the n firing rate. b Proportion of MCs with single fields (black), multiple fields (gray) and low spatial modulation (blue), across days. c Distribution of number of fields per cell for days 1–7 and days 10–20 among MCs exhibiting place fields. The number of place fields per cell was reduced across days = 2.8, p = 7e−3, two-tailed unpaired t test). d Color-coded rate maps for MCs exhibiting firing fields across days. The rows of the matrices correspond ndividual MCs and are sorted according to firing field positions. The color scale is the same as that used in (a). e Distribution of field-to-landmark ances for GC (blue) and MC (red) place fields for days 1–7 (left) and days 10–20 (right). f Average of GC (blue) and MC (red) rate maps (line, the n; shadow, s.e.m.) for days 1–7 (left, n = 1159 GCs and 181 MCs) and days 10–20 (right, n = 540 GCs and 112 MCs). Note the increase in MC (but not activity in landmark positions. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 0 40 Multiple Day 20 1 % of cells 20 a 60 0 30 30 10 20 Single Multiple 201 Position ( cm ) Low spatial modulation Trials Trials 60 1 0 b c 0 30 Single Low spatial 3 5 7 Day 1–7 Day 10–20 # of fields Hz 1 % of cells 2 3 5 7 10 13 16 2 4 6 0 Max 0 40 Multiple D 20 1 % of cells b Single Low spatial 2 3 5 7 10 13 16 x b a Trials y c 0 30 3 5 7 Day 1–7 Day 10–20 # of fields 1 % of cells 2 4 6 Day 1 2 3 5 7 10 13 16 20 0 201 cm Cells d 0 201 Position ( cm ) 0.8 0 201 Position ( cm ) Day 10–20 0 f 0 50 0 3 5 7 # of fields –20 20 0 Field-to-cue distance ( cm ) e 0.3 a.u. MC GC 1 Day 1–7 2 4 6 # of fields Day 10–20 Day 1–7 Cue MC GC ig. 6 MC overrepresentation of landmarks. a Individual cell examples for various types of MC representations. From left to right, a single-field cell, three multiple-field cells, and a cell with low spatial modulation. Top, scheme of the belt; middle, spike raster plot and color-coded firing rate map; bottom, the mean firing rate. b Proportion of MCs with single fields (black), multiple fields (gray) and low spatial modulation (blue), across days. c Distribution of he number of fields per cell for days 1–7 and days 10–20 among MCs exhibiting place fields. The number of place fields per cell was reduced across days t89 = 2.8, p = 7e−3, two-tailed unpaired t test). d Color-coded rate maps for MCs exhibiting firing fields across days. The rows of the matrices correspond o individual MCs and are sorted according to firing field positions. The color scale is the same as that used in (a). e Distribution of field-to-landmark istances for GC (blue) and MC (red) place fields for days 1–7 (left) and days 10–20 (right). f Average of GC (blue) and MC (red) rate maps (line, the mean; shadow, s.e.m.) for days 1–7 (left, n = 1159 GCs and 181 MCs) and days 10–20 (right, n = 540 GCs and 112 MCs). NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 W 600 x 3000 EC 600 GC 3000 I LV cells Grid cells 1 300 1 300 a b 123 205 280 330 367 390 409 425 442 445 1 Active GCs c 1 9 3 5 7 11 13 15 17 19 Iteration cm 0 201 0 1 Sum cm MC 0 201 a.u. cm 0 201 0 1 0 1 GC sum MC f Ej=Σ ECi. Wij i x i I=α .MC. Σ GCj j GCj=Ej-I =0 if Ej- I<0 if Ej- I<0 Active GCs 123 1 o1 o2 o3 e 0 Max Firing rate 0 1 Synaptic weight Grid o1 o2 o3 0 20 0 2 0 1 Iteration 1 50 % of GCs d Single field LV Periodic Steps for 1 iteration GC excitation GC firing rate Synaptic normalization Synaptic potentiation (1) (2) (3) (4) Wij=Wij+k . Σ ECi.GCj (5) Σ Wij=1 Fig. 7 Modeling of single-field emergence via competitive learning. a Model architecture. A total of 3000 GCs receive excitatory inputs from 300 LV cells and 300 grid cells from the EC and are subjected to feedback inhibition. The EC-to-GC synaptic weight matrix is referred to as Wij. The threshold used as feedback inhibition is referred to as I. Color coded, rate maps of EC LV cells (upper) and grid cells (lower). The firing fields were generated with Gaussian functions. LV cells encode various distances to landmarks, while grid cells have various spatial phases and the same periodicity as periodic cells. In red, the effect of MC feedforward inhibition is added to the model. The spatial modulation of MC activity is assumed to be proportional to the dynamic range of EC average activity. The color scale is the same as that used in (c). b All operations executed during one model iteration. (1) The excitation Ej received by the GCj in a given position. (2) The firing rate of GCj and (3) the feedback inhibition I in that position. The value of I is estimated numerically by finding the value among a range of I values that best satisfies relations (2) and (3). (4) The potentiation of synaptic weights, proportional to the level of cells’ co-firing throughout the belt. (5) The normalization of synaptic weights. c Transformation of spatial representations across iterations. Color-coded representation of rate maps for active GCs, across iterations. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 A total of 3000 GCs receive excitatory inputs from 300 LV cells and 300 grid cells from the EC and are subjected to feedback inhibition. The EC-to-GC synaptic weight matrix is referred to as Wij. The threshold used as feedback inhibition is referred to as I. Color coded, rate maps of EC LV cells (upper) and grid cells (lower). The firing fields were generated with Gaussian functions. LV cells encode various distances to landmarks, while grid cells have various spatial phases and the same periodicity as periodic cells. In red, the effect of MC feedforward inhibition is added to the model. The spatial modulation of MC activity is assumed to be proportional to the dynamic range of EC average activity. The color scale is the same as that used in (c). b All operations executed during one model iteration. (1) The excitation Ej received by the GCj in a given position. (2) The firing rate of GCj and (3) the feedback inhibition I in that position. The value of I is estimated numerically by finding the value among a range of I values that best satisfies relations (2) and (3). (4) The potentiation of synaptic weights, proportional to the level of cells’ co-firing throughout the belt. (5) The normalization of synaptic weights. c Transformation of spatial representations across iterations. Color-coded representation of rate maps for active GCs, across iterations. Active cells have a mean activity >0 and are sorted according to firing field positions. Note the increase in the number of active GCs (lower left numbers) and the transformation of representations from multiple to single place fields. d Evolution of the fraction of single, LV and periodic fields across iterations. e For each active GC, the color-coded representation of the total synaptic weight contributed by grid cells and by each population of LV cells (encoding a specific landmark) is shown across iterations using the same GC ordering as in (c). Note that synaptic inputs initially originate from one source and are progressively redistributed equally among grid cells and LV cells. f Effect of the distinct magnitude of MC modulation on the GC mean activity. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 6b; days 1–3 versus days 13–20, 67.3 ± 4.3% (n = 12) versus 64.5 ± 7.5% (n = 12); t22 = 0.3, p = 0.8, two-tailed unpaired t test); and for all sessions, spatially modulated MCs showed mostly multiple firing fields (Fig. 6b–d), even though the number of fields per cell decreased across days (Fig. 6c; days 1–7 versus days 10–20, 4.44 ± 0.22 versus 3.51 ± 0.25 fields per cell; n = 52 and 39, respectively, t89 = 2.8, p = 7e−3, two-tailed unpaired t test). p p ) Unexpectedly, MC firing fields were strongly modulated by the landmarks, as they were repeated at multiple landmark positions with little spatial offset from the landmarks (Fig. 6d). Accord- ingly, both the distribution of field-to-landmark distances and the averaged cell firing rate maps showed clear peaks in landmark positions for MCs, but not for GCs, an effect that was reduced but still prominent during later sessions (Fig. 6e, f; fraction y g y p Firing fields in the small subset of putative CA3 cells resembled the ones of the GC population (n = 34 single fields, n = 43 multiple unspecific fields, and n = 7 LV fields) except that we could not detect cells with periodic fields (Supplementary Fig. 5a). The proportion of cells exhibiting firing fields and the ratio of multiple versus single fields were higher than for the GC population (cells with place fields, CA3, 58.1 ± 5.6%, GCs, 7 TURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 The threshold used as feedback inhibition is referred to as I. Color coded, rate maps of EC LV cells (upper) and grid cells (lower). The firing fields were generated with Gaussian functions. LV cells encode various distances to landmarks, while grid cells have various spatial phases and the same periodicity as periodic cells. In red, the effect of MC feedforward inhibition is added to the model. The spatial modulation of MC activity is assumed to be proportional to the dynamic range of EC average activity. The color scale is the same as that used in (c). b All operations executed during one model iteration. (1) The excitation Ej received by the GCj in a given position. (2) The firing rate of GCj and (3) the feedback inhibition I in that position. The value of I is estimated numerically by finding the value among a range of I values that best satisfies relations (2) and (3). (4) The potentiation of synaptic weights, proportional to the level of cells’ co-firing throughout the belt. (5) The normalization of synaptic weights. c Transformation of spatial representations across iterations. Color-coded representation of rate maps for active GCs, across iterations. Active cells have a mean activity >0 and are sorted according to firing field positions. Note the increase in the number of active GCs (lower left numbers) and the transformation of representations from multiple to single place fields. d Evolution of the fraction of single, LV and periodic fields across iterations. e For each active GC, the color-coded representation of the total synaptic weight contributed by grid cells and by each population of LV cells (encoding a specific landmark) is shown across iterations using the same GC ordering as in (c). Note that synaptic inputs initially originate from one source and are progressively redistributed equally among grid cells and LV cells. f Effect of the distinct magnitude of MC modulation on the GC mean activity. Fig. 7 Modeling of single-field emergence via competitive learning. a Model architecture. A total of 3000 GCs rece single-field emergence via competitive learning. a Model architecture. A total of 3000 GCs receive excitatory input ll f th EC d bj t d t f db k i hibiti Th EC t GC ti i ht t i i f d t Wij Th Fig. 7 Modeling of single-field emergence via competitive learning. a Model architecture. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 Active cells have a mean activity >0 and are sorted according to firing field positions. Note the increase in the number of active GCs (lower left numbers) and the transformation of representations from multiple to single place fields. d Evolution of the fraction of single, LV and periodic fields across iterations. e For each active GC, the color-coded representation of the total synaptic weight contributed by grid cells and by each population of LV cells (encoding a specific landmark) is shown across iterations using the same GC ordering as in (c). Note that synaptic inputs initially originate from one source and are progressively redistributed equally among grid cells and LV cells. f Effect of the distinct magnitude of MC modulation on the GC mean activity. W 600 x 3000 EC 600 GC 3000 I LV cells Grid cells 1 300 1 300 a b 0 1 Sum cm MC 0 201 a.u. Ej=Σ ECi. Wij i x i I=α .MC. Σ GCj j GCj=Ej-I =0 if Ej- I<0 if Ej- I<0 o1 o2 o3 Steps for 1 iteration GC excitation GC firing rate Synaptic normalization Synaptic potentiation (1) (2) (3) (4) Wij=Wij+k . Σ ECi.GCj (5) Σ Wij=1 W 600 x 3000 EC 600 GC 3000 I LV cells Grid cells 1 300 1 300 a 0 1 Sum cm MC 0 201 a.u. o1 o2 o3 b Ej=Σ ECi. Wij i x i I=α .MC. Σ GCj j GCj=Ej-I =0 if Ej- I<0 if Ej- I<0 Steps for 1 iteration GC excitation GC firing rate Synaptic normalization Synaptic potentiation (1) (2) (3) (4) Wij=Wij+k . Σ ECi.GCj (5) Σ Wij=1 b a 0 20 0 2 0 1 Iteration 1 50 % of GCs d Single field LV Periodic 123 205 280 330 367 390 409 425 442 445 1 Active GCs c 1 9 3 5 7 11 13 15 17 19 Iteration cm 0 201 0 Max Firing rate d c cm 0 201 0 1 0 1 GC sum MC f Active GCs 123 1 e 0 1 Synaptic weight Grid o1 o2 o3 C f e Fig. 7 Modeling of single-field emergence via competitive learning. a Model architecture. A total of 3000 GCs receive excitatory inputs from 300 LV cells and 300 grid cells from the EC and are subjected to feedback inhibition. The EC-to-GC synaptic weight matrix is referred to as Wij. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 31.9 ± 4.9%, t4 = 4.9, p = 0.01; multiple over single ratio, CA3, 1.68 ± 0.34, GCs, 0.55 ± 0.04, t4 = 3.3, p = 0.03, two-tailed unpaired t test, sessions from days 13–20), consistent with previous reports13–15, and unlike the GCs, the spatial representa- tion did not increase drastically across days (151.5 ± 14.4% increase between days 1–3 and days 13–20, versus 370.1 ± 82.4% for GCs, t6 = 2.6, p = 0.06, two-tailed unpaired t test), and the LV firing fields almost vanished after a few days, consistent with the relative paucity of CA3 LV cells in familiar environments35 (Supplementary Fig. 5b, c). The average firing rate map showed only slight increases near landmark locations in early sessions (Supplementary Fig. 5d), suggesting that MC modulation by the landmarks was likely not caused by CA3 input but by other inputs such as semilunar granule cells26 or the EC27,29. field representations are generated via competitive learning18,19. To test whether competitive learning could produce the gradual increase in single-field GCs and the decrease in LV and periodic GCs, we first implemented a model of DG in which 3000 GCs received excitatory inputs from 300 EC grid cells (with spatial periodicity similar to that of the periodic cells) and 300 EC LV cells and, in addition, were subjected to feedback inhibition (Fig. 7a (gray and black); Supplementary Fig. 6a). For a given belt position, the excitation (E) received by a GC was the weighted sum of EC cell activity, the feedback inhibition (I) was proportional to the sum of GC activity, and the firing rate of a GC was equal to E–I (if E > I) or 0 (if E < I). EC-to-GC synaptic weights were initially allocated randomly according to a gamma distribution and then modified on each iteration by two operations: Hebbian synaptic potentiation, proportional to the degree of GC–EC coactivation, and normalization of the total synaptic weight for each GC42–44 (Fig. 7b (black); Supple- mentary Fig. 6b). field representations are generated via competitive learning18,19. To test whether competitive learning could produce the gradual increase in single-field GCs and the decrease in LV and periodic GCs, we first implemented a model of DG in which 3000 GCs received excitatory inputs from 300 EC grid cells (with spatial periodicity similar to that of the periodic cells) and 300 EC LV cells and, in addition, were subjected to feedback inhibition (Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 When the feedback inhibition was set too low, the fraction of GCs with place fields (single, periodic or LV) was too high. Importantly, the transformation of GC representations was accompanied by a specific reconfiguration of EC-to-GC synaptic weights. First, the overall change in synaptic weights (estimated as the vector Euclidean distance ½PðWijði þ 1Þ  WijðiÞÞ21=2 between consecutive iterations) exhibited an exponential decrease, suggesting that the plateauing of single-field GCs corresponded to a stabilization of the synaptic weights (Supple- mentary Fig. 6e, black). Importantly, the overall synaptic change measured just after the synaptic potentiation operation did not show such a drastic decrease (Supplementary Fig. 6e, red), indicating that the synaptic weights were stabilized because the effects of synaptic potentiation were progressively matched and reversed by the effects of synaptic normalization. Other parameters such as the inhibition threshold and E/I ratio (excitation divided by inhibition) did not show trends that could explain the asymptotic increase in single-field GCs (Supplemen- tary Fig. 6f, g). Second, while most excitatory input to a GC was initially contributed by one EC neuron as a result of the initial sparse input setting, excitatory input to a GC was progressively shared by multiple EC neurons, and each GC progressively received, in comparable amount, inputs from grid cells and LV cells (Fig. 7e). Hence, this simple competitive learning model could reproduce the transformation of GC representations, from periodic and LV place fields to single place fields, through the progressive integration of grid cell and LV cell inputs. g g y p p ends for single, LV, periodic and unspecific cells (Fig. 8c). trends for single, LV, periodic and unspecific cells (Fig. 8c). We next examined the predicted cell activity for the reordered and new belts. To simulate the reordered belt, the firing fields of EC LV cells were moved to the new location of the landmarks, and 40% of the non-grid/LV cells were randomly assigned new activity patterns, whereas to simulate the new belt, EC LV were randomly assigned to new object pairs, and 100% of the non-grid/ LV cells were randomly assigned new activity patterns (Fig. 8d). For both the reordered and new belts, the activity of grid cells was not altered to reflect the relative preservation of grid cell patterns across environments as opposed to the strong remapping of EC non-grid spatial cells23. Similar to the experimental data (Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 mostly periodic or LV cells, and the overall fraction of cells generating place fields was excessive (Supplementary Fig. 6). Therefore, we reasoned that unspecific place fields should be generated by a small subset of GCs under low inhibition that receive excitatory inputs from cells showing no periodic and LV activity patterns. Such a subset of excitable GCs could correspond to the small population of young adult-born GCs, which are generally more excitable and active than mature GCs45–48. On the other hand, non-periodic/LV spatial inputs could be supplied by the large population of EC non-grid spatially modulated cells reported in familiar environments23. To test this hypothesis, we added a population of 300 GCs under weak feedback inhibition and a population of 450 non-grid/LV spatially modulated EC cells to the model (the numbers of grid cells and LV cells were each reduced to 150 such that grid cells represented 20% of the EC cells23, and the EC grid:LV ratio remained 1:1, a factor critical to reproduce the initial 1:1 ratio of periodic and LV GCs; Fig. 8a). However, to reproduce the initial predominance of periodic and LV activity patterns of GCs, the number of active non-grid/LV cells was set to zero initially and was then progressively increased over iterations (a plausible phenomenon considering learning within EC and the increasingly strong spatial input feedbacked from the hippocampus; Fig. 8b top). As anticipated, the subset of excitable GCs developed multiple unspecific place fields over time (Fig. 8b), and the model could largely reproduce the experimental trends for single LV periodic and unspecific cells (Fig 8c) We found that two parameters critical to reproduce the gradual increase in single-field GCs were the initial repartition of EC-to- GC synaptic weights and the strength of the feedback inhibition. For a specific range of feedback inhibition strength and sparsity of initial inputs, the model could reproduce the initial preponderance of periodic and LV GCs and the asymptotic increase in single-field GCs (Fig. 7c, d; Supplementary Fig. 6c, d). When the sparsity of initial inputs was set too low (i.e., each GC receives substantial excitation from several EC cells), an asymptotic decrease in single-field GCs was observed over time, whereas when it was set too high (i.e., each GC is mostly excited by one EC neuron), the fraction of GCs that developed single place fields was too high. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 5b, d), the number of single-field representations was decreased for the reordered belt compared to the original belt and was further decreased for the new belt (Fig. 8e), and the number of place fields per cell was correlated with the number of belts represented (Fig. 8f). Hence, this version of the model could reproduce both the transformation of GC representations across days for the original belt and the subsequent encoding of the reordered and new belt layouts. Modeling the contribution of MCs to GC representations. Given that MC average activity was increased in landmark loca- tions and that an important contribution of MCs is believed to be feedforward inhibition27–32 (but see ref. 33), we tested how an increase of inhibition in landmark locations affects GC repre- sentations. Since MCs presumably receive EC information both directly and indirectly27, we assumed that average MC activity reflects average EC activity and modulates the threshold para- meter I of the model (Fig. 7a, b (red); Supplementary Fig. 7). y Importantly, an alternative model in which the subset of excitable GCs received excitatory inputs from the other GCs could also reproduce the diverse aspects of the experimental data (Supplementary Fig. 8). In this version of the model, EC non- grid/LV cells were not incorporated, and GC activity evolved solely from the synaptic plasticity processes. The excitable GCs generated initially periodic and LV firing fields from the sparse inputs of the other GCs and then, over time, developed multiple unspecific place fields reflecting combinations of the single place fields of the GCs. Such network configuration is plausible considering the report that mature GCs transiently provide synaptic inputs to young adult-born GC49 and thus might contribute to the emergence of GC multiple unspecific place fields. In contrast to the experimental findings, average GC activity progressively increased in landmark positions when the inhibition was not modulated by MCs (Fig. 7f; Supplementary Fig. 7c). However, this effect decreased as MC modulation of inhibition was strengthened, such that GCs could evenly represent all belt locations (Fig. 7f; Supplementary Fig. 7d–f). Hence, increasing inhibition in locations associated with larger excitation is necessary to achieve uniform mapping of the space via competitive learning, and a role of MC feedforward inhibition might be to enable the uniform mapping of the space by GCs. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 7a (gray and black); Supplementary Fig. 6a). For a given belt position, the excitation (E) received by a GC was the weighted sum of EC cell activity, the feedback inhibition (I) was proportional to the sum of GC activity, and the firing rate of a GC was equal to E–I (if E > I) or 0 (if E < I). EC-to-GC synaptic weights were initially allocated randomly according to a gamma distribution and then modified on each iteration by two operations: Hebbian synaptic potentiation, proportional to the degree of GC–EC coactivation, and normalization of the total synaptic weight for each GC42–44 (Fig. 7b (black); Supple- mentary Fig. 6b). Modeling the increase in GC single place fields. The DG has been modeled as a competitive network in which discrete place NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 W 750 x 3300 EC 750 GC 3000 + 300 I cm LV Grid 1 150 1 0 201 a Reordered New Original LV Grid 1 150 1 450 d cm 0 201 201 211 0 8 16 0 60 % of GCs Multiple Single LV Periodic Unspecific % of multiple 111315 19 Iteration 1 9 3 5 7 17 150 450 1 Non-grid/LV MC 8 16 0 0 100 1 5 50 150 Non-grid/LV Active GCs Active GCs cm 0 201 b c Iteration 1 9 3 5 7 11 13 15 17 19 1 43 123 1 30 Number of fields % of cells % of cells f e 1 5 1 5 1 5 Multiple Single Number of active belts 1 2 3 1 0 0 Iteration 1 3 5 7 40% remap 100% remap Stong inhibition Weak inhibition 0 450 Active non-grid/LV cells Reordered New Original 0H z Max Fig. 8 Modeling multiple unspecific fields and remapping experiments. a Model architecture. From the previous model design, a group of 450 spatially modulated non-grid/LV cells is added to the EC cell population, the number of Grid cells and LV cells is reduced to 150 to achieve the EC ratio of Grid cells and non-grid cells reported23, and a subgroup of 300 GCs under weak inhibition is added to the GC population. The firing fields of non-grid/LV cells were generated by the multiplication of noise and Gaussian signals to achieve lower spatial information23. The color scale is the same as that used in (b). b Transformation of spatial representations across iterations. Color-coded representation of rate maps for active GCs under high (blue) and low (green) inhibition, across iterations. In gray, the emergence of non-grid/LV cells across iterations. c Upper, proportion of GCs (irrespective of type) with a single field (black) and multiple fields (gray), across iterations. Lower, proportion of LV, periodic and unspecific GCs, among multiple-field GCs, across iterations. Note the similarity with experimental trends (Fig. 3c). d The rate map of EC LV cells (upper), grid cells (middle) and non-grid/LV cells (lower) used to model the original (left), reordered (middle) and new (right) belts. To simulate the reordered belt, the firing fields of EC LV cells are moved to the new location of the landmarks, and 40% of non-grid/LV firing fields are randomly regenerated. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 From the previous model design, a group of 450 spatially modulated non-grid/LV cells is added to the EC cell population, the number of Grid cells and LV cells is reduced to 150 to achieve the EC ratio of Grid cells and non-grid cells reported23, and a subgroup of 300 GCs under weak inhibition is added to the GC population. The firing fields of non-grid/LV cells were generated by the multiplication of noise and Gaussian signals to achieve lower spatial information23. The color scale is the same as that used in (b). b Transformation of spatial representations across iterations. Color-coded representation of rate maps for active GCs under high (blue) and low (green) inhibition, across iterations. In gray, the emergence of non-grid/LV cells across iterations. c Upper, proportion of GCs (irrespective of type) with a single field (black) and multiple fields (gray), across iterations. Lower, proportion of LV, periodic and unspecific GCs, among multiple-field GCs, across iterations. Note the similarity with experimental trends (Fig. 3c). d The rate map of EC LV cells (upper), grid cells (middle) and non-grid/LV cells (lower) used to model the original (left), reordered (middle) and new (right) belts. To simulate the reordered belt, the firing fields of EC LV cells are moved to the new location of the landmarks, and 40% of non-grid/LV firing fields are randomly regenerated. To simulate the new belt, EC LV cells are randomly assigned to new landmarks, and all non-grid/LV firing fields are randomly regenerated. The color scale is the same as that used in (b). e Fraction of GCs with single (black) and multiple (gray) place fields in each belt, across iterations (n = 3300 cells). Note the similarity with experimental trends (Fig. 5b). f The distribution of the number of fields per cell (using the belt with the largest number of fields) for the groups of GCs that are active in 1 (black), 2 (green), and 3 (purple) belts, across iterations. Note the similarity with experimental trends (Fig. 5d). encodings of other layouts, and interpreted the data in terms of learning mechanisms, information encoded and cell-specific functions. Recent studies have outlined cell-type specific differ- ences in the scale, sparseness, stability, and remapping of DG spatial representations. Our study provides data and mechanistic insights on how the spatial representations develop during learning, uniformly map the space and encode other similar environments. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 To simulate the new belt, EC LV cells are randomly assigned to new landmarks, and all non-grid/LV firing fields are randomly regenerated. The color scale is the same as that used in (b). e Fraction of GCs with single (black) and multiple (gray) place fields in each belt, across iterations (n = 3300 cells). Note the similarity with experimental trends (Fig. 5b). f The distribution of the number of fields per cell (using the belt with the largest number of fields) for the groups of GCs that are active in 1 (black), 2 (green), and 3 (purple) belts, across iterations. Note the similarity with experimental trends (Fig. 5d). W 750 x 3300 EC 750 GC 3000 + 300 I cm LV Grid 1 150 1 0 201 a 0 8 16 0 60 % of GCs Multiple Single LV Periodic Unspecific % of multiple 111315 19 Iteration 1 9 3 5 7 17 150 450 1 Non-grid/LV MC Active GCs Active GCs cm 0 201 b c Iteration 1 9 3 5 7 11 13 15 17 19 1 43 123 1 30 Stong inhibition Weak inhibition 0 450 Active non-grid/LV cells 0H z Max W 750 x 3300 EC 750 GC 3000 + 300 I cm LV Grid 1 150 1 0 201 a 150 450 1 Non-grid/LV MC Stong inhibition Weak inhibition 0 8 16 0 60 % of GCs Multiple Single LV Periodic Unspecific % of multiple 111315 19 Iteration 1 9 3 5 7 17 MC Active GCs Active GCs cm 0 201 b c Iteration 1 9 3 5 7 11 13 15 17 19 1 43 123 1 30 0 450 Active non-grid/LV cells 0H z Max b a Reordered New Original LV Grid 1 150 1 450 d cm 0 201 201 211 150 Non-grid/LV 1 0 0 40% remap 100% remap 8 16 0 % of cells e Multiple Single Iteration 1 3 5 7 Reordered New Original d e e 0 100 1 5 50 Number of fields % of cells f 1 5 1 5 1 5 Number of active belts 1 2 3 g Fig. 8 Modeling multiple unspecific fields and remapping experiments. a Model architecture. Discussion Modeling the increase in multiple unspecific GC place fields. The model did not, however, replicate the progressive emergence of multiple unspecific place fields. Reducing feedback inhibition increased the number of multiple-field cells, but these cells were Using silicon probe recording in a treadmill apparatus and neural network modeling, we identified putative GCs and MCs, mon- itored the development of spatial representations during learning of a particular layout of landmarks, investigated subsequent NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 9 9 ARTICLE NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications ARTICLE Hence, disinhibition induced by synaptic normalization might be the initial trigger for the generation of GC place fields via com- petitive learning. Hebbian synaptic plasticity might then be involved to strengthen EC inputs to GCs in the place field loca- tions. The involvement of a Hebbian form of synaptic potentia- tion is suggested by the observation that GCs receiving electrical stimulations tend to develop place fields in the stimulus loca- tions50. Note that a form of synaptic plasticity depending less on pre and postsynaptic coincident activity and more on non- linear dendritic response dynamics is believed to take place in CA1 (refs. 51–53). Over the days, the number of GC place cells progressively plateaued and the rates of place field emergences and extinctions were progressively decreased, a phenomenon reminiscent of the reduced likelihood for electrical stimuli50 and head scanning movement54 to generate place fields as environ- ments become familiar. In the model, this phenomenon was correlated with a progressive reduction of synaptic changes, as the synaptic weight matrix converged to a state where the effect of synaptic potentiation was matched and reversed by the effect of synaptic normalization. Likewise, the observed plateauing of the GC development might correspond to such equilibrium in synaptic plasticity mechanisms. Finally, increasing inhibition in landmark locations was critical to reproduce the uniform map- ping of the space by GCs. In general, a close match between feedforward inhibition and average input activity was an effective mechanism to achieve uniform mapping via competitive learning, suggesting that it might be a recurrent feature of brain networks. MCs are well suited to support such an operation, as they showed firing rate increases in landmark locations and generate feedfor- ward inhibition in GCs27–33. Hence, we suggest that one function of MCs is to sense variation in EC input via semilunar GCs and/ or direct EC afferents26,27,29 and proportionally increase inhibi- tion to ensure a uniform mapping of the space by GCs. p y We reported diverse types of firing fields in the DG and a strong effect of experience on the incidence of each type. In particular, LV and periodic cells were not reported previously, and our findings suggest that their incidence largely increases in novel environments. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 from the GCs, MCs receive external inputs from CA3, semilunar GCs and EC cells26,27. The few putative CA3 cells we monitored did not show much increased activity in the landmarks, making CA3 inputs an unlikely mechanism. On the other hand, semi- lunar GCs are especially responsive to performant path stimula- tions and generate large barrages of excitatory postsynaptic potentials in MCs26. Together with direct EC-to-MC inputs, they could relay to MCs a strong landmark-modulated signal. Second, GCs and MCs showed distinct gamma phase preferences and GCs were more modulated by gamma oscillations than MCs. While these effects were also observed in a recent study14, it is notable that the difference in spike gamma phase was larger in our study, which could reflect differences in cell classification, animal behavioral states, and/or electrode locations for gamma oscillation measurements. Third, as previously reported12–14, the fraction of active cells was lower for GCs than for MCs, and each GC preferentially exhibited a single place field once the belt layout was familiar (days 10–20) whereas MCs preferentially exhibited multiple place fields. Approximately two-thirds (64%) of the spatially modulated GCs exhibited a single place field on the familiar belt, a fraction similar to recent reports13,14 but larger than in a study where DG recordings were carried out in a progressively morphed arena11. While the lower fraction in that study might result from the lack of proper criteria to segregate GCs and MCs, another explanation could be the novelty com- ponent introduced by the progressive morphing of the arena, considering our observation that the proportion of single-field GCs was decreased in the new belt layouts. Last, like previous reports, the GCs that exhibited single and multiple place fields tended to represent single and multiple environments, respectively13,47. y g g Our findings suggest that competitive learning underlies the increase in spatial representations in DG. The model reproduced the transformation and the asymptotic increase in GC repre- sentations across days, as well as the subsequent encoding of other belt layouts. How place fields initially emerge through competitive learning is not straightforward, as cells that are silent paradoxically develop place fields as a result of Hebbian synaptic plasticity, which requires postsynaptic firing. In the model, the initial activation of silent GCs could only be elicited through disinhibition, which had to result from a local decrease in the mean GC activity following the synaptic normalization operation. ARTICLE It should be mentioned, however, that these two types of representation might also be enhanced by the oversimplification of sensory information in the treadmill35 and that their occurrence in typical maze environments might be rare. Importantly, we observed a progressive transformation of GC spatial representations over the course of several days, char- acterized by an asymptotic increase in the number of place cells, a conversion in place field types, and decreases in place field emergence and extinction rates. Another study that carried out long-term recordings of hippocampal spatial activity has emphasized a relatively high stability of GC spatial representa- tions15; however, recordings were started after >10 days of habituation in their first environment tested (by that time, the development of GC spatial representations almost reached a plateau in our study), and a progressive development of GC spatial correlations was visible in the second (novel) environment tested, consistent with the slow dynamics we observed. Further- more, the number of GCs exhibiting place fields was pro- portionally reduced in the reordered and novel belts according to the level of similarity with the original belt. A similar reduction in GC place cells was previously observed15, but the impact of context similarity was not tested. It is notable that the extent of GC remapping largely differs among studies11–15,17,47, a dis- crepancy that is probably explained by the diverse types of environments and cue alterations involved (open arena11,13,14, virtual linear track15 or treadmill12,17,47 environments; changing the room13, the layout of cues12,15,47, the arena boundaries11 or some local cues14,17). In the treadmill, the landmarks fixed on the belt provided the only environmental information useful for mapping positions on the belt, and altering the layout of land- marks is expected to have a larger impact than, for instance, altering local cues in an open arena14 where unchanged spatial information is supplied by distal cues. Last, we observed that MCs, but not GCs, overrepresented the landmarks of the belt. This observation supports the idea of a weak coupling between MC and GC activity, consistent with the weak GC-to-MC spike transmission14 and the distinct MC and GC responses to local cue manipulations14,17, but raises questions regarding the potential origin of the increase of MC activity in landmark locations. Apart pp g p y Interestingly, the development of GC spatial representations was relatively slow, plateauing after a week. NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 subpopulation of GCs, as POMC expression is limited to the 1- month period following cell mitosis and opto-tagging experi- ments were carried out 4 weeks after virus injections17. However, in addition to the overlap with opto-tagged POMC cells, the cluster of putative GCs was suggested by the non-overlap with opto-tagged DRD2 cells and the fact that putative GCs were located above putative MCs along the electrode shanks. Our data showed both consistencies and discrepancies with previous reports. First, MCs were characterized by low burst activity, consistent with in vivo intracellular recording data40, but at odd with recent studies using distinct classification and opto-tagging strategies13,14, which could reflect differences in cell classification, animal behavioral states, and/or methods to assess burst activity. Like our previous study17, we used the combination of spike autocorrelograms and spike relationships with gamma oscilla- tions and a subset of opto-tagged DRD2 and POMC cells to segregate and identify putative GCs and MCs. Opto-tagged POMC cells may have been biased toward a relatively young URE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications 10 ARTICLE Periods of rest between treadmill running sessions were likely essential to regenerate the network potency for plasticity, as the place field emergence rate progressively decreased across trials within each session (Fig. 4d). Synaptic normalization, in particular, might require sleep and sharp-wave-ripple oscillations55,56, considering the relatively slow rate of synaptic downscaling observed in vitro44. Such slow learning is in theory critical for minimizing the alteration of prior memories by new learning (the stability- plasticity dilemma57), is consistent with the high stability of DG spatial representations over time15, and should make DG infor- mation storage more specific to stable features of the environment and therefore particularly suitable for a reliable encoding of 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 spatial contexts. However, it seems at odds with the fast encoding processes typically associated with episodic memory that allow for instance to recall object locations after a quick exploration of a novel environment. This has several possible interpretations. First, spatial learning might be slower in the treadmill because of the relative sparsity of cues and the restriction of head move- ments54. In this respect, it is likely that learning dynamics also vary in natural environments depending on the abundance of cues and arousal states. Second, it is possible that fast encoding mostly takes place in the CA regions15, whereas the DG con- tributes to episodic memory mainly by supplying a reliable spatial context information and helping differentiate CA3 encodings via pattern separation6–10. Last, it should be pointed out that the plateauing of the GC development corresponded only to an optimum in information storage, in which a long time delay to reach the plateau might not be incompatible with the long time delay required to perfectly know an environment (for instance, it might take weeks to become familiar with the sequence of stores and restaurants along a street), and does not prevent that infor- mation storage occurring during single trials or days before the plateauing might already be enough to support some approx- imative memory of the belt (street) layout. patterns. This could be achieved either by adding a population of EC cells that progressively developed spatially modulated non- grid/LV firing fields23 or by using the main GC population as input to the subpopulation of excitable GCs. ARTICLE Both mechanisms are plausible considering potential learning processes within the EC, the increasingly strong spatial input feedbacked from the hippocampus, and reports that young adult-born GCs initially receive inputs mostly from MCs, the CA3 and mature GCs48,49. While the coexistence of less-excitable and more-excitable GCs could enable a differential encoding of the familiar and unique features of contexts (Fig. 5b), another possible advantage is that it allows parallel operations requiring low and high cell excitability, such as pattern separation and temporal binding of episodic information61, respectively. In this respect, it is noteworthy that the network configuration shown in Supplementary Fig. 7 could allow a temporal binding operation by young adult-born GCs that does not compromise DG pattern separation, as young adult- born GCs relay the strongly differentiated information from mature GCs. In conclusion, our findings suggest that a slow integration of spatial cell and landmark-vector cell inputs, achieved via com- petitive learning, is the mechanism underlying both the emer- gence of spatial representations and the continuous mapping of the space by GCs, while a function of MCs is to ensure a uniform distribution of GC representations. As a result, the DG may generate stable maps of the environments that embed object information, allowing the discrimination of slightly different contexts and the detection of slight changes in object layouts. Further work may test whether competitive learning mechanisms operate on the whole DG network or on the level of DG sub- networks associated with distinct information, time scales and cell morphology62. y y The conversion in place field types across days suggests the integration of diverse information. As we implemented in the model, LV and periodic GCs might be generated by strong excitatory inputs from LV cells and grid cells in the EC, respec- tively. While grid cells are typically characterized by a two- dimensional triangular grid arrangement of firing fields, they noticeably exhibited linear periodic patterns in the one- dimensional alleys of a hair-pin maze58. Such periodic activity pattern, so far unseen in the hippocampus, may have emerged in the treadmill because of the novelty of the environment (con- sidering that periodic cells almost completely disappeared after day 1), the oversimplification of the spatial information, and/or the fact that some of the landmarks on the belt were periodically interspaced (Note the periodic arrangement of every second landmarks). Methods A i l Al Animals. All experiments were conducted in accordance with institutional reg- ulations (Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology) and conformed to the Guide for the Care and Use of Laboratory Animals (NRC 2011). Four male C57BL6 mice between 6 and 7 weeks old were used. The mice were housed 2 per cage in a vivarium under a 12 h light/ dark cycle. Virus injection and preparation for head fixation. During a first surgery, under isoflurane anesthesia (supplemented by subcutaneous injections of buprenorphine 0.1 mg/kg, followed by daily subcutaneous injection of ketaprofen 5 mg/kg for 2 days), two small watch screws were driven into the bone above the cerebellum to serve as reference and ground electrodes for the recordings. A 3D printed plastic head-plate with a window opening in the center was cemented to the skull with dental acrylic. The head-plate was designed to be conveniently fixed/unfixed to a holding plate using two screws. Treadmill apparatus and behavioral training. After a post-surgery recovery period of 7 days, the mice were water restricted to 1 ml of water per day and were trained for 2 weeks (one 1-h session per day) to run on the treadmill with their heads fixed. The treadmill was not motorized but consisted of a light velvet belt laying on two 3D printed wheels, which mice moved themselves at will. Water rewards were delivered on every trial at the same position on the belt (position 0 cm) via a lick port. After behavioral learning reached an asymptote, the animals completed 100 to 150 trials in the first 45 min of each session. The quantity of water consumed on the treadmill was measured after each session, and additional water was provided such that the mice drank a total amount of 1 ml of water per day. p y For recording sessions, landmarks were fixed and interspersed on a 201-cm long belt. The landmarks consisted of arrays of vertical poles and textures fixed on the edges of the belt, which provided visual-tactile stimulation to both sides of the mice (Supplementary Fig. 4). ARTICLE A possible scenario is that the periodic GCs origi- nated from the combination of landmark-vector mechanisms and grid cell spatial distance information, the latter accounting for the selection of encoded landmarks based on landmark spatial peri- odicity rather than identity. The model largely implied an inte- gration of LV cell and spatial cell inputs, such that GC activity was contingent upon the particular alignment of object and spatial information on the belt. Such integration might underlie the elaboration of memory engrams for spatial context15,59 that incorporate object location information. Through such engrams, GCs would produce an output very specific to the spatial context and would remap when the object layout is changed17, consistent with the importance of an intact DG to discriminate contexts and detect changes in object layouts8,20,60. DG function might also be considered in terms of pattern separation, with the combination of context engram, sparse GC activity and inter-GC competition contributing to strong remapping responses by GCs17. In this respect, our findings imply that pattern separation should improve with experience as context engrams progressively develop, whereas pattern separation in a new context should be enhanced by prior experience in other similar contexts, given the generalization of learning across similar belt layouts. MMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 gamma power of a hilar LFP was computed for windows within (−10 to +10 ms) and outside (+40 to +100 ms) epochs of maximal firing activity, and the gamma coupling index was defined as the difference between the two windows divided by the sum of the two windows. The 100 largest peaks of the smoothed (using a 20 ms half-width Gaussian kernel) instantaneous firing rate vector were used as epochs of maximal firing. transformation of spatial representations. Also, modeling the experiment was straightforward. A downside of the treadmill is that some exploratory behaviors (like head scanning movements) are prevented by the head fixation. Because distal cues are missing, the treadmill belt environment might be similar to channels in high grass or underground where mice should rely mostly on local cues and path integration. transformation of spatial representations. Also, modeling the experiment was straightforward. A downside of the treadmill is that some exploratory behaviors (like head scanning movements) are prevented by the head fixation. Because distal cues are missing, the treadmill belt environment might be similar to channels in high grass or underground where mice should rely mostly on local cues and path integration. Implementation of single neuron firing rate vector. The length of the belt was divided into 100 pixels. To generate a vector of firing rates, the number of spikes discharged in each pixel was divided by the time the animal spent in the pixel. The firing rate vector was smoothed by convolving a Gaussian function (15 cm half- height width). Chronic implantation of the electrode. A 64-channel silicon probe (Neuronexus, Buzsaki64sp) was used for chronic recordings. A craniotomy (1.5 mm long and 0.5 mm wide) was performed above the hippocampus under isoflurane anesthesia35,36. The silicon probe was mounted on a custom microdrive63, coated with red- fluorescent dye (DiI, Life technologies), and lowered into the granule cell layer of the DG, which was detected by the emergence of unit activity following a ~500-µm zone of unit activity silence below the CA1 pyramidal layer. The microdrive was then cemented to the skull and head-plate. A bone wax and mineral oil mixture was used to cover the craniotomy. A plastic cap was used to protect the microdrive/ silicon probe assembly63. Place field emergence and extinction. References 1. O’Keefe, J. & Dostrovsky, J. The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res. 34, 171–175 (1971). Estimation of cell position relative to the shank. To estimate the position of a cell relative to the recording sites on a shank, we assumed that the amplitude of the spike signals are attenuated as 1/d2 (see note below), where d is the distance of the site to the cell soma, such that the amplitude measured at a given site is: 2. van Dijk, R. M., Huang, S. H., Slominaka, L. & Amrein, I. Taxonomic separation of hippocampal networks: principal cell populations and adult neurogenesis. Front. Neuroanat. 10, 22 (2016). ai ¼ A=d2 i g 3. Boss, B. D., Peterson, G. M. & Cowan, W. M. On the number of neurons in the dentate gyrus of the rat. Brain Res. 338, 144–150 (1985). g 3. Boss, B. D., Peterson, G. M. & Cowan, W. M. On the number of neurons in the dentate gyrus of the rat. Brain Res. 338, 144–150 (1985). with A being the spike amplitude at the cell position. For the many recording sites on one shank, this me with A being the spike amplitude at the cell position. For the many recording sites on one shank, this means that: gy 4. Amaral, D. G., Ishizuka, N. & Claiborne, B. Neurons, numbers and the 4. Amaral, D. G., Ishizuka, N. & Claiborne, B. Neurons, numbers and the hippocampal network. Prog. Brain Res. 83, 1–11 (1990). 4. Amaral, D. G., Ishizuka, N. & Claiborne, B. Neurons, nu 4. Amaral, D. G., Ishizuka, N. & Claiborne, B. Neurons, nu hippocampal network. Prog. Brain Res. 83, 1–11 (1990). 4. Amaral, D. G., Ishizuka, N. & Claiborne, B. Neurons, nu hippocampal network. Prog. Brain Res. 83, 1–11 (1990). A ¼ a1*d2 1 ¼ a2*d2 2 ¼ a3*d2 3 ¼ a4*d2 4 ¼ a5*d2 5: hippocampal network. Prog. Brain Res. 83, 1–11 (1990). 5. Amaral, D. G. & Lavenex, P. in The Hippocampus Book (eds Andersen, P., Morris, R., Amaral, D., Bliss, T. & O’Keefe, J.) 37–114 (Oxford Univ. Press, 2007). Therefore, to estimate the position of a cell, we simply searched for the position at which these conditions were fulfilled. ARTICLE For a given place field, the mean firing rate in a 10-cm window enclosing the field was calculated for each trial, pro- ducing a vector of firing rates. The vector was smoothed using a 3-trial half- width Gaussian kernel. The emergence of a place field corresponded to the time point when the firing rates increased and exceeded 20% of the vector peak value, while the extinction of a field corresponded to a decrease below 20% of the vector peak value. Anatomy. On the last day of recording, the animals were anesthetized at the end of the recording and perfused transcardially with 4% paraformaldehyde in phosphate buffer. The brain was removed and kept overnight in 4% paraformaldehyde solution. Coronal sections (100 µm thick) were sliced using a vibratome and were mounted on slides using Vectashield mounting medium with DAPI. Images of DAPI and DiI fluorescence were acquired separately with a Nikon FN1 microscope equipped for fluorescence imaging. The DG and the electrode signals were isolated and visualized in 3D using custom MATLAB routines. Statistics and reproducibility. All statistical analyses were performed in MATLAB (MathWorks). The number of animals and the number of recorded cells were similar to those generally employed in the field. The analysis of variance (ANOVA) was used to test mean differences between groups larger than two. Student’s t tests were used to test the sample mean. Correlations were computed using Pearson’s correlation coefficient. Behavioral control and data acquisition. The forward and backward movement increments of the treadmill were monitored using two pairs of LEDs and photo- sensors that read patterns on a disc coupled to the treadmill wheel, while the zero position was implemented by an LED and photosensor coupling that detected a small hole on the belt. From these signals, the mouse position was implemented in real time by an Arduino board (Arduino Uno, arduino.cc), which also controlled the valves for the reward delivery. Position, time and reward information from the Arduino board was sent via USB serial communication to a computer and recorded with custom LabView (National Instruments) programs. Reporting summary. Further information on research design is available in the Nature Research Life Sciences Reporting Summary linked to this article. Data availability Due to their large size, the data collected for this study are available upon reasonable request to S.K. or S.R. Source data are provided with this paper. ( ) p g Neurophysiological signals were continuously acquired at 30 kHz on a 250- channel recording system (Intan Technologies, RHD2132 amplifier board with RHD2000 USB Interface Board and a custom LabView interface). The wideband signals were digitally high-pass filtered (0.8–5 kHz) offline for spike detection and low-pass filtered (0–500 Hz) and downsampled to 1000 Hz to extract local field potentials. Spikes from each session and each shank of the silicon probe were clustered separately with automatic algorithms37 followed by manual adjustments in custom MATLAB routines implementing spike autocorrelation, cross-correlation and cluster isolation statistics. Only clusters with well-defined cluster boundaries and clear refractory periods were included in the analyses38. Code availability All the codes for data analysis and modeling are available upon reasonable request. All the codes for data analysis and modeling are available upon reasonable request. Received: 5 September 2019; Accepted: 19 August 2020; Received: 5 September 2019; Accepted: 19 August 2020; References To do this, the volume around each shank was divided into 1 µm3 pixels, and for each pixel, we computed the Euclidean distances to each recording site. Then, we defined a value S such that 6. Marr, D. Simple Memory: a theory for archicortex. Phil. Trans. R. Soc. B 262, 23–81 (1971). S ¼ X ij jai*d2 i  aj*d2 j j; 7. Rolls, E. T. The storage and recall of memories in the hippocampo-cortical system. Cell Tissue Res. 373, 577–604 (2018). 8. Kesner, R. P. & Rolls, E. T. A computational theory of hippocampal function, and tests of the theory: New developments. Neurosci. Biobehav. Rev. 48, 92–147 (2015). where i and j varied to generate all possible combinations of sites. where i and j varied to generate all possible combinations of sites The pixel with the smallest value of S was defined as the cell position. Note: Electric potential of dipoles are attenuated as 1/d2 but as 1/d for The pixel with the smallest value of S was defined as the cell positio 2 Note: Electric potential of dipoles are attenuated as 1/d2 but as 1/d for monopoles. We tested this method using either form and found that the resulting cell positions was very similar. 9. McHugh, T. J. et al. Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317, 94–99 (2007). 10. Knierim, J. J. & Neunuebel, J. P. Tracking the flow of hippocampal computation: pattern separation, pattern completion, and attractor dynamics. Neurobiol. Learn. Mem. 129, 38–49 (2016). Spike gamma phase. The LFP from a channel in the hilus was bandpass filtered between 30 and 80 Hz. A vector of instantaneous phase was derived using the Hilbert transform. The gamma phase of each spike was interpolated from the vector of the instantaneous phase. 11. Leutgeb, J. K., Leutgeb, S., Moser, M. B. & Moser, E. I. Pattern separation in the dentate gyrus and CA3 of the hippocampus. Science 315, 961–966 (2007). 12. Danielson, N. B. et al. In vivo imaging of dentate gyrus mossy cells in behaving mice. Neuron 93, 552–559 (2017). Gamma coupling index. The ‘gamma coupling index’ captures the coupling of LFP gamma power to cell activity during animal immobility. For each cell, the average 13. GoodSmith, D. et al. Spatial representations of granule cells and mossy cells of the dentate gyrus. Neuron 93, 677–690 (2017). Methods A i l Al Reducing the model inhibition threshold for a small group of GCs was critical to reproduce the emergence of unspecific GCs, suggesting that multiple unspecific firing fields were generated by more excitable GCs, possibly the young adult-born GCs that are known to exhibit multiple firing fields, low context specificity and high levels of excitability45–48. 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Priming spatial activity by single-cell stimulation in the dentate gyrus of freely moving rats. Curr. Biol. 26, 536–541 (2016). 20. Kesner, R. P., Taylor, J. O., Hoge, J. & Andy, F. Role of the dentate gyrus in mediating object-spatial configuration recognition. Neurobiol. Learn. Mem. 118, 42–48 (2015). 51. Bittner, K. C. et al. Behavioral time scale synaptic plasticity underlies CA1 place fields. Science 357, 1033–1036 (2017). 21. Hoydal, O. A. et al. ARTICLE 61. Aimone, J. B., Wiles, J. & Gage, F. H. Potential role for adult neurogenesis in the encoding of time in new memories. Nat. Neurosci. 9, 723–727 (2006). p y 30. Jinde, S. et al. Hilar mossy cell degeneration causes transient dentate granule cell hyperexcitability and impaired pattern separation. Neuron 76, 1189–1200 (2012). 31. Hsu, T. T. et al. Differential recruitment of dentate gyrus interneuron types by commissural versus performant pathways. Cereb. Cortex 26, 2715–2727 (2015). 62. Diamantaki, M. et al. Sparse activity of identified dentate granule cells during spatial exploration. eLife 5, 1109 (2016). 63. Chung, J. et al. Micro-drive and headgear for chronic implant and recovery of optoelectronic probes. Sci. Rep. 7, 2773 (2017). 32. Bui, A. D. et al. Dentate gyrus mossy cells control spontaneous convulsive seizures and spatial memory. Science 359, 787–790 (2018). 33. Ratzliff, A. D. H. et al. Rapid deletion of mossy cells does not result in a hyperexcitable dentate gyrus: implications for epileptogenesis. J. Neurosci. 24, 2259–2269 (2004). Author contributions S.K. and S.R. designed the project and performed experiments and modeling. S.K. analyzed the data. D.J. contributed to experiments, spike sorting and supplementary figures. S.K. and S.R. wrote the manuscript. 36. Fattahi, M., Sharif, F., Geiller, T. & Royer, S. Differential representation of landmark and self-motion information along the CA1 radial axis: self-motion generated place fields shift toward landmarks during septal inactivation. J. Neurosci. 38, 6766–6778 (2018). Acknowledgements h k d b This work was supported by the Korea Institute of Science and Technology Institutional Program (Project No. 2E27850) and Institute for Basic Science, grant IBS-R015-D1. This work was supported by the Korea Institute of Science and Technology Institutional Program (Project No. 2E27850) and Institute for Basic Science, grant IBS-R015-D1. 34. Royer, S. et al. Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition. Nat. Neurosci. 15, 769–775 (2012). 35. Geiller, T., Fattahi, M., Choi, J. S. & Royer, S. Place cells are more strongly tied to landmarks in deep than in superficial CA1. Nat. Commun. 8, 14531 (2017). Competing interests 37. Harris, K. D. et al. Accuracy of tetrode spike separation as determined by simultaneous intracellular and extracellular measurements. J. Neurophysiol. 84, 401–414 (2000). 38. Kadir, S. N., Goodman, D. F. M. & Harris, K. D. High-dimensional cluster analysis with the masked EM algorithm. Neural Comput. 26, 2379–2394 (2014). References 13 NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunicati Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-18351-6. 39. Barthó, P. et al. Characterization of neocortical principal cells and interneurons by network interactions and extracellular features. J. Neurophysiol. 92, 600–608 (2004). Correspondence and requests for materials should be addressed to S.R. Correspondence and requests for materials should be addressed to S.R. p y 40. Henze, D. A. & Buzsáki, G. Hilar mossy cells: functional identification and activity in vivo. Prog. Brain Res. 163, 199–216 (2007). Peer review information Nature Communications thanks Andrea Burgalossi and other, anonymous, reviewers for their contributions to the peer review of this work. Peer review reports are available. 41. Bragin, A. et al. Dentate eeg spikes and associated interneuronal population bursts in the hippocampal hilar region of the rat. J. Neurophysiol. 73, 1691–1705 (1995). Reprints and permission information is available at http://www.nature.com/reprints 42. Bliss, T. V. P. & Collingridge, G. L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39 (1993). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 43. Turrigiano, G. G. et al. Activity-dependent scaling of quantal amplitude in neocortical neurons. Nature 391, 892–896 (1998). 14 NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications ARTICLE © The Author(s) 2020 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18351-6 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 15 NATURE COMMUNICATIONS | (2020) 11:4550 | https://doi.org/10.1038/s41467-020-18351-6 | www.nature.com/naturecommunications
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Türkiye’de Bilgi ve Belge Yönetimi Bölümleri Ders Programlarının Arşivcilik ve Belge Yönetimi Eğitimi Açısından Değerlendirilmesi
Bilgi yönetimi
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Öz Bu çalışmanın amacı, Türkiye’de halen üniversite düzeyinde eğitim vermekte olan bilgi ve belge yönetimi bölümlerinin lisans ve lisansüstü güncel ders programlarını arşivcilik ve belge yönetimi dersleri açısından değerlendirmektir. Çalışma betimleme yöntemi ile gerçekleştirilmiştir. Çalışmada Türkiye’de Cumhuriyet dönemi bilgi ve belge yönetimi ve bunun içinde de arşivcilik (belge yönetimi) eğitimi hakkında kısa bir tarihçe verilmiştir. Çalışma kapsamında halen etkin olarak eğitim vermekte olan Türkiye’deki 11 Bilgi ve Belge Yönetimi Bölümü’nün güncel lisans ve lisansüstü ders programları toplam, zorunlu ve seçmeli ders sayıları, arşivcilikle ilgili toplam, zorunlu, seçmeli ders sayıları ve ders içerikleri açısından irdelenmiştir. Çalışma sonunda Türkiye’de üniversite düzeyindeki bilgi ve belge yönetimi eğitimi çerçevesinde genelde güçlü bir arşivcilik/belge yönetimi ders programının olmadığı, ders sayıları, bu derslerin zorunlu ya da seçmeli statüde oluşları açısından Bölümler arasında önemli farklar, ders içerikleri açısından ise benzerlikler olduğu anlaşılmıştır. Çalışmanın sonunda konuya ilişkin çeşitli önerilerde bulunulmuştur. Hakemli Makaleler Makale Bilgisi Gönderildiği tarih: 08.03. 2018 Kabul tarihi: 06.06. 2018 Yayınlanma tarihi: 22.06. 2018 Article Info Date submitted: 08.03. 2018 Date accepted: 06.06. 2018 Date published: 22.06. 2018 Anahtar sözcükler Bilgi ve Belge Yönetimi, Arşivcilik Eğitimi, Bilgi ve Belge Yönetimi Bölümleri- Türkiye. Keywords Information and Documentation Management, Archival Education, Departments Of Information Management, Turkey.. Keywords Information and Documentation Management, Archival Education, Departments Of Information Management, Turkey.. Evaluation of the Curriculums in Information Management Departments in Turkey in Terms of Archival and Record Management Education Bilgi Yönetimi Dergisi Cilt: 1 Sayı: 1 Yıl: 2018 Prof. Dr. Bülent YILMAZ Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü, byilmaz@hacettepe.edu.tr Prof. Dr. Bülent YILMAZ Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü, byilmaz@hacettepe.edu.tr http://dergipark.gov.tr/by Abstract The purpose of this research is to evaluate the curriculums of the undergraduate and graduate education of the Information Management Departments in Turkey, in terms of their archival and record management lessons. The study was performed by descriptive method. In the study, the information management education and archival education in the Republican Era of Turkey was briefly explained. This study aims to address: (1) 11 curriculums of the Departments were examined in terms of the total number of compulsory and elective courses and, (2) the number of compulsory and elective archival courses and, (3) the content of these courses. Findings of the research have proven that there is no powerful curriculum of the archival/documentation management education at the university level in Turkey. Findings also indicate that there are similarities in terms of the compulsory and elective course contents, although there are important differences among Departments such as the number of archival courses and whether these courses are mandatory or elective. Finally, the research makes various suggestions in the light of the results. **Çakın, 2013, 2012, 2007, 2005, 2000, 1999, 1997; Tonta, 2012a, 2012b, 2004, 2000, 1987; Tuncer, 2007; Atılgan, 1999, 2014; Baysal, 1987; Ersoy, 1962, 1965; Ersoy ve Yurdadoğ, 1963; Kum, 1970; Soysal, 1981, 1983; Akbulut, 2014; Subaşıoğlu, 2014: Yılmaz, 2014; Kandur, 2014; Keseroğlu, 2014; Odabaş, 2014; Sağsan, 2014; Bayter, 2014; Efe, Zan ve Binici, 2014; Bayram ve Zan, 2014; Baysal, 1987; Dilek, 1991; Karakaş, 1999; Koşay, 1960; Ötüken, 1957; Scepanski, 2001; Sefercioğlu, 1981; Özdemirci, 2004, 2001. 1. Giriş Arşivler bir ülkenin ve o ülkedeki kurumların belleği işlevini gören kuruluşlardır. Tarihsel ve kurumsal süreçlerde üretilen bilgi ve belgeleri toplayan, düzenleyen, koruyan ve erişime sunan kuruluşlar olarak arşivlerin bu işlevlerini yerine getirmesi birçok açıdan uygun koşullara sahip olmalarına bağlıdır. Bu koşullardan birisi de arşivlerde tüm işlem ve hizmet süreçlerini gerçekleştiren ve geliştiren profesyonel personeldir. Arşivler için profesyonel personelin önemli bir bölümü arşivci (arşivist), arşiv uzmanı ya da belge yöneticisi olarak adlandırılan uzman gruptan oluşmaktadır. Mesleki eğitimi dünyada ve Türkiye’de lisans ve lisansüstü (yüksek lisans ve doktora) düzeyde verilen arşivcilik aynı zamanda Bilgi Yönetimi 1: 1 (2018), 44-62 Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… Hakemli Makaleler bilimsel bir disiplin ve bilim alanıdır. Bir ülkede arşiv işlem ve hizmetlerinin yeterliliğini/yetersizliğini en çok belirleyen unsurlardan birisi arşivcilerin niceliği ve niteliğidir. bilimsel bir disiplin ve bilim alanıdır. Bir ülkede arşiv işlem ve hizmetlerinin yeterliliğini/yetersizliğini en çok belirleyen unsurlardan birisi arşivcilerin niceliği ve niteliğidir. Türkiye’de profesyonel arşivci üniversitelerin bilgi ve belge yönetimi bölümleri tarafından verilen eğitimle yetiştirilmektedir. Dolayısıyla arşivcilerin nitelikleri (sahip oldukları mesleki özellikler) büyük ölçüde bu Bölümlerdeki eğitim yapısı ile doğrudan ilgilidir. Bu nedenle bölümlerde arşivcilik ile ilgili eğitimin ders programları çerçevesinde incelenmesi ve çeşitli açılardan değerlendirilmesi büyük önem taşımaktadır. Çünkü başka etkenlerin yanı sıra Türkiye’deki arşivlerin bugünkü durumlarının bu kurumlarda çalışan profesyonel arşivcilerin yeterlilikleri/yetersizlikleri ile de ilgili olduğu, bunun ise son tahlilde ve büyük oranda aldıkları mesleki eğitime dayandığı söylenebilir. Bu çalışmada, Türkiye’de halen eğitim veren bilgi ve belge yönetimi bölümlerinin lisans ve lisansüstü ders programları arşivcilik ve belge yönetimi eğitimi açısından ilgili dersler bağlamında değerlendirilecektir. Özdemirci (2017, s.221)’nin de belirttiği üzere, bu iki alan yalnızca kavramsal olarak değil fakat aynı zamanda süreçsel bir birlikteliğe de sahiptir. Çalışmada arşivcilik ve belge yönetimi belirli farklılar taşıyan ancak birbirine son derece yakın iki alan olarak düşünülmüştür. Bu nedenle de her iki alan da çalışmaya dahil edilmiştir. 2. Türkiye’de Bilgi ve Belge Yönetimi Eğitimi: Kısa Tarihçe Türkiye’de bilgi ve belge yönetimi eğitiminin tarihi hakkında ya da bu konuyu da içeren çok sayıda çalışma bulunmaktadır.** Cumhuriyet Türkiye’sinde bilgi ve belge yönetimi (kütüphanecilik) eğitim tarihi literatürde genellikle dört döneme ayrılarak ele alınmaktadır. Biz de bu çalışmada literatüre dayalı yaklaşım çerçevesinde kütüphanecilik eğitimi için farklı özelliklere sahip olduğu değerlendirilen “1923- 1952, 1953-1987, 1988-2001, 2002 ve sonrası” dönemlerini temel alacağız. Yukarıda belirtilen dönemlerin kütüphanecilik eğitimi açısından öne çıkan gelişmelerini Çakın, 1999, 2005, 2013; Tonta, 2000, 2004, 2012a, 2012b; Subaşıoğlu, 2014’nun çalışmalarına dayanarak kısaca şöyle özetleyebiliriz: 1923-1952 dönemi • 1953 yılında AÜ DTCF Türk Dili ve Edebiyatı Kürsüsü’nün seçmeli olarak kabul edilen “kütüphanecilik” dersi ile “Kütüphanecilik Enstitüsü”nün temelinin atılması. • 1954-55 öğretim yılının ilk döneminde, AÜ DTCF Kütüphanecilik Enstitüsü’nde, Fakülte Profesörler Kurulu’nun almış olduğu kararla hazırlanan yönetmelikle Adnan Ötüken’in verdiği derslerle öğretime başlanması. • 1960 yılında, Üniversitelerarası Kurul’un, enstitüleri, doçentlik çalışmalarını yürütmek açısından yeterli bulmaması üzerine, Kütüphanecilik Enstitüsü’nün, “kürsü” ye dönüştürülmesine karar verilmesi. • 1960 yılında, Üniversitelerarası Kurul’un, enstitüleri, doçentlik çalışmalarını yürütmek açısından yeterli bulmaması üzerine, Kütüphanecilik Enstitüsü’nün, “kürsü” ye dönüştürülmesine karar verilmesi. • 1963’te İstanbul Üniversitesi Edebiyat Fakültesi (İÜEF) Kütüphanecilik Kürsüsü, 1972’de "bilim uzmanı" yetiştirmek amacıyla Hacettepe Üniversitesi (HÜ) "Kütüphanecilik ve Dokümantasyon Enstitüsü" kurulması ve HÜ lisans programına 1974’te başlanması. • 1987’de Marmara Üniversitesi’nde (MÜ) Arşivcilik Bölümü’nün açılması. 1988-2001 dönemi • 1988-2001 yılları arası dönemde kütüphanecilik bölümlerinin, “Kütüphanecilik”, “Arşivcilik” ve “Dokümantasyon-Enformasyon” anabilim dalları açarak yeniden örgütlenmesi. • 1994 yılında, Atatürk Üniversitesi Fen-Edebiyat Fakültesi’nde (AÜFEF) Kütüphanecilik Bölümü’nün açılması. Bölümü nün açılması. 2002 ve sonrası dönem • Türkiye’de, 2002 yılına kadar, kütüphanecilik, arşivcilik, dokümantasyon ve enformasyon anabilim dalları altında yapılandırılan kütüphanecilik bölümlerinin, YÖK’ün 29 Ocak 2002 tarihli toplantısında alınan kararla, mevcut anabilim dallarının kapatılması ve 2002-2003 akademik yılından itibaren “Bilgi ve Belge Yönetimi Bölümü” adını alması. Bölümlere yerleştirilecek öğrencilerde aranılan ÖSS puan türünün “Sözel”den "Eşit Ağırlıklı"ya dönüştürülmesi. • Bu dönemde (2018 yılı itibariyle) Kastamonu, Yıldırım Beyazıt, Çankırı Karatekin, Kıbrıs Yakın Doğu, İstanbul Medeniyet, Osmaniye Korkut Ata Üniversitelerinde bilgi ve belge yönetimi bölümleri kurularak eğitime başlanması. (Bu dönemde resmi olarak kurulan ancak henüz eğitime başlanmayan bilgi ve belge yönetimi bölümleri de bulunmaktadır.) • Bu dönemde (2018 yılı itibariyle) Kastamonu, Yıldırım Beyazıt, Çankırı Karatekin, Kıbrıs Yakın Doğu, İstanbul Medeniyet, Osmaniye Korkut Ata Üniversitelerinde bilgi ve belge yönetimi bölümleri kurularak eğitime başlanması. (Bu dönemde resmi olarak kurulan ancak henüz eğitime başlanmayan bilgi ve belge yönetimi bölümleri de bulunmaktadır.) • Yeni açılan bölümlerin öğretim elemanı gereksinimlerini karşılamaya yönelik olarak gerçekleştirilen, bazı öğrencilerin Öğretim Üyesi Yetiştirme Programı (ÖYP) çerçevesinde yeni kurulan bölümler adına diğer üniversitelerde bilgi ve belge yönetimi lisansüstü programlarına devam etmeleri ve bu öğrencilerin doktora derecelerini aldıktan sonra üniversitelerine dönerek ilgili bölümlerin kuruluşunda görev almaları. 1923-1952 dönemi 1923-1952 dönemi • 3 Mart 1924 Tevhid-i Tedrisat Kanunu’nun (Eğitimin Birliği Yasası) çıkarılması ile kütüphane ve kitapların vakıflardan Eğitim Bakanlığı’na (Maarif Vekaleti) devredilmesi. • 3 Mart 1924 Tevhid-i Tedrisat Kanunu’nun (Eğitimin Birliği Yasası) çıkarılması ile kütüphane ve kitapların vakıflardan Eğitim Bakanlığı’na (Maarif Vekaleti) devredilmesi. • Amerikalı eğitimbilimci John Dewey’nin içinde kütüphanelere ilişkin değerlendirmelerin de yer aldığı ve yurt dışına kütüphanecilik eğitimi için gönderilmesi gereken kişi sayısının belirtildiği Türk eğitim sistemi hakkındaki raporun yayımlanması. • 1925 yılında, Fehmi Ethem Karatay’ın kütüphanecilik eğitimi almak üzere Paris’e gönderilmesi. Karatay, yurda döndükten sonra İstanbul Üniversitesi Kütüphanesi’ni kurmuş ve İstanbul kütüphanelerinde çalışan kütüphanecilere yönelik olarak ilk kütüphanecilik kursunu 1925-26 yıllarında açmıştır. • 1926 yılında zamanın Maarif Vekâleti Hars Dairesi Müdürü olan Hamit Zübeyr Koşay tarafından hazırlanarak Maarif Vekaletine sunulan ve Türkiye’de kütüphaneci yetiştirilmesi sorununa da değinen halk kütüphaneleri hakkında raporun yayımlanması. • 1926 yılında zamanın Maarif Vekâleti Hars Dairesi Müdürü olan Hamit Zübeyr Koşay tarafından hazırlanarak Maarif Vekaletine sunulan ve Türkiye’de kütüphaneci yetiştirilmesi sorununa da değinen halk kütüphaneleri hakkında raporun yayımlanması. • Fehmi Ethem Karatay’dan sonra, ikinci kütüphanecilik kursunun, Ankara’da Yüksek Ziraat Enstitüsü Kütüphanesi’nin kurulmasıyla görevlendirilen, Dr. Joseph Stummvoll tarafından 1936 yılında Yüksek Ziraat Enstitüsü Kütüphanesi’nde açılması. • 1941 yılında, kütüphanelere nicelik ve nitelik yönünden yeterli kütüphaneciler yetiştirmek amacı güden bir kanun tasarısının, İstanbul Üniversitesi Rektörü’nün başkanlığında çalışan bir komisyon tarafından hazırlanarak, Kütüphaneler Müdürlüğü’ne gönderilmesi ve dönemin Kütüphaneler Müdürü Aziz Berker’in, bu tasarı ve gerekçesi ile ilgili görüşlerini yansıtan 27 45 Prof. Dr. Bülent YILMAZ Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Eylül 1941 tarihli yazıyı yayımlaması. Bu yazı ile ilk kez Aziz Berker, İstanbul Üniversitesi Edebiyat Fakültesi’ne bağlı bir Kütüphanecilik Enstitüsü’nün kurulmasını önermiştir. Eylül 1941 tarihli yazıyı yayımlaması. Bu yazı ile ilk kez Aziz Berker, İstanbul Üniversitesi Edebiyat Fakültesi’ne bağlı bir Kütüphanecilik Enstitüsü’nün kurulmasını önermiştir. • İlk sürekli ve düzenli kütüphanecilik eğitiminin, Adnan Ötüken’in girişimiyle Ankara Üniversitesi Dil ve Tarih-Coğrafya Fakültesi (AÜ DTCF) çatısı altında ve kurslar düzeyinde 1942-1952 yılları arasında gerçekleştirilmesi. Kütüphanecilik eğitimi, Adnan Ötüken’in çabaları ile ilk kez bu kurslarla üniversiteye girmiş ve üniversite kurullarında kütüphanecilik eğitimi konuşulmaya başlanmıştır. • 1952 yılında kütüphanecilik eğitiminin önemine inanan birkaç öğretim üyesinin girişim ve çabaları ile Ankara Üniversitesi Fakülte Profesörler Kurulu’nun, kütüphanecilik derslerinin DTCF Türk Dili ve Edebiyatı Kürsüsü'nde “seçmeli” statüde devam etmesine karar vermesi. 1953-1987 dönemi • 1953 yılında AÜ DTCF Türk Dili ve Edebiyatı Kürsüsü’nün seçmeli olarak kabul edilen “kütüphanecilik” dersi ile “Kütüphanecilik Enstitüsü”nün temelinin atılması. 1923-1952 dönemi (Bu uygulama 2017 yılı itibariyle sona erdirilmiştir.) Araştırma kapsamına alınan ve Türkiye’de halen eğitim verilmekte olan bilgi ve belge yönetimi bölümlerinin kuruluş yılları sırası itibariyle tarihçeleri ise kısaca şöyledir: Araştırma kapsamına alınan ve Türkiye’de halen eğitim verilmekte olan bilgi ve belge yönetimi bölümlerinin kuruluş yılları sırası itibariyle tarihçeleri ise kısaca şöyledir: Ankara Üniversitesi Bilgi ve Belge Yönetimi Bölümü, 1954-1955 öğretim yılında “Kütüphanecilik Enstitüsü” adı altında kurulmuş ve 1964 yılında “Kütüphanecilik Bölümü” adını almıştır. 1970 yılından 46 Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… itibaren programlarda bilgi bilim ile ilgili dersler yer almaya başlamıştır. 1989-1990 öğretim yılında Dokümantasyon-Enformasyon ile Arşivcilik Anabilim Dalları oluşturulmuş, böylelikle Kütüphanecilik de dâhil olmak üzere üç Anabilim Dalı bölümün bünyesinde yer almıştır. Yüksek Öğretim Kurulu’nun kararı ile yeni adı “Bilgi ve Belge Yönetimi Bölümü” olan bölüm, 2002-2003 öğretim yılından itibaren bölüm düzeyinde ve eşit ağırlıklı puan türüne göre öğrenci alınmaya başlanmıştır. Bölüm’de yüksek lisans ve doktora programları da yürütülmektedir (“Genel Bilgiler”, t.y.). itibaren programlarda bilgi bilim ile ilgili dersler yer almaya başlamıştır. 1989-1990 öğretim yılında Dokümantasyon-Enformasyon ile Arşivcilik Anabilim Dalları oluşturulmuş, böylelikle Kütüphanecilik de dâhil olmak üzere üç Anabilim Dalı bölümün bünyesinde yer almıştır. Yüksek Öğretim Kurulu’nun kararı ile yeni adı “Bilgi ve Belge Yönetimi Bölümü” olan bölüm, 2002-2003 öğretim yılından itibaren bölüm düzeyinde ve eşit ağırlıklı puan türüne göre öğrenci alınmaya başlanmıştır. Bölüm’de yüksek lisans ve doktora programları da yürütülmektedir (“Genel Bilgiler”, t.y.). İstanbul Üniversitesi Bilgi ve Belge Yönetimi Bölümü, 1963 yılında Edebiyat Fakültesi’ne bağlı “Kütüphanecilik Bölümü” adıyla kurulmuş ve 1964-1965 akademik yılında öğretime başlamıştır. Ankara Üniversitesi Dil ve Tarih-Coğrafya Fakültesi Kütüphanecilik Bölümü’nden sonra, Türkiye’de kütüphanecilik alanında açılan ikinci bölümdür. 1983′de yüksek lisans, 1984′de doktora programları açan Bölüm, 1990′da, biri ‘Kütüphanecilik’, diğeri ‘Dokümantasyon ve Enformasyon’ olmak üzere iki anabilim dalı halinde örgütlenmiş; 1995′den itibaren Kütüphanecilik Anabilim Dalı’nda, ikinci öğretim programı da açılmıştır. Bölümün adı Ankara, Hacettepe ve Başkent Üniversitelerine bağlı kütüphanecilik bölümlerininki ile birlikte Yükseköğretim Kurulu kararıyla 2002 yılında “Bilgi ve Belge Yönetimi Bölümü” olarak değiştirilmiş; ayrıca üç bölümde mevcut anabilim dalları kapatılarak, anabilim dalı yerine ‘bölüme öğrenci alma’ uygulamasına geçilmiştir. Kurulması 1985′de kararlaştırılan ve 1988-1989′dan itibaren lisans düzeyinde öğretime başlayan Arşivcilik Bölümü’nde 2002-2003 yılında yeni öğrenci alımına son verilmiş ve 2002-2003 yılında Bilgi ve Belge Yönetimi Bölümü kapsamına alınmıştır. Bölüm aynı tarihten Arşivcilik Anabilim Dalı ile Bilgi Yönetimi ve Teknolojisi Anabilim Dalı biçiminde yapılandırılmış ve halen bu yapı sürmektedir. 1923-1952 dönemi Bülent YILMAZ Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Yıldırım Beyazıt Üniversitesi Bilgi ve Belge Yönetimi Bölümü, 2011 yılında İnsan ve Toplum Bilimleri Fakültesi bünyesinde kurularak, 2014 yılında ilk lisans öğrencilerini kabul etmiş ve 2017 yılı itibariyle de Bilgi ve Belge Yönetimi Anabilim Dalı çatısı altında Bilgi Teknolojileri Anabilim Dalı ve Arşivcilik Anabilim Dalı olarak iki ayrı anabilim dalı ile lisans düzeyinde eğitim-öğretim çalışmalarını sürdürmekte olup, lisansüstü eğitim programları bulunmamaktadır (“Bölüm Hakkında”, 2017). Yakın Doğu Üniversitesi Bilgi ve Belge Yönetimi Bölümü, lisans eğitimine 2009 yılında, İnovasyon ve Bilgi Yönetimi yüksek lisans ve doktora programlarının bulunduğu lisansüstü eğitimine de 2012 yılında başlamıştır. (“Bilgi ve Belge Yönetimi Bölümü”, 2018) Yakın Doğu Üniversitesi Bilgi ve Belge Yönetimi Bölümü, lisans eğitimine 2009 yılında, İnovasyon ve Bilgi Yönetimi yüksek lisans ve doktora programlarının bulunduğu lisansüstü eğitimine de 2012 yılında başlamıştır. (“Bilgi ve Belge Yönetimi Bölümü”, 2018) İstanbul Medeniyet Üniversitesi Bilgi ve Belge Yönetimi Bölümü, 2011 yılında kurulmuş ve ilk öğrencilerini 2016-2017 öğretim yılında almıştır. Bölüm, eğitim programını lisans düzeyinde sürdürmektedir (“Tarihçe”, 2018). Osmaniye Korkut Ata Üniversitesi Bilgi ve Belge Yönetimi Bölümü, Kadirli Sosyal ve Beşeri Bilimler Fakültesi bünyesinde 2016 yılında kurulmuştur. Bölüm, ilk öğrencilerini 2017–2018 eğitim-öğretim döneminde almış olup, eğitim-öğretim halen lisans düzeyinde sürdürülmektedir (“Tarihçe”, 2016b). Osmaniye Korkut Ata Üniversitesi Bilgi ve Belge Yönetimi Bölümü, Kadirli Sosyal ve Beşeri Bilimler Fakültesi bünyesinde 2016 yılında kurulmuştur. Bölüm, ilk öğrencilerini 2017–2018 eğitim-öğretim döneminde almış olup, eğitim-öğretim halen lisans düzeyinde sürdürülmektedir (“Tarihçe”, 2016b). Bu çalışmanın içeriği açısından Türkiye’de üniversite düzeyinde verilmeye başlanan bilgi ve belge yönetimi eğitimi içinde arşivcilik/belge yönetimi eğitimi konusundaki gelişmeleri yukarıda bölümlerin tarihçesi ile ilgili açıklamalarda yer alan kaynaklara da dayalı olarak kısaca değerlendirmek ve özetlemek yararlı olacaktır. Bu açıdan önemli gelişmeler şöyle sıralanabilir: • Üniversite düzeyinde arşivcilik ile ilgili eğitim 1980’li yılların sonlarına kadar kütüphanecilik bölümü adı altında verilen bazı lisans-lisansüstü dersler çerçevesinde yürütülmüştür. • Üniversite düzeyinde arşivcilik ile ilgili eğitim 1980’li yılların sonlarına kadar kütüphanecilik bölümü adı altında verilen bazı lisans-lisansüstü dersler çerçevesinde yürütülmüştür. • 1987 yılında Marmara Üniversitesi’nde Arşivcilik Bölümü kurularak bu alanda Bölüm düzeyinde eğitim verilmeye başlanmıştır. Ancak Haziran 2002’de Arşivcilik olan bölüm adı YÖK kararı ile bilgi ve belge yönetimine dönüştürülmüştür. • Yine kurulması 1985′de kararlaştırılan İstanbul Üniversitesi Arşivcilik Bölümü’nde 1988-1989′dan itibaren lisans düzeyinde öğretime başlanmış ancak bu Bölümde de 2002-2003 yılında yeni öğrenci alımına son verilmiş ve aynı yıl bilgi ve belge yönetimi Bölümü kapsamına alınmıştır. 1923-1952 dönemi Bölüm’de yüksek lisans ve doktora programları da yürütülmektedir (“Bölüm Genel Bilgisi”, 2012). Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü, 1972 yılında "Kütüphanecilik ve Dokümantasyon Enstitüsü" adı altında kurulmuştur. Bilim uzmanlığı programı ile eğitime başlayan Bölüm, lisans programına ilk öğrencilerini 1974'te almıştır. Bir yılı İngilizce hazırlık ve dört yılı mesleki eğitim-öğretim olmak üzere toplam beş yılık lisans programı 1978/1979, 1983/1984 ve 1990/1991 eğitim dönemlerinde çeşitli düzeylerde değişikliğe uğramış, 1993 yılında ise yapısal bir değişim geçirerek, Kütüphanecilik, Arşivcilik, Dokümantasyon ve Enformasyon Anabilim dallarına ayrılmıştır. Hacettepe Üniversitesi Kütüphanecilik Bölümü akademik programında gerçekleştirilen bir diğer temel değişim, kütüphanecilik bölümlerinin yeniden yapılanmasını öngören Yükseköğretim Kurulu'nun 2002 tarihinde aldığı karar ile gerçekleşmiştir. Buna göre, 2002/2003 öğretim yılından başlayarak Kütüphanecilik, Arşivcilik, Dokümantasyon ve Enformasyon Anabilim Dalları birleştirilerek, Kütüphanecilik Bölümü yeni bir programla "Bilgi ve Belge Yönetimi Bölümü" adı altında eğitim- öğretim etkinliklerine başlamıştır. Bölüm’de lisansüstü (yüksek lisans-doktora) programı da aynı adla uygulanmaktadır (“Tarihçe”, 2016a). Ayrıca, Bölüm’ün Bilişim Enstitüsü bünyesinde açtığı Bilgi Araştırmaları Tezsiz Yüksek Lisans Programı’na 2017-2018 Güz döneminden itibaren öğrenci alınmaya başlanmıştır. Marmara Üniversitesi Bilgi ve Belge Yönetimi Bölümü, 1987’de Arşivcilik Bölümü adıyla kurulmuştur. Haziran 2002’de Arşivcilik olan Bölüm adı YÖK kararı ile Bilgi ve Belge Yönetimi’ne dönüştürülmüştür. Bölümde halen Tarihi Devlet Arşivleri ve Müessese Arşivleri olmak üzere iki anabilim dalı bulunmakta olup, Türkiyat Araştırmaları Enstitüsü bünyesinde yüksek lisans ve doktora programları da yürütülmektedir (“Genel Bilgiler”, 2018; “Anabilim Dalları”, 2018). Atatürk Üniversitesi Bilgi ve Belge Yönetimi Bölümü, resmi olarak kuruluşunu 1994 yılında tamamlamış, lisans programına 2008-2009 ve yüksek lisans programına da 2010-2011 öğretim yılından itibaren öğrenci alınmaya başlanmıştır (Odabaş, 2014, s. 131). Bölüm’de doktora programı bulunmamakta, halen yetersiz öğretim elemanı nedeniyle yüksek lisansa öğrenci de alınamamaktadır. Kastamonu Üniversitesi Bilgi ve Belge Yönetimi Bölümü, 2007’de Fen-Edebiyat Fakültesi bünyesinde kurulmuş olup, 2014-2015 eğitim öğretim yılında öğrenci almaya başlamıştır. Bölüm’de halen yüksek lisans-doktora programı bulunmamaktadır (“Bilgi ve Belge Yönetimi Genel Bilgiler”, t.y.). Çankırı Karatekin Üniversitesi Bilgi ve Belge Yönetimi Bölümü, resmi olarak 2010 tarihinde kurulmuş, ilk öğrencilerini 2013-2014 eğitim-öğretim yılında kabul etmiştir. Bölüm’de yüksek lisans eğitimine 2016 Güz döneminde başlanmıştır. Bölümde halen Kütüphanecilik, Arşivcilik ile Bilgi Yönetimi ve Teknolojisi adlarıyla üç ana bilim dalı bulunmaktadır (“Tarihçe”, t.y.). 47 Prof. Dr. * Türkiye’de arşivcilik/belge yönetimi eğitimi tarihi hakkında daha ayrıntılı bilgi için ayr. bkz. Özdemirci, t.y, 2001, 2017; Kayaoğlu, 2017. 1923-1952 dönemi • Yine kurulması 1985′de kararlaştırılan İstanbul Üniversitesi Arşivcilik Bölümü’nde 1988-1989′dan itibaren lisans düzeyinde öğretime başlanmış ancak bu Bölümde de 2002-2003 yılında yeni öğrenci alımına son verilmiş ve aynı yıl bilgi ve belge yönetimi Bölümü kapsamına alınmıştır. • 1988-2001 yılları arası dönemde kütüphanecilik bölümleri içinde, “kütüphanecilik”, “arşivcilik” ve “dokümantasyon ve enformasyon” anabilim dalları açılmıştır. Böylece doğrudan arşivcilik anabilim dalına öğrenci alınarak arşivcilik konusunda anabilim dalı çerçevesinde eğitime başlanmıştır. • 1988-2001 yılları arası dönemde kütüphanecilik bölümleri içinde, “kütüphanecilik”, “arşivcilik” ve “dokümantasyon ve enformasyon” anabilim dalları açılmıştır. Böylece doğrudan arşivcilik anabilim dalına öğrenci alınarak arşivcilik konusunda anabilim dalı çerçevesinde eğitime başlanmıştır. • 2002-2003 yılında YÖK’ün aldığı karar ile bölümlerdeki anabilim dalı uygulaması anabilim dalına öğrenci alımı açısından sona ermiş ve bölüm adları tümünde bilgi ve belge yönetimi olarak değiştirilmiştir. • 2002-2003 yılında YÖK’ün aldığı karar ile bölümlerdeki anabilim dalı uygulaması anabilim dalına öğrenci alımı açısından sona ermiş ve bölüm adları tümünde bilgi ve belge yönetimi olarak değiştirilmiştir. • 2018 yılı itibariyle eğitim verilen 11 bilgi ve belge yönetimi bölümünün bulunduğu üniversitelerden İstanbul, Çankırı Karatekin ve Yıldırım Beyazıt Üniversitelerinde Arşivcilik Anabilim Dalı, Marmara Üniversitesi’nde de Tarihi Devlet Arşivleri ve Müessese Arşivleri Anabilim Dalları bulunmaktadır. Ancak bu üniversitelerde de öğrenci alımı bölümlere yapılmakta, doğrudan ilgili anabilim dallarına öğrenci alınmamaktadır. Arşivcilik anabilim dallarının bugünkü varlığı bu Bölümlerdeki öğretim elemanlarının arşivcilik konusunda özelleşmiş bilimsel çalışmalar gerçekleştirmesine olanak sağlayan bir yapılanma olarak işlev görebilmektedir.* * Tablolarda sunulan verilerin elde edildiği kaynaklar kaynakçada ayrıca verilmiştir. 3. Türkiye’de Bilgi ve Belge Yönetimi Bölümleri Güncel Ders Programlarının Arşivcilik ve Belge Yönetimi Eğitimi Açısından Değerlendirilmesi Bu bölümde yukarıda tarihsel gelişimi kısaca verilmeye çalışılan arşivcilik ve belge yönetimi eğitimi halen 11 üniversitede uygulanmakta olan bilgi ve belge yönetimi bölümleri ders programlarındaki ders sayısı ve içerikleri itibariyle değerlendirilecektir. Sözü edilen çerçevede aşağıda yapılacak değerlendirmelerde adında doğrudan arşivcilik ya da belge yönetimi terimleri geçen (Arşiv Yönetimi, Belge Yönetimi vb. gibi), içeriği arşivcilik ve belge yönetimi ile doğrudan ilgili (Elektronik Belge Yönetim Sistemi vb. gibi) ve bu alanlarla dolaylı ilgisi olan, bu alanları besleyen dersler (Osmanlı Epigrafisi, Osmanlı Dönemi İktisat Tarihi, Osmanlıca vb. gibi) arşivcilik ve belge yönetimi dersleri olarak kabul edilmiştir. 48 Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… 4. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Toplam Ders Sayıları Açısından Değerlendirilmesi Öncelikle bölümlerde lisans ve lisansüstü (yüksek lisans-doktora) programlarda yer alan toplam, zorunlu ve seçmeli ders sayılarına bakmak programların yükü ve yapısı açısından genel bir fikir vermede yararlı olacaktır.* Tablo 1. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisans Programlarındaki Ders Sayıları Tablo 1. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisans Programlarındaki Ders Sayıla *Bölümler kuruluş tarihlerine göre sıralanmıştır. Bölümler kuruluş tarihlerine göre sıralanmıştır. ş g ş **Ortak zorunlu dersler hariç bölüm kodlu zorunlu ve seçmeli dersler. (Tüm tablolar için geçerlidir.) ***Veriler 26.2.2018 tarihinde bölüm öğretim elemanları ile yapılan görüşme yoluyla elde edilmiştir. Tablo 1 verileri incelendiğinde dikkati çeken ilk nokta, Bölümlerin ders sayısı toplamlarının 50-90 gibi geniş sayılabilecek bir aralıkta yer alıyor olmasıdır. En az ders sayısına sahip Bölüm Yakın Doğu BBY (Bilgi ve Belge Yönetimi) Bölümü (49) iken, bunu Yıldırım Beyazıt Üniversitesi BBY (53) ve Hacettepe Üniversitesi BBY Bölümü (62) izlemektedir. En fazla ders sayısına sahip Bölümler ise sırasıyla Kastamonu BBY Bölümü (90), Osmaniye Korkut Ata BBY Bölümü (82) ve Ankara Üniversitesi BBY Bölümü’dür (81). Tablo 1’e bakarak, Bölümler arasında toplam ders sayıları açısından büyük farklılıklar olduğu, örneğin 50-60 gibi belirli bir aralıkta kümelenme durumunun gerçekleşmediği görülmektedir. 49 Prof. Dr. Bülent YILMAZ Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Ders sayıları açısından ilk kurulan Bölümler ile yeni kurulan bölümler arasında anlamlı bir farklılık da göze çarpmamaktadır. Bir başka deyişle, BBY bölümlerinin eski ya da yakın tarihlerde eğitime başlamış olması ile ders sayıları arasında doğru ya da ters orantılı bir ilişki bulunmamaktadır. Örneğin yakın tarihlerde kurulmuş Korkut Ata Üniversitesi BBY Bölümü’nün ders sayısı 82 iken, yine yakın bir zamanda kurulmuş olan Yıldırım Beyazıt BBY Bölümü’nün ders sayısı 53’tür. Benzer biçimde eğitime ilk başlayan üç BBY bölümü arasında yer alan Ankara Üniversitesi BBY Bölümü toplam ders sayısı 81, Hacettepe Üniversitesi BBY Bölümü lisans ders sayısı ise 62’dir. Elbette burada sıralanan ders sayıları öğrencilerin eğitim yaşamları boyunca bölümlerinde yüklenmek zorunda oldukları toplam ders sayısı değildir. Bunlar lisans programında yer alan toplam ders sayılarıdır. Öğrencilerin mezuniyetleri için aldıkları ders sayısı buradaki sayıların altında kalmaktadır. Ancak programlarda yer alan ders sayılarının azlığı ya da yüksekliği bölümlerin eğitim-öğretim yaklaşımını/tercihini, yükünü, çeşitliliğini, ders verebilme kapasitesini ve belki de güçlüğünü göstermektedir. Son yıllarda genel olarak üniversite ve özel olarak da BBY eğitiminde daha az ders yaklaşımının benimsendiği söylenebilir.* Böyle bakıldığında, halen eğitim verilen 11 BBY bölümünün %63,6’sında (7 bölümde) toplam 70 ve üzerinde ders sayısı söz konusudur. Bölümlerde ders sayısının genel olarak yüksek olduğu söylenebilir. 4. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Toplam Ders Sayıları Açısından Değerlendirilmesi Bölümlerin lisans ders programlarında zorunlu ve seçmeli ders sayıları ve yapısı da önemli bir başka noktadır. Bu konuda da son yıllarda seçmeli ders sayısının zorunlu olanlara göre yüksek olması, en azından yarı yarıya bir oranın tutturulması yaklaşımı öne çıkmaktadır. YÖK’ün geçtiğimiz 5-10 yıllık dönemde Türkiye’deki üniversitelerde Bologna Süreci adı altında uygulamaya geçirilmesini zorunlu tuttuğu yaklaşım da bunu özendirmektedir. Ancak YÖK’ün son 1-2 yılda seçmeli dersler konusunda bu süreçteki yaklaşımından uzaklaşmakta olduğu da söylenebilir. BBY bölümlerinin lisans ders programlarında yer alan toplam derslerin ne kadarının zorunlu ve ne kadarının seçmeli statüde olduğunu gösterir veriler Tablo 1’de sunulmuştur. Bu konudaki karşılaştırmaları bölümlerin ders sayısı toplamları farklı olduğu için oranlar (%) üzerinden yapmak daha gerçekçi görünmektedir. Bölümlerin lisans ders programlarındaki zorunlu statülü ders oranları %79,2 (Yıldırım Beyazıt BBY Bölümü) ile %27,4 (Hacettepe Üniversitesi BBY Bölümü) arasında değişmektedir. Lisans ders programında en yüksek oranda zorunlu statülü derse sahip ilk üç bölüm Yıldırım Beyazıt Üniversitesi BBY Bölümü (%79,2), Atatürk Üniversitesi BBY Bölümü (%64,5) ve Yakın Doğu Üniversitesi BBY Bölümü (%61,2) iken, bu konuda en düşük oranlara sahip Bölümler sırasıyla Hacettepe Üniversitesi BBY Bölümü (%27,4), Çankırı Karatekin Üniversitesi BBY Bölümü (%29,9) ve İstanbul Medeniyet Üniversitesi BBY Bölümü (%35,6)’dür. Bu çerçevede seçmeli statüsündeki ders oranı en yüksek Bölümler (Hacettepe Üniversitesi BBY Bölümü,%72,6, Çankırı Karatekin Üniversitesi BBY Bölümü, %70,1 ve İstanbul Medeniyet Üniversitesi BBY Bölümü’dür (%64,4). Seçmeli ders oranı %50 ve üzerinde olan Bölüm sayısı 6’dır. Bu sayının düşük olduğu söylenebilir. Kısaca, incelenen bölümlerde zorunlu ders oranlarının seçmeli derslere göre daha yüksek olduğu ve bölümler arasında bu konuda önemli sayılabilecek farklılıkların bulunduğu söylenebilir. Bilgi ve belge yönetimi bölümleri bağlamında arşivcilik eğitiminin lisans öğretimi yanı sıra lisansüstü (yüksek lisans ve doktora) düzeyinde de değerlendirilmesi gerekmektedir. Bu çerçevede öncelikle lisansüstü toplam ders sayıları ve bunların zorunlu ya da seçmeli statüde olma durumlarına bakılabilir. Bu konudaki veriler Tablo 2’de yer almaktadır. 50 Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Tablo 2. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisansüstü (Yüksek Lisans-Doktora) Programlarındaki Ders Sayıları Tablo 2. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisansüstü (Yüksek Lisans-Doktora) Programlarındaki Ders Sayıları Tablo 2. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisansüstü (Yüksek Lisans-Doktora) Programlarındaki Ders Sayıları * Sadece yüksek lisans programı bulunmaktadır. ** Lisansüstü programları bulunmamaktadır. ** Lisansüstü programları bulunmamaktadır. Tablo 2’de yer alan verilere göre, lisans eğitiminin bulunduğu 11 BBY bölümünün 4 tanesinde henüz lisansüstü öğretim yapılmamakta, 2 bölümde de sadece yüksek lisans eğitimi sürdürülmektedir. 4. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Toplam Ders Sayıları Açısından Değerlendirilmesi Bölümlerin lisansüstü programlarında yer alan ders sayısı toplamları lisans programlarındaki duruma benzer biçimde Bölümler arasında önemli farklılığa sahip görünmektedir. Lisansüstü programında en fazla sayıda ders bulunan Bölüm Ankara Üniversitesi BBY Bölümü (70) iken, en az sayıda derse Atatürk Üniversitesi BBY Bölümü (23) sahiptir. Bu iki Bölüm arasında yaklaşık 3 katlık fark bulunmaktadır. Bölümlerdeki lisansüstü ders sayısı kümelenmesinin ağırlıkla 40-50 bandında olduğu söylenebilir. Burada ayrıca gösterilmemesine karşın lisansüstü derslerin daha büyük çoğunluğunun tüm Bölümlerde yüksek lisans derslerinden oluştuğu söylenebilir. Elbette Bölümler arasındaki ders sayısı farklılıkları ya da lisansüstü eğitim verip vermeme büyük ölçüde öğretim elemanı sayısına bağlı olup ve bunda Bölümlerin öncelikle lisans eğitimini belirli bir olgunluğa/yeterliliğe ulaştırma tercihi de etkili olmaktadır. Lisansüstü program dersleri Bölümlerde genellikle seçmeli statüde oluşturulan derslerdir. Ancak YÖK’ün ya da Bölümlerin araştırma yöntemleri ve bilim etiği gibi okutulmasını zorunlu gördüğü/tuttuğu dersler olabilmektedir. Nitekim Tablo 2’de zorunlu statüde görünen çok az sayıdaki derslerin büyük bölümü bu nitelikte derslerdir. Dolayısıyla BBY Bölümlerinin lisansüstü programlarındaki derslerin çok büyük bölümü seçmeli derslerden oluşmaktadır. Ancak özellikle Yakın Doğu ve Atatürk Üniversitesi BBY Bölümlerindeki zorunlu ders oranının %33,3 ve %17,4 oluşu dikkat çekicidir. 51 Bilgi Yönetimi 1: 1 (2018), 44-62 Prof. Dr. Bülent YILMAZ Hakemli Makaleler 5. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Arşivcilik ve Belge Yönetimi ile ilgili Ders Sayıları Açısından Değerlendirilmesi Türkiye’de halen lisans eğitimi sürdürülen BBY Bölümlerindeki ders programlarında yer alan arşivcilik ile ilgili ders sayılarının ve bunların zorunlu/seçmeli statülerine ilişkin veriler Tablo 3’de sunulmuştur. İ 5. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Arşivcilik ve Belge Yönetimi ile ilgili Ders Sayıları Açısından Değerlendirilmesi 5. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Arşivcilik ve Belge Yönetimi ile ilgili Ders Sayıları Açısından Değerlendirilmesi Türkiye’de halen lisans eğitimi sürdürülen BBY Bölümlerindeki ders programlarında yer alan a ile ilgili ders sayılarının ve bunların zorunlu/seçmeli statülerine ilişkin veriler Tablo 3’de sunul Tablo 3. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisans Programlarında Arşivcilik ile İlgili Ders Sayıları Tablo 3. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisans Programlarında Arşivcilik ile İlgili Ders Sayıları Bu çalışmanın odaklandığı BBY Bölümlerinin ders programlarındaki arşivcilik eğitimi boyutu ders sayıları ve statüleri bağlamında ve Tablo 3 verileri çerçevesinde değerlendirilebilir görünmektedir. Buna göre, lisans programında arşivcilik ile ilgili en çok ders sayısına sahip Bölüm İstanbul Üniversitesi BBY Bölümü’dür (29). Onu, Marmara (23) ve Atatürk Üniversitesi (21) BBY Bölümleri izlemektedir. 4. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Toplam Ders Sayıları Açısından Değerlendirilmesi Arşivcilik ile ilgili en az sayıda derse sahip Bölümler ise Yakın Doğu (1), Hacettepe (5) ve Kastamonu Üniversitesi (7) BBY Bölümleridir. Arşivcilik ile ilgili en çok ve en az ders sayısı açısından Bölümler arasında (Yakın Doğu Üniversitesi BBY Bölümü’nü ayrı tutarsak) yaklaşık 6 katlık fark görülmektedir. Ancak arşivcilik ile ilgili ders sayılarına oransal olarak bakıldığında arşivcilik ile ilgili en yoğun programa Marmara Üniversitesi BBY Bölümü’nün (%39,6) sahip olduğu görülmektedir. Onu çok yakın bir oran ile İstanbul Üniversitesi BBY Bölümü (%38,1) izlemektedir. Bu iki Bölümün derslerinin yaklaşık %40’ı arşivcilik ile ilgilidir. Bu durumun bu iki Bölümden Marmara Üniversitesi BBY Bölümü’nün Arşivcilik Bölümü olarak kurulmuş olmasının, İstanbul Üniversitesi BBY Bölümü’ne ise aynı üniversite ve fakültedeki Arşivcilik Bölümü’nün dâhil edilmesinin belirleyici etkisi olduğu söylenebilir. Genel olarak bakıldığında, programında arşivcilik ile ilgili ders oranı %10’un altında 3 Bölüm, %30’un üzerinde 2 Bölüm bulunmakta ve kalan büyük çoğunluk %20’ler (beşte bir) düzeyinde yer almaktadır. BBY Bölümlerinin arşivcilik ile ilgili ders sayıları ve oranları dışında, bu derslerin zorunlu ya da seçmeli statüde oluşları da büyük önem taşımaktadır. Arşivcilik ile ilgili derslerin zorunlu ya da seçmeli statüde 52 Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… olmaları açısından Bölümler arasında farklılıklar bulunmaktadır. Buna göre, bazı Bölümlerde arşivcilik ile ilgili zorunlu dersler bazılarında ise seçmeli dersler çoğunluktadır. 6 Bölümde zorunlu statüdeki arşivcilik ders sayısı ve oranı, 5 Bölümde ise seçmeli statüdeki ders sayısı ve oranı yüksek olup, 1 Bölümde eşit görünmektedir. Zorunlu arşivcilik ders sayısı/oranı en yüksek Bölüm Marmara Üniversitesi (%25,9) ve İstanbul Üniversitesi (%19,7) BBY Bölümleri, oransal olarak seçmeli statüde en fazla derse sahip Bölümler de İstanbul (%18,4) ve Ankara Üniversitesi (%16) BBY Bölümleridir. Genel olarak söylemek gerekirse, lisans programlarında yer alan arşivcilik ile ilgili ders sayıları/oranları ve bu derslerin zorunlu ya da seçmeli statüleri açısından BBY Bölümleri arasında farklılıklar bulunmaktadır. Arşivcilik Bölümü olarak kurulan ve Arşivcilik Bölümü kendisine dâhil edilen iki Bölüm dışındaki Bölümler arasında da bu farklılık görülmektedir. Arşivcilik eğitimine BBY Bölümlerinin lisansüstü programları açısından bakmak da gerekmektedir. Bu duruma ilişkin veriler Tablo 4’te yer almaktadır. Arşivcilik eğitimine BBY Bölümlerinin lisansüstü programları açısından bakmak da gerekmekt duruma ilişkin veriler Tablo 4’te yer almaktadır. ş ğ p g ç g duruma ilişkin veriler Tablo 4’te yer almaktadır. Tablo 4. Tablo 4. Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisansüstü Programlarında Arşivcilik ve Belge Yönetimi ile İlgili Ders Sayıları 4. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Toplam Ders Sayıları Açısından Değerlendirilmesi Türkiye’deki Bilgi ve Belge Yönetimi Bölümleri Lisansüstü Programlarında Arşivcilik ve Belge Yönetimi ile İlgili Ders Sayıları * S d ük k li b l k d D k k Tablo 4 verilerine göre, lisansüstü programa sahip 7 BBY Bölümü içinde hem sayısal hem de oransal olarak en fazla arşivcilik dersine sahip Bölüm İstanbul Üniversitesi BBY Bölümü’dür (21-%46,7). Bu Bölümün arşivcilik ile ilgili ders sayısının programda yer alan toplam ders sayısına oranı %50’ye yaklaşmaktadır. Bir başka deyişle, İstanbul Üniversitesi BBY Bölümü lisansüstü programı yarı yarıya bir arşivcilik programıdır. Bu Bölümü üçte bire yakın oranla Marmara Üniversitesi BBY Bölümü (%35) izlemektedir. Ankara ve Atatürk Üniversitesi BBY 53 Prof. Dr. Bülent YILMAZ Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Bölümleri de %20’nin üzerinde arşivcilik ders oranına sahip görünmektedir. Hacettepe, Çankırı Karatekin ve Yakın Doğu Üniversiteleri BBY Bölümlerinin lisansüstü düzeyde de arşivcilik ile ilgili yönelimlerinin zayıf olduğu söylenebilir. Arşivcilik ile ilgili lisansüstü derslerin zorunlu/seçmeli statüleri bağlamında değerlendirme yapmaya olanak sağlayacak çok fazla veri görünmemektedir. Çünkü lisansüstü dersler daha önce de belirtildiği üzere genelde seçmeli statüde oluşturulan derslerdir. Tablo 4’e bakıldığında bu durum açıkça görülmektedir. Atatürk Üniversitesi BBY Bölümü’ndeki zorunlu 1 ders dışında zorunlu arşivcilik dersine sahip Bölüm bulunmamakta, arşivcilik derslerinin tümü seçmeli statüde yer almaktadır. Bu konuda Bölümler arasında büyük bir benzerlik görülmektedir. 6. Bilgi ve Belge Yönetimi Bölümleri Lisans ve Lisansüstü Programlarının Arşivcilik ve Belge Yönetimi ile ilgili Ders İçerikleri Açısından Değerlendirilmesi Buraya kadar BBY Bölümlerinin lisans ve lisansüstü programları toplam ders sayıları, arşivcilik ile ilgili ders sayıları, bunların zorunlu ya da seçmeli statüde oluşu açılarından değerlendirilmiştir. Programlarda yer alan arşivcilik ile ilgili ders içerikleri hakkında da bazı saptamalar yapmak çalışmanın bütünlüğü açısından yararlı olacaktır. Bu çerçevede BBY Bölümlerinin lisans ve lisansüstü programlarında yer alan arşivcilik ile ilgili derslerin içerikleri konusunda bazı noktalar dikkat çekmektedir. • Buna göre, bölümlerin hem lisans hem de lisansüstü programlarında Osmanlıca, Osmanlı Paleografyası, Osmanlı İdari Yapısı, Yazma ve Nadir Eserler, Belge Yönetimi, Arşiv Hizmetleri, Belge Yönetimine Giriş, Elektronik Belge Yönetim Sistemi-EBYS, Dosyalama Sistemleri, Arşiv Kaynakları, Diplomatik, Koruma ve Restorasyon, E-Devlet Uygulamaları, Özel Arşivler gibi benzer dersler yer almaktadır. • Ancak, özellikle arşivcilik ile ilgili ders sayısı fazla olan bölümlerde diğerlerine göre Osmanlı Matbaacılığı, Arşivcilik Terminolojisi, Osmanlı Belgelerinin Sosyo-Kültürel Analizi, Tarihi Belge Türleri gibi özel uzmanlık gerektiren farklı dersler de görülmektedir. • Bazı BBY bölümleri program yapıları itibariyle ağırlıkla geleneksel arşivcilik ya da modern belge yönetimi alanına yoğunlaşmış, bazıları da her iki alana yönelik ders programları geliştirmeye çalışmıştır. • Bölümlerin lisans ve lisansüstü programları arşivcilik dersleri açısından genelde benzer yapıya sahip görünmektedir. Daha açık bir deyişle, arşivcilik dersleri açısından görece güçlü bir lisans programına sahip bölümün lisansüstü programları da bu yönüyle güçlü olabilmektedir. Ya da tam tersi durum söz konusudur. Güçsüz arşivcilik lisans programına sahip bölümün genelde lisansüstü programı da bu açıdan güçsüz olmaktadır. • Özellikle Osmanlıca ve onu bütünleyen paleografya gibi dil dersleri arşivciliğe belirli bir ağırlık verme tercihi olan tüm bölümlerde genelde zorunlu statüde yer almaktadır. • Bölümlerin lisans ve lisansüstü programlarında arşivcilik-belge yönetimi ile ilgili ders sayılarını ve içeriklerini belirleyen en önemli unsur bu konuda yetişmiş öğretim elemanı sayısı ve onların uzmanlık alanlarıdır. Bu alanda öğretim elemanı fazla olan bölümlerin arşivcilik ile ilgili açtığı ders sayısı fazla olabilmektedir. Bu konudaki ders çeşitliliği de öğretim elemanlarının uzmanlık alanlarına bağlı olarak değişebilmektedir. • Bölümlerin lisans ve lisansüstü programlarında arşivcilik-belge yönetimi ile ilgili ders sayılarını ve içeriklerini belirleyen en önemli unsur bu konuda yetişmiş öğretim elemanı sayısı ve onların uzmanlık alanlarıdır. Bu alanda öğretim elemanı fazla olan bölümlerin arşivcilik ile ilgili açtığı ders sayısı fazla olabilmektedir. Bu konudaki ders çeşitliliği de öğretim elemanlarının uzmanlık alanlarına bağlı olarak değişebilmektedir. . Bilgi ve Belge Yönetimi Bölümlerinin Öğretim Üyesi Sayıları Açısından Değerlendirilmesi 7. Bilgi ve Belge Yönetimi Bölümlerinin Öğretim Üyesi Sayıları Açısından Değerlendirilmesi BBY bölümlerinin arşivcilik/belge yönetimi programlarının niteliğini ve yürütülmesini doğrudan etkileyecek en önemli unsurlardan birisi de kuşkusuz dersler veren öğretim üyelerinin sayısıdır. Bölümlerde öğretim üyesi başına düşen ders sayısı çerçevesinde oluşturulan tablo (Tablo 5) aşağıdadır. 7. Bilgi ve Belge Yönetimi Bölümlerinin Öğretim Üyesi Sayıları Açısından Değerlendirilmesi BBY bölümlerinin arşivcilik/belge yönetimi programlarının niteliğini ve yürütülmesini doğrudan etkileyecek en önemli unsurlardan birisi de kuşkusuz dersler veren öğretim üyelerinin sayısıdır. Bölümlerde öğretim üyesi başına düşen ders sayısı çerçevesinde oluşturulan tablo (Tablo 5) aşağıdadır. 54 Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… *P f D D Öğ ti Ü i Tablo 5. Bilgi ve Belge Yönetimi Bölümlerinin Öğretim Üyesi Sayıları (2018) Tablo 5. Bilgi ve Belge Yönetimi Bölümlerinin Öğretim Üyesi Sayıları (2018) Kaynaklar: Tablo 5. Bilgi ve Belge Yönetimi Bölümlerinin Öğretim Üyesi Sayıları (2018) Kaynaklar: http://bilgibelge.humanity.ankara.edu.tr/akademik-personel-2/ http://bilgibelge.edebiyat.istanbul.edu.tr/tr/akademikkadro http://www.bby.hacettepe.edu.tr/akper.asp http://bby.fef.marmara.edu.tr/akademik-kadro/ https://atauni.edu.tr/personel/bilgi-ve-belge-yonetimi https://fef.kastamonu.edu.tr/index.php/tr/menu-personel-tr/menu-personel-akademik https://bilgibelge.karatekin.edu.tr/akademik-personel-1400-sayfasi.karatekin http://www.ybu.edu.tr/insanvetoplum/bilgi-belge/custom_page-292-akademik-kadro.html http://bby.osmaniye.edu.tr/personel-akademik Kaynaklar: http://bilgibelge.humanity.ankara.edu.tr/akademik-personel-2/ http://bilgibelge.edebiyat.istanbul.edu.tr/tr/akademikkadro http://www.bby.hacettepe.edu.tr/akper.asp http://bby.fef.marmara.edu.tr/akademik-kadro/ https://atauni.edu.tr/personel/bilgi-ve-belge-yonetimi https://fef.kastamonu.edu.tr/index.php/tr/menu-personel-tr/menu-personel-akademik https://bilgibelge.karatekin.edu.tr/akademik-personel-1400-sayfasi.karatekin http://www.ybu.edu.tr/insanvetoplum/bilgi-belge/custom_page-292-akademik-kadro.html http://bby.osmaniye.edu.tr/personel-akademik y http://bilgibelge.humanity.ankara.edu.tr/akademik-personel-2/ http://bilgibelge.edebiyat.istanbul.edu.tr/tr/akademikkadro http://www.bby.hacettepe.edu.tr/akper.asp http://bby.fef.marmara.edu.tr/akademik-kadro/ https://atauni.edu.tr/personel/bilgi-ve-belge-yonetimi https://fef.kastamonu.edu.tr/index.php/tr/menu-personel-tr/menu-personel-akademik https://bilgibelge.karatekin.edu.tr/akademik-personel-1400-sayfasi.karatekin http://www.ybu.edu.tr/insanvetoplum/bilgi-belge/custom_page-292-akademik-kadro.htm http://bby.osmaniye.edu.tr/personel-akademik Tablo 5 verilerine göre dikkat çekilecek ilk nokta öğretim üyesi sayısı açısından bölümler arasında ciddi farklılıkların olmasıdır. Bu farklılık özellikle ilk kurulan dört bölüm ile daha sonra kurulanlar arasında açık biçimde göze çarpmaktadır. Elbette öğretim üyesi başına düşen ders sayılarının genel olarak 10 ve üzeri olduğu görülmektedir. Bu sayıların yüksek olduğu söylenebilir. Lisans eğitimine en son başlayan Korkut Ata Üniversitesi BBY Bölümü’ndeki tek öğretim üyesi nedeniyle sayının 82 olduğu, bu durumun sürdürülebilir olmadığı ve değişeceği düşünülmektedir. Öğretim üyesi başına düşen lisans ve lisansüstü ortalama ders sayısının en düşük olduğu iki bölümün (Marmara Üniversitesi BBY ve İstanbul Üniversitesi) öğretim üyesi sayısı en yüksek iki bölüm olması anlaşılır bir durumdur. Kuşkusuz bu bölümlerdeki öğretim üyelerinin sayısı kadar onların ne kadarının arşivcilik/belge yönetimi alanında uzmanlaşmış oldukları da önemlidir. Bölümlerin çoğunda bu oranın çok yüksek olmadığı tahmin edilebilir. Dolayısıyla, bölümlerde arşivcilik/belge yönetimi eğitimi açısından bir olumsuzluğu ortaya koyan unsurlardan birisi de öğretim üyesi başına düşen ortalama ders sayısı ve yüküdür. 8. Sonuç ve Öneriler Çalışmada sunulan veriler çerçevesinde şu sonuçlar elde edilmiştir: Çalışmada sunulan veriler çerçevesinde şu sonuçlar elde edilmiştir: 1. Genel olarak söylemek gerekirse BBY Bölümlerinin lisans programlarında yer alan toplam ve zorunlu ders sayılarının özellikle öğretim üyesi başına düşen ortalama ders sayıları bağlamında yüksek olduğu söylenebilir. 2. BBY bölümlerinin lisans programlarında yer alan toplam ders sayıları birbirlerine göre büyük farklılıklar göstermekte ders sayısı 49 ile 90 arasında değişmektedir. Benzer durum lisansüstü programlar için de geçerli görünmektedir. Kısaca, bölümlerin gerek lisans gerekse lisansüstü programlarındaki ders sayıları farklıdır. 3. Bölümlerin lisans ders programlarındaki zorunlu ve seçmeli ders sayıları ve oranları da birbirlerine göre farklı görünmektedir. Zorunlu ders oranı %27,4 ile %79,2 arasında değişmektedir. Bu büyük oransal farklılık seçmeli ders oranları için de söz konusudur. Bu 55 Prof. Dr. Bülent YILMAZ Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler durum lisansüstü programlarda bu programların yapısı gereği farklı olup, zorunlu ders istisnai gibidir. durum lisansüstü programlarda bu programların yapısı gereği farklı olup, zorunlu ders istisnai gibidir. 4. Lisans programlarındaki toplam, zorunlu ve seçmeli ders sayıları ve oranları ile BBY bölümlerinin eski ya da yakın tarihlerde kurulmuş olmaları arasında doğru ya da ters orantılı bir ilişki bulunmamaktadır. Yani, eski ya da yeni eğitime başlanmış olması bölümlerin toplam ve zorunlu ders sayılarında belirleyici değildir. 5. 2018 yılı itibariyle BBY bölümlerinin tümünde lisansüstü eğitim başlatılamamıştır. Lisans eğitiminin bulunduğu 11 BBY bölümünün 4 tanesinde henüz lisansüstü öğretim yapılmamakta, 2 bölümde de sadece yüksek lisans eğitimi sürdürülmektedir. Bu durumun özellikle yeni kurulmuş bölümler için daha çok söz konusu olduğu söylenebilir. 6. Lisans programlarındaki arşivcilik ile ilgili ders sayıları ya da oranları açısından BBY bölümleri arasında 6 kata yaklaşan fark bulunmaktadır. Programlarında arşivciliğe “çok” ağırlık veren ya da “az” ağırlık veren bölümler vardır. Benzer durumun lisansüstü programlar için de söz konusu olduğu söylenebilir. Bu konuda bölümlerdeki öğretim üyesi sayısı ve onların uzmanlık alanları çeşitliliği en belirleyici neden olarak düşünülebilir. 7. Bölümlerin programlarında yer alan arşivcilik/belge yönetimi ile ilgili ders sayılarına, bu sayıların toplam ders sayısına oranlarına ve ders içeriklerine bakılarak arşivcilik/belge yönetimi eğitiminin oranı ve yönelimi konusunda bir tercih ya da eğilim içinde oldukları da söylenebilir. 7. Bölümlerin programlarında yer alan arşivcilik/belge yönetimi ile ilgili ders sayılarına, bu sayıların toplam ders sayısına oranlarına ve ders içeriklerine bakılarak arşivcilik/belge yönetimi eğitiminin oranı ve yönelimi konusunda bir tercih ya da eğilim içinde oldukları da söylenebilir. 8. 8. Sonuç ve Öneriler Kuruluş amaçları, geleneksel yapıları, öğretim elemanlarının uzmanlık alanları bölümler için kendilerine özgü bir “ekol” geliştirmelerine neden olabilmekte ve bu da arşivcilik eğitimine yaklaşımlarını etkilemektedir. Bu elbette bölümlerin bilinçli tercihleri sonucu da olabilir ve yanlış olarak değerlendirilemez. Hatta bölümlerin arşivcilik ile ilgili ders sayıları ve yapıları incelendiğinde arşivcilik açısından dahi farklı tercihlerin olduğu görülmekte, bazı bölümler geleneksel ve geniş çerçeveli arşivcilik alanına bazı bölümler ise kurumsal arşiv ve belge yönetimi alanına yatırım yapmayı tercih edebilmektedir. 9. Bölümler arasında arşivcilik ile ilgili derslerin zorunlu ya da seçmeli statüde olmaları açısından da önemli denilebilecek oranda farklar bulunmaktadır. Lisans programları düzeyinde bölümlerin yaklaşık yarısında zorunlu arşivcilik dersleri diğer yarısında ise seçmeli arşivcilik dersleri daha fazla görünmektedir. 10. Bir Bölümün lisans programı arşivcilik açısından güçlü ise lisansüstü programının da arşivcilik yanı güçlü görünmektedir. Bir başka deyişle, bir BBY bölümünün lisans arşivcilik programının güçlü olup da lisansüstü programının zayıf olma durumu ya da tersi genelde söz konusu olmamaktadır. Bölümlerin lisans ve lisansüstü programları arasında bu anlamda doğal olarak bir paralellik bulunmaktadır. 11. Bölümün Arşivcilik Bölümü olarak kurulmuş ve/veya BBY bölümüne dâhil edilmiş olması o Bölüm programlarındaki arşivcilik ile ilgili ders sayısını ve oranını yükseltme anlamında etkili olmaktadır. 12. Bölümlerin hem lisans hem de lisansüstü programlarında arşivcilik ile ilgili genelde benzer ad ve içerikte dersler yer almaktadır. Ancak arşivcilik ile ilgili ders programı güçlü bölümlerde farklı içerikte ve uzmanlık gerektiren dersler de bulunmaktadır. 13. Bazı BBY bölümleri program yapıları itibariyle ağırlıkla geleneksel arşivcilik ya da modern belge yönetimi alanına yoğunlaşmış, bazıları da her iki alana yönelik ders programları geliştirmeye çalışmıştır. 14. BBY bölümlerinin gerek arşivcilik ile ilgili ders sayılarını gerekse ders çeşitliliğini/içeriği belirleyen en önemli etken yeterli sayıda ve nitelikte öğretim elemanıdır. Bu konuda bölümlerin konuya yaklaşımları ve tercihleri de etkili olabilmektedir. 14. BBY bölümlerinin gerek arşivcilik ile ilgili ders sayılarını gerekse ders çeşitliliğini/içeriği belirleyen en önemli etken yeterli sayıda ve nitelikte öğretim elemanıdır. Bu konuda bölümlerin konuya yaklaşımları ve tercihleri de etkili olabilmektedir. 14. BBY bölümlerinin gerek arşivcilik ile ilgili ders sayılarını gerekse ders çeşitliliğini/içeriği belirleyen en önemli etken yeterli sayıda ve nitelikte öğretim elemanıdır. Bu konuda bölümlerin konuya yaklaşımları ve tercihleri de etkili olabilmektedir. 8. Sonuç ve Öneriler 56 Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… Kısaca, Türkiye’de BBY bölümleri arasında lisans ve lisansüstü programlarında gerek toplam ve bunun içinde zorunlu/seçmeli ders sayıları gerekse arşivcilik ve belge yönetimi ile ilgili ders sayıları ve bunların zorunlu ya da seçmeli oluşları açısından benzer bir eğilimden/yapıdan söz etmek olanaklı değildir. Bölümlerin BBY eğitimi içinde arşivcilik eğitimine ilişkin yaklaşım ve tercihleri farklıdır. Bunun olumlu ya da olumsuz oluşu ayrı bir tartışma konusudur. Ancak üniversite düzeyinde eğitim veren bölümlerin belirli bazı ortak noktalara ve temel alan bilgisini verme sorumluluğuna sahip olmakla birlikte “aynı” ya da “benzer” olmalarını beklemek/istemek olanaklı ve hatta doğru olmayacaktır. Çünkü her bölüm kendine özgü tarihsel ve yapısal nedenlerle bir ekolü temsil eder ve ders programlarında ağırlık vereceği ya da vermeyeceği konular hakkında tercih yapma hakkına sahiptir. Ayrıca, bu konuda şöyle bir tez/yaklaşım da söz konusu olabilir: Kütüphanecilik, arşivcilik ve dokümantasyon-enformasyon alanları özünde “bilgi yönetimi” olarak adlandırılan ortak bir alanın parçalarıdır. Bu nedenle bunlar için ayrı programlar geliştirilmesine gerek yoktur. Hepsi için ortak ve zorunlu bazı dersler oluşturulur, ortak alan bilgisi verilir; bu yeterlidir. Özellikle, son 20-30 yılda görülen bilgi ve iletişim teknolojileri bu üç alanı birbirine iyice yakınlaştırmış ve hatta birleştirmiştir. Dolayısıyla çok fazla sayıda ders içeren arşivcilik programlarına gerek bulunmamaktadır. Kuşkusuz bu yaklaşım da tartışılabilir görünmektedir ve nitekim Türkiye’de yıllardır da tartışılmaktadır. Ancak, yukarıda sıralanan sonuçlar çerçevesinde “Türkiye’de BBY bölümlerinde verilen arşivcilik/belge yönetimi eğitiminin ders sayısı ve içeriği açısından genel olarak zayıf olduğu” belirmesini yapmak yanlış olmayacaktır. Gerek tarihi gerek kurumsal arşivler ve gerekse belge yönetimi anlamında teknolojiye dayalı çok hızlı gelişmeler göz önüne alındığında bu alanda BBY bölümlerinden doğal olarak hazırlıklı olmaları, bu gelişme ve değişmelere programları ile yanıt verebilmeleri ve gereksinim duyulan nitelikli insan gücünü yetiştirmeleri beklenmektedir. Bunu gerçekleştirebilmeleri için bölümlerin; Gerek tarihi gerek kurumsal arşivler ve gerekse belge yönetimi anlamında teknolojiye dayalı çok hızlı gelişmeler göz önüne alındığında bu alanda BBY bölümlerinden doğal olarak hazırlıklı olmaları, bu gelişme ve değişmelere programları ile yanıt verebilmeleri ve gereksinim duyulan nitelikli insan gücünü yetiştirmeleri beklenmektedir. 8. Sonuç ve Öneriler Bunu gerçekleştirebilmeleri için bölümlerin; • Arşivcilik eğitimi konusundaki yaklaşımlarını ve tercihlerini iyi belirlemeleri ve netleştirmeleri, bu konuda bir bölüm stratejisine/politikasına sahip olmaları; • Lisans ve lisansüstü programlarını arşivcilik yönünden belirli aralıklarla gözden geçirip, güncellemeleri; • Programlarında arşivcilik açısından güçlü ve zayıf yanları belirlemeleri; • Arşivcilik eğitiminin bölüm mü yoksa anabilim dalı yapılanması içinde mi sürdürülmesinin doğru olacağını tartışmaları; • Arşivcilik konusunda öğretim elemanı/akademik uzman yetiştirme çabalarını ve bu konudaki akademik eleman sayılarını artırmaları; • Arşivcilik ve belge yönetimi ile ilgili çalışma piyasasını ve kuruluşları sürekli değerlendirmeleri; • Bu konuda yurt dışındaki gelişme ve değişmeleri izlemeleri; • Bu konuda yurt dışındaki gelişme ve değişmeleri izlemeleri; • Arşivcilik ve belge yönetimi ile ilgili kurum ve kuruluşlarla güçlü bir iletişim ve işbirliği içinde olmaları; • Arşivlere yönelik öğrenci staj programlarını sürekli değerlendirmeleri; • Kendi aralarında (bölümler arası) düzenli değerlendirme toplantıları yapmaları; • Arşivcilik/belge yönetimi konularında çeşitli düzeylerdeki akademik çalışmaları sürdürmeleri ve artırmaları; • Arşivlerle ortak projeler gerçekleştirmeleri gerekmektedir. Kuşkusuz bazı bölümler yukarıda sıralanan önerilerde dile getirilen gerekliliklere sahiptir ya da bu gereklilikleri yerine getirebilir durumda olabilir. 57 57 Prof. Dr. Bülent YILMAZ Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Arşivcilik ve belge yönetimi BBY alanı için güçlü bir gelecek vaadeden ama aynı zamanda eğer yeterli eğitim altyapısı oluşturulmazsa büyük sorunlar yaratacak/yaşatacak bir konudur. BBY bölümlerinin bu konudaki sorumluluklarının farkında olmaları ve bu doğrultuda davranmaları zorunlu görünmektedir. Kaynakça Akbulut, M. (2014). Pittsburgh Üniversitesi kütüphanecilik eğitiminin DTCF Kütüphanecilik Bölümü’ndeki eğitime yansımaları. N. Özel ve N. Er-koçoğlu (Yay. haz.). Ankara Üniversitesi Bilgi ve Belge Yönetimi Bölümü 60.Yıl Armağan Kitabı içinde (s. 15-20). Ankara: Ankara Üniversitesi DTCF Bilgi ve Belge Yönetimi Bölümü. Anabilim Dalları. (2018). Erişim adresi: http://bby.fef.marmara.edu.tr/anabilim-dallari/ Atılgan, D. (1999). 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Ankara: AÜ DTCF 60 Bilgi Yönetimi 1: 1 (2018), 44-62 Hakemli Makaleler Türkiye’de Bilgi ve Belge Yönetimi Bölümleri… Bilgi ve Belge Yönetimi Bölümü. Tonta, Y. (2012a). Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü lisans programının güncelleştirilmesi (2009-2011). Türk Kütüphaneciliği, 26(2), 291- 314. Tonta, Y. (2012b). Kütüphanecilik ve bilgibilim eğitiminde gelişmeler ve program değişiklikleri. Türk Kütüphaneciliği, 26(2), 227-261. Tuncer, N. (2007). Kütüphanecilik eğitiminde değişen ufuklar: Hacettepe Üniversitesi Kütüphanecilik Bölümünün ilk onbeş yılı. S. Kurbanoğlu, Y. Tonta ve U. Al (Yay. haz.). Değişen Dünyada Bilgi Yönetimi Sempozyumu, 24-26 Ekim 2007, Ankara, Bildiriler içinde (s. 13-18). Ankara: Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü. Yılmaz, B. (2014). Türkiye’de Bilgi ve Belge Yönetimi eğitiminde Hacettepe Üniversitesi deneyimi. N. Özel ve N. Er-koçoğlu (Yay. haz.). Ankara Üniversitesi Bilgi ve Belge Yönetimi Bölümü 60.Yıl Armağan Kitabı içinde (s. 93-108). Ankara: Ankara Üniversitesi DTCF Bilgi ve Belge Yönetimi Bölümü. Bilgi ve Belge Yönetimi Bölümü. Tonta, Y. (2012a). Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü lisans programının güncelleştirilmesi (2009-2011). Türk Kütüphaneciliği, 26(2), 291- 314. Tonta, Y. (2012b). Kütüphanecilik ve bilgibilim eğitiminde gelişmeler ve program değişiklikleri. Türk Kütüphaneciliği, 26(2), 227-261. Tuncer, N. (2007). Kütüphanecilik eğitiminde değişen ufuklar: Hacettepe Üniversitesi Kütüphanecilik Bölümünün ilk onbeş yılı. S. Kurbanoğlu, Y. Tonta ve U. Al (Yay. haz.). Değişen Dünyada Bilgi Yönetimi Sempozyumu, 24-26 Ekim 2007, Ankara, Bildiriler içinde (s. 13-18). Ankara: Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü. Yılmaz, B. (2014). Türkiye’de Bilgi ve Belge Yönetimi eğitiminde Hacettepe Üniversitesi deneyimi. N. Özel ve N. Er-koçoğlu (Yay. haz.). Ankara Üniversitesi Bilgi ve Belge Yönetimi Bölümü 60.Yıl Armağan Kitabı içinde (s. 93-108). Ankara: Ankara Üniversitesi DTCF Bilgi ve Belge Yönetimi Bölümü. Tonta, Y. (2012a). Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü lisans programının güncelleştirilmesi (2009-2011). Türk Kütüphaneciliği, 26(2), 291- 314. Tonta, Y. (2012a). Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü lisans programının güncelleştirilmesi (2009-2011). Türk Kütüphaneciliği, 26(2), 291- 314. Tonta, Y. (2012b). Kütüphanecilik ve bilgibilim eğitiminde gelişmeler ve program değişiklikleri. Türk Kütüphaneciliği, 26(2), 227-261. Kaynakça Tuncer, N. (2007). Kütüphanecilik eğitiminde değişen ufuklar: Hacettepe Üniversitesi Kütüphanecilik Bölümünün ilk onbeş yılı. S. Kurbanoğlu, Y. Tonta ve U. Al (Yay. haz.). Değişen Dünyada Bilgi Yönetimi Sempozyumu, 24-26 Ekim 2007, Ankara, Bildiriler içinde (s. 13-18). Ankara: Hacettepe Üniversitesi Bilgi ve Belge Yönetimi Bölümü. Yılmaz, B. (2014). Türkiye’de Bilgi ve Belge Yönetimi eğitiminde Hacettepe Üniversitesi deneyimi. N. Özel ve N. Er-koçoğlu (Yay. haz.). Ankara Üniversitesi Bilgi ve Belge Yönetimi Bölümü 60.Yıl Armağan Kitabı içinde (s. 93-108). Ankara: Ankara Üniversitesi DTCF Bilgi ve Belge Yönetimi Bölümü. Yılmaz, B. (2014). Türkiye’de Bilgi ve Belge Yönetimi eğitiminde Hacettepe Üniversitesi deneyimi. N. Özel ve N. Er-koçoğlu (Yay. haz.). Ankara Üniversitesi Bilgi ve Belge Yönetimi Bölümü 60.Yıl Armağan Kitabı içinde (s. 93-108). Ankara: Ankara Üniversitesi DTCF Bilgi ve Belge Yönetimi Bölümü. Tablolar için Kaynakça 61 Tablolar için Kaynakça Bilgi ve Belge Yönetimi (Yüksek Lisans/Tezli). (2017). Erişim adresi: http://bbs.karatekin.edu.tr/derskatalog.aspx?bkod=07&fkod=83 Bilgi ve Belge Yönetimi. (2017). Erişim adresi: http://bbs.karatekin.edu.tr/derskatalog.aspx?bkod=06&fkod=06 Bilgi ve Belge Yönetimi Bölümü. (2014). Erişim adresi: http://old.neu.edu.tr/tr/node/539 Bilgi ve Belge Yönetimi Bölümü Dersler. (2012). Erişim adresi: https://neu.edu.tr/akademik/fakulteler/iktisadi-ve-idari-bilimler-fakultesi/bolumler/bilgi-ve- belge-yonetimi-bolumu/dersler/?lang=tr Bilgi ve Belge Yönetimi Müfredat Dersleri ve AKTS Kredileri. (t.y.-a). Erişim adresi: http://eobs.atauni.edu.tr/Program/P3.aspx Bilgi ve Belge Yönetimi Müfredat Dersleri ve AKTS Kredileri. (t.y-b). Erişim adresi: http://eobs.atauni.edu.tr/Program/P3.aspx Bilgi ve Belge Yönetimi Müfredat Tablosu. (2017). Erişim adresi: http://www.ybu.edu.tr/insanvetoplum/bilgi- belge/contents/files/4%20y%C4%B1ll%C4%B1k%20m%C3%BCfredat%2022_09_2017.pdf Bilgi ve Belge Yönetimi, Doktora Programı, (Örgün Öğretim). (2017). Erişim adresi: http://ebs.istanbul.edu.tr/home/program?id=2153 Bilgi ve Belge Yönetimi, Lisans Programı, (Örgün Öğretim). (2017). Erişim adresi: http://ebs.istanbul.edu.tr/home/dersprogram/?id=1091&yil=2017 Bilgi ve Belge Yönetimi, Tezli Yüksek Lisans Programı, (Örgün Öğretim). (2017). Erişim adresi: http://ebs.istanbul.edu.tr/home/program?id=1694 Bilgi ve Belge Yönetimi Programı. (t.y.). Erişim adresi: http://akts.hacettepe.edu.tr/program_detay.php?birim_ref=AKDBRM_0000000000000000000000076 &birim_kod=396&prg_oid=PRGRAM_0000000000000000000000085&prg_kod=396&programd uzey=2&submenuheader=2&dil_kod=1 Ders Programı & AKTS Kredileri. (t.y.-a). Erişim adresi: http://bbs.ankara.edu.tr/Ders_Plani.aspx?bno=3515&bot=213 Ders Programı & AKTS Kredileri. (t.y.-b). Erişim adresi: http://bbs.ankara.edu.tr/Ders_Plani.aspx?bno=3515&bot=748 Ders Programı & AKTS Kredileri. (t.y.-c). Erişim adresi: http://bbs.ankara.edu.tr/Ders_Plani.aspx?bno=3516&bot=1089 61 Bilgi ve Belge Yönetimi (Yüksek Lisans/Tezli). (2017). Erişim adresi: http://bbs.karatekin.edu.tr/derskatalog.aspx?bkod=07&fkod=83 Bilgi ve Belge Yönetimi. (2017). Erişim adresi: http://bbs.karatekin.edu.tr/derskatalog.aspx?bkod=06&fkod=06 Bilgi ve Belge Yönetimi Bölümü. (2014). Erişim adresi: http://old.neu.edu.tr/tr/node/539 Bilgi ve Belge Yönetimi Bölümü Dersler. (2012). Erişim adresi: https://neu.edu.tr/akademik/fakulteler/iktisadi-ve-idari-bilimler-fakultesi/bolumler/bilgi-ve- belge-yonetimi-bolumu/dersler/?lang=tr Bilgi ve Belge Yönetimi Müfredat Dersleri ve AKTS Kredileri. (t.y.-a). Erişim adresi: http://eobs.atauni.edu.tr/Program/P3.aspx Bilgi ve Belge Yönetimi Müfredat Dersleri ve AKTS Kredileri. (t.y-b). Erişim adresi: http://eobs.atauni.edu.tr/Program/P3.aspx Bilgi ve Belge Yönetimi Müfredat Tablosu. (2017). Erişim adresi: http://www.ybu.edu.tr/insanvetoplum/bilgi- belge/contents/files/4%20y%C4%B1ll%C4%B1k%20m%C3%BCfredat%2022_09_2017.pdf Bilgi ve Belge Yönetimi, Doktora Programı, (Örgün Öğretim). (2017). Erişim adresi: http://ebs.istanbul.edu.tr/home/program?id=2153 Bilgi ve Belge Yönetimi, Lisans Programı, (Örgün Öğretim). (2017). Erişim adresi: http://ebs.istanbul.edu.tr/home/dersprogram/?id=1091&yil=2017 Bilgi ve Belge Yönetimi, Tezli Yüksek Lisans Programı, (Örgün Öğretim). (2017). Erişim adresi: http://ebs.istanbul.edu.tr/home/program?id=1694 Bilgi ve Belge Yönetimi Programı. (t.y.). Erişim adresi: http://akts.hacettepe.edu.tr/program_detay.php?birim_ref=AKDBRM_0000000000000000000000076 &birim_kod=396&prg_oid=PRGRAM_0000000000000000000000085&prg_kod=396&programd uzey=2&submenuheader=2&dil_kod=1 Ders Programı & AKTS Kredileri. (t.y.-a). Erişim adresi: http://bbs.ankara.edu.tr/Ders_Plani.aspx?bno=3515&bot=213 Ders Programı & AKTS Kredileri. (t.y.-b). Erişim adresi: http://bbs.ankara.edu.tr/Ders_Plani.aspx?bno=3515&bot=748 Ders Programı & AKTS Kredileri. (t.y.-c). Erişim adresi: http://bbs.ankara.edu.tr/Ders_Plani.aspx?bno=3516&bot=1089 Bilgi ve Belge Yönetimi (Yüksek Lisans/Tezli). (2017). Erişim adresi: Bilgi ve Belge Yönetimi (Yüksek Lisans/Tezli). (2017). Erişim adresi: Bilgi ve Belge Yönetimi Müfredat Dersleri ve AKTS Kredileri. (t.y.-a). Erişim adresi: http://eobs.atauni.edu.tr/Program/P3.aspx Bilgi ve Belge Yönetimi Müfredat Dersleri ve AKTS Kredileri. (t.y-b). Erişim adresi: htt // b t i d t /P /P3 Bilgi ve Belge Yönetimi Müfredat Tablosu. (2017). Erişim adresi: 61 Bilgi Yönetimi 1: 1 (2018), 44-62 Prof. Dr. Bülent YILMAZ Hakemli Makaleler Genel Bilgiler. (2016). Erişim adresi: http://bby.osmaniye.edu.tr/20584_genel-bilgiler.html Lisans. (2018). Erişim adresi: http://bilgibelge.medeniyet.edu.tr/tr/egitim/lisans Lisans-Fen-Edebiyat Fakültesi-Bilgi ve Belge Yönetimi. (2012). Tablolar için Kaynakça Erişim adresi: http://llp.marmara.edu.tr/organizasyon.aspx?kultur=tr- tr&Mod=1&ustbirim=1200&birim=1202&altbirim=&program=27&organizasyonId=31&muf edatTurId=932001 Lisans Ders Programı Listesi. (t.y.). Erişim adresi: https://fef.kastamonu.edu.tr/index.php/tr/anamenu- bolumler-bby-lisans-ders-ve-icerikleri-tr Lisans Programı. (2017). Erişim adresi: http://bby.fef.marmara.edu.tr/lisans-programi/ Lisans Üstü. (2018). Erişim adresi: http://bby.fef.marmara.edu.tr/lisans-ustu/ Tezli Yüksek Lisans-Türkiyat Araştırmaları Enstitüsü-Bilgi ve Belge Yönetimi. (2012). Erişim Adresi: http://llp.marmara.edu.tr/OrganizasyonInfo.aspx?kultur=tr- tr&Mod=2&ustbirim=5800&birim=5802&altbirim=- 1&program=628&organizasyonId=521&mufredatTurId=932001 Genel Bilgiler. (2016). Erişim adresi: http://bby.osmaniye.edu.tr/20584_genel-bilgiler.html Lisans. (2018). Erişim adresi: http://bilgibelge.medeniyet.edu.tr/tr/egitim/lisans Lisans-Fen-Edebiyat Fakültesi-Bilgi ve Belge Yönetimi. (2012). Erişim adresi: http://llp.marmara.edu.tr/organizasyon.aspx?kultur=tr- tr&Mod=1&ustbirim=1200&birim=1202&altbirim=&program=27&organizasyonId=31&muf edatTurId=932001 Lisans Ders Programı Listesi. (t.y.). Erişim adresi: https://fef.kastamonu.edu.tr/index.php/tr/anamenu- bolumler-bby-lisans-ders-ve-icerikleri-tr Lisans Programı. (2017). Erişim adresi: http://bby.fef.marmara.edu.tr/lisans-programi/ Lisans Üstü. (2018). Erişim adresi: http://bby.fef.marmara.edu.tr/lisans-ustu/ Tezli Yüksek Lisans-Türkiyat Araştırmaları Enstitüsü-Bilgi ve Belge Yönetimi. (2012). Erişim Adresi: http://llp.marmara.edu.tr/OrganizasyonInfo.aspx?kultur=tr- tr&Mod=2&ustbirim=5800&birim=5802&altbirim=- 1&program=628&organizasyonId=521&mufredatTurId=932001 62
https://openalex.org/W3155631951
https://medet.rsmu.press/files/issues/medet.rsmu.press/2021/1/2021-1-770_en.pdf?lang=en
Russian
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To the 75th anniversary of the beginning of the Nuremberg trials and the creation of Nuremberg codex: global significance and the enduring lessons
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К 75-ЛЕТИЮ НАЧАЛА НЮРНБЕРГСКИХ ПРОЦЕССОВ И СОЗДАНИЯ НЮРНБЕРГСКОГО КОДЕКСА: ГЛОБАЛЬНОЕ ЗНАЧЕНИЕ И НЕПРЕХОДЯЩИЕ УРОКИ А. Г. Чучалин1 , Ю. Н. Саямов2 А. Г. Чучалин1 , Ю. Н. Саямов2 1 Российский национальный исследовательский медицинский университет имени Н. И. Пирогова, Москва, Россия 2 Московский государственный университет им. М. В. Ломоносова, Москва, Россия В статье раскрывается значение Нюрнбергского процесса для переосмысления нравственных оснований медицины; рассматривается роль Нюрнбергского кодекса в становлении добровольного информированного согласия в клинической практике и в клинических исследованиях, а также его влияние на международное нормативно-правовое регулирование сферы здравоохранения. Авторы акцентируют внимание на значении уроков Нюрнберга для понимания этических вызовов, которые актуализировались в XXI веке в результате развития технологий искусственного интеллекта, редактирования генома человека и становления новых форм родительства, во многом связанных с достижениями новых репродуктивных технологий. Ключевые слова: Нюрнбергский процесс, Нюрнбергский кодекс, добровольное информированное согласие Вклад авторов: концепция и дизайн исследования — Чучалин А. Г.; написание и редактирование текста — Саямов Ю. Н. Для корреспонденции: Чучалин Александр Григорьевич ул. Островитянова, д. 1, г. Москва, 117997; Chuchаlin@inbox.ru Статья получена: 12.02.2021 Статья принята к печати: 25.03.2021 Опубликована онлайн: 31.03.2021 DOI: 10.24075/medet.2021.002 The year 2020 was marked with the 75th anniversary of the Great Victory and the subsequent anniversaries of events that could not have happened without this historic achievement. The war unprecedented in its monstrosity, victim count and scale of destruction generated a common desire and firm determination to never again allow such a catastrophe and to punish the guilty. The desire transformed into the International Military Tribunal (IMT) established in 1946 to try the ideologists and leaders of Nazism and militarism guilty of the horrific crimes. This was a trial by the peoples. The Nuremberg Trials became the starting point of a new era in the development of world civilization and global legal consciousness. They established the legal foundations and principles of the post-war world order, which were adopted by the newly created UN and essentially back the current understanding of human rights and freedoms. Nuremberg Trials formulated the concepts of crimes against peace and humanity, legally qualified and condemned the unprecedented acts by the criminal Hitlerite regime, such as the mass extermination of people in the "death factories", inhuman treatment of the civilian population and concentration camp prisoners, medical experiments involving the prisoners. ORIGINAL RESEARCH ORIGINAL RESEARCH 2 Lomonosov Moscow State University, Moscow, Russia The article reveals the significance of the Nuremberg Trials for rethinking the moral foundations of medicine, investigates the role of the Nuremberg Code in the establishment of voluntary informed consent as a mandatory component of clinical trials and procedures and assesses the impact thereof on the international legal regulation of healthcare. The authors emphasize the importance of the Nuremberg lessons for understanding the ethical challenges that came into foreground in the 21st century due to the development of artificial intelligence, human genome editing technologies and the emergence of the new forms of parenting largely backed by the new reproductive technologies. Keywords: Nuremberg trials, Nuremberg code, informed consent Author contribution: Research concept and design — Chuchalin AG; text writing and editing — Sayamov YuN. Correspondence should be addressed: Alexander G. Chuchalin Ostrovityanova st. 1, Moscow, 117997; Chuchаlin@inbox.ru Correspondence should be addressed: Alexander G. Chuchalin Ostrovityanova st. 1, Moscow, 117997; Chuchаlin@inbox.ru Correspondence should be addressed: Alexander G. Chuchalin Ostrovityanova st. 1, Moscow, 117997; Chuchаlin@inbox.ru Received: 12.02.2021 Accepted: 25.03.2021 Published online: 31.03.2021 DOI: 10.24075/medet.2021.002 ON THE 75TH ANNIVERSARY OF THE BEGINNING OF THE NUREMBERG TRIALS AND THE CREATION OF THE NUREMBERG CODE: GLOBAL RELEVANCE AND ENDURING LESSONS Chuchalin AG1 , Sayamov YuN2 1 Pirogov Russian National Research Medical University, Moscow, Russia 2 Lomonosov Moscow State University, Moscow, Russia ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ However, since most of the documents were destroyed, the researchers suggest that the number of victims of this program could have actually amounted to about 1 million people and more [2]. g g In Russia, part 2 of Article 21 of the Basic Law, the Constitution of the Russian Federation, proclaims that "No one should be subjected to torture, violence, other cruel or degrading treatment or punishment. No one can be subjected to medical, scientific or other experiments without voluntary consent" [4]. It follows from this statement that medical experiments carried out on a person fall under the category of torture, violence, cruelty and treatment that degrades human dignity. In Fundamentals of the Russian Federation Law "On the Protection of Citizens' Health", the principles of the Nuremberg Code are reflected in Article 32, which establishes the need for voluntary informed consent to any medical intervention, and in Article 43, which requires the same for medical experiments. In particular, it reads: "Any biomedical research involving a person as an object can be carried out only after obtaining the written consent of this citizen. A citizen cannot be forced to participate in biomedical research. In the context of obtaining consent for biomedical research, the citizen should be provided with information about the goals, methods, side effects, possible risks, duration and expected results of the research. The citizen has the right to refuse to participate in the research at any stage" [5]. The Nuremberg Code had a significant impact on the subsequent development of a number of international documents that played an important role in the formation of the post-war world order. One of these documents is the Universal Declaration of Human Rights adopted by the UN General Assembly on December 10, 1948 [6]. A year earlier, the global medical community united into the World Medical Association (WMA) at its first general assembly in Paris (September 1947). A kind of the world medical parliament was created, one uniting doctors of the world and setting international standards for medical activity, binding on medical professionals of all countries. The WMA Code of Medical Ethics was influenced by the Nuremberg Code; it includes the main provisions thereof. Together with the Nuremberg Code, the WMA Code supported widespread adoption and application of the principle of VIC. For more than seven decades that followed, WMA became the center spreading the ethical principles applied in healthcare and medicine. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ Another important milestone is adoption of the Declaration of Geneva, a modern version of the Hippocratic Oath, which took place at the second general assembly of the WMA in Geneva in September 1948. The Declaration of Geneva and the ethical principles applied in medicine were supplemented at the WMA assemblies in Helsinki (1964), Tokyo (1975), Venice (1983), Hong Kong (1989), South Africa (1996), Edinburgh (2000), Washington (2002), Tokyo (2004), Seoul (2008), Brazil (2013). The mere listing of these assemblies testifies to the importance of ethical issues in professional discussions held within the world medical community. The only woman among the convicts was Dr. Gertha Oberheuser, who, like Dr. Karl Brandt, the main accused, Hitler's personal doctor and curator of the inhuman medical experiments, justified her actions by orders of the leadership. In this connection, the Nuremberg Trials declared it was unacceptable to justify the cruelty, inhuman attitude of a medical worker to a patient and any person by orders that violate the "Do no harm" principle. The code adopted in Nuremberg stated that "it is a personal duty and responsibility which may not be delegated to another with impunity" [3]. The basic principle formulated by the Nuremberg Tribunal in the context of the Nuremberg Trials, is that in order to conduct an experiment on a person, a voluntary informed consent should be received from this person after he/she has been provided with full information about the nature, duration and purpose of the experiment, methods of its implementation, the alleged inconveniences and dangers associated with the experiment, and, finally, about the possible physical or mental consequences that may arise as a result of participation in the experiment [3]. It was the first time in the history of mankind when the principle of voluntary informed consent was proclaimed. In the field of medicine, paternalism was replaced by respect for dignity and human rights in making health-related decisions, this respect ensured by the process of obtaining voluntary informed consent (VIC). Medical ethics saw the doctor-patient relationship undergo qualitative changes. The Nuremberg Code and the Universal Declaration of Human Rights formed the basis for the development of the Universal Declaration on Bioethics and Human Rights, a fundamental document prepared by the UNESCO experts and adopted at the UNESCO General Conference session of October 19, 2005. The Russian philosophers B.G. Yudin and R.G. Apresyan made a great contribution to the preparation of this comprehensive document. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ The principles formulated in the Nuremberg Code became the basis for many international and national laws in the field of medical research involving human beings. 9, 1946 to August 20, 1947. The hall of the Nuremberg Palace of Justice selected for this trial was that where the key war criminals of the Third Reich were tried. Twenty most odious Nazi criminal doctors, 2 medical officials and 1 lawyer were accused at the trial, but that is not the only and, probably, not the most important fact underpinning the significance of these proceedings. There is an outcome of this trial that is at least no less significant, namely, the development of the Nuremberg Medical Code. It relied on the proceedings' results and was the first document since the works of Hippocrates to establish the principles of medical ethics, which remain current to this day. There were 1471 documents reviewed and 177 participants of the experiments [1] heard at the trial. The evidence and the testimonies revealed a horrifying picture of experiments that forcefully involved Nazi concentration camp prisoners stripped of all rights. Following requests from the air and naval forces, the participants were subjected to murderous pressure in a pressure chamber, simulating a fall from a great height, and immersed in ice-cold water to understand the limits of human survival in this environment. They were murdered for the purpose of replenishing the collection of skulls justifying superiority of the German race; forcibly sterilized, vaccinated, infected with typhus and yellow fever; had their limbs cut off and implanted to others, which ended in painful death of both the donor and the recipient. The effect of chemical weapons , phosgene and mustard gas, was investigated on living people, same as the medicinal properties of sulfonamides. For the latter purpose, test subjects had their bones crushed and phlegmons induced, broken glass and rusty nails poured into their wounds. The scope of the forced euthanasia program was terrifying. This program was aimed at "cleansing" out the people whom the Nazis considered unworthy, i.e. the disabled, people with chronic diseases, mental and other disabilities. At least 100000 people were proven violently killed under this program, with the predominant kill pattern being phenol injection to the heart. К 75-ЛЕТИЮ НАЧАЛА НЮРНБЕРГСКИХ ПРОЦЕССОВ И СОЗДАНИЯ НЮРНБЕРГСКОГО КОДЕКСА: ГЛОБАЛЬНОЕ ЗНАЧЕНИЕ И НЕПРЕХОДЯЩИЕ УРОКИ Celebrating the 75th anniversary of these events, it is necessary to dwell on the global significance and enduring lessons of the IMT's condemnation of the Nazi leaders who unleashed the world massacre, and specifically discuss the atrocities committed under their leadership, including in the field of medicine. This discussion seems especially relevant today, in the light of the ongoing processes and emergence of the new threats and challenges affecting health, safety of people and human rights. The results of the Nuremberg Trials boosted evolution of the international law and lead to a real breakthrough in the process of regulations development. They gave rise to the new legal culture that enshrined the ideas of peace, prevention of war and protection of human rights and freedoms in the national legislations. The lessons of Nuremberg include both the IMT outcomes and the results of the twelve Nuremberg trials that followed. Since Nuremberg was in the American occupation zone, the Allies agreed that the proceedings will be supported by the American military tribunal. Among them, the trial of the Nazi killer doctors occupies a special place. It lasted from December The International Military Tribunal and subsequent trials took place in the Bavarian city of Nuremberg, where Nazis held their congresses and declared the insane misanthropic ideas and the right of the Germans to dominate the world as a superior race. MEDICAL ETHICS 1, 2021 MEDET.RSMU.PRESS | | 6 6 Article 17 — Protection of the environment, the biosphere and biodiversity. The sections covering environmental ethics were included for the first time. Bioethics includes a variety of human activities and their ethical aspects: availability of medical care, clinical research, reproductive health, genome editing and human cloning, donation ethics, environmental ethics, biotechnology and nanotechnology [7]. "Voluntary" implies a deep respect for the dignity of the human person, his/her rights and freedoms, the possibility of independent choice. Article 3 of the Declaration on Bioethics and Human Rights addresses human dignity. It is an intrinsic value of a person capable of thinking, feeling, communicating verbally, choosing freely, independent behavior and creative activity. Ethics is the science studying the human being (N.A. Berdyaev); human dignity is a goal in itself. Different cultural and moral traditions and different types of societies have different understandings of the human dignity. In the academic dictionary of the Russian Academy of Sciences, dignity's definition states it is the person's awareness of his high value as a member of a group, which makes this person behave as a member of this group should behave. As an ethical concept, "voluntary" also includes the legal side. There is but a fine line between right and duty, so it is important to return to the interpretation of the person's dignity and his/her behavior as a member of the group to which he/she belongs. Intergovernmental and expert committees on bioethics were established within UNESCO. Their activity is guided by the dignity and respect principle formulated in the Nuremberg Code. It should be emphasized here that this principle underpins each fundamental international document in the field of medical ethics. Also noteworthy are the ethical principles of scientific research developed in the United States of America. In 1974, the US National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research was tasked with defining the fundamental ethical criteria for such research efforts. In 1979, the specialists that tackled the task presented the Belmont Report, the main provisions of which are: 1. Respect of persons. 1. Respect of persons. 2. Beneficence. 2. Beneficence. Voluntary decision-making implies the person making the decision is under no external pressure and can make the health-related decisions freely. Thus, "voluntary" implies respect for the person's dignity, his/her rights and obligations, and for the fact that he/she makes his decisions as a free person. Article 17 — Protection of the environment, the biosphere and biodiversity. Analyzing the problem of freedom, Fedor Dostoevsky defined it as follows: freedom is not restraining oneself, but controlling oneself. N.A. Berdyaev, the philosopher of freedom, relies on the Dostoevsky's definition of freedom and adds that ethics is the philosophy of freedom. 3. Justice. 3. Justice. Currently, the Belmont Report is the key source of ethical principles for research in the United States. Ethical problems became especially urgent in the 21st century. The agenda was extended with the ethical challenges peculiar to the present, those related to artificial intelligence, human genome editing and the new patterns of parenting. XXI. g g p p g The infectious diseases of the 21st century are of particular urgency. Human kind faced SARS (2002), MERS (2012), COVID-19 caused by SARS-COV-2 for the first time in its history. It is believed that coronavirus mutations are behind all these vital diseases. The 2009 influenza pandemic should also be mentioned in the list of this century's infectious diseases. The causative agent of that influenza was the California strain of H1N1. The pandemic was the first time when it was found to circulate in the human population. Some other diseases causing grave concern are the Ebola fever and the Zika disease, the former spread in Africa and the latter in Latin America. There is also another topic discussed hotly in the same context as the new infectious diseases: re-emergence of the "old" infections (smallpox etc). These new challenges to civilization highlight human vulnerability and raise questions about the moral foundations of the modern society. In the way of reaction thereto, in May of 2020 there was published the Guide to Informed Consent Compliance [9]. This document covers current approaches to the interpretation of the concept of VIC.XXI Finally, "voluntary" implies person's autonomy and individual responsibility. Article 5 of the Declaration on Bioethics and Human Rights covers the concepts of autonomy and responsibility. Autonomy is the person's self-determination ability, independent decisions, actions and assessments. Autonomy implies freedom from paternalistic interference, the person's ability to act on the basis of rational principles and rules in accordance with how this person understands his own good, personal dignity and happiness. According to Kant, autonomy is the ability of the will to independently establish the law of its action. Under the utilitarian approach, autonomy is the ability of a person to act in accordance with his/her own preferences. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ The Declaration considers ethical principles quite broadly, reflecting the activities of human beings all around. The key principles stated in the Declaration are covered in its main articles: МЕДИЦИНСКАЯ ЭТИКА 1, 2021 MEDET.RSMU.PRESS | | 7 ORIGINAL RESEARCH Article 3 — Human dignity and human rights. Article 3 — Human dignity and human rights. The urgency of this matter is also driven by the need for large-scale studies triggered by the COVID-19 pandemic. The scope and intensity of the relevant research efforts are unprecedented. They have to do with infectious disease epidemiology, prevention, antiviral therapy, pneumonia therapy and intensive care methods, as well as testing of the next generation vaccines. There arose a number of ethical questions that were widely discussed not only by the professional medical community but by the general public, too. In this regard, the relevance of voluntary informed consent has come to the foreground, emphasizing the need to strictly observe the traditional ethical principles of medical treatment: Primum non necere! (First of all, do no harm!) Voluntas aegroti suprema lex! (The patient's will is the highest law!) Salus aegroti suprema lex! (The patient's well-being is the highest law!) Article 4 — Benefit and harm. Article 5 — Autonomy and individual responsibility. Article 5 — Autonomy and individual responsibility Article 7 — Persons without the legal capacity to consent. Article 7 — Persons without the legal capacity to consent. Article 8 — Respect for human vulnerability and personal integrity. Article 8 — Respect for human vulnerability and personal integrity. Article 9 — Privacy and confidentiality. Article 10 — Equality, justice and equity. Article 11 — Non-discrimination and non-stigmatization. Article 12 — Respect for cultural diversity and pluralism. Article 13 — Solidarity and cooperation Article 14 — Social responsibility and health. Article 15 — Sharing of benefits. Article 16 — Protecting future generations. All activities of all medical professionals should pursue interests of the sick person. In this connection, it is also necessary to abandon paternalism for the principle of voluntary informed consent. Hereafter, we consider the semantic component of each of these keywords in detail. Article 17 — Protection of the environment, the biosphere and biodiversity. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ In ethics, the concepts of autonomy and responsibility are interconnected. If there is no responsibility, autonomy turns into arbitrariness: making a decision, a person does not take into account the interests of others. In some special cases, a life-threatening disease may necessitate VIC acceleration. This is a state-level decision made by the healthcare authorities. A case in question is that of registration of the new generation vaccines designed to prevent spread of SARS-COV-2: the circumstances called for a shorter vaccine testing procedure than is usually customary. Another special case is obtaining a VIC from persons who, for various reasons, cannot make decisions on their own. First of all, this applies to patients with impaired cognitive function. Thus, the term "voluntary" should, first of all, mean respect for the dignity of the human person, his/her rights and freedom, as well as autonomy and responsibility. The very text of the VIC is what its preparation revolves about. This document should not be considered as a piece of paper that needs a signature, i.e. as some formal procedure. Naturally, the question arises as to who is the author of this document: the sponsor, the scientific leader of the project, the organization of sick patients? Initially, the drafting of the document is initiated by the sponsor and reviewed at the topical expert meeting with participation of the scientific leader. The next important stage is the approval of the document. The preference should be given to the science and practice societies (several of them, in some cases) that can approve the VIC text. However, such a society needs to have a body authorized to approve the such a text. In the US, for example, the US Food and Drugs Administration (FDA) includes the Institution Review Board (IRB), which has approval of VIC texts among its duties. The Association of American Physicians also plays an important role. Finally, the relevant ethics committee reviews the VIC text at a meeting. The ethics committee experts review the suggested study as a whole, but they pay special attention to the said text. Practice shows that this part of a research project is the most vulnerable. The experience of international pharmaceutical companies can serve as a benchmark. As for the Russian research projects, in this part they usually contain comments from the ethics committee. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ The reason is that both the sponsor and the scientific leaders of the project typically lack the required skill, which translates into the need to train them to compile the informed consent texts. Ethical issues permeate the entire process of developing and passing the voluntary informed consent to practice. The ethical aspects of placebo and nocebo must be emphasized. The latter problem is rarely covered in Russian literature. The nocebo effect is the process of deterioration of the patient's health under the influence of information that is communicated to him/her by the doctor or medical personnel. There are following forms of nocebo distinguished: psychosomatic health deterioration caused by the expected adverse reactions; psychosomatic deterioration manifesting in the desire to have health deteriorating and, finally, actual deterioration of health of the patient due to the his/her initial attitude to such a turn of events. Unfortunately, current studies do not factor in and analyze the effects of placebo and nocebo sufficiently, which is especially relevant for the latter. It is no coincidence that the article emphasizes the historical role of the Nuremberg Code, which formed the basis of the Universal Declaration of Human Rights, the WMA International Code of Medical Ethics, the Declaration on Bioethics and Human Rights, which are the documents that largely determined the current world order. Highlighting the importance of the principle of voluntary informed consent, it is necessary to underline the need for state-level consideration of the bioethics problems. This is the only way to ensure the unity and integrity of state policy and decisions made in this important area, which stretches beyond the scopes of responsibility of individual ministries and departments. The basis element of the informed consent is the part that details purpose of the study, its duration and research procedures. An important section of the VIC is the description of the possible adverse reactions. In some studies, it is necessary to consider adverse reactions to treatment or instrumental examinations. This part of the document should be carefully analyzed by experts and explained to the patient in an understandable and accessible form. The witness is responsible for controlling how clearly the doctor explains the details of the VIC to a sick person or to his family members and other persons responsible for the sick person. The involvement of the witness, as mentioned above, is a relatively new condition for the VIC document. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ The informed consent witness must be convinced that the doctor has described the possibility of development of adverse reactions in an understandable manner, and that the patient has understood them. This is the role of the witness. He/she should note in the document that the VIC process was carried out professionally and in compliance with the established requirements. Apparently, US President Bill Clinton followed similar logic when he established the National Bioethics Advisory Commission under his auspices in 1996. In 2001, under US President George W. Bush, the status of the Commission was advanced, its powers expanded. Executive Order 13237 of November 28, 2001 transformed the National Commission into the President's Council on Bioethics. Its members and president, Edmund D. Pellegrino, the famous American scientist and theorist of bioethics, were personally appointed by the President of the United States. The council was supposed to "advise the president on bioethical issues that may arise from advances in biomedical science and technology" [9]. Barack Obama's Order 13521 of November 24, 2009, boosted the status of the body further up and turned it into the Presidential Commission for the Study of Bioethical Issues, which became interdepartmental and received powers, outgrowing its purely philosophical leadership. Amy Gutmann, President of the University of Pennsylvania, was appointed Chairman of the Commission [9]. Shortly thereafter, the information on the Commission's activities became all but inaccessible to the general public, which, according to experts, may indicate the seriousness of the issues discussed and the proposals developed for the President of the United States. The ethical challenges of the century, having shown the relevance of the lessons of Nuremberg, raised the issues of ethical education of society and ethical assessment of events unfolding in the world. The solution of the problems the society faces is largely associated with the development of bioethics that, in the conditions of the current changing world, is of particular importance to the national security of the Russian state. Barack Obama's Order 13521 of November 24, 2009, boosted the status of the body further up and turned it into the Presidential Commission for the Study of Bioethical Issues, which became interdepartmental and received powers, outgrowing its purely philosophical leadership. Amy Gutmann, President of the University of Pennsylvania, was appointed Chairman of the Commission [9]. Article 17 — Protection of the environment, the biosphere and biodiversity. The second part of Article 5 is about liability. Responsibility is a person's awareness of the duty to make decisions, act appropriately to his/her obligations, for example, to parents. MEDICAL ETHICS 1, 2021 MEDET.RSMU.PRESS | | 8 8 ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ Shortly thereafter, the information on the Commission's activities became all but inaccessible to the general public, which, according to experts, may indicate the seriousness of the issues discussed and the proposals developed for the President of the United States. When considering a research project, the doctor should also discuss with the patient the benefits that the latter receives from participating in this research or from application of the certain methods of treatment and examination. The participating person's financial interest occupies a special place. Before enrolling, he/she must receive clear and concise information regarding the financial compensation for the participation. Moreover, the participant should have the right to leave the study at any stage thereof. The ethical challenges of the century, having shown the relevance of the lessons of Nuremberg, raised the issues of ethical education of society and ethical assessment of events unfolding in the world. The solution of the problems the society faces is largely associated with the development of bioethics that, in the conditions of the current changing world, is of particular importance to the national security of the Russian state. Current requirements for the text of informed consent allow two forms thereof: a detailed text and a short form, with declaration of the project itself and the objectives of the study. МЕДИЦИНСКАЯ ЭТИКА 1, 2021 MEDET.RSMU.PRESS | | 9 ORIGINAL RESEARCH References osnovy-zdorovya-dok.html/ Russian. 1. Die Nürnberger Prozesse. Sandberg Verlag. Nürnberg. 2008. ISBN 978-3-930699-52-0. 6. Vseobshhaja Deklaracija Prav Cheloveka. https://www.un.org/ru/ documents/decl_conv/declarations/declhr.shtml/Russian. 6. Vseobshhaja Deklaracija Prav Cheloveka. https://www.un.org/ru/ documents/decl_conv/declarations/declhr.shtml/Russian. 6. Vseobshhaja Deklaracija Prav Cheloveka. https://www.un.org/ru/ documents/decl_conv/declarations/declhr.shtml/Russian. 2. Mitscherlich A, Mielke F. Medizin ohne Menschlichkeit: Dokumente des Nürnberger Ärzteprozesses, 16. Aufl., Heidelberg: Fischer 2004. ISBN 978-3-596220-03-8. 7. Vseobshhaja deklaracija o biojetike i pravah cheloveka Prinjata rezoljuciej General'noj konferencii JuNESKO 19 oktjabrja 2005 goda. https://www. un.org/ru/documents/decl_conv/declarations/bioethics_and_hr.shtml/ 8. Guide to Informed Consent Compliance. Washington, USA. Publication date: May 2020. Page count: 705 ISBN-13:978-1- 60430 136 6 htt // t t h / d t /476 id 7. Vseobshhaja deklaracija o biojetike i pravah cheloveka Prinjata rezoljuciej General'noj konferencii JuNESKO 19 oktjabrja 2005 goda. https://www. un.org/ru/documents/decl_conv/declarations/bioethics_and_hr.shtml/ 8. Guide to Informed Consent Compliance. Washington, USA. Publication date: May 2020. Page count: 705 ISBN-13:978-1- 60430-136-6 https://www.centerwatch.com/products/476-guide- to-informed-consent-compliance/ 3. Pravovye dokumenty. Njurnbergskij kodleks. http://www. psychepravo.ru/law/int/nyurnbergskij-kodeks.htm/Russian. un.org/ru/documents/decl_conv/declarations/bioethics_and_hr.shtml/ 8. Guide to Informed Consent Compliance. Washington, USA. Publication date: May 2020. Page count: 705 ISBN-13:978-1- 60430-136-6 https://www.centerwatch.com/products/476-guide- to-informed-consent-compliance/ psychepravo.ru/law/int/nyurnbergskij-kodeks.htm/Russian. 4. Konstitucija Rossijskoj Federacii, stat'ja 21, chast' 2. http://www. ОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕ consultant.ru/document/cons_doc_LAW_28399/e7fbd40d5c89 c3066eab2473bcaac30880b58eb3/Russian. 9. Prezidentskij sovet po biojetike SShA. https://ru.qaz.wiki/wiki/ The_President%27s_Council_on_Bioethics/Russian. Prezidentskij sovet po biojetike SShA. https://ru.qaz.wiki/wiki/ The_President%27s_Council_on_Bioethics/Russian. 9. Prezidentskij sovet po biojetike SShA. https://ru.qaz.wiki/wiki/ The_President%27s_Council_on_Bioethics/Russian. 5. Osnovy zakonodatel'stva Rossijskoj Federacii «Ob ohrane zdorov'ja grazhdan». Stat'i 32 i 43. https://rg.ru/1993/08/19/ shtml/ 7. Всеобщая декларация о биоэтике и правах человека Принята резолюцией Генеральной конференции ЮНЕСКО 19 октября 2005 года. https://www.un.org/ru/documents/decl_ conv/declarations/bioethics_and_hr.shtml/ 8. Guide to Informed Consent Compliance. Washington, USA. Publication date: May 2020. Page count: 705 ISBN-13:978-1- 60430-136-6 https://www.centerwatch.com/products/476-guide- to-informed-consent-compliance/ 9. Президентский совет по биоэтике США. https://ru.qaz.wiki/ Литература 6. Всеобщая Декларация Прав Человека. https://www. un.org/ru/documents/decl_conv/declarations/declhr. shtml/ 1. Die Nürnberger Prozesse. Sandberg Verlag. Nürnberg. 2008. ISBN 978-3-930699-52-0. 2. Mitscherlich A, Mielke F. Medizin ohne Menschlichkeit: Dokumente des Nürnberger Ärzteprozesses, 16. Aufl., Heidelberg: Fischer 2004. ISBN 978-3-596220-03-8. 7. Всеобщая декларация о биоэтике и правах человека Принята резолюцией Генеральной конференции ЮНЕСКО 19 октября 2005 года. https://www.un.org/ru/documents/decl_ conv/declarations/bioethics_and_hr.shtml/ 3. Правовые документы. Нюрнбергский кодекс. http://www. psychepravo.ru/law/int/nyurnbergskij-kodeks.htm/ 8. Guide to Informed Consent Compliance. Washington, USA. Publication date: May 2020. Page count: 705 ISBN-13:978-1- 60430-136-6 https://www.centerwatch.com/products/476-guide- to-informed-consent-compliance/ 4. Конституция Российской Федерации, статья 21, часть 2. http://www.consultant.ru/document/cons_doc_LAW_28399/e7f bd40d5c89c3066eab2473bcaac30880b58eb3/ 5. Основы законодательства Российской Федерации «Об охране здоровья граждан». Статьи 32 и 43. https://rg.ru/1993/08/19/ osnovy-zdorovya-dok.html/ 9. Президентский совет по биоэтике США. https://ru.qaz.wiki/ wiki/The_President%27s_Council_on_Bioethics/ 9. Президентский совет по биоэтике США. https://ru.qaz.wiki/ wiki/The_President%27s_Council_on_Bioethics/ MEDICAL ETHICS 1, 2021 MEDET.RSMU.PRESS | | 10
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https://mirtr.elpub.ru/jour/article/download/916/3547
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POINTS OF SUPPORT FOR INTELLIGENT SYSTEMS
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112 ECONOMICS 112 ECONOMICS 112 ECONOMICS ЭКОНОМИКА Точки опоры для интеллектуальных систем Ирина МИСАНОВА Irina N. MISANOVA Points of Support for Intelligent Systems (текст статьи на англ. яз. – English text of the article – ​p. 116) Ирина МИСАНОВА Ирина МИСАНОВА Мисанова Ирина Николаевна – ​доктор экономических наук, кандидат технических наук, профессор кафедры «Логистика и управление транспортными системами» Московского государственного университета путей сообщения (МИИТ), Москва, Россия. Ц Ц ель моей статьи не в том, чтобы отметить достижения и выявить недостатки соответствующих ор- ганов, отвечающих за развитие российской интеллектуальной транспортной системы. Поскольку верю, что настанет время, ког- да мы будем испытывать только гордость за нашу ИТС, а недостатки ее померкнут перед грядущими свершениями. Главное же заключается в  том, в  какой момент и какое поколение ощутит реальные плоды умов от науки, да и проявит ли наконец достойное темы любопытство «чиновни- чий класс». Ведь от этого зависят развитие экономики страны и как следствие – ​соци- ально-экономическая и  политическая устойчивость действующих общественных связей и отношений. Points of Support for Intelligent Systems (текст статьи на англ. яз. – English text of the article – ​p. 116) Points of Support for Intelligent Systems (текст статьи на англ. яз. – English text of the article – ​p. 116) Обоснованные теоретически, но остающиеся маловостребованными инновационные механизмы ищут и находят поводы заявлять о себе. В статье особое место отводится проблемам становления российских интеллектуальных транспортных систем, в особенности тех, которые позволяют в конкурентной бизнес-среде создать новые высокотехнологичные продукты. Эта тема приобрела приоритетную значимость в свете реорганизации государственного сектора экономики, необходимости обновления законодательства в интересах ускоренного развития интеллекто- содержащих средств организации и обслуживания перевозочного процесса. Вполне разделяю в этом плане взвешен- ный оптимизм по поводу форм научного участия в программах устойчивого развития российских территорий, который демон- стрирует в своей журнальной статье акаде- мик В. В. Окрепилов [1]. Идеология таких программ предполагает системную интег- рацию интеллектуальных сил и в сфере экономики, и в технико-технологической инноватике, и в мире транспорта, при этом оставляя под постоянным прицелом и со- Ключевые слова: железная дорога, законодательство, экономика, интеллектуальная транспортная система, финансовые механизмы, взаимодействие государства и бизнеса, государственные гарантии, венчурные проекты. • МИР ТРАНСПОРТА, том 14, № 2, С. 112–117 (2016) В профессорской среде транспортных вузов не раз приходилось слышать об обоснованности предложений по созда- нию с  участием думского комитета по транспорту законопроекта, определяю- щего государственную политику в отно- шении интеллектуальных транспортных систем, сущностной стороны грядущих изменений в  управлении базовой для экономики страны отрасли. Ирина МИСАНОВА Причем все очевиднее (и  названный октябрьский конгресс в этом смысле характерен), что в тесной связи сегодня находятся и тео- ретическая неготовность понятийного аппарата, без которого крайне затрудне- на формулировка статей желаемого за- кона, и непростая по целям юридическая трактовка позиций, которые относятся к задачам бюджетного финансирования исследований и проектных работ, инве- стиционной привлекательности проек- тируемых ИТС и  готовящихся парал- лельно инфраструктурных преобразова- ний. циальные интересы регионов, потребности граждан. Конечно, для любой интеллекту- альной системы управления ее консолиди- рующий смысл изначален и обязателен, но довести до этого уровня качество строящей- ся модели никому пока не удается. В октябре 2015 года состоялся 7-й рос- сийский международный конгресс по ИТС. Его целью как раз и было формирование авторитетной профессиональной платфор- мы для проработки теоретических и мето- дологических задач, обсуждение вопросов инновационного развития транспортного комплекса, ориентации интеллектуальных систем на обеспечение столь важных се- годня надежности и безопасности транс­ порта, укрепления партнерства органов государственной власти, научных и дело- вых кругов в области интеллектуализации поисковой, исследовательской и конструк- торской деятельности. Но ключевым элементом, естественно, является то, что перед государством стоит задача определения рациональной полити- ки на внутреннем и  внешнем рынках. В этих условиях возникает необходимость в установлении взаимовыгодного партнер- ства между государством и бизнесом, а за- тем, как предполагается, такой подход придаст новый импульс развитию ИТС. Впрочем, тут же возникает и вопрос, реаль- но ли осуществить полезные для страны намерения без должной законодательной базы? На протяжении семи лет участники московского и российского конгрессов докладывают о важности принятия феде- рального закона об ИТС. Пока все безре- зультатно, закона как не было, так и нет. И как следствие, инвестиционная состав- ляющая в проекты ИТС минимальна. Происходит это именно от того, что «чиновничий класс», отвечающий за раз- витие данной сферы, видимо, мыслит в русле не оправдавшей себя идеи Адама Смита о том, будто рыночная система спо- собна к саморегулированию, поскольку имеет личный интерес, связанный со стремлением к прибыли. Вопрос задается не случайно. Он впол- не созрел, чтобы стать уже не только дис- куссионным. Им озабочены и  деловые круги, и разработчики интеллектуальных систем, создатели программных продуктов. Показательно при этом, что заинтересо- ванность в законодательном закреплении идущих процессов проявляют не одни лишь представители отраслевых центров, но прежде всего компании и профильные структуры регионов, где сформировалось твердое убеждение на сей счёт. Ирина МИСАНОВА Сторонни- ки нового закона призывают своих парт- неров консолидировать позиции участни- ков рынка интеллектуальных транспорт- ных систем, чтобы получить в  общее пользование эффективные правовые ин- струменты и саму возможность форсиро- ванно развивать инновационную область научных знаний. Однако рыночная экономика не эффек- тивна без законодательного регулирования. И  естественным является обращение к историческим корням. Широко развер- нувшееся в 1860-х годах железнодорожное строительство – ​настоящая «рельсовая лихорадка» – ​дало толчок развитию других крупных отраслей промышленности. Пра- вительство Российской империи понима- ло, что для поднятия уровня народного хозяйства необходимо усиление железно- дорожного строительства, но не имело возможности строить железные дороги за государственный счёт, поэтому оно стара- лось привлекать частные капиталы (рус- 113 113 • МИР ТРАНСПОРТА, том 14, № 2, С. 112–117 (2016) • МИР ТРАНСПОРТА, том 14, № 2, С. 112–117 (2016) Мисанова И. Н. Точки опоры для интеллектуальных систем 114 ские и иностранные), гарантируя строите- лям прибыль на затраченный капитал. Откуда следует, что развитие и укрепление экономики Российской империи шло при помощи привлечения частных капиталов (инвестиций) в производство [2]. сознавать, что реализация интеллектуаль- ной транспортной системы неэффектив- на без инвестиционных вложений, без свойственных инвестициям законов, требующих точно ориентированных ис- следований, актуализаций и формализа- ций, особенно в тех секторах, где ИТС способна стать реальным «локомотивом» экономики. Сегодня российское государство, как бывало и в прежние исторические времена, доминирует в формировании экономиче- ских отношений. По большому счёту это нормальное продолжение естественной трансформации в  переходный период, когда ситуация из одного состояния плав- но перетекает в другое, а по сути, так и не может вырваться из плена концепции экономического либерализма, оставаясь в режиме «депрессивной стабилизации». То есть в том положении, когда вопреки объективным экономическим процессам допускается политика ослабления влияния государственных регуляторов экономики, вертикали управления, тормозится стиму- лирование инвестиций, либерализация внутреннего рынка сосредоточилась на приватизации собственности государства. Для этого необходимо создание понят- ных «правил игры», то есть компетентно выстроенного законодательного регулиро- вания ИТС, и полновесной системы взаи- модействия между государством и бизне- сом. В большинстве стран подобное взаи- модействие обеспечивают государственные органы федерального значения совместно со специализированными учреждениями. Инновационное развитие экономики России обречено идти по пути научно-тех- нического прогресса, одним из секторов которого являются ИТС, обеспечивающие постоянное движение вперед за счёт осно- вательной научной проработки стратеги- чески важных направлений. Это не только рост научной активности, но и совершен- ствование властных и исполнительных институтов (налоговой системы, банков- ской сферы, финансовых рынков, страхо- вания и т. п.). Мисанова И. Н. Точки опоры для интеллектуальных систем • МИР ТРАНСПОРТА, том 14, № 2, С. 112–117 (2016) Ирина МИСАНОВА Кроме того, следует отметить, что пока слабо взаимодействуют между собой бир- жи, банки, страховые компании, инвести- ционные фонды, нет единого регулирова- ния и протекционистской политики госу- дарства, основанной на взаимовыгодной экономической интеграции. Она же, в свою очередь, должна формироваться на принципах добровольности, взаимопомо- щи, правовой поддержки и гарантирован- ности демократии, общих целей и мотива- ций. И, конечно, ответственности за взя- тые обязательства. По нашему мнению, для успешного развития ИТС государству следовало бы: – подкрепить инвестиции в ИТС госу- дарственными гарантиями в венчурные проекты; – гарантировать беззалоговые кредиты частично либо полностью; – разрешить банкам относить затраты на венчурные проекты к первой группе риска. Одной из наиболее эффективных статей взаимодействия государства и  бизнеса в нормативно-законодательном регулиро- вании должны стать внешнеэкономиче- ские связи, рациональное использование финансовых ресурсов, материально-техни- ческое обеспечение производства и сбыта продукции, привлечение в  экономику иностранных инвестиций. Несомненно, чем действенней и глубже прорывы в науч- но-технической сфере и информационном обеспечении, тем больше надежд возлагают общество и бизнес непосредственно на ресурсы и институты государства [3]. Формирование венчурного капитала при непосредственном участии государст- ва – ​обязательное условие для аутсорсинга бизнеса, возможность уменьшить риски инвесторов. Но для более эффективных вложений в  ИТС-проекты государству нужно создавать и поддерживать стимули- рующие меры, как это принято в мировом сообществе. К такому стимулированию, в частности, можно отнести налоговые льготы или налоговые кредиты. Предоста- вить их было бы полезно хотя бы компани- ям, находящимся в процессе реструктури- зации, либо на начальной стадии своей Опираясь на исторический опыт, а также на практику развитых стран, надо • МИР ТРАНСПОРТА, том 14, № 2, С. 112–117 (2016) Мисанова И. Н. Точки опоры для интеллектуальных систем пресечения недобросовестной конкурен- ции в научно-технической сфере. То есть там, где практикуют интеллектуальные системы или их экспериментальные образ- цы. деятельности, поскольку в этот период они не могут обычно получить прибыль. пресечения недобросовестной конкурен- ции в научно-технической сфере. То есть там, где практикуют интеллектуальные системы или их экспериментальные образ- цы. Как правило, в реальности инвесторы не готовы вкладывать капиталы в нерефор- мированные организации по причине высоких рисков. Влияют: Многие страны идут по пути взаимодей- ствия и партнерства между государством и бизнесом, устраняя препятствия, меша- ющие инновациям, создавая стимулы для исследователей в области ИТС, активизи- руя приток инвестиций на ранней стадии и формируя условия для венчурного фи- нансирования. Россия не достигла еще сегодня оптимального уровня, отстает в  развитии не только законодательной части, но и организационно-экономиче- ской. Координаты автора: Мисанова И. Н. – kino-rodina@mail.ru. Статья поступила в редакцию 17.11.2015, принята к публикации 19.02.2016. Ирина МИСАНОВА Теоретически финансовые механиз- мы взаимодействия и  сотрудничества между государством и бизнесом, особенно в инновационной сфере, конечно, предла- гаются, но требуется еще очень серьезная работа по их практическому развитию. – незащищенность прав собственности; – значительный риск невозврата средств из-за неэффективности системы управле- ния и надзора правоприменительных ор- ганов; – непрозрачность финансового состо- яния организаций; – несовершенство бухгалтерского учета, которое, в свою очередь, влечёт за собой отсутствие достоверной информации о фи- нансово-экономическом состоянии орга- низации не только для собственников, акционеров, руководителей, возможных инвесторов и кредиторов, но и для государ- ственных органов и фискальных служб; – неразвитость механизмов гарантиро- вания исполнения контрактных обяза- тельств. ЛИТЕРАТУРА 1 О В Вывод закономерен: до тех пор, пока инвесторы не получат необходимых им гарантий и прав на интеллектуальную соб- ственность, они будут оценивать свои ри- ски как очень высокие. Ситуацию усугу- бляет и то, что пока российское законода- тельство не регулирует отношения в обла- сти конкуренции, нет и  механизмов 1. Окрепилов В. В. Повышение устойчивости развития жизни на основе экономики качества // Мир транспорта. – 2016. – № 1. – С. 6–21. 1. Окрепилов В. В. Повышение устойчивости развития жизни на основе экономики качества // Мир транспорта. – 2016. – № 1. – С. 6–21. 1. Окрепилов В. В. Повышение устойчивости развития жизни на основе экономики качества // Мир транспорта. – 2016. – № 1. – С. 6–21. р р  2. Мисанова И. Н. Основные направления совер- шенствования финансового взаимодействия государ- ства с коммерческими организациями: Моногра- фия. – М.: Дашков и Кo, 2012. – 148 с. 2. Мисанова И. Н. Основные направления совер- шенствования финансового взаимодействия государ- ства с коммерческими организациями: Моногра- фия. – М.: Дашков и Кo, 2012. – 148 с. 115 ф  Д , 3. Шамхалов Ф. И. Государство и экономика. Основы взаимодействия. – М.: Экономика, 2000. – 256 с. • 3. Шамхалов Ф. И. Государство и экономика. Основы взаимодействия. – М.: Экономика, 2000. – 256 с. • • МИР ТРАНСПОРТА, том 14, № 2, С. 112–117 (2016) Мисанова И. Н. Точки опоры для интеллектуальных систем
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Correction to: Integration of Renewable Energy in Markets: Analysis of Key European and American Electricity Markets
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Correction to: Integration of Renewable Energy in Markets: Analysis of Key European and American Electricity Markets Hugo Algarvio, Fernando Lopes, João Santana Hugo Algarvio, Fernando Lopes, João Santana To cite this version: Hugo Algarvio, Fernando Lopes, João Santana. Correction to: Integration of Renewable Energy in Markets: Analysis of Key European and American Electricity Markets. 10th Doctoral Conference on Computing, Electrical and Industrial Systems (DoCEIS), May 2019, Costa de Caparica, Portugal. pp.C1-C1, ￿10.1007/978-3-030-17771-3_37￿. ￿hal-02295246￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02295246 https://inria.hal.science/hal-02295246v1 Submitted on 24 Sep 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Correction to: Integration of Renewable Energy in Markets: Analysis of Key European and American Electricity Markets Hugo Algarvio, Fernando Lopes, and João Santana Correction to: Chapter “Integration of Renewable Energy in Markets: Analysis of Key European and American Electricity Markets” in: L. M. Camarinha-Matos et al. (Eds.): Technological Innovation for Industry and Service Systems, IFIP AICT 553, https://doi.org/10.1007/978-3-030-17771-3_28 The original version of this chapter starting on p. 321 was revised. The year mentioned in the funding information was incorrect. The original chapter has been corrected. © IFIP International Federation for Information Processing 2019 Published by Springer Nature Switzerland AG 2019 L. M. Camarinha-Matos et al. (Eds.): DoCEIS 2019, IFIP AICT 553, p. C1, 2019. https://doi.org/10.1007/978-3-030-17771-3_37 The updated version of this chapter can be found at https://doi.org/10.1007/978-3-030-17771-3_28 The updated version of this chapter can be found at https://doi.org/10.1007/978-3-030-17771-3_28 © IFIP International Federation for Information Processing 2019 Published by Springer Nature Switzerland AG 2019 L. M. Camarinha-Matos et al. (Eds.): DoCEIS 2019, IFIP AICT 553, p. C1, 2019. https://doi.org/10.1007/978-3-030-17771-3_37 © IFIP International Federation for Information Processing 2019 Published by Springer Nature Switzerland AG 2019 L. M. Camarinha-Matos et al. (Eds.): DoCEIS 2019, IFIP AICT 553, p. C1, 2019. https://doi.org/10.1007/978-3-030-17771-3_37
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A stepwise integrated risk-assessment framework in aquaculture: the case of sea lice tolerance to freshwater treatments on salmon farms
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*Corresponding author: jsanchez@upei.ca A stepwise integrated risk-assessment framework in aquaculture: the case of sea lice tolerance to freshwater treatments on salmon farms Emilie Laurin1, Marit Stormoen2, Crawford Revie1,3, Javier Sanchez1,* 1Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, C1A 4P3 Canada 2Norwegian University of Life Science, Faculty of Veterinary Medicine, Oslo, Norway 3Department of Computer and Information Sciences, University of Strathclyde, Glasgow, UK A stepwise integrated risk-assessment framework in aquaculture: the case of sea lice tolerance to freshwater treatments on salmon farms Emilie Laurin1, Marit Stormoen2, Crawford Revie1,3, Javier Sanchez1,* 1Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, C1A 4P3 Canada 2Norwegian University of Life Science, Faculty of Veterinary Medicine, Oslo, Norway 3Department of Computer and Information Sciences, University of Strathclyde, Glasgow, UK ABSTRACT: Aquaculture studies are often faced with data limitations when carrying out a quan- titative risk assessment. Consolidating results from a literature search of potentially applicable methods, we propose a stepwise integrated methods approach that incorporates foundations from an antimicrobial resistance framework, the Office International Epizooties risk model, quantita- tive microbial risk assessment and infectious disease transmission models. We suggest that an ini- tial ranking profile can be used to prioritize more in-depth qualitative and quantitative risk assess- ments, when data are available. The ranking method was done using a software that provides practical and interactive graphics for visualizing the impact of different factors and their respec- tive weights on the likelihood of undesirable events (hazards) occurring. For this step, we illus- trate how to include available data to obtain ranking results for decision makers using information from a recent sea lice freshwater tolerance literature review (Groner et al. 2019) that identified a gap in quantitative data. In our case example, for copepodid sea lice life stages, hypothetically changing how much experts believe that location and time are important factors revealed the most impact on the ranking for different degrees of freshwater tolerance evolution (no evolution, various partial options, known evolution). The factors ‘location’ and ‘time’, as well as ‘freshwater treatment’, have the greatest impact on the ranking for the preadult sea lice life-stages model. Results from our proposed ranking method can help to drive decisions around interpreting the various factors as they apply to mitigation planning and prioritizing those that should be included in further research. Additionally, we identify where quantitative data could be incorporated, as they become available, into a full risk assessment model with suggested models for a freshwater tolerance risk analysis framework. EY WORDS: Risk analysis · Risk ranking · Framework · Sea lice · Freshwater tolerance KEY WORDS: Risk analysis · Risk ranking · Framework · Sea lice · Freshwater tolerance © The authors 2020. Open Access under Creative Commons by Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. Vol. 12: 417–428, 2020 https://doi.org/10.3354/aei00373 Vol. 12: 417–428, 2020 https://doi.org/10.3354/aei00373 AQUACULTURE ENVIRONMENT INTERACTIONS Aquacult Environ Interact Published October 22 Publisher: Inter-Research · www.int-res.com 1. INTRODUCTION uncertainty about the likelihood of occurrence of a hazard (the phenomena causing the potential risk) and the associated consequences (therefore, risk = likelihood × consequences), decision makers can use RAs for evidence-based, transparent decisions and guidance documents (Vose 2001) in order to reduce the incidence of the hazard (Smith 2001, Peeler et al. Risk analyses (RAs) look at the likelihood (see Table 1 for a definition of RA terminology used) that undesirable events (hazards) will occur, together with the consequences of their occurrence (Greiner et al. 2004, Peeler et al. 2007). When faced with Publisher: Inter-Research · www.int-res.com Aquacult Environ Interact 12: 417–428, 2020 418 2007). RAs are founded on both qualitative and quan- titative methods. Semi-quantitative methods have also been proposed to provide score results when too few quantitative data are available, as is often the case for aquatic-focused RAs (Vose 2001, Peeler et al. 2007, Beaudequin et al. 2015). Once the hazard has been identified, the goal of an RA is to evaluate the risk of introducing the hazard, followed by identify- ing pathways through which the probability of expo- sure to the hazard can occur, as well as other factors that can contribute to and affect the estimation of the risk and any uncertainty related to that estimate (Travis & Hueston 2001, Peeler et al. 2007). When risks are considered harmful, the impact of mitiga- tion measures are also incorporated into the RA. The lack of information necessary to understand the impact of new management practices on cur- rent and future estimates of risk is one of the major challenges when assessing those practices in pro- duction systems, such as aquaculture, particularly where immediate harm-reduction can have nega- tive effects later. For example, in salmon aqua - culture, control of sea louse parasites is a major challenge, costing ap proximately 9% of farm rev- enues and resulting in as much as a 16% reduction in salmon biomass (Abolofia et al. 2017). Use of chemotherapeutants to control sea louse infesta- tions on salmon farms has led to the evolution of resistance to some medicinal treatments over a rel- atively short time frame (1 to 2 decades; reviewed Terminology Definition Consequence Outcome of hazard occurring Factor(s) Information that can positively or negatively impact the risk Full or complete risk analyses Complete process to assess risk estimate(s), by identifying hazards, probability of framework exposure to the hazard and subsequent consequences. 1. INTRODUCTION It can use more than one model and should identify uncertainty around the probability and risk estimate(s). It should also include risk mitigation and risk communication steps. In our proposed method, it also includes a risk ranking step Hazard Undesirable event, phenomenon causing the potential risk Likelihood Introduction and exposure assessment Measurement score, How a factor or subfactor will be measured in order to estimate the score or directional measurement scale, result of each one. Can be presented as a category (e.g. yes/no/maybe) or linear (e.g. measurement number of days) Multicriteria decision analysis A risk modelling framework for handling complex decisions, where a decision is based (MCDA) on more than one factor Multimodal More than one model Outcome The hazard defined in degrees or levels or alternatives Phi How preference is estimated. Positive flow (phi+) indicates the level to which a particular outcome is dominating all others, negative flow (phi−) indicates the level to which a given outcome is being dominated, and net flow (net phi) is used to provide an approximate measure of the overall preference of an outcome Preference, preference level, The quantitative or score value that identifies which outcome is preferred over another, preference value i.e. it is more influential on the risk estimate or score Risk Likelihood × consequence Risk estimate A quantitative estimate for the calculated risk Risk profile A schematic or outline of all the applicable factors, subfactors and weights for a unique scenario used in the ranking method to define a risk score Risk ranking, A model that ranks or prioritizes (by preference values) different degree(s) of a hazard ranking occurring Risk scores Quantitative or semi-quantitative estimates of risk (the preference values in the ranking method) as defined by a set of specific factors, subfactors and weights Subfactor(s) Further divisions of factors to define differences between conditions of a factor Uncertainty Lack of precise knowledge of input values Weight The degree of influence of a factor or subfactor(s). Combined weights refer to combining the weight of a factor and its subfactors into one weight estimate Table 1. Definition of risk analyses terminology used Table 1. Definition of risk analyses terminology used Table 1. Definition of risk analyses terminology used Complete process to assess risk estimate(s), by identifying hazards, probability of exposure to the hazard and subsequent consequences. 2. MATERIALS AND METHODS Risk ranking prioritizes current qualitative data or semi-qual- itative factors (see the ex ample of antimicrobial resistance models for knowledge data gaps as presented in CAC 2011) to drive decisions around interpreting these various factors as they apply to mitigation planning and prioritizing those that can guide re - search to ob tain the data required for a more complete quantitative RA (Peeler et al. 2007). We illustrate this ranking method using the case exam- ple of the evolution of freshwater tol- erance in sea lice. Following the ranking, we suggest a possible inte- grated RA method that would include various models that focus on both qualitative and quantitative data for the case example. 2011), which d approach focus be developed f tations, and th our case study et al. (2019). Fo ical scenarios w or less influen and how chang Fig. 1. Proposed workflow (based sea lice evolvin A literature review by Groner et al. marized current knowledge about t evolution of freshwater and low-salin in sea lice and other copepodids tha and/or farmed salmonids. However, m tative data gaps were identified that pr complete RAs. To address risk-based aquaculture when such major quan limitations exist, we propose a step-wi that uses more than one model (mul- timodal) for an integrated risk analy- sis framework. Multicriteria decision analysis (MCDA) offers a framework for handling complex decisions that need to account for conflicting objec- tives. When quantitative data are not available, risk-ranking methods can be carried out prior to a more complete RA (CAC 2011). MCDA can enable the ranking of factors that can posi- tively or negatively impact the risk of hazard(s) occurring (Fazil et al. 2008, Oidtmann et al. 2011, Martínez et al. 2015). Risk ranking prioritizes current qualitative data or semi-qual- itative factors (see the ex ample of antimicrobial resistance models for knowledge data gaps as presented in CAC 2011) to drive decisions around interpreting these various factors as they apply to mitigation planning and prioritizing those that can guide re - search to ob tain the data required for a more complete quantitative RA (Peeler et al. 2007). We illustrate this ranking method using the case exam- ple of the evolution of freshwater tol- erance in sea lice. Following the ranking, we suggest a possible inte- grated RA method that would include various models that focus on both qualitative and quantitative data for the case example. 1. INTRODUCTION It can use more than one model and should identify uncertainty around the probability and risk estimate(s). It should also include risk mitigation and risk communication steps. In our proposed method, it also includes a risk ranking step How preference is estimated. Positive flow (phi+) indicates the level to which a particular outcome is dominating all others, negative flow (phi−) indicates the level to which a given outcome is being dominated, and net flow (net phi) is used to provide an approximate measure of the overall preference of an outcome The quantitative or score value that identifies which outcome is preferred over another, i.e. it is more influential on the risk estimate or score The degree of influence of a factor or subfactor(s). Combined weights refer to combining the weight of a factor and its subfactors into one weight estimate Laurin et al.: Risk analysis framework in aquaculture 419 2. MATERIALS AND METHODS in Aaen et al. 2015). The lack of effective medicinal treatments, stricter regulations and in dustry ef forts to reduce medicine use for sea lice has pre cipitated the development of non-medicinal methods for sea louse control such as mechanical, fresh water and thermal treatments (Overton et al. 2018). RA could aid in strategizing how and when these non- medicinal methods should be used to im prove practices and avoid the evolution of treatment tolerance. We have created a step-by-step workflow for assessing the risk of sea lice developing tolerance to freshwater treatments (Fig. 1), following the regula- tory World Organization for Animal Health (Office International Epizooties; OIE 2019) RA model (which does not include a ranking method) as well as the antimicrobial RA model from the Executive Commit- tee of the Codex Alimentarius Commission (CAC 2011), which does include a ranking method. Our approach focuses on how a ranking method can first be developed for aquaculture studies with data limi- tations, and then applying that ranking method for our case study using the criteria identified by Groner et al. (2019). For our case study, we present hypothet- ical scenarios where different factors may have more or less influence (weight) on the hazard occurring and how changing the weight can change the rank- A literature review by Groner et al. (2019) sum- marized current knowledge about the potential evolution of freshwater and low-salinity tolerance in sea lice and other copepodids that infect wild and/or farmed salmonids. However, major quanti- tative data gaps were identified that preclude more complete RAs. To address risk-based de cisions in aquaculture when such major quantitative data limitations exist, we propose a step-wise approach that uses more than one model (mul- timodal) for an integrated risk analy- sis framework. Multicriteria decision analysis (MCDA) offers a framework for handling complex decisions that need to account for conflicting objec- tives. When quantitative data are not available, risk-ranking methods can be carried out prior to a more complete RA (CAC 2011). MCDA can enable the ranking of factors that can posi- tively or negatively impact the risk of hazard(s) occurring (Fazil et al. 2008, Oidtmann et al. 2011, Martínez et al. 2015). 3.1. Hypothetical examples of using the ranking method for the case study In our case example, we define the hazard as fresh- water tolerance evolution in sea lice, and we define the risk score from the ranking method here as the preference value of our outcomes (risk of evolving freshwater tolerance) occurring as defined by a com- bination of factors, subfactors and weights. For our case example, we created 2 models, using the Visual PROMETHEE software, based on 2 Lepeophtheirus salmonis sea lice life stages, namely copepodids and preadults. We did not merge them into one model for the sake of simplicity for our illustration of the method, due to numerous interactions and combina- tions that occur between life stage and various crite- ria (in particular, length of exposure time to fresh- water; see the Supplement at www.int-res.com/ articles/ suppl/ q012p417_supp.pdf). Once factors and subfactors are established and justified, we need to define how each will be meas- ured (e.g. linear scale, categories, etc.). These meas- urements should reflect evidence-based information from the risk profile and expert opinion (CAC 2011). Finally, each factor is assigned weights to reflect its impact or influence on ranking. Therefore, a combi- nation of different weights for each factor and sub- factor(s) will be used to create a risk score. The higher the score, the higher the preference of one being ranked higher than another. Our different outcomes (degrees of freshwater tol- erance occurring, described in detail in the following paragraph) provide various possibilities that are ranked (preference for one or more over another) based on the risk scores. For our example, we incor- porate factors identified in the Groner et al. (2019) review: genetics, metapopulation dynamics, and selection-strength factors such as environment and freshwater treatments (Fig. 2). These 4 broad factors were further subdivided into subfactors. In Table 2, we list our selected factors and their associated sub- factors, including how we defined them, the scale we used for measuring changes in them, and the weight of importance we assigned them in our example models. We selected the measurement scales and We chose the PROMETHEE II method (Belton & Stewart 2002, Figueira et al. 2005, Fazil et al. 2008) as the best example to generate the ranking method and assess the preferences in the light of different weights or influence of factors over others. Belton & Stewart (2002) and Figueira et al. 3. RESULTS We used MCDA to enable ranking of factors that can positively or negatively impact the risk of the hazard occurring. These factors are identified from evidence-based information and can be quantitative or semi-quantitative. A factor can be further divided into various levels of subfactors to define differences between conditions that define the factor. This method could be used as part of the framework out- lined in Fig. 1 to create a risk profile (a schematic or outline of all the applicable factors, subfactors and weights). 2. MATERIALS AND METHODS Fig. 1. Proposed workflow (based on CAC 2011) for assessment of the risk of sea lice evolving freshwater tolerance Fig. 1. Proposed workflow (based on CAC 2011) for assessment of the risk of sea lice evolving freshwater tolerance Aquacult Environ Interact 12: 417–428, 2020 420 where w is the weight of factor j, and Pj(a,b), which measures the preference for option a over option b, comes from the preference function. Ranking is gen- erated in PROMETHEE using what the methodology calls ‘positive flows’, ‘negative flows’ and ‘net flows’. The positive flow (phi+) indicates the level to which a particular outcome (in risk ranking this would be a degree of the hazard occurring) is dominating all oth- ers, the negative flow (phi−) indicates the level to which a given outcome is being dominated, and the net flow (net phi) is used to provide an approximate measure of the overall preference of an outcome. Each outcome is based on a unique pre-defined set of factors and subfactors, as well as assigned weights that are used for the preference function. ing. We include methods and examples with the case study about how to incorporate available data and visualize results of the ranking through the use of a user-friendly, graphical risk-ranking tool (Visual Promethee 1.4 software VP Solutions, 2013; www. promethee- gaia. net). Because we did not have ade- quate quantitative data available to illustrate, with the case example, a complete RA model beyond the ranking, we present instead several different model types from the literature that could be in - cluded and adapted when quantitative data become available and to help prioritize research for the case example. (1) ( , ) ( , ) 1 a b w P a b j j j k ∑ Π = = 3.1. Hypothetical examples of using the ranking method for the case study (2005) provide a detailed explanation of the mathematics and the analysis behind the preference calculations. Briefly, the degree of preference, Π(a, b), for outcome option a against outcome option b, is calculated according to the formula: (1) ( , ) ( , ) 1 a b w P a b j j j k ∑ Π = = ( , ) ( , ) 1 a b w P a b j j j k ∑ Π = = (1) Laurin et al.: Risk analysis framework in aquaculture 421 Fig. 2. Key factors identified from the recent literature review (Groner et al. 2019) that may affect the risk of sea lice evolving tolerance to freshwater treat- ments. Asterisks indicate factors with further explanatory notes as follows: location (environmental salinity, exposure to freshwater run-off, sensitivity of associated lice population to freshwater, etc.); connectivity (farm to farm, wild to farm, farm to wild, direction of water current, density of wild versus farmed hosts, etc.); duration (length of exposure, number of repeated exposures); and permanent freshwater cap (snorkel cages, skirts) weights based on information obtained from Groner et al. (2019), Ljungfeldt et al. (2017) and expert opinion (M. Andrews, Norwegian University of Life Sciences [NMBU]). Assigned weights in Table 2 are author-selected and are used to demonstrate how to incorporate overall (combined) versus individual factor and subfactor weight components. Combined weights refer to combining the weight of a factor and its subfactors into one weight estimate. We used some of the freshwater exposure time by life stage interactions described in the supplement as well as information from Groner et al. (2019), Ljungfeldt et al. (2017) and expert opinion (M. Andrews) for defining the scales, scores and weights for each of the respective sea lice life-stage models (Table 3). We created 3 degrees of sea lice evolving tolerance to freshwater treat- ments (Table 3): ‘no’ (sea lice death, therefore no tolerance), ‘partial’ (possi- ble oc currence) and ‘known’ (no change in sea lice survival and behavior, there- fore tolerance). We created several ‘partial’ options for freshwater toler- ance evolution, whereby the outcome is some degree of mortality (<30% sur- vival or >50% survival or unknown % survival) or inhibited development. These ‘partial’ degrees of freshwater tolerance were based on information from various field and experimental studies of sea lice responses to various salinity thresholds (see the sup- plement and Table 3) (Ljungfeldt et al. 3.1. Hypothetical examples of using the ranking method for the case study 2017, Crosbie et al. 2019, Sievers et al. 2019, expert opinion [M. Andrews]). In our case example, the de grees of freshwater tolerance with the highest net phi corre- sponds to a higher risk score of freshwater tolerance evolution occurring in sea lice, considering all the specific information used to define that model. Fig. 2. Key fa 2019) that ma ments. Asteri location (env associated lic to farm, farm hosts, etc.); d Fig. 2. Key factors identified from the recent literature review (Groner et al. 2019) that may affect the risk of sea lice evolving tolerance to freshwater treat- ments. Asterisks indicate factors with further explanatory notes as follows: location (environmental salinity, exposure to freshwater run-off, sensitivity of associated lice population to freshwater, etc.); connectivity (farm to farm, wild to farm, farm to wild, direction of water current, density of wild versus farmed hosts, etc.); duration (length of exposure, number of repeated exposures); and permanent freshwater cap (snorkel cages, skirts) direction of net phi value) of the different degrees of freshwater tolerance (no, various partial options, known). The subfactors ‘location’ and ‘time’, as well as ‘intervention’, had the greatest impact on the rank- ing for the preadult life-stages model. In Fig. 3, we visually present 2 different options of various changes in combined weights assigned to our factors of inter- est and the resulting effect on the ranking’s prefer- ence values for each life-stage model. The first com- bined-weight option (Fig. 3A, column 1) is the one presented in Table 2, while in the second weight option (Fig. 3A, column 2), salinity, time, and location are assigned higher combined weights. Differences in ranking (net phi value) for the degrees of fresh- water tolerance are visualized depending on the sea lice life stage (Fig. 3B,C). Variations were greater for the ‘partial’ freshwater tolerance options. The level of net phi flow (higher preference and ranking) for ‘known’ freshwater tolerance also slightly varies depending on the different weights assigned in each model. 3.1. Hypothetical examples of using the ranking method for the case study With all of these examples, we show that the analyses available through the VP software can be an effective and interactive means for visualizing the Because the influence of criteria and sub-criteria can be weighted as de scribed previously, the VP walking weights (proportion of weight assigned to each factor and subfactor[s]) allow for an interactive visualization of how the ranking of the degrees of freshwater tolerance can be influenced if the weights are changed. In our case example, for copepodid life stages, hypothetically changing the weights for the ‘location’ and ‘time’ subfactors (from those weights we assigned them as described in Table 2) had the most impact on the ranking (positive or negative Aquacult Environ Interact 12: 417–428, 2020 422 Table 2. Selected factor and subfactors (based on Ljungfeldt et al. 2017, Groner et al. 2019, Sievers et al. 2019, and expert opin- ion [M. Andrews]), for use in our Visual PROMETHEE example to evaluate risk ranking effect of these factors for sea lice evolving tolerance to freshwater (FW). Assigned weights are author-selected and are used to demonstrate how to incorporate overall (combined) versus individual factor and subfactor weight components. Combined weights refer to combining the weight of a factor and its subfactors into one weight estimate. The direction of the measurement value (either increase or decrease in number value or percent value) corresponds to an increased risk of sea lice evolving tolerance to freshwater Table 2. Selected factor and subfactors (based on Ljungfeldt et al. 2017, Groner et al. 2019, Sievers et al. 2019, and expert opin- ion [M. Andrews]), for use in our Visual PROMETHEE example to evaluate risk ranking effect of these factors for sea lice evolving tolerance to freshwater (FW). Assigned weights are author-selected and are used to demonstrate how to incorporate overall (combined) versus individual factor and subfactor weight components. Combined weights refer to combining the weight of a factor and its subfactors into one weight estimate. 3.1. Hypothetical examples of using the ranking method for the case study The direction of the measurement value (either increase or decrease in number value or percent value) corresponds to an increased risk of sea lice evolving tolerance to freshwater Factor Subfactor Combined Factor Measurement Measurement Direction (weight) (weight) weight (%) definition type definition Genetics Heritability 40 Presence of heritable (50%) (80%) genetic or epigenetic Qualitative 1 (none), 2 (present), Increase variation for FW 3 (unknown) tolerance Fitness trade-offs (20%) 10 Decreases in sea lice Qualitative 1 (none), 2 (present), Increase fitness that are associated 3 (unknown) with FW tolerance Exposure Salinity (33%) 7 Parts per thousand of Categorical Range Decrease (20%) saline concentration (0 to 30‰ salinity) measured in water Intervention (33%) 7 Type of freshwater Qualitative 1 (none), Increase treatment used 2 (>1 treatment type), 3 (wellboat), 4 (unknown) Time (33%) 7 Duration of exposure Categorical Range (0 to 7 h) Increase to hyposaline water Population/ Lice/hosts (50%) 15 Type of hosts (farm or wild) Qualitative 1 (no connectivity), Increase environment and connectivity between 2 (wild > farmed), (30%) populations 3 (farmed > wild), 4 (farmed > wild; more connectivity), 5 (unknown) Location (50%) 15 Sensitivity Qualitative 1 (more sensitivity), Increase (or decreased tolerance) 2 (less sensitivity), of a population to freshwater 3 (unknown sensitivity) treatment that is due to various environmental or other non-heritable factors (see steps in Fig. 1 and the ranking examples discussed previously). The framework integrates the traditional OIE hazard identification, release, exposure and con- sequence assessments with portions of 2 specific RA modelling foundations, namely quantitative micro- bial risk assessment (QMRA) and infectious disease transmission (IDT) models for a more inclusive and applicable framework to the diversity of data in aquaculture studies. In Table 4, we present examples from the literature for different modelling ap - proaches that can be incorporated and/or modified, including ranking and the MCDA models. Brouwer et al. (2018) define QMRA models as focusing on fac- tors in the environment that characterize pathogen dynamics and subsequent exposure. These models are based on dose−response models and are used mainly in regulatory settings, as in the case of the OIE model. In contrast, IDT models focus more on risk scores and weights of various degrees of fresh- water tolerance occurring, while allowing the user to readily make changes to factors, subfactors and weights, as these information change or become available. 3.1. Hypothetical examples of using the ranking method for the case study Results from this visual ranking method can help to drive decisions around interpreting vari- ous factors and subfactors in mitigation planning and prioritizing which ones to in clude in further RA and research (see Fig. 1). 4. DISCUSSION population factors and how pathogens are spread between and among populations (Brouwer et al. 2018). These models are based on susceptible− infectious− recovered (SIR) models to define the interacting pop- ulation groups. Outputs from these models, in partic- ular system dynamic IDT models (versus agent- based), can provide unit frequency (e.g. average population estimates) that are dependent on differ- ent conditions and can be used for determining the probability and likelihood of the hazard occurring (see examples in Greiner et al. 2013, Beaudequin et al. 2015, McEwan et al. 2016, Rico et al. 2017, Wright et al. 2018). Because it is set in a Bayesian framework to handle uncertainty, we suggest that the Aldrin et al. (2017) salmon lice (L. salmonis) model may be adapted for a quantitative RA, with factors such as temperature dependence (see also Myksvoll et al. 2018), population and individual fish demographics, transmission rates and control measures (see their papers for specific equations and descriptions of as - sumptions made within their models; e.g. a decreased effect of treatment over time due to tolerance devel- opment and maturation times). However, the Aldrin et al. (2017) model does not include salinity, as they had insufficient salinity data; yet as we are consid- ering freshwater as a treatment modality, we pro- pose adjusting their model to include freshwater treatment(s). We proposed including in the RA framework a visual method to prioritize or rank degrees of fresh- water tolerance for future research and mitigation planning, using the Visual PROMETHEE 1.4 soft- ware (VP Solutions, 2013) to analyze a risk profile classified within the MCDA framework. Our pro- posed RA framework was adapted from the import risk guidelines of the World Organization for Animal Health (OIE 2019; based on the model defined by Covello & Merkhofer 1993), by incorporating a risk- ranking step before conducting the risk assessment. The 4 steps required for the OIE RA are: (1) hazard identification (in our case example, this would be the evolution of freshwater tolerance in sea lice); (2) risk assessment (including how a hazard is introduced, pathways by which animals can be exposed to the hazard or experience it, results or consequences of having the hazard or being exposed to it, and esti- mating the risk of exposure and possible results of exposure); (3) risk management; and (4) risk commu- nication. 3.2. Proposed integrated RA framework Hosts: type of hosts (farm or wild) and connectivity between populations; location: sensitivity (or decreased tolerance) of a population to freshwater treatment that is due to various environmental or other non-heritable factors Table 3. Subfactor values for each of our suggested outcomes (degrees of freshwater tolerance evolution). We used 2 example life-stage models in Visual PROMETHEE (C: copepodid; P: preadult). n/a: not applicable. Hosts: type of hosts (farm or wild) and connectivity between populations; location: sensitivity (or decreased tolerance) of a population to freshwater treatment that is due to various environmental or other non-heritable factors Outcome Life Subfactor (freshwater stage Heritability Fitness Salinity Intervention Time Hosts Location tolerance) trade-offs (psu or ppt) (h) No C No No 30 None 0.0 None More P No No 30 None 0.0 None More Known C Yes Yes 0 Wellboat 7.0 Farm > wild; Less more connectivity Less P Yes Yes 0 Wellboat 7.0 farmed > wild; more connectivity Partial >50% C Yes Yes 13 Unknown 1.0 Farm > wild Less lice survival 1 P Yes Yes 5 Unknown 1.0 Farm > wild More Partial >50% C Yes Yes 23 Unknown 1.0 Farm > wild More lice survival 2 P n/a n/a n/a n/a n/a n/a n/a Partial <30% C n/a n/a n/a n/a n/a n/a n/a lice survival P Yes Yes 0 Unknown 1.0 Unknown Unknown Partial ?% C Yes Yes 7 >1 0.5 Wild > farm Unknown lice survival P Yes Yes 5 >1 n/a Wild > farm Unknown Partial inhibited C Yes No 25 Unknown 0.5 Unknown Unknown lice development P n/a n/a n/a n/a n/a n/a n/a 3.2. Proposed integrated RA framework We also propose an integrated RA framework (Fig. 4). This could be developed pending available data and resources, to support the particular goals of an RA team. It is also dependent on the outcomes and applicability of the risk ranking related to those goals Laurin et al.: Risk analysis framework in aquaculture 423 population factors and how pathogens are spread between and among populations (Brouwer et al. 2018). These models are based on susceptible− infectious− recovered (SIR) models to define the interacting pop- ulation groups. Outputs from these models, in partic- ular system dynamic IDT models (versus agent- based), can provide unit frequency (e.g. average population estimates) that are dependent on differ- ent conditions and can be used for determining the probability and likelihood of the hazard occurring (see examples in Greiner et al. 2013, Beaudequin et al. 2015, McEwan et al. 2016, Rico et al. 2017, Wright et al. 2018). Because it is set in a Bayesian framework to handle uncertainty, we suggest that the Aldrin et al. (2017) salmon lice (L. salmonis) model may be adapted for a quantitative RA, with factors such as temperature dependence (see also Myksvoll et al. 2018), population and individual fish demographics, transmission rates and control measures (see their papers for specific equations and descriptions of as - sumptions made within their models; e.g. a decreased effect of treatment over time due to tolerance devel- opment and maturation times). However, the Aldrin et al. (2017) model does not include salinity, as they had insufficient salinity data; yet as we are consid- ering freshwater as a treatment modality, we pro- pose adjusting their model to include freshwater treatment(s). 4. DISCUSSION We proposed including in the RA framework a visual method to prioritize or rank degrees of fresh- water tolerance for future research and mitigation planning, using the Visual PROMETHEE 1.4 soft- ware (VP Solutions, 2013) to analyze a risk profile classified within the MCDA framework. Our pro- posed RA framework was adapted from the import risk guidelines of the World Organization for Animal Health (OIE 2019; based on the model defined by Covello & Merkhofer 1993), by incorporating a risk- ranking step before conducting the risk assessment. 3.2. Proposed integrated RA framework The 4 steps required for the OIE RA are: (1) hazard identification (in our case example, this would be the evolution of freshwater tolerance in sea lice); (2) risk assessment (including how a hazard is introduced, pathways by which animals can be exposed to the hazard or experience it, results or consequences of having the hazard or being exposed to it, and esti- mating the risk of exposure and possible results of exposure); (3) risk management; and (4) risk commu- nication. These formal RAs can range from including qualitative to semi-quantitative to full quantitative parameter estimates for probability and likelihood outcomes. It is important that transparency around assump- tions and uncertainty be maintained, including inter- 423 Laurin et al.: Risk analysis framework in aquaculture Outcome Life Subfactor (freshwater stage Heritability Fitness Salinity Intervention Time Hosts Location tolerance) trade-offs (psu or ppt) (h) No C No No 30 None 0.0 None More P No No 30 None 0.0 None More Known C Yes Yes 0 Wellboat 7.0 Farm > wild; Less more connectivity Less P Yes Yes 0 Wellboat 7.0 farmed > wild; more connectivity Partial >50% C Yes Yes 13 Unknown 1.0 Farm > wild Less lice survival 1 P Yes Yes 5 Unknown 1.0 Farm > wild More Partial >50% C Yes Yes 23 Unknown 1.0 Farm > wild More lice survival 2 P n/a n/a n/a n/a n/a n/a n/a Partial <30% C n/a n/a n/a n/a n/a n/a n/a lice survival P Yes Yes 0 Unknown 1.0 Unknown Unknown Partial ?% C Yes Yes 7 >1 0.5 Wild > farm Unknown lice survival P Yes Yes 5 >1 n/a Wild > farm Unknown Partial inhibited C Yes No 25 Unknown 0.5 Unknown Unknown lice development P n/a n/a n/a n/a n/a n/a n/a Table 3. Subfactor values for each of our suggested outcomes (degrees of freshwater tolerance evolution). We used 2 example life-stage models in Visual PROMETHEE (C: copepodid; P: preadult). n/a: not applicable. Hosts: type of hosts (farm or wild) and connectivity between populations; location: sensitivity (or decreased tolerance) of a population to freshwater treatment that is due to various environmental or other non-heritable factors Table 3. Subfactor values for each of our suggested outcomes (degrees of freshwater tolerance evolution). We used 2 example life-stage models in Visual PROMETHEE (C: copepodid; P: preadult). n/a: not applicable. Fig. 3. Changes in net phi values (prefer- ence) by sea lice life stage: (B) copepod; (C) preadult, of different degrees of freshwater tolerance occurring, according to weights presented in (A). The 2 result options are based on 2 different combined-weighting schemes assigned to the following factors and subfactors. 1: genetics = 50% (with her- itability = 80% and fitness trade-offs = 20%), exposure = 20% (with salinity, intervention, and time each 33%) and population/envi- ronment = 30% (with lice/hosts and location each 50%); 2: genetics = 40% (with heri- tability = 75% and fitness trade-offs = 25%), exposure = 20% (with salinity and time each 40% and intervention = 20%) and popula- tion/environment = 40% (with lice/hosts = 25% and location = 75%) Fig. 3. Changes in net phi values (prefer- ence) by sea lice life stage: (B) copepod; (C) preadult, of different degrees of freshwater tolerance occurring, according to weights presented in (A). The 2 result options are based on 2 different combined-weighting schemes assigned to the following factors and subfactors. 1: genetics = 50% (with her- itability = 80% and fitness trade-offs = 20%), exposure = 20% (with salinity, intervention, and time each 33%) and population/envi- ronment = 30% (with lice/hosts and location each 50%); 2: genetics = 40% (with heri- tability = 75% and fitness trade-offs = 25%), exposure = 20% (with salinity and time each 40% and intervention = 20%) and popula- tion/environment = 40% (with lice/hosts = 25% and location = 75%) 25% and location = 75%) 4. DISCUSSION These formal RAs can range from including qualitative to semi-quantitative to full quantitative parameter estimates for probability and likelihood outcomes. It is important that transparency around assump- tions and uncertainty be maintained, including inter- Aquacult Environ Interact 12: 417–428, 2020 424 actions between factors that can affect the interpretation of risk estimates (CAC 2011). For example, interactions between farmed and wild fish as well as environmental factors can impact fish stress and increase RA model un - certainty in aquaculture studies (Travis & Hueston 2001, Sievers et al. 2019). Kristoffersen et al. (2018) and Myksvoll et al. (2018) discussed how various environmental factors (e.g. tempera- ture, location, presence of farmed fish, etc.) can change the behaviour of sea lice and/or fish, depending on whether the fish are wild or farmed. In addition, the length and number of treatments, salinity and the potential for reattach- ment of sea lice could all affect the preference of one degree of freshwater tolerance over another (Wright et al. 2018, Crosbie et al. 2019, Sievers et al. 2019). There is a greater risk of the hazard occurring as there is more uncertainty around an unknown pres- ence or quantity of a factor or subfac- tor. Uncertainty due to interaction(s) between factors can be mitigated by applying different weighting schemat- ics to key factors and subfactors. However, it can be difficult to obtain quantitative data for selecting weights. Expert opinion or literature review information can be used, in which case this should be reported (Fazil et al. 2008). Groner et al. (2019) identified several gaps in available knowledge for each of the selected factors for our 25% and location = 75%) Laurin et al.: Risk analysis framework in aquaculture 425 4. Suggested risk analysis framework incorporating quantitative methods (Brouwer et al. 2018, Kristoffersen additional risk and quantitative examples for characterizing the risk of sea lice evolving tolerance to freshw represent the OIE risk model categories 4. Suggested risk analysis framework incorporating quantitative methods (Brouwer et al. 2018, Kristoffersen e additional risk and quantitative examples for characterizing the risk of sea lice evolving tolerance to freshwa represent the OIE risk model categories Fig. 4. Suggested risk analysis framework incorporating quantitative methods (Brouwer et al. 2018, Kristoffersen et al. 2018) and additional risk and quantitative examples for characterizing the risk of sea lice evolving tolerance to freshwater. 4. DISCUSSION Linear model Atlantic salmon altered the detachment and survival (2018) of gill-isolated Neoparamoeba perurans Probabilistic model − Antibiotic Risk of antibiotic resistance development Rico et al. exposure concentrations concentrations in environmental compartments (2017) to antibiotics in pond water in aquaculture production systems QMRA and BN Foodborne and Evaluation of the capabilities Beaudequin et al. waterborne pathogens and challenges of current (2015) QMRA methods and BN models QMRA and BN Food safety Explore Bayesian computational Greiner et al. statistics in the context of QMRA. (2013) Explore the analogy between Monte Carlo modelling and Bayesian inference Hydrodynamic model Salmon lice Hydrodynamic ocean model system, Myksvoll et al. particle tracking model (infestation pressure) (2018) and Bayesian statistical model Spatial−temporal linear Sea lice To assess relative spatial and temporal Kristoffersen et al. mixed model risks associated with farm-origin salmon lice. (2018) Qualitative and quantitative, Foodborne Discuss the formal OIE risk analysis CAC (2011) including risk ranking and antimicrobial approach, as well as qualitative steps, OIE approaches resistance quantitative steps and risk ranking steps for evaluating the risk of foodborne antimicrobial resistance in humans MCDA Food safety To demonstrate the use of ranking methods Fazil et al. (PROMETHEE) in microbial food safety decision making to (2008) reduce microbial risks MCDA −additive Fish farm To rank freshwater salmonid fish farms in a Oidtmann et al. risk score model microbiological risks country or region free of a specified pathogen (2011) for the likelihood of disease introduction and spread of the specified pathogen Decision chart Antibiotic resistance, Risks associated with genes that are present Martínez et al. human pathogens in environmental resistomes (likelihood of their (2015) introduction into human pathogens, and the consequences for the treatment of infections) and their weights, then the factors for the ‘known’ degree of freshwater tolerance are more likely to fail (i.e. copepodid sea lice are more likely to evolve freshwater tolerance), followed by those that cause inhibited development. On the other hand, if we look at the preadult model, ‘<30% survival’ had the high- est ranking (i.e. the most likely to see freshwater tol- erance evolution), followed closely by ‘known’, and then less likely (larger gap in net phi values) were the remaining ‘partial’ degrees of freshwater toler- ance (i.e. these are better choices if we want to avoid freshwater tolerance occurring). 4. DISCUSSION Ovals represent the OIE risk model categories case example that either increased uncertainty or led to non-accessibility of risk estimates. water tolerance that are more or less likely to occur over others will help decision makers to determine those that are to be preferred over others as a first- choice management strategy in order to decrease the likelihood of sea lice evolving tolerance to freshwater treatments. For instance, for the hypothetical cope- podid model, the ‘known’ degree of freshwater toler- ance had the highest net phi value, while the ‘partial’ freshwater tolerance degrees were grouped lower, mostly in pairs evenly spaced, and specifically ‘inhib- ited development’ was second in preference to ‘known’. Practically, if treatment and management protocols include the defined factors and subfactors The degrees of the hazard occurring that are used can be changed based on the set of factors, subfac- tors and weights used as new empirical information becomes available, or if the effect of various rankings (for example, midpoint versus extreme values) may need to be evaluated depending on the purposes of the RA (see Fig. 1). The practical interpretation of ranking results is dependent on the inclusion of fac- tors and up-to-date information that will define dif- ferent management or mitigation protocols. The ranking (net phi value) of some degrees of fresh- Aquacult Environ Interact 12: 417–428, 2020 426 Table 4. Various modelling approaches discussed that can be incorporated or modified for a full risk analysis framework, in- cluding risk ranking, for evaluation of the risk of freshwater tolerance evolution occurring in sea lice. QMRA: quantitative mi- crobial risk assessment; SD: system dynamic; BN: Bayesian network; OIE: World Organization for Animal Health; MCDA: multi-criterion decision analysis Table 4. Various modelling approaches discussed that can be incorporated or modified for a full risk analysis framework, in- cluding risk ranking, for evaluation of the risk of freshwater tolerance evolution occurring in sea lice. QMRA: quantitative mi- crobial risk assessment; SD: system dynamic; BN: Bayesian network; OIE: World Organization for Animal Health; MCDA: multi-criterion decision analysis Modelling approach Species/uses Main goal of models Citation QMRA and SD Waterborne bacteria Review to look at integration of QMRA Brouwer et al. and SD for: risk of waterborne outbreaks (QMRA) (2018) and quantification of transmission pathways (SD) SD Gill disease in Assess how freshwater exposure duration Wright et al. 4. DISCUSSION For trans- parency, RAs also need to describe all factors that contribute to uncertainty (‘lack of precise knowledge of input values’) around the risk estimate and as - sumptions made in lieu of available data (Travis & Hueston 2001). For example, some of the factors from the Tasmanian study by Wright et al. (2018) could be used in a quantitative RA model (e.g. time limits, sublethal or lethal outcomes, salinity doses, etc.). For another example, Rico et al. (2017) looked at a prob- abilistic approach to the development of antimicro- bial resistance in aquaculture and tolerance to antibiotic treatments; it may be possible to alter their calculations for exposure concentration, probability, minimum selective concentration (this would only be applicable if looking at ranges of salinity, not just freshwater) and tolerance development risk for toler- ance to freshwater treatments instead of antibiotic treatments. One of the major differences, however, is that chemical resistance frequently evolves from a single-point mutation, whereas freshwater tolerance may be polygenic (reviewed in Groner et al. 2019). Rico et al. (2017) suggests that the tolerance develop- ment risk ‘can be used to derive tolerance threshold concentrations’, but they caution that the model needs to ‘adequately define exposure standards pro- tective of background tolerance levels…’ (based on factors including amount of exposure, length of ex - posure time, dose, environmental factors, susceptibil- ity factors in fish and fish biomass in environments). However, because of limitations and un certainties, they suggest their approach should only be used for the initial phase of RA. with currently available data from recent freshwater tolerance research and literature reviews. In addi- tion, we present a flowchart that can assist future in- depth and quantitative RAs, particularly for fresh- water tolerance evolution in sea lice as additional data become available. Acknowledgements. This project was funded in kind by the Canada Excellence Research Chair in Aquatic Epi- demiology programme (Atlantic Veterinary College, Prince Edward Island), the University of Prince Edward Island, and the Norwegian Seafood Research Fund (FHF; Project Num- ber 901438). We thank Melanie Andrews from the Norwe- gian University of Life Sciences for her expert opinion. We also thank Maya L. 4. DISCUSSION In our case example, we were not able to consider the uncertainty of the output of closer-ranked de - grees of freshwater tolerance for simplicity’s sake, but as data become more available, then a subse- quent quantitative model can help to handle the uncertainty. In general, quantitative RA probabilities and sensitivity analyses are identified mathemati- cally through stochastic simulation models using Laurin et al.: Risk analysis framework in aquaculture 427 software, such as @RISK, to perform Monte Carlo simulations (Greiner et al. 2004, Peeler et al. 2007). All model assumptions for each step need to be trans- parently described (Greiner et al. 2004). For trans- parency, RAs also need to describe all factors that contribute to uncertainty (‘lack of precise knowledge of input values’) around the risk estimate and as - sumptions made in lieu of available data (Travis & Hueston 2001). For example, some of the factors from the Tasmanian study by Wright et al. (2018) could be used in a quantitative RA model (e.g. time limits, sublethal or lethal outcomes, salinity doses, etc.). For another example, Rico et al. (2017) looked at a prob- abilistic approach to the development of antimicro- bial resistance in aquaculture and tolerance to antibiotic treatments; it may be possible to alter their calculations for exposure concentration, probability, minimum selective concentration (this would only be applicable if looking at ranges of salinity, not just freshwater) and tolerance development risk for toler- ance to freshwater treatments instead of antibiotic treatments. One of the major differences, however, is that chemical resistance frequently evolves from a single-point mutation, whereas freshwater tolerance may be polygenic (reviewed in Groner et al. 2019). Rico et al. (2017) suggests that the tolerance develop- ment risk ‘can be used to derive tolerance threshold concentrations’, but they caution that the model needs to ‘adequately define exposure standards pro- tective of background tolerance levels…’ (based on factors including amount of exposure, length of ex - posure time, dose, environmental factors, susceptibil- ity factors in fish and fish biomass in environments). However, because of limitations and un certainties, they suggest their approach should only be used for the initial phase of RA. software, such as @RISK, to perform Monte Carlo simulations (Greiner et al. 2004, Peeler et al. 2007). All model assumptions for each step need to be trans- parently described (Greiner et al. 2004). LITERATURE CITED Aaen SM, Helgesen KO, Bakke MJ, Kaur K, Horsberg TE (2015) Drug resistance in sea lice: a threat to salmonid aquaculture. Trends Parasitol 31: 72−81 Abolofia J, Asche F, Wilen JE (2017) The cost of lice: quanti- fying the impacts of parasitic sea lice on farmed salmon. Mar Resour Econ 32: 329−349 Aldrin M, Huseby RB, Stien A, Grøntwedt RN, Viljugrein H, Jansen PA (2017) A stage-structured Bayesian hierarchi- cal model for salmon lice populations at individual salmon farms—estimated from multiple farm data sets. Ecol Model 359: 333–348 Beaudequin D, Harden F, Roiko A, Stratton H, Lemckert C, Mengersen K (2015) Beyond QMRA: modelling micro- bial health risk as a complex system using Bayesian net- works. Environ Int 80: 8−18 Belton V, Stewart T (2002) Multiple criteria decision analy- sis: an integrated approach. Kluwer Academic Publish- ers, Boston Brouwer AF, Masters NB, Eisenberg JNS (2018) Quantita- tive microbial risk assessment and infectious disease transition modeling of waterborne enteric pathogens. Curr Environ Health Rep 5: 293−304 4. DISCUSSION Groner for her expert opinion and guid- ance with the research development; her contribution was carried out while serving as a visiting scientist with the US Geological Survey (any use of trade, firm, or product names is for descriptive purposes only and does not imply endorse- ment by the US Government). 5. CONCLUSION CAC (Codex Alimentarius Commission) (2011) Guidelines for risk analysis of foodborne antimicrobial resistance (CAC/ GL 77-2011). CAC, Rome. www. codexalimentarius.net/ download/standards/11776/CXG_077e.pdf As an aid to focusing future research for aqua - culture studies with quantitative data limitations, we propose including a risk ranking method in an in tegrated RA framework that can assist decision makers, dependent on their goals, as well as data and funding availabilities. We suggest that an initial ranking method can be used to prioritize more in- depth qualitative and quantitative RAs, using a soft- ware that provides practical and interactive graphics for visualizing the impact of different factors and their respective weights on the risk of the hazard occurring. We have presented hypothetical examples around risk ranking for 2 different sea lice life stages, Covello VT, Merkhofer MW (1993) Risk assessment meth- ods: approaches for assessing health and environmental risks. Plenum Publishing, New York, NY Crosbie T, Wright DW, Oppedal F, Johnsen IA, Samsing F, Dempster T (2019) Effects of step salinity gradients on salmon lice larvae behaviour and dispersal. Aquacult Environ Interact 11: 181−190 Fazil A, Rajic A, Sanchez J, McEwen S (2008) Choices, choices: the application of multi-criteria decision analy- sis to a food safety decision-making problem. J Food Prot 71: 2323−2333 Figueira J, Greco S, Ehrgott M (2005) Multiple criteria decision analysis: state-of-the-art surveys. Springer, New York, NY Aquacult Environ Interact 12: 417–428, 2020 428 OIE (2019) Import risk analysis. OIE Aquatic animal health code, Chapter 2.1. OIE, Paris OIE (2019) Import risk analysis. OIE Aquatic animal health code, Chapter 2.1. OIE, Paris Greiner M, Paisley L, Hostrup-Pedersen J, Lo Fo Wong DMA and others (2004) Principles, application areas and an example of risk assessment conducted at the Danish Institute for Food and Veterinary Research. Berl Munch Tierarztl Wochenschr 117: 177−181 Overton K, Dempster T, Oppedal F, Kristiansen TS, Gis- mervik K, Stien LH (2019) Salmon lice treatments and salmon mortality in Norwegian aquaculture: a review. Rev Aquacult 11:1398–1417 Tierarztl Wochenschr 117: 177−181 Greiner M, Smid J, Havelaar AH, Müller-Graf C (2013) Graphical models and Bayesian domains in risk model- ling: application in microbiological risk assessment. Prev Vet Med 110: 4−11 Peeler EJ, Murray AG, Thebault A, Brun E, Giovaninni A, Thrush MA (2007) The application of risk analysis in aquat ic animal health management. 5. CONCLUSION Prev Vet Med 81: 3−20 Groner ML, Laurin E, Stormoen M, Sanchez J, Fast M, Revie CW (2019) Evaluating the potential for sea lice to evolve freshwater tolerance as a consequence of freshwater treatments in salmon aquaculture. Aquacult Environ Interact 11: 507−519 Rico A, Jacobs R, Van den Brink PJ, Tello A (2017) A proba- bilistic approach to assess antibiotic tolerance develop- ment risks in environmental compartments and its appli- cation to an intensive aquaculture production scenario. Environ Pollut 231: 918−928 Kristoffersen AB, Qviller L, Helgesen KO, Vollset KW, Vilju- grein H, Jansen PA (2018) Quantitative risk assessment of salmon louse-induced mortality of seaward-migrating post-smolt Atlantic salmon. Epidemics 23: 19−33 Sievers M, Oppedal F, Ditria E, Wright DW (2019) The effec- tiveness of hyposaline treatments against host-attached salmon lice. Sci Rep 9: 6976 Smith P (2001) Assessing the risks associated with the use of antimicrobial agents in aquaculture. In: Rodgers CJ (ed) Proceedings of risk analysis in aquatic animal health. World Organisation for Animal Health (OIE) conference in Paris, France, February 2000. OIE, Paris, p 175−184 Ljungfeldt LER, Quintela M, Besnier F, Nilsen F, Glover KA (2017) A pedigree-based experiment reveals variation in salinity and thermal tolerance in the salmon louse, Lep- eophtheirus salmonis. Evol Appl 10: 1007−1019 p pp Martínez JL, Coque TM, Baquero F (2015) What is a resist- ance gene? Ranking risk in resistomes. Nat Rev Micro- biol 13: 116−123 Travis D, Hueston W (2001) Factors contributing to uncer- tainty in aquatic animal risk analysis. In: Rodgers CJ (ed) Proceedings of risk analysis in aquatic animal health. OIE conference in Paris, France. February 2000. OIE, Paris, p 27−35 McEwan GF, Groner ML, Burnett DL, Fast MD, Revie CW (2016) Managing aquatic parasites for reduced drug resistance: lessons from the land. J R Soc Interface 13: 20160830 Vose D (2001) Qualitative versus quantitative risk analysis and modelling. In: Rodgers CJ (ed) Proceedings of risk analysis in aquatic animal health. OIE conference in Paris, France, February 2000. OIE, Paris, p 19−26 Myksvoll MS, Sandvik AD, Albretsen J, Aspin L and others (2018) Evaluation of a national operational salon lice monitoring system — from physics to fish. PLOS ONE 13: e0201338 Wright DW, Nowak B, Oppedal F, Crosbie P, Stien LH, Dempster T (2018) Repeated sublethal freshwater expo- sures reduce the amoebic gill disease parasite, Neop- aramoeba perurans, on Atlantic salmon. Submitted: January 21, 2020; Accepted: August 17, 2020 Proofs received from author(s): October 13, 2020 Editorial responsibility: Bengt Finstad, Trondheim, Norway 5. CONCLUSION J Fish Dis 41: 1403−1410 Oidtmann BC, Crane CN, Thrush MA, Hill BJ, Peeler EJ (2011) Ranking freshwater fish farms for the risk of patho- gen introduction and spread. Prev Vet Med 102: 329−340 Editorial responsibility: Bengt Finstad, Trondheim, Norway
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Furniture design based on parent-child interaction experience
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1 Introduction Parent-child interactive furniture is an innovative design concept in today's home furnishing industry. The core lies in the design of home products that can meet the needs of parents and children's interaction. It fits the lifestyle and promotes children's physical development, mental health and emotional exchanges between parents and children. Because parents are children's first teachers, furthermore, they are the closest friends of children. Furniture is a necessary product in family daily life, and it is the carrier of various activities and behaviors[1]. The guiding concept of furniture design has gone through various stages, from the initial "product service to function". The concept of furniture design will go through all stages. The original form is summarized as "product serves function". And then after the arts and crafts thought in the late 19th century and pop art in the 20th century, it has developed into a diversified modern design theory. We can see that with the improvement of people's living standard and value orientation, the aesthetic and connotation of furniture design has changed significantly. However, in most families in the city, many parents are busy in work. When the parents return home, they are entangled in family chores and lack of time and energy to get along with the children. In addition, electronic products emerge in an endless stream in the information age. In fact, many adults addicted to their mobile phones, children are also attracted by online games and mobile apps. In the remote countryside, there are many left behind children who are separated from their parents for a long time, and the communication and emotional communication between parents and children are increasingly reduced. This kind of life style separated from parent-child interaction not only causes physical fatigue of children, but also can not stimulate children's desire to explore the environment and knowledge, which is not conducive to the healthy development of children's body and mind. The concept of interactive design is a study that emphasizes the interaction between people and objects[2]. In the furniture industry, the concept of interactive design is a very valuable guiding concept, which can guide designers to combine people's life style with furniture production. It can promote the communication and interaction between people and furniture, and improve people’s family life taste. Furniture design based on parent-child interaction experience Jingyuan Zhu1, Qing Wu2 1Art and design College, Wuhan University of Science and Technology, Wuhan, Hubei province, 430065, China 2Art and design College, Wuhan University of Science and Technology, Wuhan, Hubei province, 430065, China Abstract. To explore how children's furniture can become a tool of parent-child interaction, so that furniture can not only satisfy children's curiosity, but also bring new feelings and experiences to parents and children. It is necessary to establish a bond of love between parents and children by adding parent-child interaction projects. This children’s furniture will give children a warm family life experience. Background analysis of parent-child interaction furniture design was conducted by means of data search and market research to explore children's physiological and psychological characteristics. It is necessary to analyze parent-child interaction mode and its elements and try to make compound chairs have interactive functions so that compound chairs can bring good interaction experience to parents and children. As a result, this compound chair can meet user needs. The design features of parent-child interactive furniture are analyzed with the physiological characteristics of children as the design elements. Personally interactive furniture can create an interactive world for children and parents in a limited living space. It is able to promote the emotional exchange between parents and children, create a space conducive to children's physiological and psychological growth. Ultimately, this kind of interactive furniture can promote the physical and mental health of children. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: 961349008@qq.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: 961349008@qq.com E3S Web of Conferences 179, 02098 (2020) EWRE 2020 E3S Web of Conferences 179, 02098 (2020) EWRE 2020 https://doi.org/10.1051/e3sconf/202017902098 © The Authors, published by EDP Sciences. This is an open access article distributed License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction Interactive design concept can promote the innovation of home design, mainly reflected in the following aspects: The concept of interactive design emphasizes theme and innovation in the forms of expression. The way of interaction between people and furniture and the spiritual E3S Web of Conferences 179, 02098 (2020) EWRE 2020 https://doi.org/10.1051/e3sconf/202017902098 A children's furniture brand called ABC has designed a bed for parents and children, which is different from the conventional bunk beds. This kind of furniture not only has the function of bed, but also has the function of multiple closets and storage cabinets, which can be divided and combined, and can effectively solve the problem of children's separation when they grow up. A children's furniture brand called ABC has designed a bed for parents and children, which is different from the conventional bunk beds. This kind of furniture not only has the function of bed, but also has the function of multiple closets and storage cabinets, which can be divided and combined, and can effectively solve the problem of children's separation when they grow up. connotation to be conveyed are also varied. Thus, the design of interactive products should be presented with a certain themes, with emphasis and flexibility. Theme design reflects the designers' thinking and exploring process in interactive experience. Furniture design not only meets the needs of basic family activities, but also extends the value beyond the functional design to expand the innovative elements of furniture in a diversified and personalized form. Fig1. Parent-child bunk The concept of interactive design emphasizes sensory and psychological activities in the way of interaction. Furniture is not only an objective functional product, but also has vitality. Through the interaction and experience of vision, auditory, touch and other multiple senses, it creates a harmonious sense between people and environment. The concept of interactive design emphasizes the user oriented theory in the service object. Traditional furniture design is endowed with functions by designers, and users passively accept the service provided by designers. In the concept of interactive design, designers and users no longer draw a clear line. Furniture users are the second designers of furniture design. They can feedback, optimize and even recreate the use of products. Fig1. Parent-child bunk However, these furniture brand products are usually only designed for children and their functional structure is relatively single, which can not provide a good atmosphere and platform for parent-child interaction. 1 Introduction In this context, parent-child interactive furniture came into being. It provides interactive facilities for parents and children in the family residence like a small "playground" at home. It sends thoughts on the design of parent-child interactive furniture, which can not only meet the needs of daily home, but also cultivate children's hands-on and brain skills. It has good entertainment and education expansion functions. It can guide parents and children to communicate and interact, so that parents and children can fully feel the interaction of family affection, and establish a bond of love between parents and children. The concept of interactive design emphasizes emotional communication in spiritual connotation. With the accelerated pace of life, people are suffering from heavy work pressure and life pressure, and there are less and less communication between people. Therefore, the interactive design of furniture can promote the emotional communication between family members. On this basis, in-depth dialogue and communication can be carried out between relatives, which can shorten the distance between people’s hearts [2]. 3 Market background and research of parent-child interactive furniture In order to make the society's views on parent-child interactive furniture understood better and prospect of parent-child interactive furniture. I compiled an online survey questionnaire which collected nearly 200 valid data from families. In the conclusion of Figure 2, nearly 90% of parents have a strong interest in parent-child interactive furniture, which comes from the internal demand for children to obtain family happiness. At the same time, figure 3 shows that most parents lack in understanding of parent-child interactive furniture. At present children's consumption market is a huge market with great business opportunities and vitality. Consumption of children plays an important role in the whole family[3]. According to experts' prediction, in the future, Children's consumption will have a market scale of 100 billion. Therefore, in recent years, parent-child interaction and parent-child education have become more and more important in the society and family, and the related research is very extensive. Environment and facilities that meet the needs of parent-child activities have been derived at home and abroad. From the perspective of the market, there are many household brands for children's design. These brand furniture can start from the characteristics of children, meet the design of ergonomics and the daily life needs of ordinary families. For example, The survey shows that parent-child interactive furniture has a good social demand and market prospect. While it puts forward higher requirements for furniture designers to conceive and design more innovative and dynamic interactive home products. 2 2 E3S Web of Conferences 179, 02098 (2020) EWRE 2020 E3S Web of Conferences 179, 02098 (2020) EWRE 2020 https://doi.org/10.1051/e3sconf/202017902098 Fig 2. Research on the demand Fig 3. Research on the understanding Fig 2. Research on the demand Fig 3. Research on the understanding Fig 2. Research on the demand Fig 3. Research on the understanding Fig 2. Research on the demand 4 Elements of parent-child interactive furniture design Children are the main part of parent-child interaction. The design of parent-child furniture should be based on children's nature and conform to the general law of children's physical and mental development[5]. Access to relevant information, the physiological and psychological characteristics of children aged 3-6 are shown in Table 1. The design feature of parent-child interactive furniture is to establish a household system in line with the characteristics of children. It is adopt interactive form, and taking into account the family applicability of the family[4]. The core advantage is to build a bridge between parents' and children's love in daily home life, which could give children the inspiration of entertainment and the soul of growth. Table 1. Psychological and physiological characteristics of children aged 3-6 psychological characteristics Physiological characteristics Have a strong thirst for knowledge and curiosity. They learned to walking, running and jumping, these movements are gradually coordinated. Sensitive to color and shape, start to master cognitive methods and have certain thinking ability. Be able to understand most of the dialogue, listen to songs and speak simple sentences. Have preliminary aesthetic consciousness. Can consciously carry out artistic conception [6]. Like to imitate other people's actions, can use tableware to eat. Have their own opinions and independence. Be able to follow the rules and participate in game activities. Can have some restraint in the mood and produce the psychology of fear under certain situation. Children can balance themselves when jumping from a certain height and stand still. They like the outdoor environment. The initial formation of personality, plasticity. Children can draw pictures, fold papers, match building blocks and climb with both their hands and brains. Table 1. Psychological and physiological characteristics of children aged 3-6 3 E3S Web of Conferences 179, 02098 (2020) EWRE 2020 https://doi.org/10.1051/e3sconf/202017902098 The following principles should be considered in the parent-child interactive furniture design, as shown in Figure 4. 4.2 Principles of parent-child interactive furniture design Fig 4. Design principles of parent-child interactive furniture   Principles of parent-child interactive furniture Practicability Safety Particularity Interaction Practicability Safety Principles of parent-child interactive furniture Fig 4. Design principles of parent-child interactive furniture Safety: safety is the most important element of design. Children have a lively and active nature, nevertheless their self-protection awareness is far from enough, which is also the point that many parents are most concerned about. Hence safety is the premise of all design. 4 Elements of parent-child interactive furniture design As a furniture, it must have a stable structure and sufficient strength which can ensure that children do not topple or break during the play. Secondly, the shape of the furniture should be safe, without sharp corners and the elements such as glass and mirror that may cause harm to children should be eliminated [7]. In addition, choose material safety also is very important, the material of children furniture must pass the national green safety certification to be green and pollution-free. abandon the attributes of furniture. Therefore, not only the interaction between parents and children should be fully considered, but also the cost performance, such as occupation area and time limit are the key indicators of practicality. Interactivity: furniture is a material carrier which plays an intermediary role. By using parent-child interactive furniture, we can create opportunities for parent-child communication and cooperation, and increase the tacit understanding between them. The most common parent- child interactive furniture can be divided into sports type, game type, leisure type and activity type. See figure 5. Practicability: the design of parent-child interactive furniture should take practicality into account, and do not Practicability: the design of parent child interactive furniture should take practicality into account, and do not Fig 5. Classification of parent-child interaction Interactive mode Sports type  Game type  Leisure type  Active type Interactive mode Interactive mode Fig 5. Classification of parent-child interaction with sports style is designed. Since children like outdoor exploration games, they can learn from such game mode to play simple game at home. Product which meets the basic seat function and provides the parent-child interaction space, while let parents and children get the sense of participation and warm experience of daily family activities. Particularity: boys and girls have different ways of living and interaction. Boys prefer logical, three- dimensional and interesting interactive furniture design, while girls focus on sensory, color and richness of furniture design. In addition, children's personality and hobbies are also special factors of parent-child interactive furniture design. 5 Practice of parent-child interactive furniture design Based on the above design concept and design elements of parent-child interaction, a parent-child interactive seat 4 https://doi.org/10.1051/e3sconf/202017902098 E3S Web of Conferences 179, 02098 (2020) E3S Web of Conferences 179, 02098 (2020) EWRE 2020 5.1 Design orientation of parent-child interactive furniture The design orientation of parent-child interactive furnit in this design should consider various comprehens factors, as shown in Figure 6. Fig 6. Design orientation of parent-child interactive furniture Design position Price Users Material Form Function 5.1 Design orientation of parent-child interactive furniture The design orientation of parent-child interactive furniture in this design should consider various comprehensive factors, as shown in Figure 6. parent-child The design orientation of p in this design should co factors, as shown in Figure Fig 6. Design orientation of parent-child interactive furniture Design position Price Users Material Form Function Function Price Material Design position Users Form Fig 6. Design orientation of parent-child interactive furniture In the design process, it is considered to take the seat as the carrier, integrate into environmental exploration projects such as climbing, drilling caves, etc., so as to establish personalized and versatile seat products. Safety is the basic requirement of design, so the form design must not be too complex, and the final form of design should have a simple and harmonious warm family style. The basic material of the seat is pine, which is determined as environmental protection furniture by the Ministry of culture and culture of the United Nations. It is solid, light and durable [8].All edges and corners of wood must be rounded and smoothed to prevent scratches. In addition, felt and cloth are added to package in the design process, so that both safety and comfort are taken into consideration. The diversity and individuation of functions determine the price ceiling of products. The process of children’s development is divided into different stages. The target users of this design are 3-6 years old children in early childhood. During this period, children are active and do not have heavy homework, which is the key period of character cultivation. Furniture positioning belongs to mid-range household products, with a price of 1000 yuan. Most families can afford it. parent-child interactive seat, with the appearance dimension of 1.2 * 0.7 * 1.0m. Fig 7. Basic structure of parent-child interactive seat Fig 7. 5 Practice of parent-child interactive furniture design Basic structure of parent-child interactive seat The design includes many elements such as light, sound, electricity, mechanics, etc., which could increase the fun and diversity of parent-child interaction from the perspective of vision, auditory and other senses [10].Visually, the decorative light string adopts LED light strip, which can select a variety of colors to make the seat appear fashionable and colorful. LED with low voltage and small size is relatively safe. In terms of hearing, Bluetooth speaker is hidden in the dark design of wood board. You can choose music, spoken English or fairy tales to bring better hearing experience to children. 5.2 Details of parent-child interactive furniture design The seats reserve space for expansion. Parents and children can act as second designers to give their own parent-child interactive furniture unique inspiration and design elements. Dynamically, the interactive furniture adopts a folding and extending design architecture, which can have a variety of shapes[11]. Statically, the DIY function of painting and weaving can be added. When parents are reading books or watching videos, children can draw pictures and play small games. "Indoor amusement park" is the theme of this interactive furniture. The design inspiration comes from the children's entertainment facilities in the shopping mall, which integrates the innovative elements of rock climbing and cave, making children feel novel. The back of the seat is made of rattan and rope, green and environmental protection. This product uses wood panel for splicing, which is stronger and more stable than ordinary seats. Shape is simple without sharp corners. Children's sensory system is very sensitive, bright colors are more likely to arouse their interest[9], so the color of the rope is design to be red. The seat cushion and the corner are pasted with the dirty resistant gray felt. The gray felt and the red rope complement each other. The joint of the wood board is pasted with the Red Lace felt, which is full of children's interest. There is also a high and large cave for children behind the chair. Figure 7 is the structural diagram of From the emotional point of view of parent-child interaction, rope is like a bond of love to weave parent- child families. All kinds of entertainment facilities facilitate warm interaction and play between parents and children. And speaker devices enable children to get education in happiness and interest[12]. A little bit of starlight adds a touch of warmth to it, immerses children in their own small world, and makes the family 5 https://doi.org/10.1051/e3sconf/202017902098 E3S Web of Conferences 179, 02098 (2020) EWRE 2020 environment warm, colorful and enjoyable. Figure 8 shows the effect of parent-child interaction. Fig 8. Effect picture of parent-child interactive seat environment warm, colorful and enjoyable. Figure 8 shows the effect of parent-child interaction. 9. ZHANG Ying. (2007 )Material Factors In Color Expression[J]. East China Normal University. 9. ZHANG Ying. (2007 )Material Factors In Color Expression[J]. East China Normal University. 9. ZHANG Ying. (2007 )Material Factors In Color Expression[J]. East China Normal University. 10. Otaka Maisel. (2011) Chair Design[M]. Shandong Pictorial Publishing. 10. Otaka Maisel. 6 Summary It is concluded that parent-child interactive furniture is a kind of functional and interactive household product, which can create a good parent-child interactive experience in the limited family space. This product could not only let children get the inspiration of entertainment and learning, but also deepen the feelings between family members in the interaction. The designer needs to take children as the design goal and design products that conform to children's naive and lively personality from the aspects of safety, practicality, interest, etc. The detachable versatile parent-child interactive seat can not only provide a comfortable seat environment for parents, but also provide diversified entertainment items for children. It is also an exercise tool to practise the children's senses of their visual, auditory, tactile. Furthermore, it can create a growth environment beneficial to children's physical and mental health. 5.2 Details of parent-child interactive furniture design (2011) Chair Design[M]. Shandong Pictorial Publishing. Fig 8. Effect picture of parent-child interactive seat 11. WANG LiYing, YU Li, DENG YingHua. (2017) Early childhood product design based on preschool children's life form[J]. Packaging Engineering. 11. WANG LiYing, YU Li, DENG YingHua. (2017) Early childhood product design based on preschool children's life form[J]. Packaging Engineering. 12. MAN XiaoMan. (2019) Research on parent-child interactive product design based on emotional interaction model[J]. Shandong University. Fig 8. Effect picture of parent-child interactive seat 10. Otaka Maisel. (2011) Chair Design[M]. Shandong Pictorial Publishing. References 1. Jim Postell. (2014) furniture design[M]. U.S.A. 1. Jim Postell. (2014) furniture design[M]. U.S.A. 2. ZENG Rong. (2011) The Applied Research On Interactive Design Concept In Furniture[J]. Jiangnan University. 3. Rena A. Hallam.(2016) The Role of Home Visits in Promoting Positive Parent-Child Interaction [M]. U.S.A. 4. ZHU Hong. (2019) Children's Furniture Design Based On Parent-child Interaction Experience[J] .Craft Building. 5. Donald Norman. (2005) Emotional Design[M]. Beijing: Electronic Industry Press. 6. NIE Qian. (2015) Emotional Design Of Parent- child Products[J]. Art Design Research. 7. LUO ShiJian, YING FangTian.(2011) Children's product design[M]. Beijing: Mechanical Industry Press. 8. ZHANG RuoYun.(2018) Parent Child Interactive Product Design Based On Kansei Engineering[J]. NCEPU. 6 6
https://openalex.org/W2539775685
https://journals.vgtu.lt/index.php/MLA/article/download/2511/2033
Lithuanian
null
NOISE EMISSION CAUSED BY TRANSPORT IN TRAKAI CITY MODELLING AND EVALUATION / TRANSPORTO SUKELIAMO TRIUKŠMO SKLAIDOS TRAKŲ MIESTE MODELIAVIMAS IR VERTINIMAS
Mokslas - Lietuvos ateitis
2,016
cc-by
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Transporto sukeliamo triukšmo sklaidos Trakų mieste modeliavimas ir vertinimas Tomas VILNIŠKIS1, Tomas JANUŠEVIČIUS2 Vilniaus Gedimino technikos universitetas, Vilnius, Lietuva El. paštas: 1tomasviln@gmail.com; 2tomas.januševičius@vgtu.lt Santrauka. Viena didžiausių problemų miestuose – tai vis didėjančių transporto srautų sukeliamas triukšmas ir jo sklaida aplinkoje. Yra nustatyta, kad aplinkos triukšmas gali sukelti ne tik nepatogumų, tačiau ir kenkti sveikatai. Todėl triukšmo sklaida aplinkoje yra aktuali aplinkosaugos problema. Šiame straipsnyje vertinama transporto triukšmo sklaida, atsižvelgiant į transporto srautus Trakų mieste. Parinktose vietose šiaurinėje ir pietinėje miesto dalyse yra išmatuojami ekvivalentiniai ir didžiausieji garso lygiai. Pagal apskaičiuotus transporto srautus dienos, vakaro ir nakties metu, naudojant CadnaA programą, sudaromas triukšmo sklaidos žemėlapis. Žemėlapiuose pavaizduota triukšmo sklaida dienos, vakaro ir nakties metu. Atlikus matavimus, buvo nustatyta, jog didžiausios triukšmo vertės yra prie pagrindinių kelių. Pietinėje miesto dalyje, šalia Gedimino gatvės, ekvivalentinis garso lygis dienos metu siekia 60 dB, vakaro – 54 dB, nakties – 48 dB. Šiaurinėje miesto dalyje didžiausi triukšmo lygiai buvo prie Karaimų gatvės, kur ekvivalentinis garso lygis dienos metu siekė 59 dB, vakaro metu buvo 54 dB, nakties metu 51 dB. Atlikus modeliavimą, gautos garso lygio vertės buvo panašios kaip ir išmatuotos, skirtumai tarp sumode- liuotų ir išmatuotų verčių buvo iki 4 dB. Reikšminiai žodžiai: garso lygis, transporto triukšmas, triukšmo sklaida, modeliavimas. 2016 © Straipsnio autoriai. Leidėjas VGTU leidykla „Technika“. Šis straipsnis yra atvirosios prieigos straipsnis, turintis Kūrybinių bendrijų (Creative Commons) licenciją (CC BY-NC 4.0), kuri leidžia neribotą straipsnio ar jo dalių panaudą su privaloma sąlyga nurodyti autorių ir pirminį šaltinį. Straipsnis ar jo dalys negali būti naudojami komerciniams tikslams. MOKSLAS – LIETUVOS ATEITIS SCIENCE – FUTURE OF LITHUANIA ISSN 2029-2341 / eISSN 2029-2252 http://www.mla.vgtu.lt MOKSLAS – LIETUVOS ATEITIS SCIENCE – FUTURE OF LITHUANIA ISSN 2029-2341 / eISSN 2029-2252 http://www.mla.vgtu.lt MOKSLAS – LIETUVOS ATEITIS SCIENCE – FUTURE OF LITHUANIA APLINKOS APSAUGOS INŽINERIJA ENVIRONMENTAL PROTECTION ENGINEERING ISSN 2029-2341 / eISSN 2029-2252 http://www.mla.vgtu.lt http://dx.doi.org/10.3846/mla.2016.956 2016 8(4): 431–437 2016 © Straipsnio autoriai. Leidėjas VGTU leidykla „Technika“. Įvadas kelio tipas, jo sudėtis, transporto greitis ir intensyvumas (Gražulevičienė, Bendokienė 2009). Prie transporto suke- liamo triukšmo taip pat gali būti priskiriamas geležinkelių bei orlaivių triukšmas. Garso sklidimui įtakos turi šaltinio tipas, atstumas nuo triukšmo šaltinio, teritorijos paviršius, meteorologinės sąlygos (vėjas, temperatūra, drėgmė, kri- tuliai), atspindinčios kliūtys, ekranavimas (kliūtys kaip barjerai ir pastatai) (Lekavičiūtė 2007; Jensen et al. 2002). Aplinkos triukšmas pastaraisiais metais yra vis svarbes- nė pasaulinė problema – daugiausia dėl didelių transporto srautų miestuose. Transportas prisideda ne tik prie augančių šiltnamio efektą sukeliančių dujų emisijų, bet ir vis didėjan- čios triukšmo sklaidos miestuose (Bastian-Monarca et al. 2016). Transporto srautų didėjimas yra opi problema dau- gelyje miestų. Automobilių transportas yra specifinis taršos šaltinis, jis yra dinamiškas, todėl apima visas miesto terito- rijas, gyvenamąsias ir pramonines, miestų centrus, ligoninių ir sanatorijų teritorijas, taip pat rekreacines zonas (Vaišis, Januševičius 2009). Daugelyje miestų vidutinis triukšmo lygis pakyla 1–3 dB per metus ir prognozuojama, kad per 15 metų jis padvigubės (Baltrėnas et al. 2010). Transporto keliai yra sudėtingi inžineriniai statiniai, jiems keliami vis didesni reikalavimai. Kai kuriose šalyse nuo 2000 m. dėl technologinių sprendimų, kelio dangos keitimo, triukšmo barjerų įrengimo ir transporto srautų reguliavimo triukšmo lygis sumažėjo. Nepaisant to, manoma, kad triukšmo lygiai artimiausiais dešimtmečiais dėl transporto tik didės, nebent būtų imtąsi papildomų priemonių (Staatsen et al. 2004). Per 30 proc. Europos Sąjungos populiacijos (apie 120 milijonų gyventojų) yra veikiama didesnio nei 55 dB(A) ke- lių transporto triukšmo. Daug žmonių miego metu yra vargi- nami tokių garso lygių (Lekavičūtė 2007). Ekvivalentiniai triukšmo lygiai dienos metu, viršijantys 65 dBA, yra nepri- imtini. Kadangi daugelis gyvenamųjų rajonų yra pastaty- ti toliau nuo kelių, ten triukšmo lygis svyruoja tarp 55 ir 65 dBA (Zannin et al. 2013). Dėl nuolatinio triukšmo gali sutrikti miegas, sumažėti darbingumas (Stanfeld et al. 2000), pablogėti psichinė ir fizinė žmonių sveikata (Babisch 2005), kilti stresas. Minėti nuolatinio triukšmo sukelti padariniai turi įtakos įvairių žmogaus organizmo reakcijų kitimams, tarp jų ir homeostazės pakitimams. Ilgainiui atsiranda širdies ritmo, raumenų tonuso, smegenų elektrinio aktyvumo pokyčių, di- dėja emocinė įtampa, kurią lydi hipertenzija ir išeminė šir- dies liga (Tamošiūnas et al. 2005). Triukšmo įtaka žmogaus Pagrindiniai transporto priemonių triukšmo šal- tiniai yra varikliai, padangos, besiliečiančios su asfaltu, jėgos perdavimo mechanizmai, daug įtakos triukšmui turi Šiaurinėje miesto dalyje matavimai buvo atlikti įver- tinant Karaimų gatve važiuojančio transporto keliamo triukšmo sklaidą į gyvenamąją teritoriją. Triukšmo ly- giai buvo išmatuoti šiose vietose (2 pav.): Karaimų g. 95, Karaimų g. Įvadas 90, Karaimų g. 79, Karaimų g. 74, Karaimų g. 60 ir Karaimų g. 51. organizmui priklauso nuo triukšmo intensyvumo, dažnio, veikimo trukmės, taip pat nuo individualių žmogaus savy- bių – amžiaus, sveikatos, triukšmo jautrumo (Paulauskas, Klimas 2011). Pasak Pasaulinės sveikatos organizacijos pa- rengtos ataskaitos, triukšmo tarša po oro taršos yra antra didžiausia aplinkosauginė problema, kenkianti sveikatai ir yra atsakinga už 50 000 žmones ištinkančių širdies smūgių Europoje kiekvienais metais (WHO 2011). 2 pav. Triukšmo matavimo vietos šiaurinėje miesto dalyje Fig. 2. Noise level measurement points in the northern part of the city Trakai – miestas Vilniaus apskrityje. Trakų mies- to plotas – 11,5 km2. 2011 metų duomenimis, Trakuose gyveno 4933 žmonės. Gyventojų tankumas – 429 žm/km2. Triukšmingiausi transporto mazgai yra Trakų miesto terito- rijos pietinėje dalyje. Šioje teritorijoje yra traukinių stotis, taip pat eina magistralinis kelias A16, jungiantis Vilnių ir Marijampolę, bei krašto kelias 220, jungiantis Trakus ir Alytų. Didelė dalis transporto yra nukreipiama į krašto kelią 107, jungiantį Trakus ir Vievį. 2 pav. Triukšmo matavimo vietos šiaurinėje miesto dalyje Fig. 2. Noise level measurement points in the northern part of the city Metodika 30 LAeq,nT 44 39 32 LAmax 56 53 49 2. Gedimino g. 23 LAeq,nT 60 54 48 LAmax 73 67 62 3. Šilo g. 8 LAeq,nT 58 53 47 LAmax 70 66 59 4. Statybininkų g. 11 LAeq,nT 47 43 39 LAmax 60 57 55 5. Babruko g. 9 LAeq,nT 44 40 37 LAmax 57 54 52 6. Gedimino g. 26 LAeq,nT 52 46 41 LAmax 69 65 58 1 lentelė. Garso slėgio lygio matavimo rezultatai pietinėje mies- to dalyje Trakų miesto teritorijoje, kurioje buvo atlikti matavimai, vyrauja privatūs 2–3 aukštų namai. Jų aukštis siekia 7–10 metrų. Pagrindinės gatvės, kuriose vyksta intensyviausias eismas, buvo dviejų juostų. Gedimino gatvės plotis yra 11 metrų, Karaimų gatvės plotis 9 metrai. Kitos gatvės buvo siauresnės, jų plotis siekė 5–7 metrus. Transporto srautas Gedimino gatvėje dienos metu per vieną valandą buvo 120 automobilių, iš kurių 15 % sunkiasvorių, vakaro metu 34 automobiliai, iš kurių 12 % sunkiasvorių, ir nakties metu 19 automobilių, iš kurių 2 % sunkiasvorių. Karaimų gat- vėje transporto srautas per valandą dienos metu buvo 36 automobiliai, iš kurių 4 % sunkiasvorių, vakaro metu 17 automobilių, iš kurių 3 % sunkiasvorių, nakties metu 7 automobiliai, iš kurių 1 % sunkiasvorių. Krašto kelio 220 intesyvumas dienos metu buvo 80 automobilių, iš kurių 10 % sunkiasvorių, nakties metu 30 automobilių ir 8 % sunkiasvorių, nakties metu 15 automobilių, iš kurių 8 % sunkiasvorių. Kitose gatvėse eismo intensyvumas yra la- bai menkas, transporto srautai dienos metu neviršija 10 automobilių, vakaro metu ne daugiau kaip 5 automobi- liai, nakties metu 1–2 automobiliai. Matavimai buvo at- likti spalio mėnesį. CadnaA programoje gali būti įvertinta ir aprašyta iki 16 mln. objektų. Triukšmas normuojamas pagal ES direktyvas. Gauti triukšmo sklaidos rezultatai gali būti pavaizduojami trimačiu vaizdu. Darbo metu ga- lima remtis esamais triukšmo žemėlapiais. Skaičiavimo metodą gali nusistatyti vartotojas. Galima nustatyti, kokiu atstumu nuo taško – gavėjo ar nuo triukšmo šaltinio – bus skaičiuojamas triukšmo sklidimo modelis ir kokia tvarka. Nustačius laiko intervalus (diena, vakaras, naktis) ir pa- rametrų ribines reikšmes, gali būti taikomas visokių rūšių triukšmo šaltinių reguliavimas (Baltrėnas, Puzinas 2009). Modeliavimas atliekamas 4 m aukštyje nuo žemės pavir- šiaus. Gavus rezultatus, buvo nustatyta, kokie garso lygiai veikia gyventojus, nustatytos vietos, kur triukšmo sklaida nuo transporto yra didžiausia, ir ar yra nustatyti viršijimai, lyginant su Lietuvos higienos norma HN 33:2011. taip pat buvo išmatuotos didžiausios vertės vakaro ir nakties metu, atitinkamai 54 dB ir 48 dB. Metodika Triukšmo tyrimų tikslas – išanalizuoti triukšmo sklaidą Trakų miesto teritorijoje ir atlikti triukšmo sklaidos modeliavimą. Trakų miesto triukšmo sklaidai matuoti ir modeliuoti buvo pasirinktos pietinė ir šiaurinė miesto dalys. Pietinėje daly- je matavimai buvo atlikti prie krašto kelio 220, jungiančio Trakus ir Alytų. Triukšmo matavimo vietos buvo išdėstytos 6 vietose, siekiant įvertinti transporto sukeliamo triukšmo sklaidą į gyvenamąją teritoriją. Triukšmas vertintas šiose vietose (1 pav.): Gedimino g. 30, Gedimino g. 23, Šilo g. 8, Statybininkų g. 11, Babruko g. 9 ir Gedimino g. 26. Triukšmo lygiai buvo matuojami po 20 minučių kiekvienoje vietoje, skaičiuojant lengvųjų ir sunkiasvorių automobilių kiekį. Matavimai buvo atlikti dienos, vakaro ir nakties metu. Garso slėgio lygių matavimų rezultatai gyve- namoje teritorijoje palyginami su leidžiamomis triukšmo lygio vertėmis, nurodytomis Lietuvos higienos normoje HN 33:2011. Gyvenamųjų pastatų (namų) ir visuomeninės paskirties pastatų (išskyrus maitinimo ir kultūros paskir- ties pastatus) aplinkoje, veikiamoje transporto sukeliamo triukšmo, ekvivalentinio garso slėgio lygio vertės negali viršyti 65 dB dienos metu, 60 dB vakaro metu ir 55 dB nakties metu. Didžiausiojo garso slėgio lygio vertės negali viršyti 70 dB dienos metu, 65 dB vakaro metu ir 60 dB nakties metu. 1 pav. Triukšmo matavimo vietos pietinėje miesto dalyje Fig. 1. Noise level measurement points in the southern part of the city Pradedant triukšmo matavimus, nustatomos meteoro- loginės sąlygos – temperatūra, oro drėgmė, slėgis, vėjo greitis ir vėjo kryptis. Esant didesniam nei 1 m/s vėjo grei- čiui, ant mikrofono turi būti uždedamas specialus gaubtas. Triukšmo matavimams naudojamas danų gamybos, pirmos klasės precizinis garso analizatorius Bruel&Kjaer 2260. Jis gali matuoti ekvivalentinio bei plačiajuosčio triukšmo parametrus. Prietaisu registruojamas nuo 6,3 Hz iki 20 kHz dažnio triukšmas (Baltrėnas, Puzinas 2009). Santykinė matavimo paklaida ±1,5 %. Matavimai atliekami 4 metrų aukštyje. Triukšmo sklaidai modeliuoti buvo naudojama DataKustik kompanijos sukurta programa CadnaA. Kiekvienoje dalyje yra pažymimi namai, nurodomi jų aukščiai, pažymimos gatvės, nurodant kelio tipą, jo cha- rakteristikas bei lengvojo ir sunkiasvorio transporto kiekius. 1 pav. Triukšmo matavimo vietos pietinėje miesto dalyje Fig. 1. Noise level measurement points in the southern part of the city 432 1 lentelė. Garso slėgio lygio matavimo rezultatai pietinėje mies- to dalyje Table 1. The sound pressure level of the measurement results in the southern part of the city Eil. nr. Matavimo vieta Išmatuotas garso slėgio lygis, dB Ld Lv Ln Didžiausi leidžiami triukšmo ribiniai dydžiai HN 33:2011 LAeq,nT 65 60 55 LAmax 70 65 60 1 Gedimino g. Metodika Mažiausiosios triukšmo lygio vertės buvo nustatytos prie Gedimino g. 30 esančio pastato. Šioje vietoje ekvivalentinis nuolatinis garso slėgio lygis dienos metu buvo 44 dB, vakaro metu – 39 dB, nakties metu – 32 dB. Lyginant didžiausio akimirkinio garso slėgio ver- tes su Lietuvos higienos normoje nustatytomis vertėmis, galima pastebėti, jog prie Gedimino g. 23 esančio namo išmatuotos vertės viršijo normas. Dienos metu išmatuota vertė buvo 73 dB, vakaro metu – 67 dB, nakties metu – 62 dB. Taip pat didžiausia garso slėgio vertė vakaro metu buvo viršyta Šilo g. 8 esančioje matavimo vietoje, kur ji siekė 66 dB. Didžiausiųjų reikšmių viršijimas šiose vietose buvo nustatytas todėl, kad pagrindiniu keliu važiuojantys sunkieji automobiliai dėl nepakankamai lygios kelio dan- gos sukeldavo akimirkinį triukšmo lygį, kuris arčiausiai gatvės esančiose matavimo vietose viršijo didžiausiąsias leistinas vertes. Kitose vietose triukšmo lygio vertės nevir- šijo Lietuvos higienos normoje nustatytų verčių, nes kitos vietos buvo parinktos toliau nuo pagrindinės Gedimino gatvės. Triukšmo modeliavimas Remiantis matavimų metu surinktais duomenimis, panau- dojant suskaičiuotus lengvųjų bei sunkiųjų automobilių kiekius buvo atliktas triukšmo sklaidos į gyvenamąją teri- toriją modeliavimas. Žinant visų gatvių transporto srautus, pastatų aukščius, CadnaA programa buvo sumodeliuoti triukšmo žemėlapiai dienos, vakaro, nakties metu bei bend- ros triukšmo sklaidos Ldvn žemėlapis. Remiantis šiais žemė- lapiais galima prognozuoti, kaip sklis triukšmas į skirtingas teritorijas. Atlikus pietinės miesto dalies modeliavimą, aiškiai pa- stebima, kad triukšmą sukelia Gedimino gatve važiuojantis transportas. Šia gatve transporto intensyvumas yra didelis, tačiau jo sukeliamas garso lygis, lyginant su Lietuvos hi- gienos normoje pateiktais didžiausiais leidžiamais triukšmo ribiniais dydžiais, naudojamais triukšmo strateginio kar- tografavimo rezultatams įvertinti, neviršija normų. Buvo sudaryti 4 triukšmo sklaidos žemėlapiai, kuriuose įvertinta triukšmo sklaida dienos, vakaro, nakties metu ir bendra triukšmo sklaida. Dienos metu (3 pav.) didžiausiosios vertės nustatytos prie pastatų, esančių arčiausiai Gedimino gatvės. Palyginus išmatuotas garso slėgio lygio vertes su pateiktomis Lietuvos higienos normoje, ekvivalentinio nuolatinio garso slėgio lygio LAeq,nT vertės viršijimo ne- buvo nustatyta. Didžiausieji lygiai buvo užfiksuoti prie Karaimų g. 95 esančio namo, kur garso slėgio lygio vertės dienos metu siekė 59 dB, vakaro metu – 54 dB, nakties metu – 51 dB. Didžiausiosios vertės šioje vietoje buvo nustatytos todėl, jog ši vieta yra arčiausiai krašto kelio 107, kur yra didesnis transporto srauto intensyvumas. Mažiausiosios ekvivalentinio garso slėgio lygio ver- tės buvo užfiksuotos prie Karaimų g. 79 esančio namo. Dienos metu užfiksuota vertė buvo 46 dB, vakaro metu – 41 dB, nakties metu – 39 dB. Ši vieta yra toliausiai nuo Karaimų gatvės, todėl čia yra mažiausiai jaučiama trans- porto sukeliamo triukšmo įtaka gyvenamojoje teritorijoje. Šalia gatvės garso lygis yra tarp 57–59 dB. Tolstant nuo pagrindinės Gedimino gatvės, triukšmo lygiai mažėja ir siekia 43–50 dB. Vakaro (4 pav.) ir nakties (5 pav.) metu tendencijos išlieka tokios pačios, pagrindiniai automobilių srautai yra Gedimino gatvėje, todėl didžiausieji triukšmo lygiai užfiksuoti šalia jos. Toliau nuo gatvės garso lygis sumažėja, nes dalis jo yra sulaikoma arčiau gatvės esančių namų. Vakaro metu triukšmo lygiai yra 52–55 dB. Tolstant nuo pagrindinės Gedimino gatvės, dėl arčiau esančių namų, kurie ekranuoja triukšmą, garso lygiai sumažėja ir siekia 39–45 dB. 3 pav. Triukšmo sklaida pietinėje miesto dalyje dienos metu Fig. 3. Noise dispersion in the southern part of the city during daytime Lyginant didžiausiojo akimirkinio garso slėgio vertes, buvo nustatyta, kad Karaimų g. 95 esančioje matavimo vietoje dienos ir vakaro metu garso slėgio lygio vertės buvo viršytos. Rezultatai ir jų analizė Pietinėje miesto dalyje, prie krašto kelio 220, triukšmo ma- tavimai buvo atlikti dienos, vakaro ir nakties metu. Buvo iš- matuotas ekvivalentinis nuolatinis garso slėgio lygis LAeq,nT ir didžiausias akimirkinis garso slėgis LAmax parinktuose taškuose. Rezultatai pateikti 1 lentelėje. Šiaurinėje miesto dalyje matavimai buvo atlikti Karaimų gatvėje, kuri yra šiaurinėje miesto dalyje ir jungiasi su krašto keliu 107. Šioje miesto dalyje taip pat buvo išmatuoti ekvivalentiniai nuolatinio garso slėgio lygiai LAeq,nT ir didžiausi akimirkinio garso slėgio lygiai LAmax parinktose matavimo vietose. Rezultatai pateikti 2 lentelėje. Išmatavus triukšmo lygius buvo nustatyta, kad nė vienoje matavimo vietoje ekvivalentinis nuolatinis garso slėgio lygis LAeq,nT nebuvo viršytas. Didžiausia vertė dienos metu buvo nustatyta prie Gedimino g. 23 esančio namo, kur triukšmas, krintantis į fasadą, siekė 60 dB. Šioje vietoje 433 2 lentelė. Garso slėgio lygio matavimo rezultatai šiaurinėje miesto dalyje Table 2. The sound pressure level of the measurement results in the northern part of the city Eil. nr. Matavimo vieta Išmatuotas garso slėgio lygis, dB Ld Lv Ln Didžiausi leidžiami triukšmo ribiniai dydžiai HN 33:2011 LAeq,nT 65 60 55 LAmax 70 65 60 1 Karaimų g. 95 LAeq,nT 59 54 51 LAmax 72 66 59 2. Karaimų g. 90 LAeq,nT 49 45 41 LAmax 63 59 57 3. Karaimų g. 79 LAeq,nT 46 41 39 LAmax 59 57 56 4. Karaimų g. 74 LAeq,nT 53 50 44 LAmax 68 63 60 5. Karaimų g. 60 LAeq,nT 50 48 40 LAmax 64 61 57 6. Karaimų g. 51 LAeq,nT 51 49 43 LAmax 66 63 59 Triukšmo modeliavimas Dienos metu išmatuota vertė buvo lygi 72 dB, vakaro metu – 66 dB. Šioje vietoje triukšmo vertės buvo viršytos todėl, kad matavimo vieta buvo arti 107 krašto kelio, kur transporto intensyvumas yra didelis ir sunkaus transporto kiekis jame yra iki 15 procentų, todėl sunkusis transportas dėl kelio nelygumų sukėlė akimirkinį garso lygį, kuris viršijo didžiausiąsias vertes. Kitose vietose di- džiausiosios vertės neviršijo higienos normoje nustatytų verčių. 3 pav. Triukšmo sklaida pietinėje miesto dalyje dienos metu Fig. 3. Noise dispersion in the southern part of the city during daytime 434 Nakties metu garso lygiai yra 46–49 dB. Toliau nuo gatvės garso lygis siekia 34–39 dB. Kitomis gatvėmis va- žiuojančio transporto įtaka menka, nes transporto srautai yra maži. Vertinant bendrą triukšmo sklaidos Ldvn žemėlapį (6 pav.), viršijimai taip pat nenustatyti. Arčiausiai Gedimino gatvės esančių pastatų aplinkoje triukšmo lygis yra 58–61 dB. Toliau nuo Gedimino gatvės triukšmo lygiai siekia 48–53 dB. Atlikus modeliavimą, gali- ma pastebėti, kad, tolstant nuo pagrindinės gatvės, triukšmo lygis mažėja dėl arčiau gatvės esančių pastatų, kurie sulaiko nuo Gedimino gatvės sklindantį triukšmą ir yra 5–10 dB mažesni. Lyginant modeliuojant gautus rezultatus su išma- tuotomis vertėmis, jos skiriasi labai nežymiai, apie 2 dB. 4 pav. Triukšmo sklaida pietinėje miesto dalyje vakaro metu Fig. 4. Noise dispersion in the southern part of the city in the evening Atlikus šiaurinės miesto dalies modeliavimą, buvo nustatytos panašios tendencijos, kaip ir pietinėje miesto da- lyje. Didžiausieji triukšmo lygiai nustatyti šalia pagrindinės Karaimų gatvės bei 107 krašto kelio, kur transporto inte- nsyvumas yra didžiausias. Dėl mažesnio transporto srauto negu pietinėje miesto dalyje čia sumodeliuotos triukšmo sklaidos vertės yra mažesnės ir higienos normoje pateiktų verčių neviršija. Didžiausieji triukšmo lygiai yra nustatyti prie krašto kelio 107, tačiau šalia jo yra vos keli gyvenamie- ji namai, todėl bendrai triukšmo sklaidai jis didelės įtakos neturi. Pagrindinė triukšmo sklaida kyla nuo Karaimų gatve važiuojančio transporto. Pietinėje dalyje buvo sumodeliuo- ti triukšmo žemėlapiai, kurie vaizduoja triukšmo sklaidą dienos (7 pav.), vakaro (8 pav.) ir nakties (9 pav.) metu bei bendrą triukšmo sklaidą Ldvn (10 pav.). 4 pav. Triukšmo sklaida pietinėje miesto dalyje vakaro metu Fig. 4. Noise dispersion in the southern part of the city in the evening evening 5 pav. Triukšmo sklaida pietinėje miesto dalyje nakties metu Fig. 5. Noise dispersion in the southern part of the city at night 6 pav. Bendra triukšmo sklaida pietinėje miesto dalyje Ldvn Fig. 6. Išvados 1. Išmatavus triukšmo lygius pietinėje miesto dalyje buvo nustatyta, kad nė vienoje matavimo vietoje ekvivalen- tinis nuolatinis garso slėgio lygis LAeq,nT nebuvo viršy- tas. Didžiausioji vertė dienos metu buvo nustatyta prie Gedimino g. 23 esančio namo – 60 dB. Šioje vietoje taip pat buvo išmatuotos didžiausiosios vertės vakaro ir nakties metu, atitinkamai 54 dB ir 48 dB. 9 pav. Triukšmo sklaida šiaurinėje miesto dalyje nakties metu Fig. 9. Noise dispersion in the northern part of the city at night 9 pav. Triukšmo sklaida šiaurinėje miesto dalyje nakties metu Fig. 9. Noise dispersion in the northern part of the city at night night 10 pav. Bendra triukšmo sklaida šiaurinėje miesto dalyje Ldvn Fig. 10. General noise dispersion in the northern part of the city Lden 10 pav. Bendra triukšmo sklaida šiaurinėje miesto dalyje Ldvn Fig. 10. General noise dispersion in the northern part of the city Lden 2. 2. Lyginant didžiausiąsias garso slėgio vertes su Lietuvos higienos normoje nustatytomis vertėmis, galima matyti, jog prie Gedimino g. 23 esančio namo išmatuotos ver- tės viršija normas. Dienos metu išmatuota vertė buvo 73 dB, vakaro metu – 67 dB, nakties metu – 62 dB. Taip pat didžiausioji garso slėgio vertė vakaro metu buvo viršyta Šilo g. 8 esančioje matavimo vietoje, kur ji siekė 66 dB. Viršijimai buvo nustatyti dėl sunkiojo tran­sporto, kuris važiuodamas dėl kelio nelygumų sukėlė akimirkinį triukšmą. 2. Lyginant didžiausiąsias garso slėgio vertes su Lietuvos higienos normoje nustatytomis vertėmis, galima matyti, jog prie Gedimino g. 23 esančio namo išmatuotos ver- tės viršija normas. Dienos metu išmatuota vertė buvo 73 dB, vakaro metu – 67 dB, nakties metu – 62 dB. Taip pat didžiausioji garso slėgio vertė vakaro metu buvo viršyta Šilo g. 8 esančioje matavimo vietoje, kur ji siekė 66 dB. Viršijimai buvo nustatyti dėl sunkiojo tran­sporto, kuris važiuodamas dėl kelio nelygumų sukėlė akimirkinį triukšmą. 10 pav. Bendra triukšmo sklaida šiaurinėje miesto dalyje Ldvn Fig. 10. General noise dispersion in the northern part of the city Lden 3. 3. Išmatavus triukšmo lygius šiaurinėje miesto dalyje, didžiausieji lygiai buvo užfiksuoti prie Karaimų  g. 95 esančio namo, kur garso slėgio lygio vertės dienos metu buvo 59 dB, vakaro metu – 54 dB, nakties metu – 51 dB. Dienos metu prie pastatų, esančių arčiausiai gatvės, garso lygis svyruoja tarp 47–50 dB. Toliau nuo gatvės triukšmo lygiai siekia 40–45 dB, nes dalis triukšmo yra sulaikoma arčiau gatvės esančių pastatų, taip pat triukšmas sklisdamas tolyn slopsta. 4. Triukšmo modeliavimas General noise dispersion in the southern part of the city Lden 5 pav. Triukšmo sklaida pietinėje miesto dalyje nakties metu Fig. 5. Noise dispersion in the southern part of the city at night 7 pav. Triukšmo sklaida šiaurinėje miesto dalyje dienos metu Fig. 7. Noise dispersion in the northern part of the city during daytime 5 pav. Triukšmo sklaida pietinėje miesto dalyje nakties metu Fig. 5. Noise dispersion in the southern part of the city at night 5 pav. Triukšmo sklaida pietinėje miesto dalyje nakties metu Fig. 5. Noise dispersion in the southern part of the city at night 7 pav. Triukšmo sklaida šiaurinėje miesto dalyje dienos metu Fig. 7. Noise dispersion in the northern part of the city during daytime 7 pav. Triukšmo sklaida šiaurinėje miesto dalyje dienos metu Fig. 7. Noise dispersion in the northern part of the city during daytime 6 pav. Bendra triukšmo sklaida pietinėje miesto dalyje Ldvn Fig. 6. General noise dispersion in the southern part of the city Lden 6 pav. Bendra triukšmo sklaida pietinėje miesto dalyje Ldvn Fig. 6. General noise dispersion in the southern part of the city Lden y 8 pav. Triukšmo sklaida šiaurinėje miesto dalyje vakaro metu Fig. 8. Noise dispersion in the northern part of the city in the evening 8 pav. Triukšmo sklaida šiaurinėje miesto dalyje vakaro metu Fig. 8. Noise dispersion in the northern part of the city in the evening 6 pav. Bendra triukšmo sklaida pietinėje miesto dalyje Ldvn Fig. 6. General noise dispersion in the southern part of the city Lden 435 9 pav. Triukšmo sklaida šiaurinėje miesto dalyje nakties metu Fig. 9. Noise dispersion in the northern part of the city at night 10 pav. Bendra triukšmo sklaida šiaurinėje miesto dalyje Ldvn Fig. 10. General noise dispersion in the northern part of the city Lden 9 pav. Triukšmo sklaida šiaurinėje miesto dalyje nakties metu Fig. 9. Noise dispersion in the northern part of the city at night 9 pav. Triukšmo sklaida šiaurinėje miesto dalyje nakties metu Fig. 9. Noise dispersion in the northern part of the city at night pietinėje miesto dalyje triukšmo lygiai yra 7–8 dB didesni. Triukšmo modeliavimas Žinant, kad 3 dB triukšmo pasikeitimas jau yra jaučiamas žmogaus ausiai, 5 dB pokytis sukelia žymų pasikeitimą, o 10 dB triukšmo pakitimas yra suvokimas kaip dvigubai gar- sesnis triukšmas, galima teigti, kad šiaurinėje miesto dalyje dėl mažesnio transporto srauto garso lygis į gyvenamąją teritoriją yra žymiai mažesnis negu pietinėje miesto dalyje. Išvados Lyginant didžiausiąsias garso slėgio vertes su Lietuvos higienos normoje nustatytomis vertėmis matyti, kad Karaimų  g. 95 esančioje matavimo vietoje dienos ir vakaro metu garso slėgio lygio vertės buvo viršytos. Dienos metu išmatuota vertė buvo lygi 72 dB, vakaro metu – 66 dB. Viršijimai buvo nustatyti šalia 107 krašto kelio, kur yra didelis sunkiojo transporto intensyvumas. Kitose vietose didžiausiosios vertės neviršijo higienos normoje nustatytų verčių. Vakaro metu garso lygis yra 45–47 dB. Toliau nuo Karaimų gatvės, kur transporto srautai yra mažesni, garso lygis yra 35–41 dB. Nakties metu garso lygis yra 39–42 dB. Tolstant nuo Karaimų gatvės, triukšmo lygis yra 32–38 dB, nes kitose gatvėse eismo intensyvumas yra mažas, todėl triukšmo sklaida yra menka, o garsas, kuris sklinda nuo pagrindinės gatvės, yra sulaikomas arčiau jos esančių namų. 5. Atlikus pietinės dalies modeliavimą buvo nustaty- ta, jog didžiausieji garso lygiai yra šalia pagrindinės Gedimino gatvės. Čia triukšmo lygiai dienos metu siekė 57–59 dB, vakaro metu 52–55 dB, nakties metu 46–49 dB. Tolstant nuo pagrindinės gatvės dėl šalia jos stovinčių namų garso sugėrimo ir ekranavimo triukšmo lygiai buvo 5–10 dB mažesni. Vertinant bendros triukšmo sklaidos Ldvn žemėlapį, triukšmo lygis prie gatvės yra 49–53 dB. Dėl mažo trans- porto intensyvumo kitose gatvėse, jų įtaka triukšmo sklaidai yra labai menka, todėl toliau nuo Karaimų gatvės esančiuose gyvenamuosiuose pastatuose triukšmo lygis yra 5–9 dB ma- žesnis negu prie gatvės. Lyginant modeliuojant metu gautus duomenis su išmatuotais, skirtumas yra apie 3 dB. 6. Atlikus šiaurinės dalies modeliavimą buvo nustatyta, kad didžiausieji triukšmo lygiai yra šalia Karaimų gat- Lyginant šiaurinės ir pietinės miesto dalies žemė- lapius, galima teigti, jog dėl didesnio transporto srauto 436 vės ir krašto kelio 107. Čia garso lygiai siekė 47–50 dB dienos metu, 45–47 dB vakaro metu ir 39–42 nakties metu. Toliau nuo pagrindinių gatvių triukšmo lygis su- mažėjo 5–9 dB, nes dalį triukšmo sulaikė šalia pagrin- dinės gatvės stovintys namai. related noise exposures. Topic paper noise. Transport related health effects with particular focus on children. Toward an integrated assessment of their costs and benefits. State of the art knowledge, methodological aspects and policy direction. RIVM report 815120002/2004. 76 p. Stanfeld, S.; Haines, M.; Brown, B. 2000. Noise and health in the urban environment, Reviews on Environmental Health 15: 43–82. 7. Lyginant šiaurinės ir pietinės miesto dalies žemėlapius, galima teigti, jog dėl didesnio transporto srauto pietinė- je miesto dalyje triukšmo lygiai yra 7–8 dB didesni. Tamošiūnas, A.; Rėklaitienė, R.; Domarkienė, S. 2005. Literatūra Vaišis, V.; Januševičius, T. 2009. Modelling of noise level in the northern part of Klaipeda city, Journal of Environmental Engineering and Landscape Management 17(3): 181–188. http://dx.doi.org/10.3846/1648-6897.2009.17.181-188 Babisch, W. 2005. Noise and health, Environmental Health Perspectives 113(1): A14–15. http://dx.doi.org/10.1289/ehp.113-a14 World Health Organization (WHO). 2011. Burden of disease from environmental noise quantification of healthy life years lost in Europe. 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Assessment of methods for simplified traffic noise mapping of small cities: Casework of the city of Valdivia, Chile, Science of the Total Environment 550: 439–448. Išvados Prevalence of risk factors and risk of mortality in relation to occupational group, Medicina 41(8): 705–712. T. Vilniškis, T. Januševičius Gražulevičienė, R.; Bendokienė, I. 2009. Influence of truck traffic on acoustic pollution in Kaunas districts crossed by highwa- ys, Journal of Environmental Engineering and Landscape Management 17(4): 198–204. http://dx.doi.org/10.3846/1648-6897.2009.17.198-204 Abstract One of the biggest problem in most cities – noise emissions in the environment caused by transport. It is found that environ- ment noise can cause not only discomfort, but it is also harmful to health. Therefore, noise emissions in the environment is topical environmental problem. In this article the vehicle noise emissions are evaluated, depending on traffic in Trakai city. At selected  locations  in the northern  and  southern  parts of the city equivalent and maximum noise levels are measured. According to estimates of traffic day, evening and night, using CadnaA program, noise maps are simulated. The noise map will show noise emissions day, evening and night. Measurement have shown, that maximum noise values was near main roads. In the south of the city, near Gediminas street, equivalent sound level during daytime was 60 dB, evening 54 dB, night – 48 dB. In the north of the city, near Karaimai street equivalent sound lever during daytime was 59 dB, evening  – 54 dB, night  – 51 dB. The simulations generated sound level values were similar to measured, the differences between the simulated and measured values were up to 4 dB. HN 33:2011 Triukšmo ribiniai dydžiai gyvenamuosiuose ir visuomeninės paskirties pastatuose bei jų aplinkoje, Valstybės žinios, 75 – 3638. Jensen, A.; Ballisager,  S.; Sørensen,  P. L.; Baltmiškienė,  I.; Švarplienė, A.; Jankauskienė, N. 2002. Triukšmo mažinimo užtvarų vadovas. TetraPlan A/S, Acoustica, Carl Bro ajs, Transporto ir kelių tyrimo institutas. 79 p. Lekavičiūtė, J. 2007. Transporto triukšmas Kauno mieste ir jo įtaka miokardo infarkto rizikai: daktaro disertacija. Vytauto Didžiojo universitetas, Kaunas, Lietuva. Paulauskas, L.; Klimas, L. 2011. Modeling of the spread of motor transport noise in Šiauliai city, Journal of Environmental Engineering and Landscape Management 19(1): 62–70. http://dx.doi.org/10.3846/16486897.2011.557249 Staatsen, B. A. M.; Nijland, H. A.; Kempen, E. M. M.; Hollander, A. E. M.; Franssen, A. E. M.; Kamp, I. 2004. Assessment of health impacts and policy options in relation transport- Keywords: noise level, transport noise, noise emissions, sim- ulation. 437
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[The process if teaching-learning in blood pressure monitoring].
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RELATED TEACHING-LEARNING PROCESS TO THE TECHNIQUE OF VERIFICATION OF THE ARTERIAL PRESSURE EL PROCESO ENSENANZA -APRENDIZAJE EN LA VERIFICACIÓN DE LA PRESIÓN ARTERIAL Thereza Maria Magalhães Moreira! Taciana Cavalcante de Oliveira 2 Thelma Leite de Araújo' Thereza Maria Magalhães Moreira! Taciana Cavalcante de Oliveira 2 Thelma Leite de Araújo' RESUMO: A enfermeira é considerada a profissional que obtém dados mais fidedignos nas medidas indiretas de pressão arterial (PA) por induzir menos reaçOes de alerta no cliente, geradoras de valores falsamente elevados. Porém, muitas vezes, ela apresenta lacunas no conhecimento do assunto, tanto nos aspectos técnicos, como nos anátoma-fisiológicos. Isso prioriza o desenvolvimento de estratégias de ensino, visando minimizar falhas e tornar o processo de aprendizagem efelivo. Objelivando promover o conhecimento da técnica de aferição da PA junto a um grupo de pesquisa. realizou-se uma reciclagem e aplicou-se questionário avaliativo do conhecimento do grupo antes e após a mesma. Evidenciou-se a eficácia desse método, pois 100% da amostra (nove) referiu contribuição para aprimoramento da fundamentação teórico-prática, diminuindo dificuldades oriundas da verificação da PA. Conclui-se que, sendo as ações de educação em saúde extremamente válidas e necessárias, o enfermeiro, como educador que é, necessita reciclar seu conhecimento e aprimorar suas técnicas continuamente. PALAVRAS-CHAVE: ensino-aprendizagem, pressão arterial, Enfermagem. , Mes/randa em Enfermagem pela Universidade Federal do Ceará, bolsista da CAPES. 2 Doutora em Enfermagem, Adjunto do Deparlamento de Enfermagem da Universidade Federal do Ceará e Coordenadora do Projeto ·Cuidando de Pessoas com Alterações da Pressão Arleriar J Especializanda em Enfermagem, bolsista de aperfeiçoamento do CNPq. , Mes/randa em Enfermagem pela Universidade Federal do Ceará, bolsista da CAPES. INTRODUÇÃO Um dos grandes problemas de saúde pública no Brasil, nos dias atuais, é a hipertensão arterial (HA), tanto pela sua prevalência no país, como pela detecçao quase sempre tardia da doença. Seu controle constitui um problema complexo devido a magnitude e seriedade do assunto e por representar um grande desafio ao nosso sistema de saúde, ainda muito alicerçado no modelo curativo e farmacêutico. A hipertensão arterial é uma condiçao clinica de natureza multifatorial, caracterizada por níveis de pressão arterial sistólica elou diastólica elevada. Seus nlveis variam entre as pessoas e oscilam durante o dia devido a fatores pasturais, sensoriais, metabólicos, musculares, térmicos e etários (HAS, 1996, Porto; Rassi, 1990). ( , , ; , ) A pressão arterial (PA) é elemento fundamental na dinâmica da circulação sangüinea, pois mantém o aparte de oxigénio necessário à nutrição dos tecidos, dos 6rgãos e da própria vida humana. Mas, em excesso, a PA pode provocar enormes danos à saúde e, inclusive, a R. Bras. Enferm., Brasilia, v. 52, n. 1, p. 67-78, jan.lmar. 1999 67 MOREIRA, Thereza Maria Magalhães et ai. morte. Medidas para seu controle têm importante papel na detecção precoce. necessária á prevenção de sua morbi-mortalidade e das complicações, diminuindo custos com tratamentos crónicos e melhorando a qualidade de vida dos acometidos. morte. Medidas para seu controle têm importante papel na detecção precoce. necessária á prevenção de sua morbi-mortalidade e das complicações, diminuindo custos com tratamentos crónicos e melhorando a qualidade de vida dos acometidos. q A primeira detecção da existência de pressão arterial data de 1733 e foi realizada pelo reverendo inglês Stephen Hales, que canulou a car6tida de uma jumenta e observou o sangue subir até 290 cm de altura. Mas foi em dezembro de 1896 (123 anos depois) que Scipione Riva Rocei apresentou ao mundo o que chamou de "nuovo sphygmomano-metro", semelhante aos instrumentos de verificaç:io da PA atuais. Portanto, faz mais de 100 anos a invenção que revolucionou as ciências da saúde, A criação de Rocei foi aperfeiçoada em 1905 por Nicolai Korotkoff, que introduziu o sistema auscultatório para as medidas de pressao sist61ica e diastólica, Em 1939, surgiu o conceito de hipertensão arterial como fator de risco independente para as doenças cardiovasculares (DCV). Essa concepção é fruto da introdução da verificação da pressão arterial como rotina em uma seguradora em 1917. No entanto. INTRODUÇÃO foi somente no final da década de 40 e inicio dos anos 50, que os primeiros estudos epidemiológicos sobre hipertensão arterial começaram a ser realizados pelos Ministérios da Saúde da Europa e Estados Unidos da América (EUA) com o objetivo de organizar programas de saúde pública, sendo as campanhas de conscientização iniciadas na década de 60 (Lolio 1990; Araújo; Arcuri; Martins, 1998). Até hoje são realizados estudos visando compreender a melhor maneira de verificação da PA. Zanella et ai. (1992) afirmam que a classificação da hipertensão quanto à gravidade, utilizando-se referenciais numéricos, é útil porque, em geral, as complicações ocorrem na proporção direta dos n!veis tensionais. No entanto, a ausência ou presença de lesão nos órgãos- alvo (coração, rins. cérebro. artérias e retina) é ainda muito importante na avaliação clínica da severidade do processo hipertensivo. Evidentemente. um paciente que apresente proteinúria e hipertrofia cardfaca apresenta uma forma severa de hipertensao arterial, mesmo que os níveis tensionais não estejam tão elevados. Por outro lado, um paciente com n!veis muito elevados de pressão arterial deve também ser considerado como portador de forma severa da hipertensão e receber tratamento intensivo, pela grande posssibilidade de lesão nos órgãos-alvo, Normalmente, nos quadros de hipertensão arterial, ambas as pressões, sistólica e diastólica, estão elevadas, porém o achado de hipertensão sistólica isolada não é incomum e deve ser tratado. A importãncia da enfermeira na aferição da PA vem sendo reconhecida hã mais de 50 anos, Nos últimos anos esse papel se ampliou muito além do simples procedimento de verificação da pressão, pois diversos estudos têm demonstrado que tanto no desenvolvi-mento de protocolos de pesquisa, como na prática de atendimento à saúde, os niveisde PA. aferidos pelas enfermeiras sofrem menos variações que os obtidos por médicos. ç q p Araújo (1994) em pesquisa desenvolvida com enfermeiros da área de cardiologia. identificou lacunas importantes no conhecimento dessa categoria sobre a medida indireta da PA. Cita, inclusive. que enfermeiras que trabalham com clientes portadores de alterações cardiovasculares, nos quais o controle fidedigno da PA assume um aspecto importante, desconhecem aspectos relativos à técnica, ao instrumental e aos aspectos anátoma-fisiológicos envolvidos. Questiona. ainda, a responsabilidade das escolas e dos programas de educação continuada, sugerindo a necessidade do desenvolvimento de novas formas de ensino-aprendizagem. A atualização dos conhecimentos inclui, em especial, o acompanhamento dos estudos relativos às recomendações sobre a medida da pressão arterial. R. Bras. Enferm., Brasilia, v. 52. n. 1. p, 67-78. jan.lmar. 1999 ESPECIFICOS: • Aprimorar a técnica de verificação da PA em um grupo de pesquisa: p ç g p p q · Avaliar o conhecimento apreendido sobre verificação da pressão arterial pela clientela em estudo em dois momentos distintos: pré e pós·treinamento, comparando o conhecimento obtido nos dois momentos. · Avaliar o conhecimento apreendido sobre verificação da pressão arterial pela clientela em estudo em dois momentos distintos: pré e pós·treinamento, comparando o conhecimento obtido nos dois momentos. GERAL: • Promover o conhecimento da técnica de aferição da pressão arterial junto a um grupo de pesquisa. INTRODUÇÃO A discussão dos aspectos polêmicos envolvidos na técnica de mensuração não deve ser restrita ao meio acadêmico, mas envolver profissionais que atuam junto à clientela que apresenta alteraçOes da PA. O processo ensino-aprendizagem mostra-se complexo por sofrer influência de fatores externos, dentre eles: metodologia de ensino, relação aluno- professor e a própria estrutura R. Bras. Enferm., Brasilia, v. 52. n. 1. p, 67-78. jan.lmar. 1999 68 o Processo Ensino·Aprefldizagem ... fisica das instituições de ensino. Sabemos das dificuldades sobre·humanas que as universidades públicas, em especial. vêm enfrentando com a desvalorização do ensino, os baixos sala rios dos professores e as parcas condições de trabalho em que estes desempenham suas atividades cotidianas. fisica das instituições de ensino. Sabemos das dificuldades sobre·humanas que as universidades públicas, em especial. vêm enfrentando com a desvalorização do ensino, os baixos sala rios dos professores e as parcas condições de trabalho em que estes desempenham suas atividades cotidianas. Para Mizukami (1986) o ensino e a aprendizagem têm significado amplo, não havendo restrições as suas situaçOes formais, desta forma acredita·se que o ensino não deve ser realizado apenas nas salas de aula. p No tocante a técnica de verificação da PA, essa realidade não é diferente. Ha a necessidade fremente de estimular o desenvolvimento profissional capacitado para a detecção precoce e acompanhamento da hipertensão. Diante de todo o exposto no inicio deste trabalho, entende·se ser importante a realização de estudos que visem a detecção precoce da doença, seu acompanhamento e evolução. Sentiu· se a necessidade de treinar um modelo de ensino do procedimento da medida da pressão arterial a ser desenvolvido com acadêmicos de enfermagem e enfermeiros. Resolveu·se, então, testar um modelo ou uma estratégia de ensino com a finalidade de aprofundar e promover o conhecimento da técnica de aferição da pressão arterial junto a um grupo de pesquisa. R. Bras. Enferm., Brasília, v. 52, n. 1, p. 67·78, jan.lmar. 1999 o PROCESSO ENSINO-APRENDIZAGEM A aprendizagem é um processo integrado no qual o eu hoJistico é mobilizado de forma orgânica qualitativa, e pelo qual a pessoa fica melhor preparada para novas aprendizagens, não havendo, portanto, um aumento quantitativo de conhecimentos, mas uma transformação estrutural da inteligência da pessoa. O aluno é o agente essencial desse processo. Já o processo de ensino é pragmático, ou seja, é um mecanismo pelo qual se tenta alcançar determinados objetivos através de estratégias seqüenciais. Dessa maneira, o processo de ensino consiste em planejar, orientar e acompanhar a aprendizagem do aluno (Bordenave; Pereira, 1977). Os alunos são diferentes entre si e necessitam de diferentes tipos de aprendizagem para seu próprio desenvolvimento. Para que se obtenha uma aprendizagem eficaz, é indispensável que se considere as capacidades de cada um ao selecionar e organizar os procedimentos de ensino, o que é uma tarefa diflcil e complexa (Turra et aI., 1975). Mas Novaes (1992) afirma que os processos criativos poderiam ser implantados em todas as disciplinas, proporcionando ao aluno uma abordagem inovadora do conteúdo. A aprendizagem é uma mudança na disposição ou capacidade do homem, modificação essa que pode ser inferida comparando·se o comportamento posslvel antes e após o individuo ser colocado em uma situação de aprendizagem. Dessa maneira, o processo de aprendizagem se realiza quando a situação estimuladora afeta de tal maneira o aprendiz que seu desempenho R. Bras. Enferm., Brasília, v. 52, n. 1, p. 67·78, jan.lmar. 1999 69 MOREIRA, Thereza Maria Magalhlies et aI. é modificado após o cantata com essa situação. ~ importante considerar as habilidades ja adquiridas antes mesmo do processo de aprendizagem (Gagné, 1973). As diversas crises econOmicas, mudanças nas politicas de saúde, novas necessidades da demanda, desenvolvimento cientlfico-tecnol6gico, novos instrumentos, além de outros, foram integrando o cotidiano da enfermagem e imprimindo alteraçOes em sua pratica assistencial e no ensino. Para que os docentes e a equipe de enfermagem que atua na assistência possam elaborar e trocar conhecimentos entre si visando atingir um cuidado qualitativamente mais apropriado, têm que se atentara dinâmica das relaçOes que vão se estabelecendo no cotidiano profissional, as tradições e novas tecnologias, materiais e intelectuais que instrumentalizam o processo de trabalho e às necessidades apresentadas nesse ambiente. o PROCESSO ENSINO-APRENDIZAGEM E diz que é mister saber desenvolver as técnicas visando a superação do efeito tecnicista da própria atividade, numa intemalização da mesma, uma vez que as técnicas são um conteúdo fundamental na formação da enfermeira, pois compOem um instrumento de seu trabalho (CoI/et; Rocha, 1996). R. Bras. Enferm., Brasília, v. 52, n. 1, p. 67-78, jan./mar. 1999 TECNICA • Colocaçao inadequada do manguito causa erro de medida. A - Verificaçao no braço: A Verificaçao no braço: Colocar a braçadeira do esfigmomanOmetro com o manguito totalmente vazia, ao redor do braço livre de roupas. Deve ficar bem ajustada mais sem comprimir. A borda da braçadeira deve ficar 2 a 2,5 cm acima do espaço antecubilal (menos nas crianças) e o centro do manguito deve estar direlamente sobre a artéria braquial. • Este método previne erros de medida que podem ocorrer por inflaçao insuficiente do manguito ou por causa da presença de hiatos ou buracos auscultatórios . A inflaçao excessiva é referida como desconfortável. Quando a pressao habitual do cliente é conhecida, este passo pode ser omitido. B - Avaliar a pressao sistótica (PAS) pela palpaçao; • Palpar a artéria radial com as polpas dos dedos de sua mao nao dominante. • Inflar o manguito e simultaneamente palpar a artéria, até o desaparecimento do pulso radial. Anotar este ponto. • Esvaziar o manguito, colocar o diafragma do estetoscópio sobre a artéria braquial, esperar de 1 a 2 minutos e inflar até 30 mmHg acima do ponto de desaparecimento do pulso radial. • Soltar o ar mais lentamente (de 2 a 3 mmHg por segundo). No ponto de inicio dos sons de Korotkoff determinar a PAS e no ponto de desaparecimento dos sons, determinar a pressao arterial diastólica (PAD). Caso a manobra deva ser repetida, aguardar de 1 a 2 minutos. • O ponto de desaparecimento do pulso representa o ponto da PAS. • Deflaçao rápida gera erro de medida. • Quando existe diferença de tempo significativa entre o abafamento dos sons e seu desaparecimento, registrar os dois valores. • Consideram-se normal diferenças de 5 a 10 mmHg entre os braços. • Consideram-se normal diferenças de 5 a 10 mmHg entre os braços. • Na primeira consulta a medida deve ser realizada em ambos os braços. • Na primeira consulta a medida deve ser realizada em ambos os braços. ENFERMAGEM NAASSISTIÔNCIAAOS HIPERTENSOS A enfermagem no controle da HA vem prestando cuidados aos individuas com hipertensao com o intuito de controlar a PA e evitar posslveis complicaçOes. Além disso, para indivlduos que apresentam fatores de risco e déficit de conhecimento que contribuem para o desencadeamento da hipertensao, a enfermeira mantém, através do sistema apoio-educaçao, esclarecimentos, possibilitando a promoçao e recuperação da saúde do cliente. O déficit de conhecimento da técnica de aferição da PA prejudica no tratamento dos hipertensos, pois diante de técnicas inadequadas poderemos ter valores incorretos, dificultando a avaliação fidedigna da situaçao di nica do individuo. R. Bras. Enferm., Brasilia, v. 52, n. 1, p. 67-78, jan./mar. 1999 PADRONIZAÇAo NA MEDIDA DA PRESsAo ARTERIAL • Soltar o ar mais lentamente (de 2 a 3 • Deflaçao rápida gera erro de medida. mmHg por segundo). No ponto de inicio dos sons de Korotkoff determinar a PAS e no ponto • Quando existe diferença de tempo de desaparecimento dos sons, determinar a significativa entre o abafamento dos sons e pressao arterial diastólica (PAD). Caso a seu desaparecimento, registrar os dois manobra deva ser repetida, aguardar de 1 a 2 valores. minutos. • Na primeira consulta a medida deve ser • Consideram-se normal diferenças de 5 a 10 realizada em ambos os braços. mmHg entre os braços. PADRONIZAÇAo NA MEDIDA DA PRESsAo ARTERIAL Sempre houve uma preocupação dos estudiosos referente à padronização na medida da PA. Araújo (1994) cita que a necessidade de tomara método não invasivo auscultat6rio mais preciso incentivou a formação de especialistas, designados pela Associação Americana de Cardiologia para a construção de padrOes utilizados na mensuração da PA A primeira publicação contou com a participação de vários estudiosos renomados e abordou temáticas relativas à padronização do instrumental, posição do cliente e método de adaptação do manguito. Este trabalho ficou conhecido como padronização da medida da pressão arterial, foi publicado em 1939 e avaliado nos Estados Unidos e Europa em décadas seguintes, considerando o conhecimento ao longo dos anos, relativo as passiveis fontes de erro na aferição da PA, referente ao observador, ao cliente e ao instrumental. A padronização tem incorporado este conhecimento e as revisões têm divulgado as novas determinaçOes. g ç A seguir encontra-se um quadro expositivo da técnica de verificação da PA recomendada pela última revisão publicada pela American Heart Association (Perloff, 1993). 70 R. Bras. Enferm., Brasília, v. 52, n. 1, p. 67-78, jan./mar. 1999 o Processo Ensino-Aprendizagem ... VERIFICANDO A PA TECNICA JUSTIFICATIVAS A - Verificaçao no braço: • Colocaçao inadequada do manguito causa Colocar a braçadeira do esfigmomanOmetro erro de medida. com o manguito totalmente vazia, ao redor do braço livre de roupas. Deve ficar bem ajustada mais sem comprimir. A borda da braçadeira deve ficar 2 a 2,5 cm acima do espaço antecubilal (menos nas crianças) e o centro do manguito deve estar direlamente sobre a artéria braquial. B - Avaliar a pressao sistótica (PAS) pela • Este método previne erros de medida que palpaçao; podem ocorrer por inflaçao insuficiente do • Palpar a artéria radial com as polpas dos manguito ou por causa da presença de hiatos dedos de sua mao nao dominante. ou buracos auscultatórios . A inflaçao • Inflar o manguito e simultaneamente palpar excessiva é referida como desconfortável. a artéria, até o desaparecimento do pulso Quando a pressao habitual do cliente é radial. Anotar este ponto. conhecida, este passo pode ser omitido. • Esvaziar o manguito, colocar o diafragma do estetoscópio sobre a artéria braquial, • O ponto de desaparecimento do pulso esperar de 1 a 2 minutos e inflar até 30 mmHg representa o ponto da PAS. acima do ponto de desaparecimento do pulso radial. ENFERMAGEM E A TECNICA DE VERIFICAÇÃO DA PA (esfigmomanometria) A investigação no campo da esfigmomanometria vem mostrando que a enfermeira é o 71 R. Bras. Enferm., Brasilia, v. 52, n. 1, p. 67-78, jan./mar. 1999 MOREIRA, Thereza Maria Magalhães et ai. elemento da equipe de saúde mais indicado para executar a medida da PA. O fato da enfermeira causar menor reaçao de alerta no cliente é fundamentado nos estudos realizados por Mancia et aI. (1987), que compararam, em pacientes internados, o aumento da PA provocado pela entrada do médico na enfermaria com o provocado pela enfermeira. A técnica indireta de aferiçtlo da PA pode se tornar mais imprecisa devido à reaçao de alerta. A pseudo hipertensao durante medida casual em consultórios, clinicas e enfermarias em alguns clientes ou pacientes é fenómeno conhecido e demonstrado. As enfermeiras vêm constantemente se firmando como responsàveis pela detecçao precoce da HA desconhecida e nao controlada e pelo controle do quadro hipertensivo jà conhecido, sendo particularmente atuantes na educação dos pacientes, reforçando a importância do controle contínuo e a longo prazo da PA (Hill; Mccombs, 1981). Assim, a enfermeira tem tido um papel preponderante nos programas de hipertensão arterial, estando perfeitamente apta a tratar com pacientes hipertensos pela sua formação acadêmica, que requer o trabalho com grupos, pela a observância de principias como interdisciplinaridade e multiprofissionalismo, além da regulamentação pelo Conselho Federal de Enfermagem (COFEN) da consulta e prescrição de enfermagem no cuidado a pacientes hipertensos em acompanhamento (Conselho Regional de EnfermagemlCOREN-CE. 1994: Lei 7.498/86, regulamentada pelo Decreto N.o 94.406/87). Acredita-se que muito importam todas as açOes desenvolvidas pela enfermagem no ãmbito da hipertensão, assim como seus registras e teorizações. A necessidade da enfermeira detectar dados hemodinãmicos precisos e de interpretá- los com segurança é indiscutivel, o que requer sólido conhecimento teórico e prático no campo da esfigmomanometria a fim de garantir a tomada de decisão e encaminhamentos corretos. No enlanlo, a medida da PA por ser considerada um procedimento corriqueiro hã anos, não é tema dos programas de educaçao continuada. Sabe-se que até recentemente, a despeito do desenvolvimento tecnológico e das pesquisas no assunto, pouco foi acrescentado ao conhecimento adquirido nos cursos de graduação. O pobre conhecimento no campo da esfigmomanometria já foi referido por Areuri (1989), ao identificar um desconhecimento preocupante por parte dos profissionais da área de saúde. ENFERMAGEM E A TECNICA DE VERIFICAÇÃO DA PA (esfigmomanometria) Isso foi mais recentemente confirmada por Araujo (1994), que constatou importante lacuna no conhecimento da medida indireta da pressão arterial, entre enfermeiros atuantes na área de cardiologia. Considerando a hipertensão como um dos grandes fatores de risco para doenças cardiovasculares, um dos importantes agravantes no perfil de morbi-mortalidade em todas as regiOes do mundo e que a medida precisa da PA é um dos parãmetros essenciais a sua detecção precoce, considera-se fundamental a atualização da enfermagem neste ãmbito. METODOLOGIA o estudo foi desenvolvido no Departamento de Enfermagem da Universidade o estudo foi desenvolvido no Departamento de Enfermagem da Universidade Federal do Ceará PropOe-se uma abordagem exploratório-descritiva. Ceará PropOe-se uma abordagem exploratório-descritiva. A população constou dos quinze integrantes de um projeto de pesquisa, que tem, entre outros, o objetivo de: A população constou dos quinze integrantes de um projeto de pesquisa, que tem, entre outros, o objetivo de: - Identificar casos novos de hipertensão, relacionando os níveis de PA com a presença de fatores de risco e variáveis constitucionais, sociais, étnicas e económicas. - Identificar casos novos de hipertensão, relacionando os níveis de PA com a presença de fatores de risco e variáveis constitucionais, sociais, étnicas e económicas. - Desenvolver açOes educativas-assistenciais para clientes com diagnóstico de HA e para os que pertencem a grupos de risco, contribuindo para a adesão ao tratamento proposto. - Desenvolver açOes educativas-assistenciais para clientes com diagnóstico de HA e para os que pertencem a grupos de risco, contribuindo para a adesão ao tratamento proposto. p p A amostra foi representada por nove integrantes do Projeto cuidando de pessoas com p p A amostra foi representada por nove integrantes do Projeto cuidando de pessoas com R. Bras. Enfeml .. Brasília, v. 52, n. 1. p. 67-78, jan./mar. 1999 72 o Processo EnsitJo-Aprendizagem ... o Processo EnsitJo-Aprendizagem ... alterações da pressão arterial presentes a reunião do dia 22 de abril de 1996 alterações da pressão arterial presentes a reunião do dia 22 de abril de 1996 O i di f i li d t é d l itlV di ã alterações da pressão arterial presentes a reunião do dia 22 de abril de 1996 O processo ensino-aprendizagem foi realizado através de aula expositlVa, discussão em grupo, bem como aplicação prática da técnica aluai de mensuração da PA O processo ensino-aprendizagem foi realizado através de aula expositlVa grupo, bem como aplicação prática da técnica aluai de mensuração da PA rocesso ensino-aprendizagem foi realizado através de aula expositlVa, discussão em m como aplicação prática da técnica aluai de mensuração da PA g p , p ç p ç A fonte de coleta de dados foi do tipo primária, sendo o levantamento de dados ocasional. ANÁLISE DE DADOS GRÁFICO 1: Distribuição da amostra, segundo nível de escolaridade e tempo no projeto. Departamento de EnfermagemlUFC. Fortaleza - CE. Abril de 1998. GRÁFICO 1: Distribuição da amostra, segundo nível de escolaridade e tempo no projeto. Departamento de EnfermagemlUFC. Fortaleza - CE. Abril de 1998. o ,'" Mestranda Tempo no "'o Doutora '" ,'" ::I", -O'" 7' II 01 mês '" ::I ~-o 1102 meses 0>", Q) ~ 5' z'(l [J 12 meses '" '" ~ '8. 3' Q) (fJ l ' n° de essoas Diante dos dados, percebe-se que boa parte da amostra é composta por acadêmicos de enfermagem do 7° semestre, correspondendo aproximadamente a 33,3% do total, estando estes tambêm com maior tempo de atuação dentro do projeto (12 meses). Além disso, é notável que o projeto é composto por alunos de diversos níveis, ínclusive da pós-graduação e que oito (87,7%) da amostra estão inseridos no projeto há um ano ou menos, o que é considerado pouco tempo, embora, segundo Campos (1987) a maneira de aprender e o ritmo do processo seja variável de indivIduo para individuo, frente ao aspecto pessoal da aprendizagem. Isso significa que as características intelectuais do grupo em questão poderão trazer um ritmo diferenciado na execução do desenvolvimento educativo, considerando a experiência anterior de cada membro. o ,'" Mestranda Tempo no "'o Doutora '" ,'" ::I", -O'" 7' II 01 mês '" ::I ~-o 1102 meses 0>", Q) ~ 5' z'(l [J 12 meses '" '" ~ '8. 3' Q) (fJ l ' n° de essoas o ,'" Mestranda Tempo no "'o Doutora '" ,'" ::I", -O'" 7' II 01 mês '" ::I ~-o 1102 meses 0>", Q) ~ 5' z'(l [J 12 meses '" '" ~ '8. 3' Q) (fJ l ' n° de essoas Diante dos dados, percebe-se que boa parte da amostra é composta por acadêmicos de enfermagem do 7° semestre, correspondendo aproximadamente a 33,3% do total, estando estes tambêm com maior tempo de atuação dentro do projeto (12 meses). Além disso, é notável que o projeto é composto por alunos de diversos níveis, ínclusive da pós-graduação e que oito (87,7%) da amostra estão inseridos no projeto há um ano ou menos, o que é considerado pouco tempo, embora, segundo Campos (1987) a maneira de aprender e o ritmo do processo seja variável de indivIduo para individuo, frente ao aspecto pessoal da aprendizagem. METODOLOGIA O processo de coleta foi do tipo interrogatório, na forma de questionário tipo cédula, aplicado antes e após o desenvolvimento de um processo de reciclagem explicativa, que abordou o método atual de aferição da pressão arterial (PA). O instrumento utilizado foi do tipo semi- estruturado. Os dados apurados foram expostos em tabelas, gráficos e demonstrados em discursos. Para evidenciar melhor a experiência de aprendizado foram feitas comparações entre as informaçOes fornecidas antes e depois do processo. Os integrantes são identificados pela letra I acrescido de um numeral: '" 12, 13, I~ e assim por diante. R. Bras. EtJfenn., Brasilia, v. 52, n. 1, p. 67-78, jan.Jmar. 1999 ANÁLISE DE DADOS Isso significa que as características intelectuais do grupo em questão poderão trazer um ritmo diferenciado na execução do desenvolvimento educativo, considerando a experiência anterior de cada membro. R. Bras. EtJfenn., Brasilia, v. 52, n. 1, p. 67-78, jan.Jmar. 1999 73 MOREIRA, Thereza Maria Magalhães et ai. TABELA 1: Definição de PA no 1° e 2° momentos do processo de ensino-aprendizagem. Departamento de Enfermagem/UFC. Fortaleza - CE. Abril de 1998. TABELA 1: Definição de PA no 1° e 2° momentos do processo de ensino-aprendizagem. Departamento de Enfermagem/UFC. Fortaleza - CE. Abril de 1998. orreta Parcialmente correta ota 01 Período D e f . d e P A C o r r e t a P a r c i a l m e n t e c o r r e t a T o t a l 1º 2º 0 9 0 8 0 1 0 9 09 - A tabela descreve a correção ou não da definição de PA no 1 ° e 2° momentos do processo de ensino-aprendizagem. A orientação da aprendizagem tem como meta primordial alterar o comportamento do educando. Tal propósito leva, necessariamente, a uma tomada de decisão sobre a natureza da mudança que se pretende realizar (Turra et aI., 1975). Seguindo a linha de raciocínio dos autores e com os dados supracitados podemos inferir que houve mudança de comportamento, visto que, um dos participantes definiu PA no 1 ° momento de forma parcialmente correta e no 2° momento o fez de forma correta. TABELA 2: Distribuição da amostra, segundo verificação da PA. Departamento de Enfermagem/UFC. Fortaleza - CE. Abril de 1998. VenfIcação da PA SIm, raras vezes Sim, freqüentemente Não, nunca Total N° 02 06 01 09 % 22 67 11 100 V e r i f i c a ç ã o d a P A Nº % Sim, r a r a s v e z e s S i m , f r e q ü e n t e m e n t e N ã o , n u n c a Total 0 2 0 6 0 1 09 1 0 0 1 1 6 7 22 A tabela acima retrata que 89% da amostra teve alguma experiência prévia na técnica de verificação da PA. Sendo que destes, 67% verificavam freqüentemente e 22% esporadicamente. Situação contribuidora para o processo ensino-aprendizagem da técnica correta de verificação da PA. R. Bras. Enferm., Brasília, v. 52, n. 1, p. 67-78, jan.lmar. 1999 R. Bras. Enferm., Brasílía, v. 52, n. 1, p. 67-78, jan.lmar. 1999 ANÁLISE DE DADOS Campos (1987) refere que cada nova aprendizagem soma novos elementos à experiência prévia, num contexto gradativo e ascendente. Com essa reflexão, queremos enfatizar que a vivência do grupo no que tange a aferição da pressão arterial foi facilitadora do processo de ensino-aprendizagem desenvolvido. Dentro deste mesmo contexto, Ou Gas (1988) retrata que a aprendizagem é possibilitada quando o aprendiz entende as definições fundamentais em que é baseado o tema a ser aprendido. 74 R. Bras. Enferm., Brasília, v. 52, n. 1, p. 67-78, jan.lmar. 1999 o Processo Ensino-Aprendizagem. .. QUADRO 1: Fatores de erro na verificação da PA citados pelos respondentes antes e após a reciclagem. Departamento de Enfermagem/UFC. Fortaleza - CE. Abril de 1998. Inquietude/ tensão (I 1) Agitação (I2) Álcool, fumo, café, ambiente, estresse, exercícios físicos, bexiga cheia, tamanho do manguito (l 3) Ansiedade, hora, posição, roupas apertadas, alimentação (I 4) Alimentação, gravidez, posição, exercícios físicos, estresse e obesidade Emoção, estresse, tensão e obesidade (I 6) Fatores relacionados ao examinador, ao cliente e aparelhos (l 7) Estresse, barulho, agitação, roupas apertadas e pessoas conversadeiras (l 8) Alimentação, medicamentos, estado emocional, estresse e fumo. (I 9) Alimentação, medicamentos, estado emocional, estresse e fumo. (I 9) Estresse, barulho, lugares agitados, roupas apertadas e pessoas que gostam de conversar (I8) Fatores relacionados ao examinador, ao cliente e aparelhos (l 7) Material utilizado, emoção, estresse (I 6) Fumo, alimentação, posição, exercícios físicos, gravidez, estresse, obesidade, álcool e outras drogas (I 5) Ambiente, posição, horário, ansiedade, roupas apertadas (l 4) Tamanho do manguito, altura do braço, ansiedade, fumo, café, hora, ambiente, estresse, exercícios físicos, bexiga cheia (I 3) Fumo, álcool, sal, lipídeos (I 2) Estado do paciente, a maneira como ele está vestido, posição da pessoa (l 1) (I5) o Processo Ensino-Aprendizagem. .. QUADRO 1: Fatores de erro na verificação da PA citados pelos respondentes antes e após a reciclagem. Departamento de Enfermagem/UFC. Fortaleza - CE. Abril de 1998. Inquietude/ tensão (I 1) Agitação (I2) Álcool, fumo, café, ambiente, estresse, exercícios físicos, bexiga cheia, tamanho do manguito (l 3) Ansiedade, hora, posição, roupas apertadas, alimentação (I 4) Alimentação, gravidez, posição, exercícios físicos, estresse e obesidade Emoção, estresse, tensão e obesidade (I 6) Fatores relacionados ao examinador, ao cliente e aparelhos (l 7) Estresse, barulho, agitação, roupas apertadas e pessoas conversadeiras (l 8) Alimentação, medicamentos, estado emocional, estresse e fumo. (I 9) Alimentação, medicamentos, estado emocional, estresse e fumo. ANÁLISE DE DADOS Período Dificuldades Sim Não Não respondeu Total 09 09 --- 01 04 04 05 04 Antes Depois TABELA 3: Distribuição do grupo, segundo dificuldade na venficaçao da PA, considerando o momento pré e pós recIclagem. Departamento de EnfermagemlUFC. Fortaleza - CE. Abril de 1998. TABELA 3: Distribuição do grupo, segundo dificuldade na venficaçao da PA, considerando o momento pré e pós recIclagem. Departamento de EnfermagemlUFC. Fortaleza - CE. Abril de 1998. Período Dificuldades Sim Não Não respondeu Total 09 09 --- 01 04 04 05 04 Antes Depois Diante dos resultados acima, podemos demonstrar que antes da realização do processo quatro pessoas do grupo demonstraram dificuldades na aferiçao da PAe posteriormente cinco. Resultado esse que nos revela que a partir do processo ensino- aprendizagem é posslvel ocorrer dúvidas e questionamentos. Contribuindo, assim, para o aprimoramento do processo, com envolvimento bilateral de educando e educador. Com base nestas considerações observa-se que no momento do aprendizado ocorreu uma busca ascendente de conhecimento relativo as dificuldades encontradas na aferição da pressão arterial. Considerando Du Gas (1 988), a aprendizagem provoca mudança nos processos de pensamento, ações ou atitudes do aprendiz. TABELA 4: Dificuldades encontradas pelo grupo, antes e após a realização do processo de ensino-aprendizagem. Departamento de EnfermagernlUFC. Fortaleza -CE. Abril de 1998 TABELA 4: Dificuldades encontradas pelo grupo, antes e após a realização do processo de ensino-aprendizagem. Departamento de EnfermagernlUFC. Fortaleza -CE. Abril de 1998 Dificuldades Não consegue ouvir a diastólica Diferença entre o ensinado e o utilizado na prática Conversas paralelas, pressa e cansaço Aparelhos não calibrados Achar o pulso radial/pulsação Total 04 05 - 02 01 01 01 01 01 01 01 - n n Antes Anos ANÁLISE DE DADOS (I 9) Estresse, barulho, lugares agitados, roupas apertadas e pessoas que gostam de conversar (I8) Fatores relacionados ao examinador, ao cliente e aparelhos (l 7) Material utilizado, emoção, estresse (I 6) Fumo, alimentação, posição, exercícios físicos, gravidez, estresse, obesidade, álcool e outras drogas (I 5) Ambiente, posição, horário, ansiedade, roupas apertadas (l 4) Tamanho do manguito, altura do braço, ansiedade, fumo, café, hora, ambiente, estresse, exercícios físicos, bexiga cheia (I 3) Fumo, álcool, sal, lipídeos (I 2) Estado do paciente, a maneira como ele está vestido, posição da pessoa (l 1) (I5) O quadro acima demonstra fatores de erro na verificação da PA, citados pela clientela antes e após o processo de ensino-aprendizagem. Vale salientar que utilizamos a identificação referida anteriormente para categorização do grupo. É importante observar também que a numeração indica coincidência da clientela no primeiro e segundo momento. A aferição da PA é um procedimento simples e fácil de ser realizado. Contudo, determinados princípios devem ser atendidos no momento da técnica, evitando-se, assim, fatores de erro. Para a medida precisa da PA e , conseqüentemente, o diagnóstico correto da hipertensão , é imprescind ível adotar os cuidados e os critérios relacionados ao observador, ao paciente, ao ambiente e ao equipamento (Pierin; Santello; Mion Jr., 1997). Diante desta realidade, observa-se que a clientela, de maneira geral, citou alguns elementos que interferem, ou melhor, que podem causar erros na técnica de verificação da PA. Percebe-se, contudo, que dentre os fatores de erro relacionados à medida da PA o mais referido foi relativo ao paciente. É importante que se considere também o observador, o ambiente e o equipamento. Apesar de haver padronização na medida da PA e ser uma prática rotineira, ainda, ocorre erro na aferição, visto que, muitos profissionais a fazem de maneira incorreta. Fazendo uma análise comparativa com o quadro do "antes", pode-se perceber, de maneira geral, uma complementariedade de conhecimentos. Havendo referência mais significativa ao ambiente, cliente e equipamento. Contudo, observa-se, ainda uma lacuna relativa ao observador, que foi citado por apenas uma pessoa, tanto antes quanto após o processo de ensino- aprendizagem. R. Bras. Enferm., Brasílía, v. 52, n. 1, p. 67-78, jan.lmar. 1999 75 MOREIRA, Tllereza Maria Magalllães et aI. TABELA 3: Distribuição do grupo, segundo dificuldade na venficaçao da PA, considerando o momento pré e pós recIclagem. Departamento de EnfermagemlUFC. Fortaleza - CE. Abril de 1998. Dificuldades A tabela revela as dificuldades encontradas pelo grupo no 1° momento. As dificuldades relatadas pela clientela mostraram mais uma vez que os fatores relacionados ao ambiente, ao observador e ao equipamento. stlo aqueles que podem causar erros na aferição da PA, e consequentemente dificultam o processo de mensuração. !:: valido ressaltar que no 2° momento. tivemos a manutençtlo de três dificuldades, correspondendo a "dIferença entre o ensinado e o aprendido·, "conversas paralelas, pressa e cansaço· e "aparelhos ntlo calibrados". Além destas surgiram mais duas: "achar o pulSO radial" e "achar pulsação". R. Bras. Enfenn .. Brasília, v. 52, n. 1, p. 67-78, jan.fmar. 1999 76 o Processo Ensino-Aprendizagem ... o Processo Ensino-Aprendizagem ... o Processo Ensino-Aprendizagem ... Observa-se com este resultado que o processo de aprendizagem é algo mUito complexo e sua avaliação algo bastante complicado. TABELA 5: Contribuição do processo utilizado para o aprimoramento da técnica de verificação da PA. Departamento de Enfermagem/UFC. Fortaleza - CE. Abril de 1998. Contribuição Sim Não Total 09 --- 09 Nº Podemos referir que o processo educativo aplicado foi importante para a clientela envolvida, visto que a lolalidade dos participantes avaliou a reCiclagem como válida para o aprimoramento adequado do conhecimento relativo à técnica de venficação da PA. A avaliação é a maneira de pesquisar a distância percorrida pelo aprendiz, levando em consideraçao objetivos preestabelecidos. Se os objetivos foram enuciados especificamente em termos do que se espera que o educando seja capaz de realizar ao término do ensino, a avaliaçao de sua aprendizagem torna-se relativamente simples (Ou Gas, 1988). CONSIDERAÇÕES FINAIS Diante do exposto, conclui-se que: KEYWQRDS: leaching-Iearning. arterial pressure, nurSlng. R. Bras. ElJfenn., Brasília, v. 52, n. 1, p. 67-78, jan.lmar. 1999 Diante do exposto, conclui-se que: - A técnica de verificação da PA no grupo de pesquisa em esludo foi aprimorada; -O conhecimento apreendido sobre verificação da PA pela clientela mos incompleto e difuso no momento anterior ao processo de ensino aprendizagem -Considerando o momento posterior ao processo desenvolvido, pode-se dizer que houve um aperfeiçoamento do conhecimento da técnica de verificação da PA, mas é válido ressaltar que tal capacitação não pode ser entendida como totalizadora, ou seja, com o domínio completo da técnica, pela própria característica inacabada do processo ensino-aprendizagem e postura reflexiva do individuo. ABSTRACT: lhe nurse is considered lhe prolessional Ihal oblains more Iruslworthy dala in lhe measures insmualions 01 arterial pressure for inducmg less reacllons 01 alert in lhe customu, generating 01 values lalsely high. Even $O. a 101 01 limes. she presenlS spa ces ln lhe knowledge 01 lhe sUbject, so much ln lhe technical aspecls, as in lhe anatomo-physiologic ones. That prioritizes lhe developmenl 01 leaching slralegies, seeking lo minimize Ilaws and lo lurn lhe effectlve learning processo Objeclilying to promote lhe knowledge 01 lhe technique 01 measuremenl of lhe arterial pressure elose lo a research group, iI took place a reciclahon and avalialion queslionnaire 01 lhe knowledge of lhe group was applied belore and after lhe sarne. The effecliveness 01 Ihal melhod was evidenced, because 100% 01 lhe sam pIe (nine) Ihey referred conlribution lor beller 01 lhe lundamenlalion Iheorelical-praclice, decreasing difficulties originating Irom 01 lhe verification 01 lhe arterial pressure. II is ended Ihal, beíng lhe education aclions in exlremely valid and necessary heatth, lhe male nurse, as educalor Ihal is, needs lo recyele ils knowledge and ils belter techniques conlinually. R. Bras. ElJfenn., Brasília, v. 52, n. 1, p. 67-78, jan.lmar. 1999 77 MOREIRA, TI/Breza Maria Maga/Mes Bt ai. RESUMEN : la enfermera es considerada la profesional que obtiene datos mâs fidedignos en las medidas indirectas de presión arterial (PA) por inducir menos reacciones de alerta en el cliente, generadoras de valores falsamente elevados. Sin embargo, muchas veces, elJa presenta lagunas en el conocimiento dei asunto, tanto en los aspectos técnicos, como en los anatómicos y fisiológicos. Esto toma prioritario el desarrolJo de estrategias de ensei'ianza, con miras a disminuir los errores y volver efectivo el proceso de aprendizaje. Diante do exposto, conclui-se que: Objetivando promover el conocimiento de la técnica de medida de la presión arterial (P.A.) junto a un grupo de pesquisa, se realizó un reciclaje y se aplicó un cuestionario de evaluación dei grupo antes y después dei mismo. Se puso en evidencia la eficacia dei método. pues 100% de la mueslra (nueve) refirió contribución para la mejoria de los fundamentos teóricos y prácticos. disminuyendo las dificultades originadas de la verificación de la presión arterial. Se concluye que, siendo las acciones de educaciOn en salud de extrema necesidad y validez, el enfermero, como educador que es, necesita reciclar su conocimiento y mejorar sus técnicas continuamente. PALAVRAS llAVE: ensei'ianza - aprendizaje. presión arterial, enfermeria. REFERÊNCIAS BIBLIOGRÁFICAS ARAUJO, T. L. de. Medida indireta da pressão arterial: caracterização do conhecimento_do enfermeiro. São Paulo, 1994. Tese (Doutorado). Escola de Enfermagem. Universidade de São Paulo. ARAUJO, T. L. de. Medida indireta da pressão arterial: caracterização do conhecimento_do enfermeiro. São Paulo, 1994. Tese (Doutorado). Escola de Enfermagem. Universidade de São Paulo. g ARAUJO. T. L. de., ARCURI. E.A .M., MARTINS, E. 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TURRA, C.M.G. el aI. Planejamento de ensino e avaliação. 4. ed. Porto Alegre: PUC, Emma. REFERÊNCIAS BIBLIOGRÁFICAS 1975. p. 123-155. p ZANEllA. M.T. el. aI. Hipertensão arterial essencial: conceito e etiopatogenia. RSCESP (São Paulo), II. 2, n. 4, p. 18-22 , jullago. 1992. R. Bras. Enferm., Brasília, v. 52, n. 1. p. 67-78, jan.lmar. 1999 7.
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Assessing the potential for non-turbulent methane escape from the East Siberian Arctic Shelf
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Correspondence: Matteo Puglini (matteo.puglini@mpimet.mpg.de) As a consequence, we conclude that previously published esti- mates of ocean–atmosphere CH4 fluxes from the ESAS can- not be supported by non-turbulent, benthic methane escape. Correspondence: Matteo Puglini (matteo.puglini@mpimet.mpg.de) Correspondence: Matteo Puglini (matteo.puglini@mpimet.mpg.de) Received: 2 July 2019 – Discussion started: 8 July 2019 Revised: 17 May 2020 – Accepted: 19 May 2020 – Published: 26 June 2020 Received: 2 July 2019 – Discussion started: 8 July 2019 Revised: 17 May 2020 – Accepted: 19 May 2020 – Published: 26 June 2020 Received: 2 July 2019 – Discussion started: 8 July 2019 Revised: 17 May 2020 – Accepted: 19 May 2020 – Published: 26 June 2020 Abstract. The East Siberian Arctic Shelf (ESAS) hosts large yet poorly quantified reservoirs of subsea permafrost and as- sociated gas hydrates. It has been suggested that the global- warming induced thawing and dissociation of these reser- voirs is currently releasing methane (CH4) to the shallow coastal ocean and ultimately the atmosphere. However, a ma- jor unknown in assessing the contribution of this CH4 flux to the global CH4 cycle and its climate feedbacks is the fate of CH4 as it migrates towards the sediment–water interface. In marine sediments, (an)aerobic oxidation reactions gener- ally act as a very efficient methane sink. However, a number of environmental conditions can reduce the efficiency of this biofilter. Here, we used a reaction-transport model to assess the efficiency of the benthic methane filter and, thus, the po- tential for benthic methane escape across a wide range of en- vironmental conditions that could be encountered on the East Siberian Arctic Shelf. Results show that, under steady-state conditions, anaerobic oxidation of methane (AOM) acts as an efficient biofilter. However, high CH4 escape is simulated for rapidly accumulating and/or active sediments and can be further enhanced by the presence of organic matter with in- termediate reactivity and/or intense local transport processes, such as bioirrigation. In addition, in active settings, the sud- den onset of CH4 flux triggered by, for instance, permafrost thaw or hydrate destabilization can also drive a high non- turbulent methane escape of up to 19 µmol CH4 cm−2 yr−1 during a transient, multi-decadal period. This “window of opportunity” arises due to delayed response of the resident microbial community to suddenly changing CH4 fluxes. A first-order estimate of non-turbulent, benthic methane efflux from the Laptev Sea is derived as well. We find that, under present-day conditions, non-turbulent methane efflux from Laptev Sea sediments does not exceed 1 Gg CH4 yr−1. 1 Introduction The Siberian shelf represents the largest shelf on Earth (∼ 3 millions km2 Wegner et al., 2015) and spreads from the Kara Sea to the Laptev, East Siberian and Chuckhi seas. The East Siberian Arctic Shelf (ESAS) corresponds to the broad area beneath the shallow (∼45 m water depth, James et al., 2016) Laptev and East Siberian Arctic Sea (Romanovskii et al., 2004; Shakhova et al., 2010a) and represents the largest region on the Siberian shelf (Romanovskii et al., 2005), cov- ering about 25 % of the total Arctic shelf (Shakhova et al., 2010a). Although similar in many aspects to other shelf environ- ments, a distinguishing feature of the ESAS is the presence of subsea permafrost and associated gas hydrates buried in the sediment (Sloan and Koh, 2007; Romanovskii et al., 2005). Subsea permafrost is a terrestrial relict that mainly formed during glacial periods, when retreating sea levels (with a minimum of 120 m below the current level around the Last Glacial Maximum) exposed Arctic shelves (Fairbanks, 1989; Bauch et al., 2001). Under these conditions, permafrost ag- graded on the shelf and was subsequently submersed (Ro- manovskii and Hubberten, 2001; Romanovskii et al., 2005) by rising sea levels during the Holocene sea transgression Assessing the potential for non-turbulent methane escape from the East Siberian Arctic Shelf Matteo Puglini1,2, Victor Brovkin1,3, Pierre Regnier2, and Sandra Arndt2 1Land in the Earth System, Max Planck Institute for Meteorology, Hamburg, Germany 2BGeosys, Department Geoscience, Environment & Society (DGES), Université Libre de Bruxelles, Brusse 3CEN, Universität Hamburg, Hamburg, Germany g p p from the East Siberian Arctic Shelf Matteo Puglini1,2, Victor Brovkin1,3, Pierre Regnier2, and Sandra Arndt2 1Land in the Earth System, Max Planck Institute for Meteorology, Hamburg, Germany 2BGeosys, Department Geoscience, Environment & Society (DGES), Université Libre de Bruxelles, Brussels, Belgium 3CEN, Universität Hamburg, Hamburg, Germany Matteo Puglini1,2, Victor Brovkin1,3, Pierre Regnier2, and Sandra Arndt2 1Land in the Earth System, Max Planck Institute for Meteorology, Hamburg, Germany 2BGeosys, Department Geoscience, Environment & Society (DGES), Université Libre de Bruxelles, Brussels, Belgium 3CEN, Universität Hamburg, Hamburg, Germany Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. M. Puglini et al.: Assessing the potential for non-turbulent methane escape M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3248 (12–5 kyr BP) (Bauch et al., 2001). Gas hydrates are solid, methane-concentrated formations in which a gas molecule is trapped in a cage of water molecules (Ruppel and Kessler, 2017). They are thermodynamically stable under specific temperature–pressure–salinity in the ocean floor, including areas beneath the subsea permafrost (Sloan and Koh, 2007). and, ultimately, methane–climate feedbacks remains poorly quantified (James et al., 2016; Saunois et al., 2016). As a consequence, it has not received much attention in the re- cent IPCC special report (Masson-Delmotte et al., 2018). At present, a major unknown is the strength of methane sinks in Arctic sediments and waters and their influence on methane emissions (Ruppel and Kessler, 2017). Therefore, improved assessments of the present and future climate impact of per- mafrost thaw and hydrate destabilization require not only a better knowledge of the Arctic subsea permafrost and hydrate distribution, reservoir size and characteristics, but also a bet- ter quantitative understanding of Arctic methane sinks. p Little is known about the total amount of carbon stored in subsea permafrost or its original extent. Published estimates of carbon reservoir sizes diverge by orders of magnitude. For instance Shakhova et al. (2010a) estimate that 1175 Pg C are locked in subsea permafrost on the ESAS alone, while McGuire et al. (2009) calculate that, across the entire Arc- tic shelf, 9.4 Pg C reside in upper sediments and 1.5–49 Pg C (2–65 Pg CH4) in methane gas hydrates. Thus, the size of the Arctic subsea permafrost reservoir, its spatial distribution, as well as its biogeochemical and physical characteristics re- main poorly known. In marine sediments, upward migrating methane is gen- erally efficiently consumed by the anaerobic oxidation of methane (AOM) and, to a lesser extent, the aerobic oxidation of methane (AeOM) (Hinrichs and Boetius, 2002; Reeburgh, 2007; Knittel and Boetius, 2009). Although the exact AOM process has not been fully understood yet (James et al., 2016; McGlynn et al., 2015; Milucka et al., 2012; Wegener et al., 2015; Dean et al., 2018), it is thought that AOM is mediated by a consortium of methane-oxidizing Archaea and sulfate- reducing bacteria (Boetius et al., 2000) according to the re- action (Hinrichs and Boetius, 2002; Krüger et al., 2003) p y These knowledge gaps are critical as climate change is am- plified in polar regions. M. Puglini et al.: Assessing the potential for non-turbulent methane escape The Arctic is currently warming at a rate twice as fast as the global mean (Trenberth et al., 2007; Bekryaev et al., 2010; Jeffries and Richter-Menge, 2012; Christensen et al., 2013). Recent observations indicate that bottom water temperatures in the coastal and inner shelf re- gions of the ESAS (water depth < 30 m, Dmitrenko et al., 2011) are rising, while the central shelf sea may be subject to intense episodic warming (Janout et al., 2016). The increas- ing influx of warmer Atlantic water into the Arctic Ocean – the so-called Atlantification (Polyakov et al., 2017; Bar- ton et al., 2018) – will not only further enhance this warm- ing, but will also influence circulation and salinity patterns on the shelf (Carmack et al., 1995; Zhang et al., 1998; Bias- toch et al., 2011). At the same time, it has been long recog- nized that the Arctic is a potential hotspot for methane emis- sions. Extensive methane gas bubbling has been observed in the Laptev Sea and has been directly linked to these envi- ronmental changes (Shakhova et al., 2010b, 2014). Shakhova et al. (2014) suggest that warming induced subsea permafrost thaw and hydrate destabilization may support methane emis- sions of up to 17 Tg CH4 yr−1 from the ESAS alone. Pro- jected change in temperature (Shakhova et al., 2017, 2019) due to climate change is expected to further destabilize Arc- tic subsea permafrost and gas hydrate reservoirs and might thus enhance further methane emissions (Piechura and Wal- czowski, 1995; Westbrook et al., 2009; Reagan and Moridis, 2009; Biastoch et al., 2011; Hunter et al., 2013; Drake et al., 2015; Ruppel and Kessler, 2017). However, a number of re- cent studies have questioned the significance of subsea per- mafrost thaw and hydrate destabilization for methane efflux from Arctic sediment (Thornton et al., 2016; Ruppel and Kessler, 2017), for methane concentrations in Arctic Ocean waters (Overduin et al., 2015; Sapart et al., 2017) and, ulti- mately, for methane emissions from the Arctic waters (Rup- pel and Kessler, 2017; Sparrow et al., 2018). Thus, the con- tribution of subsea permafrost thaw and gas hydrate destabi- lization to methane emissions from the warming Arctic shelf CH4 + SO2− 4 →HCO− 3 + HS−+ H2O. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.1 BRNS: reaction-transport model Therefore, we here use a one-dimensional reaction- transport model approach to understand and quantify the effi- ciency of the AOM biofilter and its influence on the potential benthic release of methane in response to a plausible range of upward migrating dissolved methane fluxes from thawing permafrost and/or dissociating methane gas hydrates on the warming ESAS. The developed model accounts for the most pertinent primary and secondary redox processes, as well as mineral precipitation, methane gas formation and fast equi- librium reactions. Both active sites (characterized by an up- ward water flow) and passive sites (without an upward water flow) are investigated. We limit our model analysis to non- turbulent methane efflux, because methane in gaseous form is not directly accessible for the AOM community. As a con- sequence, free gas bubbles are less prone to be consumed by AOM and methane gas either is trapped in the sediments or rapidly migrates upwards through cracks, faults or fractures (Boudreau, 2012), bypassing the AOM biofilter. The Biogeochemical Reaction Network Simulator (BRNS) (Regnier et al., 2002; Aguilera et al., 2005; Centler et al., 2010) – an adaptive simulation environment suitable for sim- ulating large, mixed kinetic-equilibrium reaction networks in porous media (e.g. Jourabchi et al., 2005; Thullner et al., 2005; Dale et al., 2009) – is used to quantitatively explore the fluxes and transformations of methane in a sediment col- umn representative for ESAS conditions. For this purpose, we set up a reaction network (Tables S1, S2 in the Supple- ment), model parameters (Table S6), as well as boundary conditions (Table S7) that cover the conditions encountered on the present-day Siberian shelf. In the BRNS, the general mass conservation for each solid and dissolved species is described by a set of cou- pled advection–diffusion–reaction equations in porous me- dia which are solved simultaneously (e.g. Berner, 1980; Boudreau, 1997; note that dependencies on z and t have been omitted for simplicity): The model is forced with a set of boundary conditions that are broadly representative of the conditions potentially encountered on the ESAS. It is applied to conduct a com- prehensive one-at-a-time, steady-state sensitivity study over the entire plausible range of (1) sedimentation rates, (2) ac- tive fluid flow velocities, (3) AOM rate constants, (4) organic matter reactivity and (5) non-local transport activity encoun- tered on the ESAS. M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3249 methane gas accumulates in the sediments. When free gas pockets grow enough, methane tends to migrate upwards along pathways with higher permeability or where fractures occur (Yakushev, 1989; Boudreau et al., 2005; Wright et al., 2008; Shakhova et al., 2014, 2015, 2017; Leifer et al., 2017) and might even crack the sediments themselves (O’Connor et al., 2010; Overduin et al., 2016; Yao et al., 2019; Stranne et al., 2019). However, despite a wealth of AOM-related re- search, a holistic, quantitative evaluation of the most impor- tant environmental controls on the efficiency of the AOM biofilter and its impact on methane escape from marine sedi- ments is currently lacking. Thus our ability to understand and quantify AOM sink in ESAS sediments and thus the climate impact of subsea permafrost thaw and gas hydrate destabi- lization is seriously compromised. methane gas accumulates in the sediments. When free gas pockets grow enough, methane tends to migrate upwards along pathways with higher permeability or where fractures occur (Yakushev, 1989; Boudreau et al., 2005; Wright et al., 2008; Shakhova et al., 2014, 2015, 2017; Leifer et al., 2017) and might even crack the sediments themselves (O’Connor et al., 2010; Overduin et al., 2016; Yao et al., 2019; Stranne et al., 2019). However, despite a wealth of AOM-related re- search, a holistic, quantitative evaluation of the most impor- tant environmental controls on the efficiency of the AOM biofilter and its impact on methane escape from marine sedi- ments is currently lacking. Thus our ability to understand and quantify AOM sink in ESAS sediments and thus the climate impact of subsea permafrost thaw and gas hydrate destabi- lization is seriously compromised. fluxes originating from thawing subsea permafrost or desta- bilizing methane gas hydrates under a plausible range of en- vironmental conditions encountered on the present and fu- ture Siberian shelf. Model results are then used to (3) iden- tify environmental conditions (and thus areas on the ESAS) that favour non-turbulent dissolved methane fluxes across the sediment–water interface and (4) derive transfer functions that allow estimation of the potential for non-turbulent CH4 escape from ESAS sediments, thus providing first-order con- straints on the Arctic methane budget. M. Puglini et al.: Assessing the potential for non-turbulent methane escape (1) (1) A recent assessment indicates that, in global sediments, around 45–61 Tg CH4 yr−1 (Egger et al., 2018) are consumed by AOM, thus significantly reducing previously published estimates of 320–360 Pg CH4 yr−1 (Hinrichs and Boetius, 2002; Reeburgh, 2007). AOM generally acts as a particularly efficient biofilter for upward migrating methane and oxidizes up to 100 % of the methane flux coming from below (e.g. Regnier et al., 2011). However, a number of environmental conditions can reduce the efficiency of this AOM biofilter, allowing methane to es- cape from the sediment (Iversen and Jorgensen, 1985; Piker et al., 1998; Jørgensen et al., 2001; Treude et al., 2005; Knab et al., 2008; Dale et al., 2008c; Thang et al., 2013; Egger et al., 2016). It has been shown that, in particular, high sed- imentation rates (Egger et al., 2016), slow microbial growth (Dale et al., 2006, 2008c) or the accumulation of free gas can promote methane efflux from the sediment. These find- ings are particularly relevant for potential methane escape from Arctic shelf sediments. The Siberian shelf is the largest sedimentary basin in the world (Gramberg et al., 1983) and shelf areas close to the large Arctic rivers reveal sedimen- tation rates than can be up to 5 times faster than rates that are typically observed in the ocean (Leifer et al., 2017). In addition, the Arctic shelf is subject to large seasonal, as well as climate-induced long-term changes in environmen- tal conditions, namely SO2− 4 concentration in seawater and availability of CH4 in the sediments coming from deeper strata. These factors may influence the efficiency of the AOM biofilter through their effect on microbial biomass dynam- ics. Finally, observations from the ESAS also indicate that https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 2.1.1 Transport The effective diffusion coefficients Di are determined by correcting the diffusion coefficients in free solution D0 i (Boudreau, 1997) for tortuosity θ and temperature. Tortuos- ity is calculated by means of porosity ϕ according to a mod- ified Weissberg relation (Boudreau, 1997): θ = 1 −ln(ϕ2). Note that the effective diffusion coefficients used in the model neglect pressure effects. Following Dale et al. (2008a), migration of methane gas is simply parameterized via a pseudo-diffusive term, with an apparent gas diffusion coef- ficient, DCH4(g). Bioturbation in the upper decimetres of the sediment is simulated using a diffusive term (e.g. Boudreau, 1986), with a constant bioturbation coefficient, D0 b. The model assumes that bioturbation ceases at the bioturbation depth, zbio (Boudreau, 1997). Bioirrigation is included in the mass conservation equation as a source or a sink function analogous to a kinetic rate. It is calculated as the product of the irrigation intensity, α (α = 0 for all solids), and the differ- ence in concentration of species i relative to the concentra- tion at the SWI, Ci(0). The bioirrigation rate α, is evaluated from the bioirrigation coefficient at the sediment surface (α0) and the bioirrigation attenuation depth (zirr) and is given by Eq. (S9) in the Supplement. Porosity is assumed to decrease with depth according to an exponential decay (Athy, 1930): As described above, methane is produced during organic matter degradation by methanogens in deeper sediment lay- ers, once all TEAs are depleted (Table S2, R6). If the con- centration of dissolved methane exceeds the saturation con- centration [CH4]∗, methane gas forms. The transfer rate of methane between the dissolved and gaseous phase is linearly controlled by the departure of the simulated dis- solved methane concentration from the saturation concen- tration (Haeckel et al., 2004; Hensen and Wallmann, 2005; Tishchenko et al., 2005; Mogollón et al., 2009; Graves et al., 2017). [CH4]∗is calculated according to Dale et al. (2008a), derived from the formulation proposed by Duan et al. (1992) for which [CH4]∗depends on in situ salinity, pressure and temperature. Here, we assume that the formed methane gas is inaccessible to microbial activity and hence bypasses anaer- obic and/or aerobic oxidation zones. In contrast, dissolved methane can be consumed by anaerobic (AOM) or aero- bic oxidation of methane (AeOM). Free gas can re-dissolve into porewater once porewater methane concentration falls below the saturation level and may then become available to methanotrophs. M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3250 is written as 1991) that describes compound-specific reactivities (Tesi et al., 2014) and, thus, captures the widely observed decrease in apparent organic matter reactivity with degradation state. The relative importance of each metabolic pathway is simu- lated through a series of kinetic limitation terms, reflecting their sequential utilization in the order of their decreasing Gibbs energy yields (Table S1). After all terminal electron acceptors (TEAs) are consumed, the remaining organic mat- ter may be degraded by methanogenesis. The rates of sec- ondary redox reactions (Table S3), are described by bimolec- ular rate laws (e.g. Wang and Van Cappellen, 1996). Adsorp- tion reactions are considered as fast equilibrium processes (Table S3, R28–R31 in the Supplement). Mineral precipita- tion rates are simulated according to kinetic–thermodynamic rate laws (Table S3, R16–R24). 1991) that describes compound-specific reactivities (Tesi et al., 2014) and, thus, captures the widely observed decrease in apparent organic matter reactivity with degradation state. The relative importance of each metabolic pathway is simu- lated through a series of kinetic limitation terms, reflecting their sequential utilization in the order of their decreasing Gibbs energy yields (Table S1). After all terminal electron acceptors (TEAs) are consumed, the remaining organic mat- ter may be degraded by methanogenesis. The rates of sec- ondary redox reactions (Table S3), are described by bimolec- ular rate laws (e.g. Wang and Van Cappellen, 1996). Adsorp- tion reactions are considered as fast equilibrium processes (Table S3, R28–R31 in the Supplement). Mineral precipita- tion rates are simulated according to kinetic–thermodynamic rate laws (Table S3, R16–R24). (3) Si = X j λijRj, (3) where λij are the stoichiometric coefficients of all reaction rates Rj that affect species i. where λij are the stoichiometric coefficients of all reaction rates Rj that affect species i. 2.1.1 Transport AeOM rate is simply described by a bi- molecular rate law (Table S3, R14). The description of AOM depends on the model scenario. For steady-state simulations, we apply a simple bimolecular rate: ϕ(z) = ϕ0e−c0z, (4) ϕ(z) = ϕ0e−c0z, (4) with ϕ0 the porosity at the SWI and c0 the typical length scale for compaction. with ϕ0 the porosity at the SWI and c0 the typical length scale for compaction. 2.1 BRNS: reaction-transport model In addition, we also evaluated the influ- ence of (1) seasonal variability and (2) idealized, projected climate change on the efficiency of the AOM biofilter and po- tential non-turbulent methane escape from the ESAS under transient conditions. For this purpose, the model is extended by adopting an explicit description of AOM biomass dynam- ics and a bioenergetic rate law for AOM (Dale et al., 2006, 2008c, b). Finally, the results of all sensitivity study runs are used to identify the most important controls on methane ef- flux and derive a transfer function that allows establishment of a first-order estimate of methane escape from the ESAS. ∂ξCi ∂t = ∂ ∂z  (Di + Db,i)ξ ∂Ci ∂z  −∂ ∂z(vξCi) + αiξ(Ci(0) −Ci) + Si. (2) (2) Ci is the concentration of the species i (mass per pore- water volume for dissolved species or mass per solid matrix volume for a solid species); ξ i.e. the porosity ξ = ϕ for dis- solved species and ξ = ϕs = 1−ϕ for solid species. Di is the effective diffusion coefficient for species i and is affected by salinity, temperature and tortuosity (see Table S6). Db de- notes the bioturbation coefficient and v is the advective ve- locity. For solid species v = ω with ω being the burial rate, while the advective velocity for dissolved species is given by the sum of the burial rate and an advective flow velocity, vup, i.e. v = ω + vup. A site where vup ̸= 0 is defined as an active site, while a site with no advective upward water flow is de- fined as passive. αi is the bioirrigation coefficient (αi = 0 for solid species) and Ci(0) is the concentration of the species i at the sediment–water interface (SWI). The reaction term Si The specific aims of this work are thus (1) to identify and quantitatively understand the most important environmental controls on the efficiency of the AOM biofilter, as well as (2) its significance in reducing upward migrating methane https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 2.2 Model evaluation To evaluate the performance of the BRNS set-up in captur- ing the main diagenetic patterns observed in Arctic shelf sediments we run the model for two case study sites in the area of interest: (1) a site offshore Kotelny Island in the cen- tral region of the Laptev Sea, north of the Lena River delta (76.171◦N, 129.333◦E, 56 m water depth) collected dur- ing the SWERUS-C3 expedition in summer 2014 (Brüchert et al., 2018; Brüchert, 2020).    1 −exp  1Gr+1GBQ χRT  , if 1Gr+1GBQ χRT < 0 0, if 1Gr+1GBQ χRT > 0, (8) (8) (8) where R is the gas constant, T is the absolute temperature, χ is the average number of electrons transferred per reac- tion per mole of ATP produced (Jin and Bethke, 2005), 1Gr is the Gibbs free energy of the reaction and 1GBQ = 20 kJ (mol e−)−1 is the minimum energy needed to support syn- thesis of ∼1 3 −1 4 mol ATP (Dale et al., 2008c). In or- der to be thermodynamically favourable, the total energy 1Gr + 1GBQ has to be negative, meaning that Gibbs free energy provided by the catabolic reaction is sufficient to sus- tain the microbial biomass growth. 1Gr is given by Although observations are merely available for the first 22 cm, the first 3 m of sediment are simulated to allow for the full development of the early diagenetic network, thus also accounting for biogeochemical processes (e.g. methano- genesis) in deeper sediment layers that potentially affect bio- geochemical cycling in the shallower sediment. Observations at the site indicate the absence of active flow and the advec- tive velocity vup is thus set to zero. Upper boundary condi- tions and model parameters are constrained on the basis of the observations reported (Brüchert, 2020) (Table S4). The observed organic carbon profile is imposed in the first 19 cm (Table S5) and organic carbon contents in deeper sediments are calculated on the basis of the reactive continuum model for organic matter degradation (described in Sects. S2 and S3) and the deepest observed value. M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3251 performing biomass (B) can be written as performing biomass (B) can be written as 2.1.3 Boundary conditions dB dt = µgB · FK · FT −µdB2, (6) dB Boundary conditions place the model in its environmental context. For dissolved species, constant bottom water con- centrations (Dirichlet boundary conditions) are applied at the sediment–water interface, while a known flux condi- tion (Neumann boundary condition) are applied for solid species. At the lower boundary, a zero gradient flux boundary condition (∂C/∂z = 0) is considered for all species except methane, for which a Dirichlet condition is specified to ac- count for methane supplied from thawing permafrost and/or dissociating gas hydrates below. (6) where µg is the growth rate and µd is the decay rate. FK is the kinetic constraint given by where µg is the growth rate and µd is the decay rate. FK is the kinetic constraint given by FK = [CH4] KCH4 m + [CH4] · h SO2− 4 i K SO2− 4 m + h SO2− 4 i, (7) (7) with K SO2− 4 m half saturation constant of SO2− 4 and KCH4 m half saturation constant of CH4, according to a typical Michaelis– Menten formulation for enzymatically catalysed reactions. FT represent the thermodynamic limitation and is given by 2.1.2 Biogeochemical network The reaction network implemented here (33 species, 37 re- actions) encompasses the most pertinent primary and sec- ondary redox reactions, equilibrium reactions and mineral precipitation and adsorption reactions. A summary of the re- actions, their stoichiometry and their rate formulations can be found in Tables S2 and S3. The following section provides a short description of the implemented reaction network, as well as a more detailed description of the reactions that af- fect the production/consumption of methane. A complete de- scription can be found in the Supplement. rateAOM = kAOM[CH4][SO2− 4 ]. (5) (5) It is the simplest and most commonly used formulation of the AOM rate in reaction-transport models (e.g. Regnier et al., 2011). It accounts for kinetic controls and assumes that, un- der steady-state conditions, bioenergetic controls are negligi- ble (Dale et al., 2006; Regnier et al., 2011). For transient model simulations, we apply a bioenergetic rate law in combination with an explicit description of the AOM-performing biomass (Dale et al., 2006, 2008c). It has been shown that the rates of redox reactions, whose en- ergy yield is used by micro-organisms to grow, can be cou- pled to biomass growth rates via a kinetic Monod term and a thermodynamic Boltzmann term (e.g. Rittmann and VanBriesen, 2019). Hence, the time derivative of AOM- The BRNS model accounts for the degradation of or- ganic matter by aerobic degradation, denitrification, man- ganese oxide reduction, iron reduction, sulfate reduction and methanogenesis (Table S2). Organic matter degradation is described by means of the reactive continuum model (RCM) (Aris, 1968; Ho and Aris, 1987; Boudreau and Ruddick, https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 2.3.1 Steady-state sensitivity analysis To evaluate the main physical and biogeochemical controls on the efficiency of the AOM biofilter and its impact on non- turbulent methane emission from deep methane sources such as dissociating permafrost and/or disintegrating methane gas hydrates in ESAS sediments, we conduct a comprehensive, steady-state sensitivity study. For this purpose, we design a set of two baseline scenarios that are broadly representa- tive for environmental conditions encountered on the shallow ESAS: 1. Sedimentation rate ω. The sedimentation rate is var- ied over 2 orders of magnitude (0.03–0.123–0.17– 1.5 cm yr−1). Maximum values are comparable to ter- restrial sediment accumulation rates in the Lena River delta (Bolshiyanov et al., 2015), fast marine sedimen- tation rates during the early Holocene sea transgression (Bauch et al., 2001) and marine accumulation on subsea permafrost deposit in Buor Khaya Bay (∼1.1 cm yr−1, inferred from Overduin et al., 2015), while minimum values are representative of sedimentation rates found in the East Siberian Arctic Sea (Stein et al., 2001 in Levitan and Lavrushin, 2009). The baseline value of ω is calculated based on the empirical global relationship proposed by Burwicz et al. (2011). 1. a passive case, i.e. vup = 0 cm yr−1; 2. an active case, i.e. with vup = 1 cm yr−1, a value which falls within the range of fluid flow velocities vup = 0.005−30 cm yr−1 observed across a wide range of dif- ferent active environments (Regnier et al., 2011). 2. an active case, i.e. with vup = 1 cm yr−1, a value which falls within the range of fluid flow velocities vup = 0.005−30 cm yr−1 observed across a wide range of dif- ferent active environments (Regnier et al., 2011). For both baseline scenarios, we assume a water depth of 30 m, which is slightly shallower than the average water depth of the ESAS ∼45 m (James et al., 2016), since we are here interested in the shallow, near-coastal part of the shelf that potentially hosts large subsea permafrost reservoirs and is most affected by the warming. Temperature is set equal to 0◦C and is thus similar to the yearly average of −0.79◦C ob- served in the Laptev Sea at a depth of about 30 m (Dmitrenko et al., 2011). The bioturbation coefficients D0 b and bioirriga- tion coefficients α0 (Thullner et al., 2009) are then derived from global empirical relationships according to Middelburg et al. (1997) and Thullner et al. M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3252 variation in sediment porosity, LaRowe et al., 2017). It can thus only cause uniform shifts in the [NH+ 4 ] profile, but does not affect the overall shape of the NH+ 4 depth profile. Sim- ilarly, PO3− 4 is only consumed by fluorapatite precipitation (R22) and adsorption processes (R29 and R31). Fluorapatite precipitation controls maximum dissolved PO3− 4 concentra- tions, while the mineral adsorption process (R29) exerts a homogeneous influence and the interaction with Fe(OH)3 is expected to be minor and mainly affects PO3− 4 within the iron reduction zone. by observations. They are chosen to be broadly representa- tive of the wider Siberian shelf environment. Each sensitivity study run is forced with a range of dif- ferent dissolved [CH4] concentrations at the lower model boundary, mimicking different methane fluxes from thaw- ing subsea permafrost and/or disintegrating methane gas hydrates at depth. The applied set of methane concen- trations at the lower boundary range from zero to the methane gas saturation concentration [CH4]−= 0–20–100– 330–1169–5455 µM and also include the highest methane concentration that has been to date observed in ESAS cores (Overduin et al., 2015) ([CH4]−= 1.169 mM). OM reactivity parameters (a and ν), bottom methane con- centration ([CH4]−) and reaction rates are varied to find the best fit between observed and simulated profiles. Methane concentrations at the bottom of the model domain can also exceed the saturation concentration [CH4]∗= 14 mM (esti- mated according to the value reported in Dale et al., 2008a) to include the possibility of methane in gaseous form. Table 1 and Table S6 summarize the parameters applied in the baseline simulation and Table S7 provides an overview of the applied upper boundary conditions. To assess the influence of environmental conditions on the efficiency of the AOM biofilter and its influence on non- turbulent methane emission from dissociating permafrost and/or disintegrating methane gas hydrates in ESAS sedi- ments a set of five “one-at-time” parameter variation exper- iments is designed. It encompasses the most important con- trols on benthic methane cycling (Regnier et al., 2011; Meis- ter et al., 2013; Egger et al., 2018) and parameter variation experiments are performed for both the passive as well as active baseline scenarios. 2.2 Model evaluation In addition, the possibil- ity of a source of methane is implemented at the bottom of the modelled sediment column by applying a Dirichlet boundary condition, thus taking into account the possible presence of methane seeping from deep sediments as results of destabi- lizing gas hydrates/subsea permafrost – a distinguishing fea- ture of the ESAS sediments. The methane boundary condi- tion is determined by model fitting (see below). 1Gr = 1G0 r + RT ln γ [HS−] · [HCO− 3 ] [CH4] · [SO2− 4 ] ! , (9) (9) with 1G0 r the standard free energy of the reaction, the second term the deviation from standard conditions (temperature and reaction quotient) on Gibbs free energy, and γ a parameter representing departure from ideal behaviour. The link between substrate consumption and microbial growth (anabolism) is given by Dale et al. (2006) 13.8SD · SO2− 4 + 14.3SD · CH4 + 0.2SD · NH+ 4 + 0.3SD · H+ →0.2B + 13.3SD · HCO− 3 + 13.8SD · HS−, (10) (10) assuming that the cellular composition of the biomass B is equal to C5H7O2N (Bruce and Perry, 2001; Dale et al., 2006, 2008c; Rittmann and McCarty, 2012). SD = (1−ϕ)/ϕ is the conversion factor between dissolved and solid species, here represented by micro-organisms (which are assumed to be at- tached to the solid matrix). Catabolism is linked to biomass growth (anabolism) through the growth yield. We apply a yield of 0.0713 (Dale et al., 2006), which falls at the up- per end of reported AOM growth yields, i.e. 0.05–0.07 (Dale et al., 2006; Nauhaus et al., 2007). When evaluating model performance, particular attention is given to sulfate, methane, ammonium (NH+ 4 ), phosphates (PO3− 4 ) and dissolved inorganic carbon (DIC) depth profiles. While the former two species are of main interest for eval- uating simulated AOM dynamics, the remaining three serve as indicators for OM degradation dynamics since they are metabolic byproducts of degradation (see Table S2). More- over, NH+ 4 is only affected by nitrification (R7) and ad- sorption (R28). The latter, although important, acts homo- geneously throughout the sediment (considering the slight https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 1. a passive case, i.e. vup = 0 cm yr−1; 2.3.1 Steady-state sensitivity analysis 5) is thus varied over the range kAOM = 5×102–5×103–5×104–5×105–5×106– 5 × 107 M−1 yr−1. thic AOM filter and facilitate methane escape due to the delayed response of the microbial community to changing conditions. Therefore, in addition to the steady-state sensi- tivity study, we also performed a series of transient simula- tions to explore the impacts of seasonal and projected cli- mate change on benthic methane effluxes on the ESAS in re- sponse to changing upward methane fluxes from dissociating permafrost and/or disintegrating methane gas hydrates. Tran- sient simulations were run with a bioenergetic rate law for AOM (Eq. 6) and an explicit description of AOM biomass. Simulation results from the passive steady-state baseline run with [CH4]−= 0 mM were used as initial conditions for the transient experiments. Four different transient environmental perturbation scenarios that reflect seasonal (1, 2) as well as idealized future (3, 4) environmental variability on the ESAS were run with three different values of vup = 0–1–5 cm yr−1 over a period of 200 years. 4. Organic matter reactivity (i.e. RCM parameters). Al- though the apparent OM reactivity is controlled by a combination of two parameters (a and ν), previous stud- ies indicate a less pronounced variability in ν (Arndt et al., 2013; Sales de Freitas, 2018), as well as a strong control of a on the sulfate–methane transition zone (SMTZ) depth (Regnier et al., 2011; Meister et al., 2013). Thus, ν was kept constant, while a was var- ied over the entire range of previously published val- ues a = 0.1–1–10–100–500–1000 years (Arndt et al., 2013). Studies about ESAS organic matter degradation show a reactivity of deposited organic matter which is compatible with the RCM parameters we explored. For instance, Bröder et al. (2016) found a half life for the organic carbon in the East Siberian Arctic Shelf of 19– 27 years, which would correspond to a = 3.4–4.8 years, with ν = 0.125. 1. Seasonal CH4: seasonal change in methane supply from permafrost thaw and/or hydrate destabilization. CH4 concentration at the bottom of the sediment column: null for 6 months, then increasing up to a peak of [CH4]−(20–100–330–1169–5455 µM) for the remain- ing 6 months of the year and again back to null concen- tration. 5. Bioirrigation coefficient α0. Bioirrigation activity re- mains largely unconstrained on the Siberian shelf due to the scarcity of observational data (Teal et al., 2008). 2.3.1 Steady-state sensitivity analysis (2009), respectively. The methane saturation concentration [CH4]∗is calculated on the basis of the relationship proposed by Dale et al. (2008a) as- suming a soil matrix density of 2.41 g cm−3. Values of ϕ0 and c0 (see Eq. 4) are determined based on LaRowe et al. (2017). Boundary conditions are reported in Table S7 and informed 2. Active fluid flow vup. Buoyancy-induced motion (Baker and Osterkamp, 1988), water streams channeled through fault lines or groundwater discharge (Charkin et al., 2017) can cause active fluid flow in Arctic shelf sediments underlain by subsea permafrost or gas hydrates (Judd and Hovland, 2009; Semenov et al., 2019). Therefore, vup is varied from 0–0.3–0.5–1–3–7– 10 cm yr−1. This interval falls in the range of reported upward advective water velocities in marine sediments 0.005–30 cm yr−1 (Regnier et al., 2011). 2. Active fluid flow vup. Buoyancy-induced motion (Baker and Osterkamp, 1988), water streams channeled through fault lines or groundwater discharge (Charkin et al., 2017) can cause active fluid flow in Arctic shelf sediments underlain by subsea permafrost or gas hydrates (Judd and Hovland, 2009; Semenov et al., 2019). Therefore, vup is varied from 0–0.3–0.5–1–3–7– 10 cm yr−1. This interval falls in the range of reported upward advective water velocities in marine sediments 0.005–30 cm yr−1 (Regnier et al., 2011). 3. AOM constant kAOM. Rate constants implicitly account for factors that are not explicitly described in the model and thus tend to show a strong variability between sites. A comprehensive compilation of published model AOM https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3253 M. Puglini et al.: Assessing the potential for non-turbulent methane escape Table 1. Model parameters changed in the “one-at-time” sensitivity studies. Reported values are for the baseline simulations Quantity Meaning Value Units Reference ω Sedimentation rate 0.123 cm yr−1 Burwicz et al. (2011) a Average lifetime of reactive OM 10 year This study vup Upward water velocity 0, 1 cm yr−1 This study α0 Bioirrigation coefficient 99.5 yr−1 Thullner et al. (2009) kAOM AOM rate constant 5.0 × 103 M−1 yr−1 Regnier et al. (2011) [CH4]− CH4 lower boundary condition 0–5.455 mM This study meters changed in the “one-at-time” sensitivity studies. Reported values are for the baseline simulations. rate constants (Regnier et al., 2011) reveals a variability of over 6 orders of magnitude (10–107 M−1 yr−1). The AOM rate constant kAOM (Eq. 2.3.1 Steady-state sensitivity analysis However, environmental stressors, such as ice scour- ing (e.g. Shakhova et al., 2017, and references therein) and trawling, which can dig furrows up to few me- tres (Shakhova et al., 2017) are detrimental to the lo- cal fauna, thus suggesting a low bioirrigation intensity. However, observations from other polar sites indicate that although biological diversity and activity is often low, it might be locally enhanced (Clough et al., 1997). In addition, ice scouring might also enhance non-local transport seasonally. We therefore varied α0 over the entire range of plausible values: 0–33–66–99.5–120– 240 yr−1 (Thullner et al., 2009). 2. Seasonal CH4 + SO2− 4 : seasonal freshening of waters due to riverine discharge and sea ice melt. During win- ter, higher bottom salinity (Dmitrenko et al., 2011) results in higher sulfate concentration (Dickson and Goyet, 1994), while lower salinities and thus sulfate concentrations characterize the melt season. The bottom boundary condition for methane [CH4]−follows an op- posite trend: it is set to zero during the winter months and increases in Arctic summer. 3. Linear CH4: slow increase in methane supply from per- mafrost thaw and/or hydrate destabilization. A linear in- crease in the bottom boundary methane concentration [CH4]−(from 0 up to the peak) over 200 years is ap- plied. 3. Linear CH4: slow increase in methane supply from per- mafrost thaw and/or hydrate destabilization. A linear in- crease in the bottom boundary methane concentration [CH4]−(from 0 up to the peak) over 200 years is ap- plied. 2.3.2 Transient sensitivity study 4. Sudden CH4: abrupt increase in methane supply from permafrost thaw and/or hydrate destabilization. An in- stantaneous change in bottom boundary methane con- 4. Sudden CH4: abrupt increase in methane supply from permafrost thaw and/or hydrate destabilization. An in- stantaneous change in bottom boundary methane con- Dale et al. (2008c) showed that temporally varying envi- ronmental conditions may reduce the efficiency of the ben- 3.1 Case study: sediment core on the Laptev Sea shelf Figure 1 compares simulated and observed depth profiles for site 14-3. Cores were retrieved during the SWERUS-C3 cam- paign (Miller et al., 2017; Brüchert et al., 2018; Brüchert, 2020). Simulation results show an overall good agreement with measurements, but also reveal a slight overestimation of NH+ 4 . Data–model fitting reveals that reconciling simu- lated and observed CH4 and SO2− 4 depth profiles requires a diffusive flux of CH4 through the lower model boundary (i.e. a bottom boundary concentration of [CH4]−= 16 mM). Nei- ther higher marine OM contents in sediment layers below the first 22 cm for which observations are available, nor higher reactivities results, can satisfactorily reproduce the observed sulfate depletion and observed gradients. Model–data fitting thus not only highlights the important role of AOM in con- trolling the SMTZ, but also indicates that upward migrating methane from deep, pre-Holocene sources, such as subsea permafrost in the sediment might be an ubiquitous feature on the Siberian shelf. 3 However, results also show that, under a specific set of environmental conditions that lower the efficiency of the AOM biofilter (see detailed discussion below), non-turbulent CH4 escape from ESAS sediments can reach values of up to 27 µmol CH4 cm−2 yr−1. Simulation analysis shows that these high effluxes and, thus, low AOM biofilter efficien- cies are generally obtained for environmental conditions that cause a shallow location of the SMTZ (< 18 cm) and that they are very sensitive to changes in environmental condi- tions that would cause a deepening of the SMTZ. For in- stance, a deepening of the SMTZ from 18 to 26 cm re- sults in a rapid increase in AOM efficiency from 1 % to 98 % (Fig. 2a). Furthermore, results indicate that, for SMTZ depths larger than 26 cm, AOM remains an efficient bar- rier across the full spectrum of investigated environmental conditions (Fig. 2). The observed link between AOM fil- ter efficiency and SMTZ is reflected in the strong (semilog) linear relationship between methane flux at the SWI and the SMTZ depth (Fig. 2b). 3.2 Main physical and biogeochemical controls on potential non-turbulent methane flux from ESAS sediments 2. the depth of the SMTZ; 3. the efficiency (η) of the AOM biofilter (see Appendix A for the exact definition of AOM applied here). 2.3.3 Analysed output For each simulation we evaluate the effect of the respective parameter change on 1. the non-turbulent (i.e. not-ebullition-driven) flux of methane from the sediments into the water column; M. Puglini et al.: Assessing the potential for non-turbulent methane escape centration – from 0 to one of the peak value [CH4]−– is applied. is observed for NH+ 4 . Observed NH+ 4 concentrations first in- crease to a maximum at about 6 cm depth and the slightly de- crease in the lower sediment layers, whereas simulated NH+ 4 show an asymptotic increase in NH+ 4 concentrations. The ob- served NH+ 4 profile might either indicate changes in OM re- activity and/or characteristics or spatially heterogeneous ad- sorption/desorption dynamics. Such heterogeneity is not in- corporated into the model, and accounting for it would re- quire additional information. 3.2.1 General patterns of methane and sulfate cycling on the ESAS In addition, fluxes of SO2− 4 and CH4 at the SMTZ, the maximum and integrated AOM rate and the Damköhler num- ber (Da) for AOM and methanogenesis are also calculated. The Damköhler number is defined as Eq. (B4) (see Ap- pendix B) and sets the ratio between the typical transport timescale and the typical reaction timescale. If Da < 1, the reaction timescale is longer than transport timescale (i.e. the reaction is slower) and the process is reaction-limited. If Da > 1 the process is transport-limited. Finally, for transient simulations, the integrated AOM-performing biomass (6B) was also analysed. The comprehensive ensemble of all sensitivity experiments allows exploration of the general patterns of methane and sul- fate cycling under a range of environmental conditions that is broadly representative for conditions encountered on the ESAS at present (Fig. 2). Model results confirm that AOM is an efficient sink for the diffusive CH4 supply from be- low. For most of the investigated environmental conditions (95 % of the runs), 95 %–99.9 % of the upward diffusing CH4 is consumed within the SMTZ, resulting in very small or negligible methane effluxes (≤10−2 µmol CH4 cm−2 yr−1) from the sediment. If upscaled to the total area of the ESAS (∼1.485 × 106 km2, Wegener et al., 2015), for which methane outgassing estimates have been published, the smallest simulated non-turbulent methane flux (i.e. 1.4 × 10−13 µmol cm−2 yr−1, Fig. 2b) would sum up to a total flux of 2.1 mmol CH4 yr−1, resulting in a negligible role of non- turbulent, benthic methane fluxes to the Arctic methane bud- get. https://doi.org/10.5194/bg-17-3247-2020 https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 3254 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3255 Figure 1. Pore water concentration profiles for O2, SO2− 4 , CH4, NH+ 4 , PO3− 4 and DIC at site 14-3 on the Laptev Sea (76.171◦N, 129.333◦E, 56 m water depth). Dots represents the measurements and continuous lines the simulated results. The boundary conditions and model pa- rameters employed in the model are reported in Table S4, the measured organic carbon content in Table S5. For O2 no measured profile is available. Figure 1. Pore water concentration profiles for O2, SO2− 4 , CH4, NH+ 4 , PO3− 4 and DIC at site 14-3 on the Laptev Sea (76.171◦N, 129.333◦E, 56 m water depth). Dots represents the measurements and continuous lines the simulated results. The boundary conditions and model pa- rameters employed in the model are reported in Table S4, the measured organic carbon content in Table S5. For O2 no measured profile is available. rors the empirically found linear log–log relationship be- tween measured CH4 fluxes at the SMTZ and the SMTZ depths (Fig. S5 in the Supplement) by Egger et al. (2018). Maximum simulated CH4 effluxes are thus comparable in magnitude to fluxes reported from other settings potentially sensible for CH4 emissions. These include mud volcanoes, e.g. in the Gulf of Cadiz: 2.1–40.7 µmol CH4 cm−2 yr−1 (Niemann et al., 2006a); Håkon Mosby mud volcano in the Barents Sea: 0.03 µmol CH4 cm−2 yr−1 (Niemann et al., 2006b) and coastal settings, e.g. a Dutch coastal reser- voir (20–80 µmol CH4 cm−2 yr−1, Egger et al. (2016)) or tidal flats (4–800 µmol CH4 cm−2 yr−1 Borges and Abril, 2011). Upscaling the highest simulated non-turbulent flux (27.48 µmol CH4 cm−2 yr−1) to the ESAS results in a to- tal efflux of 0.408 Tmol CH4 yr−1 = 6.52 Tg CH4 yr−1. This value represents an estimated upper limit which, for com- parison, equals ∼10 % of global marine seepage at seabed level (Saunois et al., 2016) and is in magnitude similar to the global methane efflux that has been estimated for up- per continental slope sediments on a centennial timescale (4.73 Tg CH4 yr−1, Kretschmer et al., 2015). consistent with the fact that methanogenesis rates are merely supported by the slow influx and transport of OM by burial and bioturbation. In contrast, high DaAOM values (DaAOM =32–2.78×105 – Fig. M. Puglini et al.: Assessing the potential for non-turbulent methane escape S2, orange circles), show that AOM is transport-limited, suggesting a sensitive role of transport parameters in de- termining AOM efficiency and in controlling methane flux across the SMTZ and subsequently the SWI. 3.2.2 Environmental controls and mechanisms of methane escape from ESAS sediments The simulated general patterns of methane and sulfate cy- cling on the ESAS broadly corroborate previous findings re- garding the dominant environmental controls on AOM biofil- ter efficiency and SMTZ depth (Regnier et al., 2011; Eg- ger et al., 2018; Meister et al., 2013; Winkel et al., 2018). However, they also challenge intuitive views on the factors that favour high CH4 escape through the SWI. In particu- lar, they highlight the essential link between AOM efficiency and SMTZ depth and the central importance of environmen- tal conditions that control the depth of the SMTZ. In addition, they suggest that transport processes play a dominant role for non-turbulent methane effluxes from ESAS sediments. The following sections explore the role of each of the investigated environmental conditions on methane efflux in more detail and also shed light on the mechanisms behind non-turbulent methane escape from ESAS sediments. Further insights into the general drivers that control methane dynamics in ESAS sediments are provided by Damköhler numbers. Damköhler numbers for simulated methanogenesis (DaMG) and AOM (DaAOM) are reported in Fig. S2. DaMG (purple circles) are < 1, span a range of ∼0.0021–0.43 and are thus comparable to previously re- ported DaMG of 0.22 for methane gas hydrate bearing sites, such as the Hydrate Ridge and Kithley Canyon (Chatter- jee et al., 2011). They reveal that methanogenesis is always slower than methane transport and that CH4 dynamics driven by methanogenesis are thus reaction-limited. This result is 3.1 Case study: sediment core on the Laptev Sea shelf Such a relationship reveals the pivotal connections between these two quantities and mir- The simulated PO3− 4 and DIC profiles are in good agree- ment with data, suggesting that the degradation dynamics of marine organic matter and adsorption are well captured by the model – although the maximum concentration of PO3− 4 at depth is mostly controlled by the saturation value of [PO3− 4 ]. The largest discrepancy between data and modelling results Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape Biogeosciences, 17, 3247–3275, 2020 3.2.3 Role of advective transport 3c) from 5.5 × 10−15 µmol CH4 cm−2 yr−1 for low sedimentation rates (ω = 0.03 cm yr−1) to values as high as 27.5 µmolCH4 cm−2 yr−1 for high sedimentation rates (ω = 1.5 cm yr−1). Accordingly AOM acts as an efficient filter for upward diffusing methane (with η ∼100 %, see Fig. S4), in slowly accumulating sedi- ments. ments. In contrast, the efficiency of the AOM biofilter drops to 50 %–0 % for high sedimentation rates. The main driver be- hind the simulated high CH4 fluxes and low AOM efficien- cies in these rapidly accumulating sediments, are enhanced methanogenesis rates. High sedimentation rates not only fa- cilitate the supply of organic matter to the methanogenic zone of the sediment, but also reduce residence times in the upper sediment layer, resulting in a lower OM age (see Eqs. S14 and S16 in the Supplement) or degradation state (see Eq. S12) within the methanogenic zone. The enhanced supply of reactive OM to anoxic sediment layers supports higher methanogenesis rates, resulting in higher methane porewater concentrations and an upward shift of the SMTZ. In addition, the presence of active fluid flow further en- hances methane efflux. The CH4 fluxes from below adds complexity to the overall methane dynamics and this effect is investigated further by contrasting Damköhler numbers for passive and active settings on the shelf. Table S8 shows that for low to intermediate sedimentation rates, DaAOM val- ues significantly decrease with vup, indicating that less and less methane consumption occurs within the typical transport timescale τT, thus leading to a reduction in AOM biofilter ef- ficiency. In contrast, the efficiency of the AOM biofilter drops to 50 %–0 % for high sedimentation rates. The main driver be- hind the simulated high CH4 fluxes and low AOM efficien- cies in these rapidly accumulating sediments, are enhanced methanogenesis rates. High sedimentation rates not only fa- cilitate the supply of organic matter to the methanogenic zone of the sediment, but also reduce residence times in the upper sediment layer, resulting in a lower OM age (see Eqs. S14 and S16 in the Supplement) or degradation state (see Eq. S12) within the methanogenic zone. The enhanced supply of reactive OM to anoxic sediment layers supports higher methanogenesis rates, resulting in higher methane porewater concentrations and an upward shift of the SMTZ. In addition, the presence of active fluid flow further en- hances methane efflux. 3.2.3 Role of advective transport Figure 3a illustrates the effects of sedimentation rate ω on the flux of methane across the SWI. For both ac- https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 3256 M. Puglini et al.: Assessing the potential for non-turbulent methane escape Figure 2. Aggregation of all the simulation performed for the “one-at-time” sensitivity study. (a) AOM efficiency versus the depth of the SMTZ. (b) Scatter plot and semi-log fit of the methane flux (JCH4) at the SWI versus SMTZ depth. M. Puglini et al.: Assessing the potential for non-turbulent methane escape M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3256 Figure 2. Aggregation of all the simulation performed for the “one-at-time” sensitivity study. (a) AOM efficiency versus the depth of the SMTZ. (b) Scatter plot and semi-log fit of the methane flux (JCH4) at the SWI versus SMTZ depth. Maximum simulated flux differences between active and passive settings can reach up to 10 orders of magnitude. However, flux differences quickly decrease with increasing sedimentation rates. Rapidly accumulating sediments show almost no difference in efflux between active and passive sites (Fig. 3a). In contrast to sedimentation rates, the mecha- nism behind the control of vup on non-turbulent methane ef- flux is straightforward and self-evident. Active flow enhances the upward transport of CH4, shifting the SMTZ upwards and, thus, increasing CH4 concentrations at shallow sediment depths (see Fig. 3d). The apparent paradox of the CH4 efflux insensitive to fluid flow in fast accumulating sediments can be resolved by examining the dissolved CH4 depth profiles (Fig. 4). Simulated depth profiles are nearly identical and reveal CH4 concentrations at or near the saturation concen- tration. In fast accumulating sediments, high methanogene- sis rates result in an over-saturation of porewaters directly below the generally shallow SMTZ. High methanogenesis rates thus support the build-up of methane gas. Methane gas formation also explains why, in these cases, integrated methanogenesis exceeds non-turbulent CH4 fluxes by up to 6 times. In rapidly accumulating, active and passive sedi- ments, non-turbulent CH4 fluxes are thus essentially iden- tical. However, active settings will be characterized by the additional build-up of gaseous CH4 and its potential escape through the sediment–water interface – a process not simu- lated in the present study. tive (vup = 1 cm yr−1) and passive (vup = 0 cm yr−1) set- tings, simulated CH4 effluxes increase as a power with sed- imentation rate (log–log linear; see Fig. 3.2.3 Role of advective transport The CH4 fluxes from below adds complexity to the overall methane dynamics and this effect is investigated further by contrasting Damköhler numbers for passive and active settings on the shelf. Table S8 shows that for low to intermediate sedimentation rates, DaAOM val- ues significantly decrease with vup, indicating that less and less methane consumption occurs within the typical transport timescale τT, thus leading to a reduction in AOM biofilter ef- ficiency. Model results thus show that the dominant mechanism be- hind the observed transport-control on non-turbulent CH4 efflux is an overall increase in CH4 concentration and an upward shift of the SMTZ rather than an increasing rela- tive contribution of advective transport processes to the to- tal efflux. In fact, a comparison of the different methane Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3257 gure 3. (a) Barplot of the methane flux at the SWI versus ω for passive case (plain style) and active case (pattern style) and the [CH4]− ported in the text. The squared value of ω is the reference value. (b) Semilog plot of methane flux at SWI versus vup for the different CH4]−reported in the text. (c) Log–log plot of methane efflux at SWI versus ω for passive case (diamonds) and active case (circle). The g–log fit is also displayed. (d) Log–log plot of SMTZ depth versus ω for passive case (diamonds) and active case (circle) with log–log fit. he red line is the trend found by Egger et al. (2018) (the term log(100) is to take into account unit conversion). Figure 3. (a) Barplot of the methane flux at the SWI versus ω for passive case (plain style) and active case (pattern style) and the [CH4]− reported in the text. The squared value of ω is the reference value. (b) Semilog plot of methane flux at SWI versus vup for the different [CH4]−reported in the text. (c) Log–log plot of methane efflux at SWI versus ω for passive case (diamonds) and active case (circle). The log–log fit is also displayed. (d) Log–log plot of SMTZ depth versus ω for passive case (diamonds) and active case (circle) with log–log fit. The red line is the trend found by Egger et al. (2018) (the term log(100) is to take into account unit conversion). Figure 4. Porewater profiles in the case of ω = 1.5 cm yr−1 for CH4 (a), SO2− 4 (b) and gaseous CH4 (c). Dashed lines are simulation in passive scenario with [CH4]−= 0 mM, while continuous lines simulations display active scenario with [CH4]−= 5.455 mM, corresponding to the saturation concentration in the environmental conditions considered for the representative profile. Figure 4. Porewater profiles in the case of ω = 1.5 cm yr−1 for CH4 (a), SO2− 4 (b) and gaseous CH4 (c). Dashed lines are simulation in passive scenario with [CH4]−= 0 mM, while continuous lines simulations display active scenario with [CH4]−= 5.455 mM, corresponding to the saturation concentration in the environmental conditions considered for the representative profile. M. Puglini et al.: Assessing the potential for non-turbulent methane escape M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3258 transport processes across the SWI (i.e. molecular diffu- sion, bioturbation-induced diffusion, bioirrigation and advec- tive transport, as explained in Sect. S1 in the Supplement) shows that the relative contribution of both the advection and molecular diffusion flux to the total flux is small and further decreases with increasing vup (Fig. S3). High non- turbulent methane effluxes in rapidly accumulating and/or active settings are thus largely driven by the non-local irri- gation flux (see Sect. 3.2.5 for more details on the role of irrigation). With increasing ω or vup, the SMTZ shifts up- wards, resulting in higher methane concentrations at shallow sediment depths and thereby reinforcing the relative contri- bution of non-local transport for CH4 fluxes, as well as low- ering the efficiency of the AOM barrier from η ∼100 % to η ∼78 %. The important role of the SMTZ location as a key control on CH4 efflux is further confirmed by the ob- served power-law relationship between the location of the SMTZ and ω (Fig. 3d). This result is qualitatively in agree- ment with the global compilation of empirical data by Eg- ger et al. (2018), which reveals the same log–log decreasing trend between SMTZ and sedimentation rate. Our results are also consistent with observations from brackish sediments that show that sedimentation rates > 10 cm yr−1 give rise to high non-turbulent CH4 fluxes (20–80 µmol CH4 cm−2 yr−1) and a high OM burial efficiency (∼78 %, Egger et al., 2016). Egger and co-workers explained these findings by the slow growth of AOM micro-organisms and the resulting inability of the microbial community to consume all of the CH4 pro- duced. However, our results show that the same pattern can be observed without having to invoke a low AOM efficiency. Our simulations thus indicate that the rapid burial of reactive organic matter to deeper sediment layers in rapidly accumu- lating sediments is sufficient to explain high CH4 effluxes. organic matter quality is high (a < 10 years), methanogene- sis becomes substrate limited due to the rapid degradation of organic matter through energetically more favourable degra- dation pathways in the shallow sediments. In turn, if organic matter quality is low (a > 100 years), methanogenesis be- comes reactivity limited. And this is especially true for ESAS sediments at depth. 3.2.5 Role of non-local transport The analysis of the influence of bioirrigation on non- turbulent CH4 efflux from the ESAS (Fig. S12) shows that such a process enhances methane efflux in sediments that are characterized by a shallow SMTZ. However, bioirriga- tion exerts a limited effect under a range of environmental conditions that favour a deep or shallow SMTZ location, re- spectively. In passive settings, changes in bioirrigation coefficient (α0) exert a limited control on CH4 effluxes. For most pas- sive scenarios, the SMTZ is located well below the sediment layer affected by bioirrigation (zirr = 3.5 cm, which implies that bioirrigation is strongly suppressed below 15 cm) and, thus, changes in α0 have no effect on methane efflux. M. Puglini et al.: Assessing the potential for non-turbulent methane escape Therefore, the ideal combinations of or- ganic matter reactivity and sedimentation rate that result in maximum methane effluxes correspond to conditions char- acterized by OM that is (i) sufficiently reactive to support enhanced methanogenesis rates and thus an accumulation of CH4 at depth, but (ii) sufficiently unreactive (in comparison to the burial rate) to escape the complete degradation in non- methanogenic sediments. BRNS outcomes show that the on- set of active fluid flow and an enhanced methane supply from below (i.e. higher CH4 concentration at the lower boundary) increase the CH4 efflux through the SWI without altering the overall patterns (see Fig. 5a–b versus 5c–d). https://doi.org/10.5194/bg-17-3247-2020 https://doi.org/10.5194/bg-17-3247-2020 https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape ini et al.: Assessing the potential for non-turbulent methane escape 3259 Figure 5. Flux of methane at the SWI as dependent on a and ω. For [CH4]−= 0 mM (a, c) and [CH4]−= 5.455 mM (b, d), and passive (a, b) and active (c, d) case. The circle with pattern corresponds to the baseline simulation. Figure 5. Flux of methane at the SWI as dependent on a and ω. For [CH4]−= 0 mM (a, c) and [CH4]−= 5.455 mM (b, d), and passive (a, b) and active (c, d) case. The circle with pattern corresponds to the baseline simulation. Figure 5. Flux of methane at the SWI as dependent on a and ω. For [CH4]−= 0 mM (a, c) and [CH4]−= 5.455 mM (b, d), and passive (a, b) and active (c, d) case. The circle with pattern corresponds to the baseline simulation. kAOM, on non-turbulent methane effluxes. However, mod- elling reveals that kAOM only plays a minor role for non- turbulent methane fluxes across the SWI (see Figs. S14, S15). An increase in kAOM can reduce methane effluxes from pas- sive shelf sediments by up to 5 orders of magnitude. Still, its effect remains small compared, for instance, to the re- sponse to variations in sedimentation rate, which can change methane efflux by up to 14 orders of magnitude. The most important effect of increasing kAOM is the increasing linear- ity of the [CH4] and [SO2− 4 ] profiles around the SMTZ and the concurrent narrowing and downward movement of the SMTZ, which can result in a reduction in methane efflux. Simulations thus show that the AOM biofilter and, as a conse- quence, non-turbulent methane effluxes from sediments, are not affected by the exact value of the kinetic rate constant, at least in the range we analysed. This is in disagreement with results by Dale et al. (2008c), which show that, in dy- namic settings subject to large methane fluxes, an increase of 3 orders of magnitude in kAOM (from 102 to 105 M−1 yr−1) leads to a reduction in steady-state methane fluxes below 10−2 µmol CH4 cm−2 yr−1. However, this discrepancy might sites despite the differences in dominant transport mech- anism (diffusion when α0 = 0 yr−1; irrigation when α0 ̸= 0 yr−1). Under these conditions (i.e. high vup and/or high ω), the SMTZ is located close to the SWI. M. Puglini et al.: Assessing the potential for non-turbulent methane escape In such settings, non-local transport becomes the dominant transport process in bioirrigated sediments (see Sect. 3.2.3 and Fig. S3) be- cause it weakens concentration gradients near the SWI and, thus, contributes to a substantial reduction in the gradient- driven, diffusive transport terms. As a consequence, simu- lated CH4 efflux at the SWI are broadly similar for all of the investigated α0 ̸= 0 yr−1 (Fig. 6b, c, d). It is worth notic- ing that, independently on the α0, CH4 efflux for ω = 0.03 and vup = 10 cm yr−1 is ∼1 µmol CH4 cm−2 yr−1 – a value almost identical to the one reported in Luff and Wallmann (2003) – 1.4 µmol CH4 cm−2 yr−1 – for sediments character- ized by vup = 10 and ω = 0.0275 cm yr−1. 3.2.4 Role of organic matter quality In contrast to passive settings, active settings reveal a rapid increase in methane efflux with the onset of bioirriga- tion activity. Methane effluxes first increase by up to 5 or- ders of magnitude from α0 = 0 yr−1 to α0 = 5 yr−1, reach- ing maximum effluxes of ∼0.02 µmol CH4 cm−2 yr−1, be- fore remaining almost constant with a further increase in bioirrigation coefficients (up to 240 yr−1). The simulated in- crease in methane efflux is a direct effect of the transport pro- cess itself, which enhances the upward transport of methane accumulating in the upper sediment layers, including layers below the generally shallow SMTZ. The quality of organic matter deposited onto the sediment ex- erts an additional control on CH4 efflux. Figure 5 illustrates the influence of organic matter quality (as a function of OM degradation model parameter a; see Eq. S12) and sedimen- tation rate ω on non-turbulent methane efflux for both active and passive settings, as well as different methane fluxes from below. Results corroborate the dominant influence of sedi- mentation rates on methane efflux, while organic matter qual- ity exerts a secondary control. This also means that, in order to assess the main features of possible CH4 efflux in terms of modelling, capturing the details of organic matter qual- ity is not fundamental. Maximum fluxes are generally sim- ulated for rapidly accumulating sediments ω > 0.5 cm yr−1 that receive organic matter of intermediate quality (a = 10– 100 years). These results are corroborated by the concomitant analysis of CH4 dynamics over the three-dimensional transport coef- ficient ω, vup and α0 space shown in Fig. 6. A comparison between simulations with α0 = 0 yr−1 and α0 ̸= 0 yr−1 (α0 = 5, α0 = 10 and α0 = 33 yr−1) shows that irrigation increases the CH4 efflux at low to intermediate sedimentation rates and/or high vup (lower-left corner of the phase space in both plots). However, maximum methane ef- fluxes that are simulated for high sedimentation rates or vup are almost identical between bioirrigated and non-irrigated These findings are in agreement with previously published studies (Regnier et al., 2011; Meister et al., 2013) and can be explained with the fact that high methanogenesis rates re- quire a supply of reactive OM to the methanogenic zone. If Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 3.2.6 AOM rate constant Given its crucial role in AOM biogeochemistry, one would expect a pronounced influence of the kinetic rates constant, 3.2.7 Summary of steady-state experiments be ascribable to the high water flow velocity employed in their simulation (vup = 10 cm yr−1), 10 times higher than the one we considered in our active simulations. Finally, on the shallow ESAS, dissolved methane concentrations are limited by the comparably low gas saturation concentration, result- ing in a minor influence of kAOM on methane fluxes (as the AOM rate is proportional to the CH4 concentration). An in- direct support to our findings regarding the secondary role of kAOM on the AOM itself comes from Luff and Wallmann (2003). They showed that, as long as it is not null, the ac- tual value of kAOM is unimportant for the precipitation of authigenic carbonate. Since the authigenic carbonate precip- itation is largely driven by alkalinity produced during AOM (e.g. Aloisi et al., 2004; Luff et al., 2005; Karaca et al., 2010; Pierre et al., 2012; Crémière et al., 2016b, a; Meister et al., 2018), the observed independence of precipitation rates from kAOM bolsters our conclusion. be ascribable to the high water flow velocity employed in their simulation (vup = 10 cm yr−1), 10 times higher than the one we considered in our active simulations. Finally, on the shallow ESAS, dissolved methane concentrations are limited by the comparably low gas saturation concentration, result- ing in a minor influence of kAOM on methane fluxes (as the AOM rate is proportional to the CH4 concentration). An in- direct support to our findings regarding the secondary role of kAOM on the AOM itself comes from Luff and Wallmann (2003). They showed that, as long as it is not null, the ac- tual value of kAOM is unimportant for the precipitation of authigenic carbonate. Since the authigenic carbonate precip- itation is largely driven by alkalinity produced during AOM (e.g. Aloisi et al., 2004; Luff et al., 2005; Karaca et al., 2010; Pierre et al., 2012; Crémière et al., 2016b, a; Meister et al., 2018), the observed independence of precipitation rates from kAOM bolsters our conclusion. The results of the steady-state sensitivity study indicate that, under environmental conditions that are broadly rep- resentative for the ESAS, low AOM efficiencies and thus high non-turbulent CH4 effluxes from thawing subsea per- mafrost and/or dissociating methane gas hydrates (larger than 4 µmol CH4 cm−2 yr−1) are promoted by intense advec- tive transport (sedimentation rate ω > 1 cm yr−1, active fluid flow vup > 7 cm yr−1). https://doi.org/10.5194/bg-17-3247-2020 https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape of methane at the SWI as dependent on vup and ω for α0 = 0 (a), α0 = 5 (b), α0 = 10 (c) and α0 = 33 yr−1 (d). Circles ions outcomes. Results for α0 ̸= 0 yr−1 are almost the same. The lower boundary condition for methane is [CH4]−= ee-dimensional version of the plot is reported in Fig. S13. M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3260 Figure 6. Efflux of methane at the SWI as dependent on vup and ω for α0 = 0 (a), α0 = 5 (b), α0 = 10 (c) and α0 = 33 yr−1 (d). Circles represent simulations outcomes. Results for α0 ̸= 0 yr−1 are almost the same. The lower boundary condition for methane is [CH4]−= 1.169 mM. A three-dimensional version of the plot is reported in Fig. S13. 3.2.8 Geographic pattern and potential for non-turbulent methane emissions from Laptev Sea sediments Model results indicate that sedimentation rate exerts the dominant control on benthic escape of methane from thaw- ing subsea permafrost and/or dissociating methane gas hy- drates on the ESAS. The functional relationship between sed- imentation rate and methane flux across the SWI reported in Fig. 3c thus allows estimation of a potential non-turbulent, benthic methane efflux derived from deep sources for a given sedimentation rate. Thus, if the spatial distributions of these environmental controls on methane efflux are known, a first- order geographical distribution of potential non-turbulent methane escape from the Siberian shelf can be derived. How- ever, the availability of observational data from the Siberian shelf is extremely scarce. Therefore, we here focus on the Laptev Sea – a comparable well-studied part of the Siberian shelf. The Laptev Sea is well known for its subsea permafrost and gas hydrate content and subject to large riverine inputs from the Lena River. To derive a map of sedimentation rates for Laptev Sea shelf sediments, we use published linear sedi- mentation rates (Table S9) and extrapolate these values to the entire region by applying a simple three-dimensional krig- ing method (see Fig. 7a), using the International Bathymetric Chart of the Arctic Ocean (IBCAO) (Jakobsson et al., 2012) and employing longitude, latitude and water depth as predic- tors for ω. Higher advective fluid flow velocities, intermediate or- ganic matter reactivity and/or a more intense macrobenthic biological activity could increase these estimates of non- turbulent methane escape from the Laptev Sea shelf. Higher advective fluid flow velocities (i.e. vup > 1 cm yr−1), pos- sibly in connection with active seepages, groundwater dis- charges and fault lines (the latter follow parallel pattern in Laptev Sea (Drachev et al., 1998) on the direction SW–NE from the west of Lena delta up to the little Lyakhovsky and Kotelny islands), could result in methane effluxes of up to 10–101.3 µmol CH4 cm−2 yr−1 (see Figs. 5 and 6). However, such high fluid flow velocities would be only found locally and would thus merely give rise to a number of methane emission hotspots that would not change the overall non- turbulent methane flux budget. 3.2.8 Geographic pattern and potential for non-turbulent methane emissions from Laptev Sea sediments Table 2. Estimated flux of CH4 at SWI in mol yr−1 for different depth regions of Laptev Sea in a passive (vup = 0 cm yr−1) and ac- tive (vup = 1 cm yr−1) case. vup Region (water depth, area) 0 1 0–10 m, 7.7 × 104 km2 6.5 8.9 × 105 10–80 m, 4.5 × 105 km2 296.2 8.5 × 106 One strength of models is that they can provide the explo- rative means to assess dynamics at spatial/temporal scales that cannot easily be assessed by observations alone. In par- ticular, transfer functions, simple look-up tables and neural networks that are derived from, or trained on, a large ensem- ble of individual model simulations over a broad range of plausible boundary conditions have been frequently and suc- cessfully used to investigate regional and even global dynam- ics (Gypens et al., 2008; Marquardt et al., 2010; Dale et al., 2015, 2017; Capet et al., 2016; Bowles et al., 2014). Such a quantitative framework for first-order estimates of poten- tial non-turbulent methane escape from ESAS sediments can also be derived from the results of the sensitivity study we ran. tribution of sedimentation rates with values around 0.03– 0.06 cm yr−1. Estimated non-turbulent methane effluxes corresponding to the highest measured sedimentation rates close to the Lena mouth do not exceed 1.57×10−1 µmol CH4 cm−2 yr−1 assuming the presence of active fluid flow and 2.25 × 10−5 µmol CH4 cm−2 yr−1 for passive settings. These find- ings are not surprising as steady-state sensitivity results in- dicate that high CH4 efflux requires sedimentation rates of ω > 1 cm yr−1. The regional non-turbulent CH4 efflux bud- get for different depth sections of the Laptev Sea assuming the absence of active fluid flow in Laptev Sea shelf sediments (see Table 2) thus indicates that non-turbulent CH4 efflux is negligible. Even if we assume the omnipresence of an active fluid flow of vup = 1 cm yr−1, the estimated non-turbulent methane efflux merely sums up to 9.39 × 106 mol CH4 yr−1 (∼0.1 Gg CH4 yr−1) over the entire Laptev Sea area of 527.4 × 103 km2. Such small effluxes would most likely be subject to further oxidation in the water column, thus limiting any potential impact on atmospheric methane concentrations and climate. 3.2.7 Summary of steady-state experiments Under these conditions, CH4 efflux can be further enhanced by moderate OM reactivity (a = 10– 102 years) and intense non-local transport processes, such as bioirrigation (irrigation constant α0 > 0 yr−1). Overall, non- turbulent benthic escape of CH4 from deep sources appears to be mainly controlled by the concurrent effects of ω, vup and α0. In contrast, maximum AOM rates, kAOM, exert no influence on the AOM filter efficiency. Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 3261 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3262 Figure 7. (a) Values of the sedimentation rate extrapolated for the whole Laptev Sea via a simple kriging method. The reference values (circles) are the ones reported in Table S9. (b, c) (Log) values of the potential non-turbulent dissolved methane emissions at the SWI considering the relationship presented in Fig. 3c for passive (b) and active (c) cases. Figure 7. (a) Values of the sedimentation rate extrapolated for the whole Laptev Sea via a simple kriging method. The reference values (circles) are the ones reported in Table S9. (b, c) (Log) values of the potential non-turbulent dissolved methane emissions at the SWI considering the relationship presented in Fig. 3c for passive (b) and active (c) cases. Figure 7. (a) Values of the sedimentation rate extrapolated for the whole Laptev Sea via a simple kriging method. The reference values (circles) are the ones reported in Table S9. (b, c) (Log) values of the potential non-turbulent dissolved methane emissions at the SWI considering the relationship presented in Fig. 3c for passive (b) and active (c) cases. estimated methane outgassing fluxes of few teragrams of CH4 yr−1 (Berchet et al., 2016; Thornton et al., 2020) or even tens of teragrams of CH4 yr−1 (Shakhova et al., 2014). If such outgassing were to be supported by methane efflux from Laptev Sea sediments, it would require the build-up of CH4 gas reservoirs in Laptev Sea sediments of at least similar or larger size than the evaded amount, as well as the preferential and rapid transport of this CH4 gas to the atmosphere. Nev- ertheless, model results also suggest that projected trends of terrestrial permafrost thawing and coastal permafrost degra- dation (Vonk et al., 2012) might increase the importance of non-turbulent methane escape for the Arctic’s methane budget by potentially increasing sedimentation rates through coastal erosion (vast amount of debris and terrigenous ma- terial) and increased riverine inputs (Guo et al., 2007); ac- tive fluid flow through permafrost and methane gas hydrate degradation (James et al., 2016; Ruppel and Kessler, 2017); organic matter reactivity through an enhanced delivery of more reactive permafrost organic matter (Wild et al., 2019) and/or an enhanced macrobenthic activity through warming only change efflux by less than an order of magnitude as- suming both a = 100 years or a = 1 years. 3.2.8 Geographic pattern and potential for non-turbulent methane emissions from Laptev Sea sediments In addition, intermediate or- ganic matter reactivity, in particular in the fast accumulat- ing sediments close to the coastline and the Lena River delta that receive more reactive organic matter from thawing ter- restrial permafrost (Wild, 2019) could result in a higher esti- mated non-turbulent methane escape. However, our sensitiv- ity study results show that OM reactivity merely plays a sec- ondary role, suggesting that changes in OM reactivity would Observations indicate that sedimentation rates are high- est (ω = 0.45 cm yr−1) close to the mouth of the Lena River and Moustakh Island in the Buor-Khaya Gulf. As a conse- quence, the vicinity of the river mouth, as well as the area along the shallow bathymetric profile towards the NE of the Lena delta are characterized by comparably high sedimen- tation rates (ω = 0.27–0.42 cm yr−1). The relatively shallow areas (∼10 m deep) around the New Siberian islands reveal intermediate values (ω = 0.06–0.12), while minimum sedi- mentation rates (∼0.002–0.03 cm yr−1) roughly follow the 55 m isobath down to the continental slope at 100 m. Deeper shelf areas are characterized by a more homogeneous dis- https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape M. Puglini et al.: Assessing the potential for non-turbulent methane escape influence on non-turbulent methane efflux. Conversely, both microbial growth dynamics and the presence/absence of ac- tive fluid flow (Table 3) largely control the transient response to environmental change. The reasons for this are 2-fold. First, the response time of the resident AOM community is longer than the characteristic timescales under investiga- tion, thus smoothing out the impact of environmental per- turbations. Second, active fluid flow enhances the impact of the perturbation by triggering a significant upward shift of the SMTZ. In particular, the initial movement of the SMTZ prevents the establishment of an efficient AOM community at the SMTZ: this creates a “window of opportunity” for methane escape. In contrast, the comparably slow and lim- ited movements of the SMTZ in passive settings (Fig. S18) enables the establishment of an AOM community that acts as an efficient biofilter for upward-migrating methane. and Atlantification. However, the magnitude of these pro- jected environmental changes and thus their effect on non- turbulent methane escape from ESAS sediments is difficult to assess. and Atlantification. However, the magnitude of these pro- jected environmental changes and thus their effect on non- turbulent methane escape from ESAS sediments is difficult to assess. 3.3.1 Window of opportunity Figure 8 illustrates the temporal evolution of the simulated (a) filter efficiency and AOM rate, (b) CH4 efflux, (c) SMTZ depth and (d) AOM biomass for transient scenario 4 in the case of vup = 1 cm yr−1 and [CH4]−= 1.169 mM. The on- set of a sudden methane flux from thawing permafrost and/or dissociating methane gas hydrates below the sediment col- umn triggers the rapid movement of the SMTZ. Simulation outputs show that the velocity at which the SMTZ moves upward (vSMTZ) is solely controlled by vup, as evident from the constant vSMTZ ∼2.46 cm yr−1 for all the transient sce- narios with vup = 1 cm yr−1 (Fig. S18). The initial upwards movement of the SMTZ delays the microbial response since the transient dynamics inhibits the establishment of a res- ident AOM community sufficiently large to consume up- ward migrating methane. The AOM rate, and thus the fil- ter efficiency, is controlled by the AOM biomass dynamics (Eq. 6), which in turn is determined by the kinetic (FK, Eq. 7) and thermodynamic (FT, Eq. 8) constraints. Figure 10 illus- trates the typical depth profiles of the thermodynamic and kinetic terms in the bioenergetic AOM formulation (Eq. 6), as well as their evolution in response to the onset of a sud- den methane flux from below. Initially, although kinetically possible (i.e. FK ̸= 0), AOM is inhibited by thermodynamic constraints (i.e. FT = 0). For vup = 1 cm yr−1 during the first In contrast, active settings (i.e. vup = 1 cm yr−1) exhibit an initial increase in CH4 fluxes to maxima of 0.55– 0.83 µmol CH4 cm−2 yr−1 over the first 50 years. This growth coincides with a rapid upward shift of the SMTZ by 100 cm. Methane escape then temporarily drops by 17 %– 20 % until years 70–75, when it begins to increase again un- til the end of the simulation. During this second phase (i.e. after the first 50 years), the SMTZ remains stationary. Tem- porally integrated methane efflux (over 200 years) increases with active fluid flow rate from 66–121 µmol CH4 cm−2 for vup = 1 cm yr−1 to ∼0.95−1.154 mmolCH4 cm−2 for vup = 5 cm yr−1. A large fraction of these emissions (30 % and 48 %–87 %, respectively) occurs in the first 100 years after the perturbation. 3.3 Methane efflux dynamics in response to seasonal and long-term environmental variability The steady-state sensitivity results reveal that, under steady- state conditions, AOM represents an efficient biofilter for upward migrating methane from thawing permafrost and/or dissociating methane gas hydrates on the ESAS. However, transient dynamics induced by, for instance, seasonally or climate-change-driven variability in environmental condi- tions, may weaken the efficiency of the AOM biofilter. There- fore, we additionally explore the potential for non-turbulent methane escape from thawing subsea permafrost and/or dis- sociating methane gas hydrate in ESAS sediments under transient conditions. Table 3 summarizes the maximum sim- ulated non-turbulent methane fluxes for two kinds of environ- mental change scenarios: seasonal and long-term. With the former, we explore seasonal changes in deep methane flux and seasonal freshening of bottom waters. With the latter in- stead, we investigate the impacts of a slow linear increase and a sudden maximum increase in deep methane flux (see Sect. 2.3.2). The following sections explore the factors that control the creation of such a window of opportunity and discusses the mechanisms behind the simulated methane escape. Given the overall similar transient response of non-turbulent methane fluxes to different environmental scenarios (Figs. S17, S18), we will base the following discussion on scenario 4, namely a step-like CH4 forcing with vup = 1 cm yr−1 and a specific bottom concentration ([CH4]−= 1.169 mM). The reason for selecting this scenario is simple. In contrast to the other sce- narios, scenario 4 allows for a straightforward definition of the initial and final state, which facilitates the attribution of a typical response timescale for the system. Results reveal that the transient response of simulated non-turbulent methane efflux is similar for all environmen- tal scenarios, but instead significantly differs for passive and active sites. In general, passive settings do not al- low for significant methane escape (Fig. S17). Although transient methane efflux monotonously increases over the simulated period, it only reaches a maximum value of 0.03–0.05 µmol CH4 cm−2 yr−1 (Fig. S17). Similarly, the simulated SMTZ depth merely migrates 11.5–29 cm up- wards (Fig. S18). Over the simulated 200 years, the inte- grated non-turbulent methane escape from passive settings for all environmental change scenarios barely reaches 3– 4 µmol CH4 cm−2. M. Puglini et al.: Assessing the potential for non-turbulent methane escape Changes in bioir- rigation intensity would exert merely a limited effect on ef- flux estimates, as bioirrigation has already been included in the estimate calculations. The absence of bioirrigation, which is known to be patchy in Arctic sediments, could act both in the direction of further reducing (limiting the bioirrigated flux from the sediments) or increasing (by limiting the flux of TEAs from the seawater and therefore oxidation) the esti- mated non-turbulent methane efflux. Additional physical re- working, such as ice scouring or dredging, may also have such an opposite effect: it could reduce the methane efflux (by enhancing the flux of TEAs into the sediments) but it could also intensify it (by removal of the upper sediment layer). Model results thus show that, under present-day, steady- state environmental conditions, AOM acts as an efficient biofilter for potential non-turbulent methane fluxes in Laptev Sea sediments. The estimated non-turbulent methane escape from Laptev Sea shelf sediments cannot support previously https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 3263 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3264 Table 3. Maximum of methane fluxes (in µmol cm−2 yr−1) at SWI for the four analysed transient scenarios. Values in round parenthesis indicate the year after the beginning of simulation corresponding to the reported maximum. Table 3. Maximum of methane fluxes (in µmol cm−2 yr−1) at SWI for the four analysed transient scenarios. Values in round parenthesis indicate the year after the beginning of simulation corresponding to the reported maximum. [1. Seasonal CH4] [2. Seasonal CH4 + SO2− 4 ] vup (cm yr−1) vup (cm yr−1) 0 1 5 0 1 5 CH4 (µM) 20 0.030 (200) 0.550 (50) 12.7 (17.5) 0.059 (200) 0.772 (51) 13.7 (18) 100 0.029 (200) 0.550 (50) 12.7 (17.5) 0.058 (200) 0.753 (51) 13.7 (18) 330 0.030 (200) 0.552 (49.5) 12.8 (18) 0.058 (200) 0.775 (51) 13.8 (18) 1169 0.031 (200) 0.558 (49.5) 12.9 (18) 0.059 (200) 0.783 (51) 14.0 (18) 5455 0.034 (200) 0.577 (49) 14.0 (19) 0.062 (200) 0.832 (50) 15.2 (19) [3. Linear CH4] [4. Sudden CH4] vup (cm yr−1) vup (cm yr−1) 0 1 5 0 1 5 CH4 (µM) 20 0.029 (200) 0.550 (50) 11.7 (20) 0.029 (200) 0.550 (50) 12.7 (18) 100 0.030 (200) 0.550 (50) 11.7 (20) 0.030 (200) 0.552 (50) 12.7 (18) 330 0.030 (200) 0.550 (50) 11.7 (20) 0.031 (200) 0.557 (50) 12.9 (18) 1169 0.032 (200) 0.550 (50) 11.7 (20) 0.033 (200) 0.565 (49.5) 13.4 (18) 5455 0.036 (200) 0.560 (50) 11.8 (20) 0.040 (200) 0.639 (47) 18.8 (23) 23 years, AOM biomass thus remains largely constant (Figs. 8d and S21a) and, as a consequence, AOM rate and filter efficiency are zero. In this period, aerobic methane oxida- tion represents the only barrier to upward diffusing methane. However, this barrier is weak due to the limited availability of oxygen and the competition with aerobic organic matter degradation as well as additional secondary redox reactions that also consume oxygen (see Table S3). As a consequence, CH4 efflux increases. The initial methane efflux is largely supported by in situ methanogenesis since the advective transport of methane (occurring at vup −ω = 0.877 cm yr−1, corresponding to 20.17 cm in 23 years) is too slow to allow methane from below 3 m to reach the sediment–water inter- face. ever, the duration of this initial “window of opportunity” for methane escape decreases with increasing vup. M. Puglini et al.: Assessing the potential for non-turbulent methane escape In general, simulated maximum methane fluxes fall within the range of previous models applied to different environments (Sommer et al., 2006; Dale et al., 2008c) but do not reach the high val- ues measured in other settings (Linke et al., 2005; Regnier et al., 2011). After the initial “window of opportunity” (i.e. 23+30.1 = 53.1 years), the effect of an efficient methane consumption at the SMTZ starts to reduce the non-turbulent methane efflux at the SWI (Fig. 8b). This reduction lasts until the upward movement of the SMTZ slows down. At this point, the AOM filter efficiency reaches a quasi-stationary level of ∼85 % (as Fig. 8a). Meanwhile, in situ methanogenesis continues to produce methane, which is not entirely consumed by the AOM community that already reached its full capacity. As a consequence, methane fluxes at SWI increase again until a new steady state is reached. After the first 23 years, thermodynamic constraints ease and AOM begins to efficiently consume upward migrating methane at the SMTZ by 40 % (Fig. 8a). However, as con- sumption occurs at the SMTZ (for the specific case at a sed- iment depth of 100.4 cm), it does not immediately affect the methane efflux at the SWI. The time required for the con- sumption signal to propagate to the SWI with velocity v = vSMTZ +vup −ω = 3.337 cm yr−1 is therefore 100.4 cm 3.337 cm yr−1 = 30.1 years. Consequently, methane efflux further increases. This methane efflux is now also supported from deep sources such as thawing permafrost and/or dissociating methane gas hydrates, which have started to contribute to methane ef- flux between years 7 and 20 (assuming typical values of vup reported for active marine sediments of 0.5–5 cm yr−1). Methane efflux typically peaks 2–3 decades after the onset of methane supply. Maximum methane efflux increases with vup: from 0.5–0.6 µmol CH4 cm−2 yr−1 for vup = 1 cm yr−1 to 11–19 µmol CH4 cm−2 yr−1 for vup = 5 cm yr−1. How- 3.3.1 Window of opportunity Model results thus indicate that the exact temporal char- acter of environmental changes does not exert an important https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 3.3.2 Final new steady state The final new steady-state value of methane efflux (Fig. 8b, S17 and S19) is generally in good agreement with Dale et al. (2008c), who reported an efflux of the same order of magni- tude (3 µmol CH4 cm−2 yr−1) for the new steady state at the end of a transient run with vup = 10 cm yr−1 and [CH4]−= 70 mM. Simulations with vup = 5, ω = 0.123 cm yr−1 and [CH4]−= 1.169 mM (Fig. S19) offer a better understand- ing of the model. In this case, the final new steady state is about 2 orders of magnitude larger than the efflux of ∼ 0.1 µmol CH4 cm−2 yr−1 simulated in the steady-state simu- https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape M. Puglini et al.: Assessing the potential for non-turbulent Figure 8. Time evolution over 200 years for the case of an ac- tive set-up with vup = 1 cm yr−1 and a step-like methane forcing from below from 0 to [CH4]−= 1.169 mM. (a) AOM vertically integrated rate (blue) and AOM efficiency (red). (b) CH4 flux at SWI. (c) SMTZ depth. (d) Vertically integrated biomass (number of cells). 3265 Figure 10. Vertical profile of FT, FK, FTot = FK ·FT and the AOM (scaled to the maximum) for three instants in time: 8 years (a), 19 years (b) and 200 years (c) of simulation, for the case [CH4]−= 1.169 mM and vup = 5 cm yr−1. Figure 10. Vertical profile of FT, FK, FTot = FK ·FT and the AOM (scaled to the maximum) for three instants in time: 8 years (a), 19 years (b) and 200 years (c) of simulation, for the case [CH4]−= 1.169 mM and vup = 5 cm yr−1. The reason for this discrepancy can be clarified by plotting the apparent kAOM for transient simulations. Such a value is calculated by computing an apparent bimolecular rate con- stant kAOM (as in Eq. 5) from the transient bioenergetic sim- ulations for the new final steady state. Results are shown in Fig. 9. Figure 9a illustrates that the concentration product [CH4] · [SO2− 4 ] is wider than the AOM rate profile (Fig. 9b, blue curve). Figure 9c also shows that the apparent kAOM is not uniform: it reaches a maximum value of 109 M−1 yr−1, but remains well below 100 M−1 yr−1 at most depths. 3.3.2 Final new steady state Com- pared to the values typically applied for bimolecular rate laws (i.e. kAOM = 102–107 M−1 yr−1), these values are rather low and reflect the ongoing thermodynamic limitation of AOM. FT remains the main constraint on AOM throughout the sim- ulation (Fig. 10c). A more uniform sulfide concentration – [HS−] enters in defining FT – in lower sediments combined with the upward movement of the SMTZ pushes the maxi- mum of FT upwards, thus limiting the zone where AOM is thermodynamically favourable (∼13 cm deep). Figure 8. Time evolution over 200 years for the case of an ac- tive set-up with vup = 1 cm yr−1 and a step-like methane forcing from below from 0 to [CH4]−= 1.169 mM. (a) AOM vertically integrated rate (blue) and AOM efficiency (red). (b) CH4 flux at SWI. (c) SMTZ depth. (d) Vertically integrated biomass (number of cells). Figure 9. Vertical profiles at the end of transient simulation (af- ter 200 years) with bioenergetic AOM formulation for the case [CH4]−= 1.169 mM and vup = 5 cm yr−1. (a) Bimolecular prod- uct [CH4]·[SO2− 4 ]. (b) AOM rate according to the bioenergetic for- mulation (blue) and, for comparison, according to bimolecular for- mulation used for the steady-state simulations (red). (c) Apparent kAOM, estimated from Eq. (5). Integrated biomass 6B ranges from ∼1.2×1010 to 3.5× 1011 cells cm−2 (except for simulation with vup = 5 cm yr−1 and [CH4]−= 5.455 mM, whose 6B= 1.2 × 1012). These values are comparable with AOM biomass reported in Treude et al. (2003) (1.5–1.8 × 1010 cells cm−2) or with values sim- ulated in Dale et al. (2008c) (3.7×1011 cells cm−2 for vup = 5 cm yr−1). In addition, the maximum simulated biomass for active settings (0.5–2.5 × 1010 cells cm−3) agrees well with previously reported values, ranging from 0.27 to 7.4 × 1010 cells cm−3 (Dale et al., 2008c). Integrated AOM rates (6AOM) are instead smaller then previously published rates for shallow, active sites above the shelf break (Boetius et al., 2000; Haese et al., 2003; Luff and Wallmann, 2003; Linke et al., 2005; Wallmann et al., 2006b; Dale et al., 2008c), but comparable to those observed in active sites below the shelf break (Aloisi et al., 2004; Wallmann et al., 2006a; Maher et al., 2006) or in passive settings (Borowski et al., 1996; Martens et al., 1998; Fossing et al., 2000; Jørgensen et al., Figure 9. lations, with bimolecular rate law, under identical environ- mental conditions (inferred from Fig. 6). https://doi.org/10.5194/bg-17-3247-2020 4 Conclusions In this study, we evaluate the potential for non-turbulent, ben- thic methane escape from thawing subsea permafrost and/or dissociating methane gas hydrates in both passive as well as active settings and under a range of environmental conditions that are broadly representative for conditions encountered on the present and future East Siberian Arctic Shelf (ESAS). We identify the most important biogeochemical and phys- ical controls on non-turbulent methane escape from those sediments under steady-state conditions, as well as in re- sponse to environmental variability on seasonal and centen- nial timescales. Based on model results, we derive a simple transfer function that allows establishment of a first-order re- gional estimate of (not-turbulent) methane efflux and of po- tential methane consumption in Laptev Sea sediments. AOM generally acts as an efficient biofilter for upward mi- grating CH4 under environmental conditions that are repre- sentative for the present-day ESAS with potentially impor- tant yet unquantified implications for the Arctic ocean’s alka- linity budget and, thus, CO2 fluxes. Our results thus suggest that previously published fluxes estimated from ESAS wa- ters to the atmosphere cannot be supported by non-turbulent methane efflux alone. p p Model results reveal that AOM is an efficient sink for up- ward migrating, dissolved methane in ESAS sediments. Sim- ulated non-turbulent methane effluxes are negligible for a broad range of environmental conditions under both steady- state and transient conditions. Since AOM is a transport- limited process, transport parameters exert a dominant con- trol on the efficiency of the AOM biofilter and, ultimately, on the methane efflux at the SWI. Both steady-state and tran- sient model results confirm the key role of advective trans- port (mainly sedimentation and active fluid flow) in sup- porting methane escape from Arctic shelf sediments. Un- der steady-state conditions, high methane effluxes (up to 27.5 µmol cm−2 yr−1) are generally found for sediments that are characterized by high sedimentation rates and/or active fluid flow (sedimentation rate ω > 0.7 cm yr−1, active fluid flow vup > 6 cm yr−1). Under these conditions, methane ef- flux can be further enhanced by intermediate organic matter reactivity (RCM model parameter a = 10–102 years) even though the control exerted by organic matter is only sec- ondary with respect to the transport parameters. Finally in- tense local transport processes, such as bioirrigation (irri- gation constant α0 > 1 yr−1), do also contribute to larger methane effluxes. M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3266 2001; Dale et al., 2008c). The discrepancy may be due to different environmental conditions encountered at these sites. For instance, Dale et al. (2008c) applied an advective veloc- ity of vup = 10 cm yr−1 and [CH4]−= 60 mM. While differ- ences in vup affect the 6AOM, its effect on 6B is negligible since an efficient AOM microbial filter has to account for at least > 1010 cells cm−3 (Lösekann et al., 2007; Knittel and Boetius, 2009). 2001; Dale et al., 2008c). The discrepancy may be due to different environmental conditions encountered at these sites. For instance, Dale et al. (2008c) applied an advective veloc- ity of vup = 10 cm yr−1 and [CH4]−= 60 mM. While differ- ences in vup affect the 6AOM, its effect on 6B is negligible since an efficient AOM microbial filter has to account for at least > 1010 cells cm−3 (Lösekann et al., 2007; Knittel and Boetius, 2009). response to the onset or increase in permafrost thawing or CH4 gas hydrate destabilization. High methane escape (up to 11–19 µmol CH4 cm−2 yr−1 corresponding to 2.6–4.5 TgCH4 yr−1 if upscaled to the ESAS) can occur during a transient period following the on- set of methane flux from the deep sediments. Under these conditions, substantial methane escape from sediments re- quires the presence of active fluid flow that supports a sig- nificant and rapid upward migration of the SMTZ in re- sponse to the onset of CH4 flux from below. Such rapid and pronounced movements create a window of opportunity for non-turbulent methane escape by inhibiting the accumula- tion of AOM-performing biomass within the SMTZ – mainly through thermodynamic constraints – thereby perturbing the efficiency of the AOM biofilter. The magnitude of methane effluxes, as well as the duration of this window of oppor- tunity, is largely controlled by the active flow velocity. In addition, results of transient scenario runs indicated that the characteristic response time of the AOM biofilter is of the or- der of few decades (20–30 years), thus exceeding seasonal– interannual variability. Consequently, seasonal variation of bottom methane and seawater sulfates exert a negligible ef- fect on methane escape through the sediment–water inter- face. 3.3.2 Final new steady state Vertical profiles at the end of transient simulation (af- ter 200 years) with bioenergetic AOM formulation for the case [CH4]−= 1.169 mM and vup = 5 cm yr−1. (a) Bimolecular prod- uct [CH4]·[SO2− 4 ]. (b) AOM rate according to the bioenergetic for- mulation (blue) and, for comparison, according to bimolecular for- mulation used for the steady-state simulations (red). (c) Apparent kAOM, estimated from Eq. (5). lations, with bimolecular rate law, under identical environ- mental conditions (inferred from Fig. 6). https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 Biogeosciences, 17, 3247–3275, 2020 4 Conclusions Our results indicate therefore that present methane efflux from ESAS sediments can be supported by methane gas escape and non-turbulent CH4 efflux from rapidly accumulating and/or active sediments (e.g. coastal settings, portions close to river mouths or submarine slumps). In particular, active sites sediments may release methane in A regional upscaling of non-turbulent methane efflux for the Laptev Sea shelf using a model-derived transfer function that relates sedimentation rate and methane efflux merely sums up to ∼0.1 GgCH4 yr−1. Nevertheless, it also suggests that the evaluation of methane efflux from Siberian shelf sediments should pay particular attention to the dynamic and rapidly changing Arctic coastal areas close to big river mouths, as well as areas that may favour preferential methane gas release (e.g. rapidly eroding coastlines, fault lines or shal- low sea floors, i.e. < 30 m). In addition, our findings call for more data concerning sedimentation and active fluid flow rates, as well as the reactivity of depositing organic matter and bioirrigation rates in Arctic shelf sediments. In conclusion, we argue that the evaluation of projected subsea permafrost thaw and/or hydrate destabilization im- pacts on the Arctic environment requires models that include an explicit description of (1) methane gas, (2) AOM biomass, as well as (3) the entire network of the most pertinent bio- geochemical reactions. Such approaches, valid globally for all the shelves underlain by methane reservoirs (e.g. conti- nental slopes), are even more recommended in order to en- able a robust quantification of methane escape from the Arc- tic shelf to the Arctic ocean, settings even more sensible to https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape the rapidly changing environmental conditions. Finally such refined modelling will also help evaluate the impact of sub- sea permafrost thaw and methane destabilization on Arctic alkalinity and biogeochemical cycling. 3267 https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape the rapidly changing environmental conditions. Finally such refined modelling will also help evaluate the impact of sub- sea permafrost thaw and methane destabilization on Arctic alkalinity and biogeochemical cycling. Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape M. Puglini et al.: Assessing the potential for non-turbulent methane escape M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3268 https://doi.org/10.5194/bg-17-3247-2020 Appendix A: AOM efficiency η If we identify the SMTZ region as the portion of the sediment column where the rate of AOM is 1 % of the maximum, we can define the efficiency of the AOM filter η as η(%) = 1 − J + CH4 J − CH4 ! · 100, (A1) (A1) where J + CH4 is the methane flux at the shallowest point where the AOM rate is 1 % of the maximum (upper dashed line in Fig. A1), and J − CH4 is methane flux at the deepest point where the AOM rate is 1 % of the maximum (lower dashed line in Fig. A1). Figure A1. Typical sediment profile of [SO2− 4 ], [CH4] and AOM rate. Units are mM for concentration and mM yr−1 for rate. The re- gion between the two dashed lines represents the zone where AOM rate is larger than 1 % of it its maximum and defines the sulfate– methane transition zone (SMTZ). The fluxes J − CH4 and J + CH4 are the fluxes used in the definition of η of Eq. (A1). Figure A1. Typical sediment profile of [SO2− 4 ], [CH4] and AOM rate. Units are mM for concentration and mM yr−1 for rate. The re- gion between the two dashed lines represents the zone where AOM Figure A1. Typical sediment profile of [SO2− 4 ], [CH4] and AOM rate. Units are mM for concentration and mM yr−1 for rate. The re- gion between the two dashed lines represents the zone where AOM rate is larger than 1 % of it its maximum and defines the sulfate– methane transition zone (SMTZ). The fluxes J − CH4 and J + CH4 are the fluxes used in the definition of η of Eq. (A1). Figure A1. Typical sediment profile of [SO2− 4 ], [CH4] and AOM g yp p 4 4 rate. Units are mM for concentration and mM yr−1 for rate. The re- gion between the two dashed lines represents the zone where AOM rate is larger than 1 % of it its maximum and defines the sulfate– methane transition zone (SMTZ). The fluxes J − CH4 and J + CH4 are the fluxes used in the definition of η of Eq. (A1). Biogeosciences, 17, 3247–3275, 2020 Biogeosciences, 17, 3247–3275, 2020 Biogeosciences, 17, 3247–3275, 2020 3269 https://doi.org/10.5194/bg-17-3247-2020 Biogeosciences, 17, 3247–3275, 2020 M. Puglini et al.: Assessing the potential for non-turbulent methane escape 3270 Code and data availability. Primary data needed to reproduce the analyses presented in this study are archived by the Max Planck Institute for Meteorology are available upon request (publica- tions@mpimet.mpg.de). Athy, L. F.: Density, porosity, and compaction of sedimentary rocks, AAPG Bull., 14, 1–24, 1930. Baker, G. and Osterkamp, T.: Implications of salt fingering pro- cesses for salt movement in thawed coarse-grained subsea per- mafrost, Cold Reg. Sci. Technol., 15, 45–52, 1988. Barton, B. I., Lenn, Y.-D., and Lique, C.: Observed Atlantification of the Barents Sea causes the Polar Front to limit the expansion of winter sea ice, J. Phys. Oceanogr., 48, 1849–1866, 2018. Supplement. 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Appendix B: Damköhler number The Damköhler number Da is a dimensionless quantity which relates timescales typical of transport processes to timescales typical of chemical reactions. It compares the con- sumption/production rate with the advective transport and is defined as (B1) Da = τT/τR, (B1) Da = τT/τR, where τT is the advective timescale and τR is the reaction timescale. τR is defined as 1/KR where KR is the reaction rate of AOM or methanogenesis. If we call R the reaction rate then KR reads as KR = 1 L Z L R [CH4]dz, (B2) (B2) where L is the width where the reaction rate is larger than 1 % of the maximum rate. τT is instead defined as τT = L |vup −ω|, (B3) (B3) where vup −ω is the effective advective velocity. Da can be the expressed by Da = τT τR = 1 |vup −ω| Z L R [CH4]dz. (B4) (B4) Biogeosciences, 17, 3247–3275, 2020 https://doi.org/10.5194/bg-17-3247-2020 M. 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SOLVING NON-STANDARD TASKS AS AN EDUCATIONAL SITUATION IN MATHEMATICS EDUCATION
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1 INTRODUCTION Although mathematics, including language and natural sciences, are the main areas of each education system, those areas do not guarantee the development of all skills (competences) that today's pupils will need in adult life. Many of the world's leading experts agree that the core of key competencies for life and work in the future world is not only the acquisition of basic literacy (i.e. linguistic, mathematical, science), but (above all) the ability to solve non-standard tasks and problems in which elements and concepts are interconnected not only from mathematics but from different fields, requiring different representations and different solutions. For this reason, although non-standard tasks and problems are primarily conceived as part of mathematical education in some curricular documents, their application is often universally interdisciplinary. It does not mean there would be a little scope to solve non-standard problems in school subjects, but that the solution to these problems is nowadays increasingly seen as a separate area of human activity, which can be well distinguished from discipline-related areas (primarily mathematics) with which specific skills are associated. [1] Abstract Mathematics is a frequent subject of many debates, especially in relation to the process of education. One of the questions in the mathematics education is working with pupils who require some educational or pedagogical attention. An example may be pupils who are mathematically gifted or talented. Working with these pupils is generally very demanding because it requires specific educational help. The interest in educating such pupils is still increasing, as it is logical that the development of their talents is essential for society as a whole. One of the ways how to develop these skills is through non-standard types of mathematics tasks. These tasks develop pupils' logical and combinatorial thinking, promote their perception of spatial relationships and point out the context of mathematics with everyday life. The intention to include such tasks in the teaching of mathematics is also to point out the usability, playfulness and interestingness of mathematics, thus encouraging pupils to enjoy mathematical discovery. Solving non-standard mathematics tasks is not always dependent on knowledge so it is a new educational situation for pupils who are usually used to the familiar algorithmic solution of common tasks. Similarly, the tasks require more difficult cognitive operations and place high demands on the competences of the teacher, who must be able to direct pupils to the solution in the spirit of constructivism and give them an opportunity and space to find their own solution. Describing of the educational situation when pupils dealing non-standard mathematics tasks, is an important issue of this paper as well as describing teachers' approach to work with this kind of pupils. Keywords: mathematics, educational, solution, non-standard, tasks, situation, competences, p ds: mathematics, educational, solution, non-standard, tasks, situation, competences, pupils. SOLVING NON-STANDARD TASKS AS AN EDUCATIONAL SITUATION IN MATHEMATICS EDUCATION Marek Skultety Palacký University in Olomouc (CZECH REPUBLIC) 2.1 Characteristics and typology of mathematical problems Due to the terminological inconsistency of individual authors in the use of terms, the term problem must be understood subordinate to the term of mathematical problem, or mathematical learning problem. The term problem in mathematical teaching refers to the task of a problematic character, which assumes a greater share of activity and ingenuity. Solving the problem (problem, "non-standard" tasks) requires a creative approach to solving the problem. There are usually three phases of problem resolution: orientation-analytical (active processing, problem situation modelling), strategic-operational (hypothesis formation, method, solution strategy) and synthetic-verification (hypothesis validating). [5] The teacher knows from his teaching experience that learning tasks and their solutions are an integral part of teaching that is applied at all stages of the learning process to achieve learning goals. [6] There are several typologies of mathematical learning tasks in the literature. In accordance with the classification of tasks in terms of the level of creativity of the solver in the solution, we can distinguish (with some simplification), standard mathematical learning tasks – where known formulas, rules or procedures (algorithms) are used to solve – and non-standard (problem) learning tasks where known methods and algorithms are not enough to solve them. In the context of labelling of non-standard task, similar attributes are often offered as unusual, interesting, motivating, challenging, and innovative. The term “non-standard” may lie in a delay between the description of a particular type of task and its general acceptance by the teaching public and subject-related didactics. Non-standard tasks can be viewed from two perspectives: Non-standard tasks can be viewed from two perspectives: • A non-standard task, whose content corresponds to the reality of society, technology and level of knowledge; • A non-standard task indented solely for motivation, for the purposes of supplementary materials or mathematical olympiads, competitions and various circles and other activities for gifted children. In general, non-standard tasks in mathematical education represent an unconventional topic that aims to develop logical and combinatorial thinking of pupils, to promote their perception of spatial relationships and to point out the usability, playfulness and interestingness of mathematics, and encourage pupils to enjoy mathematical discovery. [7] The solution of non-standard tasks and problems represents the ability of an individual to use cognitive processes to solve real cross-curricular situations in which the solution is not immediately apparent. 2 THEORETICAL BASIS In the theoretical context, we must define the concept of an educational situation that we understand as "a specifically defined compliance of external circumstances, time-limited, during which an individual is exposed to particular influences on which responds in some way". [2] An educational situation represents a specific time and place constraint in which an individual is exposed to the teacher's influences having their specific learning objective, to which the pupil responds in some way. Similarly, we can cite a definition as "part of the learning process defined by time, place, and content of teaching". [3] e of any educational situation is usually a learning task that can be defined as a “didactic ct, whose assignment encourages the pupil to solve a particular problem to lead the pupil's Proceedings of EDULEARN19 Conference 1st-3rd July 2019, Palma, Mallorca, Spain Proceedings of EDULEARN19 Conference 1st-3rd July 2019, Palma, Mallorca, Spain ISBN: 978-84-09-12031-4 4154 learning and develop his competences”. [3] Defining the role of teaching can generally be regarded as a "challenge to action". In this case, the mathematical learning task is a challenge to mathematical action. For example, a mathematical learning task is a linear algebraic equation with one variable, a word problem task with a subject from the field of financial mathematics, or a non-standard (problem) task requiring the discovery of a solution strategy, etc. [4] learning and develop his competences”. [3] Defining the role of teaching can generally be regarded as a "challenge to action". In this case, the mathematical learning task is a challenge to mathematical action. For example, a mathematical learning task is a linear algebraic equation with one variable, a word problem task with a subject from the field of financial mathematics, or a non-standard (problem) task requiring the discovery of a solution strategy, etc. [4] 2.1 Characteristics and typology of mathematical problems Such tasks focus on assessing skills rather than knowledge, and except interconnections from different subjects, they also place emphasize on linking between school curriculum and real-life situations that pupils encounter in everyday life. [1] In addition, these tasks are set in situations that are usually not part of the curriculum and are not commonly found in school textbooks. Therefore, since solving non-standard types of tasks in mathematics may not always be dependent on the knowledge and skills of school mathematics in specific educational situations, these mathematical problems lead to capture and activity of less successful pupils in mathematics and, on the other hand, more complex and challenging problems motivate the gifted pupils. [7] 2.2 Solving non-standard mathematical tasks One of the possible educational situations that a teacher can create is an educational situation focused on solving non-standard mathematical tasks. When creating or selecting specific non- traditional tasks, the teacher should pay close attention to the fact that the tasks are not unilaterally focused and cover, as far as possible, the full range of the most common problem situations and skills needed to successfully solve them. [1] The pupils then have to solve the (mathematical) problem, search for and discover the method, the solution method, because their previous experience of solving the task is not possible or sufficient. The process of solving a task is usually not known, the solver is often looking for a way to the result in an original way. Solution of non-standard, resp. problematic 4155 tasks requires deep concentration, invention and time. According to the authors [8], the motivational aspect is especially important. In terms of humanistic education, it contributes to the creation of a stimulating learning environment, promotes and develops student self-confidence and self- confirmation, prefers positive evaluation by both teachers and classmates. Therefore, motivational issues as a challenge to creativity are an important factor in the effectiveness of mathematical education. tasks requires deep concentration, invention and time. According to the authors [8], the motivational aspect is especially important. In terms of humanistic education, it contributes to the creation of a stimulating learning environment, promotes and develops student self-confidence and self- confirmation, prefers positive evaluation by both teachers and classmates. Therefore, motivational issues as a challenge to creativity are an important factor in the effectiveness of mathematical education. Solutions and the creation of non-standard tasks are usually considered suitable for working with gifted or extraordinarily gifted pupils, because they develop knowledge, metacognition and motivation. [9] The “Level of Heuristic” of non-standard tasks is a typical example, when it is necessary to consider not only the task itself but also the competence of the solver. For some pupils (given the age, individual abilities, level and scope of preliminary mathematical knowledge and the means by which they are equipped), a certain task is non-standard – "heuristic", for others its solution is only a routine matter. A task one pupil solves based on a known algorithm or a known theory, may require creative effort of another pupil. 2.2 Solving non-standard mathematical tasks Non-standard tasks and problems in mathematical education do not mean tasks as difficult but as unusual (both by entering and by way of solution) and are also suitable for pupils' research activities. During solving such tasks, the pupils' individual solutions should be respected and appreciated if the solutions are correct and, in an appropriate way (e.g. supplementary questions), correct pupils' practices that are not correct or accurate. Also, complex tasks from practical life have a non-standard character in which different areas of mathematics are interlinked. In solving practical tasks, we use specific experiences of pupils from everyday life as well as from different areas of their interests (sport, technology, nature, art, etc.), which increase the motivational value of tasks. The authors [10] also point out that tasks (e.g. cascades), in which difficulty is increased gradually, also have their room in educational practice. Pupils' competence to work independently with data, their ability to use mathematical tools to solve real life situations, is seen as a significant manifestation of mathematical literacy. Estimating, proving, modelling – used to interpret natural phenomena and predict their development – should become the core of mathematical education already at primary school as an important component of the development of mathematical thinking. [11] Polya [12] presents four basic stages of the problem-solving process that can be suitably applied to solving non-standard tasks. We distinguish: a) understanding the issue that it considers to be key: „It is foolish to answer a question that you do not understand“; b) linking individual information and data in a task assignment that aims to create a "solution plan"; c) the solution itself (the implementation of this set plan); d) Evaluating or interpreting the result in terms of "view / step back". According to Hejný [13], individual stages of solving a non-standard problem can be named as a level. There are five of them: the level of grasping the situation, the level of gaining insight into the situation of the task, the level of finding and determining the strategy of the solution, the level of implementation of the solution (calculation) and the level of interpretation of the result. According to Hejný [13], individual stages of solving a non-standard problem can be named as a level. 2.2 Solving non-standard mathematical tasks Evaluation and decision-making processes are also significant in solving divergent tasks. [15] Zelina & Zelinová draw attention to the paradox in this context, when very many tasks and situations we solve in practical life – from building relationships, through solving work tasks to everyday tasks such as dressing, leisure time, etc. – are divergent in nature; in contrast, convergent tasks dominate school education. According to Scholtz [16], typical non-standard learning tasks are combinatorial tasks. These tasks aim to develop key competencies, especially problem-solving competencies, by leading the pupil to "developing combinatorial and logical and critical thinking and comprehensible and substantive argumentation through solving mathematical problems". Dvořáková further explains [17] that combinatorial tasks can also be used to realize and develop interdisciplinary contexts, such as ciphers, Morse code, flag colourfulness, coin changing, dressing or classroom children seating. In this context, Makrides [18] uses the term open-ended tasks and emphasizes the importance of using them in working with gifted pupils (according to this author, their typical feature is an above- average ability to understand mathematics and to think mathematically, not only the ability to perform mathematical calculations and getting good grades from mathematics). Another important characteristic of appropriate tasks for gifted pupils is the difficulty, which is a challenge for these pupils. It is necessary to present challenging tasks that develop knowledge, metacognition and motivation. One of the forms of work with non-standard learning tasks, especially with gifted pupils, is the variation and alone creation of tasks. In accordance with Tollinger [19], asking questions and formulating tasks classifies to the highest category of her taxonomy, in a group of tasks requiring creative thinking. School practice confirms that the creation or modification of tasks is one of the activities by which mathematical teaching can be significantly enriched. Sarrazy [20] states in this context that "belief in pupil's creativity is essential for teachers, but this pedagogical humanist belief often leaves them helpless when it comes to creating conditions for mathematical creation: mere will is an ineffective weapon to fight with ignorance and pupils’ misunderstanding”. In doing so, it is possible to use different ways to specify pupils' assignments for task creation. Pupils can create tasks that contain predefined numbers (tasks with a given mathematical model), can create tasks related to a given topic [21], tasks related to the story, a given image, or a real situation. 2.2 Solving non-standard mathematical tasks There are five of them: the level of grasping the situation, the level of gaining insight into the situation of the task, the level of finding and determining the strategy of the solution, the level of implementation of the solution (calculation) and the level of interpretation of the result. Hejný and Stehlíková [14] cite that in a situation of insufficient understanding of the task assignment one of the following ways of responding of the pupil usually occurs: Hejný and Stehlíková [14] cite that in a situation of insufficient understanding of the task assignment one of the following ways of responding of the pupil usually occurs: • resignation – the pupil will give up the solution, does not try the solution at all; • cheating – the pupil tries to get the solution without his own efforts, for example to copy it from his classmates; • random calculation – the pupil takes (sometimes only some) numbers from the assignment and "does something with them randomly"; • alternative grasp – the pupil's solution is based on "external motives". The motive is that a pupil receives some additional information, whether from a teacher or found in a task assignment ("signal", which is a word or phrase that has the character of an idiom that in the pupil's consciousness associates a certain calculation procedure). Non-standard learning tasks often have a divergent character, as opposed to convergent tasks, which predominate in traditional textbooks (90-95% according to Zelina and Zelinová [15]). Convergent tasks Non-standard learning tasks often have a divergent character, as opposed to convergent task predominate in traditional textbooks (90-95% according to Zelina and Zelinová [15]). Converge 4156 require thought processes that use perception, differentiation, cognition, memory, analysis, synthesis, induction and deduction at the level of specific relationships, applying knowledge to a particular situation that is characteristic of the logical and algorithmic process leading to the correct conclusion. require thought processes that use perception, differentiation, cognition, memory, analysis, synthesis, induction and deduction at the level of specific relationships, applying knowledge to a particular situation that is characteristic of the logical and algorithmic process leading to the correct conclusion. Solving divergent tasks requires divergent thinking that does not lead to one correct answer, but requires to generate as many suggestions, alternatives, or possible solutions strategies as possible. Divergent thinking, in addition to producing more alternatives, requires consideration of the implications of proposals, their value and accuracy. 2.2 Solving non-standard mathematical tasks [22] According to Novotná [23], in the actual creation of tasks, pupils get into a new role when they become the creators from passive recipients of assigned tasks by teachers or textbooks and approximates the very nature of mathematical activity. Tasks are given to pupils by their mathematics teachers, but they can also be used in independent homework. Above all, students with talent for mathematics can choose for themselves, estimate their possibilities and confront the chosen solutions. Attempting to detect some of the typical strategies that pupils have used to solve and the mistakes they have made can inspire teachers to reflect on pupil performance and correct the simplified teacher's judgments about their abilities and skills. Subsequent analysis of pupils' solutions non-standard tasks can become a new instrument of a more accurate, apposite evaluation based on a thorough knowledge of pupil's individuality. It can help the teacher to diagnose hitherto undetected potencies and correct teachers present' ideas. [24] 3 RESEARCH Mathematical literacy in PISA research Several recent research [27] focused on the mathematical literacy of the PISA program showed that pupils of OECD countries handle algorithmic mathematical tasks, but the problem of many countries' pupils (e.g. the Czech Republic) is such mathematical tasks as non-standard, problematic, with cross- curricular connections and requiring creative solutions. One of the reasons for the failure of many pupils in problematic mathematical problems is the approach of pupils to mathematics, which is rather negative in the Czech Republic. This fact is often associated with an overall low willingness of pupils to solve these problematic tasks. [28] The problem, and at the same time the possible cause, of the low success rate of pupils (several OECD countries) in these tasks is that mathematical education is very often unilaterally oriented primarily on the formation and subsequent consolidation of algorithmic practices that pupils learn on model examples (standard tasks). The learned template methods then lead pupils to a thoughtless application of the method being taught. The teacher deduces the algorithm on the model example (at best in cooperation with pupils) and the pupils acquires the solution of several similar tasks. The teacher deliberately chooses type-like tasks to consolidate a given algorithm for the faster acquisition. 3 RESEARCH There are various international researches that evaluates learning outcomes of pupils. One of the researches, whose main focus is on the skills and knowledge that students will need for life in modern society (in a way, success of solving non-standard tasks and problems) is the PISA (Programme for International Student Assessment). The program takes place at international level and examines the level of learning outcomes of 15-year-old pupils (i.e., pupils at the age where they usually end their compulsory mathematics education). The research is conducted under the auspices of the OECD and, at regular three-year intervals, identifies the level of reading, mathematical and science literacy of pupils. 4157 4157 The PISA mathematical literacy assessment focus mainly on the extent to which pupils are able to use mathematics to solve a variety of situations from everyday life. Their successful solution assumes knowledge of mathematical terminology, facts and procedures, and the ability to perform mathematical operations precisely because there are usually tasks where mathematical content is not immediately obvious, requiring creative combining of individual elements of mathematical subject, depending on the requirements of the particular situation. [25] Specifically, mathematical literacy was defined as „the ability of an individual to identify and understand the role that mathematics plays in the world, and to make the well-founded judgements and decisions required in life by constructive, engaged and reflective citizens “. [25] Table 1 shows that the average of achieved results in mathematical literacy in OECD countries is gradually decreasing. [26] The range of mathematical literacy in PISA research is divided into six levels. These levels reflect how advanced mathematical skills pupils have and how difficult tasks they can handle. Based on PISA, the range of points 483–544 (in which the OECD average) means “the ability of pupils to execute clearly describe procedures, including those that require sequential decisions. They can select and apply simple problem-solving strategies. Pupils at this level can interpret and use representations based on different information sources and reason directly from them. They can develop short communications reporting their interpretations, results and reasoning”. [25] [25] Table 1. Mathematical literacy in PISA research Research OECD average 2000 500 2003 500 2006 500 2009 496 2012 494 2015 490 Table 1. A 17 B 20 C 21 D 23 E 25 Also, a closed task with a choice of 5 answers. The correct answer is D based on an arithmetic progression with common difference 4 (7; 6+5; 5+5+5; 4+5+5+5; …). The solution of the problem is based on a (thought) experiment, which can be accompanied by a written record of the individual steps of replacement the cards. Characteristic is the absence of mathematical concepts and known numerical procedures, but also the "arrangement" of a task with a graphical representation of the initial situation. Replacing three cards (answer C) lead to the correct solution, which can be written down as follows: 5 1 4 3 2 ® 2 1 4 3 5 ® 1 2 4 3 5 ® 1 2 3 4 5. The divergent nature of the task provides an opportunity for finding and discovering, following discussion and argumentation of the pupils (e.g. can there be other solutions?). Task 2: With which square do you have to swap the question mark, so that the white area and the black area are the same size? Figure 2. Task 2. This is a closed task with a choice of 5 answers. The correct answer is B based on a comparison of the black and white squares – to the three white ones, a third black one should be added. Task 3: Thor has seven stones and a hammer. Every time he hits a stone with the hammer it breaks into exactly five smaller stones. He does this several times. Which of the following numbers could be the number of stones he may end with? 3.1 Examples of non-standard mathematical tasks Within this chapter, we can present non-standard learning tasks [29] in which pupils use logical reasoning and combination judgment and find different solutions to the presented or examined situations. The tasks are based on the International Mathematical Kangaroo Competition: Task 1: Five cards are lying on the table in the order 5, 1, 4, 3, 2. You must get the cards in the order: 1, 2, 3, 4, 5 (look at the picture). Per move, any two cards may be interchanged. How many moves do you need at least? A 5 B 4 C 3 D 2 E 1 4158 4 CONCLUSION The non-standard task aims to motivate pupils and develop their thinking and mathematical skills. These tasks go beyond the classical (framework) teaching of mathematics. Finding methods of solution can be more valuable to developing pupil's thinking than the correctness of the solution. This is interconnected with the assessment of the pupil's solution, which should be solved by the self- reflection of the pupil or by the method of verbal evaluation, by means of which it is possible to focus more on the evaluation of the process itself than the final result. Regular inclusion of non-standard tasks in mathematical teaching is a suitable instrument for the development of pupils' personality. Also, non-standard tasks are used to identify gifted pupils for mathematics in order to maintain interest in mathematics and to develop the mathematical abilities of talented pupils, for example for further study. This may lead to an improvement in the school climate and to increasing the popularity of mathematics as a school subject. [29] The primary aim of this paper was to define the basic concepts related to solving non-standard problems in mathematical education through the educational situation. The paper was complemented by the results of the PISA research, which regularly evaluates the level of education in specific literacy 4159 of 15-year-old pupils in OECD countries, and sample examples of non-standard tasks. Although it is primarily up to the teachers, how non-standard tasks will be included in mathematical education, we can conclude that all world authors generally agree on the need to include these types of tasks in teaching and their generally positive impact on pupil motivation. of 15-year-old pupils in OECD countries, and sample examples of non-standard tasks. Although it is primarily up to the teachers, how non-standard tasks will be included in mathematical education, we can conclude that all world authors generally agree on the need to include these types of tasks in teaching and their generally positive impact on pupil motivation. REFERENCES [1] V. Tomášek, E. Potužníková, Netradiční úlohy: Problémové úlohy mezinárodního výzkumu PISA. Praha: Ústav pro informace a vzdělávání, 2004. [2] J. Pelikán, Výchova jako teoretický problem. Ostrava: Amosium servis. 1995. [3] J. Slavík, T. Janík, “Kvalita výuky: obsahově zaměřený přístup ke studiu procesů vyučování a učení” in Pedagogika, vol. 62, no. 3, pp. 276-279, Praha, 2012. [4] F. Kuřina, Matematika a řešení úloh. České Budějovice: Jihočeská univerzita, 2011. [5] J. Vyšín, Metodika řešení matematických úloh. Praha: SPN, 1962. [6] B. Novák, Řešení matematických úloh. Olomouc: Univerzita Palackého, 2013. [7] J. Houska, K. Nemčíková, “Netradiční úlohy ve výuce matematiky,“ in Metodický portál RVP, 2009. Retrieved from https://clanky.rvp.cz/clanek/c/zvb/3002/netradicni-ulohy-ve-vyuce- matematiky.html/ [8] O. Šedivý, J. Fulier, Úlohy a humanizácia vyučovania matematiky. Nitra: Univerzita Konstantina Filozofa, 2004. [9] D. Malinová, Mimořádně nadaný žák v primárním matematickém vzdělávání. Olomouc: Univerzita Palackého, 2014. [10] H. Lišková, P. Rezek, “Tematický okruh Nestandardní aplikační úlohy a problémy” in Metodické komentáře ke Standardům pro základní vzdělávání Matematika a její operace, pp. 101-128, Praha: NÚV, 2015. [11] P. Boero, “Students´ everyday experience and teaching and learning of mathematics” in M. Uhlířová (Ed.), Acta Univ. Palacki. Olom.: Matematika 2, pp. 10-12, Olomouc: Univerzita Palackého, 2006. [12] G. Polya, How to solve it. Princeton: Princeton University Press, 1957. [13] M. Hejný, A. Michalcová, Skúmanie matematického riešitelského postupu. Bratislava: Metodické centrum, 2001. [14] M. Hejný, N. Stehlíková, Číselné představy dětí. Praha: Univerzita Karlova, 1999. [15] M. Zelina, M. Zelinová, Rozvoj tvorivosti detí a mládeže. Bratislava: SPN, 1990. [16] I. Scholtzová, Integrácia poznatkou z kombinatoriky do vyučovania matematiky na základnej škole – pohĺad druhý. 2006. [17] H. Dvořáková, “Propedeutika kombinatoriky na 1. stupni ZŠ” in N. Stehlíková & L. Tejkalová (Eds.), Dva dny s didaktikou matematiky, pp. 25-28, Praha: Univerzita Karlova, 2010. [18] G. Makrides, et. al., Objevování, motivace a podpora matematických talent na evropských školách. Praha: MATHEU, 2006. [19] D. Tollingerová, “Úvod do teorie a praxe programované výuky a výcviku” in Odborná výchova, vol. 21, no. 5, 1971. [20] B. Sarrazy, “Tvorba v matematice: nezbytná iluze?” in Š. Pěchoučková (Ed.), Tvořivost v počátečním vyučování matematiky, pp. 24-37, Plzeň: ZČU, 2011. 1] J. Bureš, J. Novotná, “Žákovská tvorba úloh” in M. Lávička & B. Bastl (Eds.), 11. Setkání učite matematiky všech typů a stupňů škol, pp. 71-75, Plzeň: JČMF, 2008. 2] V. Palková, A. Prídavková, et. al., Matematika pre život: Zbierka úloh na rozvoj matematickej gramotnosti žiakov primárnej školy. Prešov: Prešovská univerzita, 2011. [23] J. REFERENCES Novotná, Analýza řešení slovních úloh. Praha: Univerzita Karlova, 2000. 4160 [24] B. Novák, Sbírka nestandardních úloh, Olomouc: Univerzita Palackého, 2006. [25] V. Tomášek, E. Potužníková, Netradiční úlohy: Matematická gramotnost v mezinárodním výzkumu PISA. Praha: Ústav pro informace a vzdělávání, 2006. [26] OECD. PISA: Program for International Student Assessment. Retrieved from: https://oecd.org/pisa/ [27] OECD, PISA 2015: Results in Focus, 2018. Retrieved from: https://oecd.org/pisa/pisa-2015- results-in-focus.pdf [28] M. Hejný, J. Novotná, N. Vondrová, Dvacet pět kapitol z didaktiky matematiky. Praha: Univerzita Karlova, 2004. [29] K. Bártek, R. Dofková, et. al., Reflexe vzdělávacích potřeb učitelů matematiky jako východisko jejich profesního rozvoje. Olomouc: Univerzita Palackého, 2018. 4161
https://openalex.org/W4238144747
https://www.nss-journal.org/articles/nss/pdf/1993/04/nss19930104p298.pdf
French
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Commentaires
Natures sciences sociétés/Natures sciences sociétés
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cc-by
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COMMENTAIRES . JEAN-MARIE LEGAY ARTICLES On peut sans doute dans l’histoire des relations de l’Homme avec les autres êtres vivants, végétaux ou animaux, pro- poser, au moins comme base de discussion, l’existence de trois grandes périodes. ont servi de contexte, de cadre, si ce n'est de déterminants complé­ mentaires. génétiques d’importance ont été, au début, obtenus sur des orga­ nismes cultivés ou élevés dans un contexte agricole : pois, tabacs, maïs, pétunias, tomates, lapins, cailles, etc. Mais en même temps les plantes cultivées et les animaux domestiques prenaient leur auto­ nomie en s’éloignant de plus en plus de leurs parents sauvages, et, en acquérant une identité locale ou régionale, se diversifiaient en races ou populations nettement dis­ tinctes. d’apporter un commencement de réponses et d’établir quelques pistes de recherches dans ce domaine, en un mot de contribuer à ouvrir un sujet. Les progrès récents les plus importants concernent, un peu paradoxalement, la première période, la plus lointaine. Grâce à l’imagination des chercheurs enga­ gés sur ce sujet (et en particulier celle des archéologues et des eth­ nologues), grâce à des progrès tech­ niques décisifs (en particulier de datation, d’exploitation des objets et des données), on sait mieux aujourd’hui ce qui s’est passé aux origines. Le glissement d’une définition locale des races à une définition régionale, puis à un statut prati­ quement international est parti­ culièrement intéressant à suivre. L’estimation du poids des contraintes socio-économiques par rapport à celui des contraintes éco­ logiques, le passage d’un milieu écologique à un milieu d’élevage et à un environnement humain, la construction d’un troupeau fina­ lisé à partir du matériel génétique disponible, sont autant de thèmes à poursuivre et à approfondir. En outre, on peut débattre de ces ques­ tions en référence à l’évolution des systèmes agraires, mais on pourrait le faire aussi en fonction des espèces ; ce que l’auteur de l’article nous invite à voir chez les bovins est-il généralisable aux autres espèces animales et végétales ? La première fut une longue phase d’exploration, de cueillette plus ou moins discriminante, d’essais culturaux, qui vit son apo­ gée avec la révolution néolithique. Une sélection largement empi­ rique, et souvent inconsciente, n’en créa pas moins une distance entre plantes et animaux “domes­ tiques” et leurs ancêtres et, encore souvent, cousins sauvages. Jean-Marie Legay : Université Claude Bernard - Lyon I - 43, boulevard du 11 -Novembre, 69622 Villeurbanne Cedex. COMMENTAIRES . JEAN-MARIE LEGAY La troisième période débute à peine : à l’inverse des phases pré­ cédentes où la pratique précédait largement la théorie, ce sont main­ tenant les connaissances scienti­ fiques acquises au niveau cellulaire et moléculaire qui sont à l’origine de tentatives diverses d’interven­ tions et par suite de modifications des plantes cultivées et, plus diffi­ cilement ailleurs, des animaux domestiques. Par contre la deuxième période, qui est pourtant celle sur laquelle nous vivons actuellement, a fait l’objet de peu de recherches proprement scientifiques et reste donc très mal connue. Il existe évi­ demment de nombreuses publica­ tions et quantité de documents d’archives, mais les premières appartiennent pour l’essentiel à la littérature grise (ou à très faible diffusion) et sont difficiles d’accès ; les secondes, très dispersées, n’ont pratiquement pas été explorées, faute de main-d’œuvre. Et surtout pour développer ces recherches, on manque, semble-t-il, d’idées directrices et de méthodes. L’inté­ rêt des réflexions proposées par Bertrand Vissac est précisément La deuxième période qui s’étala à son tour sur des millé­ naires fut dominée par une sélec­ tion consciente, bien entendu accompagnée dans chaque cas de sélections inconscientes, et mar­ quée en tout état de cause par des interactions fortes entre protago­ nistes. Son apogée commença à la fin du siècle dernier, c’est-à-dire à un moment où la prise de conscience des phénomènes héré­ ditaires et de leur support maté­ riel allait conduire à ce que l’on peut qualifier de révolution géné­ tique. On peut d’ailleurs noter au passage que de nombreux résultats NATURES - SCIENCES - SOCIÉTÉS, 1993,1 (4) Étant donné l’énorme impor­ tance économique et sociale prise par ces organismes devenus auxi­ liaires de l’Homme, bases de son alimentation, et matière première d’industries diverses, on ne peut que s’étonner du peu d’études concernant leur histoire, les méca­ nismes qui ont présidé à leur évo­ lution, l’ensemble des facteurs qui Plusieurs champs semblent devoir s’ouvrir à la discussion. D’abord celui des interactions per­ manentes entre l’évolution des races et celle des systèmes agraires. Ensuite celui de la comparaison race-population : « une race ani- Jean-Marie Legay : Université Claude Bernard - Lyon I - 43, boulevard du 11 -Novembre, 69622 Villeurbanne Cedex. nants qui conduit à la décision ne peut être ignoré. nants qui conduit à la décision ne peut être ignoré. male avec son cortège de pratiques d’élevage » est-elle assimilable à une population avec son cortège de contraintes écologiques ? COMMENTAIRES . JEAN-MARIE LEGAY Et puis encore, la crise actuelle (à la fin de ce que j’ai appelé la deuxième période) est-elle un bon révélateur des relations historiques de l’éle­ vage à la société ? Si les articles scientifiques sont si rares quant à l’évolution récente de nos animaux domestiques et de nos plantes cultivées, ce n’est pas seulement en raison de la com­ plexité du sujet, de la fragilité de certaines de nos hypothèses, et peut-être des risques d’une réflexion à implications sociales et politiques, c’est aussi par paresse intellectuelle : des relations sim­ plistes, souvent économistes, ont servi à masquer une situation d’une richesse exceptionnelle. Dans les relations de l’Homme à ses ani­ maux domestiques et à ses plantes cultivées, il faut partir à la chasse aux preuves et aux documents, et ne pas se contenter d’une logique probable et d’un itinéraire vrai­ semblable. Si l’on veut écrire une histoire, il nous faut des faits. Cela devrait tenter de jeunes chercheurs. Enfin, ne faudrait-il pas profi­ ter de l’occasion qu’offre cet article pour aborder une question de fond ? On a un peu trop l'habitude de considérer la plurisdisciplina- rité comme une forme d’approche que nous aurions choisie dans l’organisation de nos recherche et dont on pourrait se passer ; c’est certainement vrai dans certains cas. Mais dans d’autres, la pluri­ disciplinarité est essentielle : c’est le sujet qui est plurisdisciplinaire, avant même la manière de le trai­ ter. Un objectif de sélection, par exemple, est fondamentalement pluridisciplinaire. Comment parler de qualité de lait sans penser simul­ tanément au fromage qui en sortira, si ce n’est aux filières commerciales qui s’en suivront et aux habitudes alimentaires qui l’accompagneront. Même si la traduction technique finale (et froide) de toutes ces considérations doit s’exprimer par une simple teneur en sucres ou en protéines, le faisceau de détermi­ J.-M. Legay NATURES - SCIENCES - SOCIÉTÉS, 1993,1 (4)
https://openalex.org/W2612791592
https://www.nepjol.info/index.php/JAIM/article/download/18313/14879
English
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Coexistence of Pulmonary tuberculosis and lung cancer
Journal of advances in internal medicine
2,017
cc-by
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This work is licensed under a Creative Commons Attribution 3.0 Unported License. This work is licensed under a Creative Commons Attribution 3.0 Unported License. tests and Renal function tests were within normal limits. Keywords Pulmonary Tuberculosis can co-exist with lung malignancy masking the under- lying disorder leading to delay in diagnosis and management. Here we present an interesting case of a 72 year old female who on initial presentation was diagnosed with tuberculosisbut later developed plural effusion during treat- ment and on investigation was found to have an underlying lung malignancy. Lung carcinoma,Tuberculosis Sandeep Mahajan,1 Vijay Gotwal,2 Neeraj Lata,3* Nisanth. PS.2 Assistant Professor, Department of chest and Tuberculosis1 Junior Resident, Department of chest and Tuberculosis,2 Senior Resident, Department of Pediatrics,3 Government Medical College, Amritsar Mahajan S, et al. Coexistence of Pulmonary tuberculosis| Case Report Coexistence of Pulmonary tuberculosis and lung cancer Sandeep Mahajan,1 Vijay Gotwal,2 Neeraj Lata,3* Nisanth. PS.2 Assistant Professor, Department of chest and Tuberculosis1 Junior Resident, Department of chest and Tuberculosis,2 Senior Resident, Department of Pediatrics,3 Government Medical College, Amritsar Mahajan S, et al. Coexistence of Pulmonary tuberculosis| Case Report Citation Citation Sandeep Mahajan, Vijay Gotwal, Neeraj Lata, Nisanth. Coexistence of Pulmonary tuberculosis and lung cancer. Journal of Advances in Internal Medicine 2017;06(01):11-13. Introduction:Pulmonary Tuberculosis and lung carcinoma are the two co mor- bidities that have been shown in many studies to coexist. Many of Signs and symptoms of Pulmonary Tuberculosis mimics lung carcinoma and coexistence increases mortality and morbidity.So we are presenting a case which had pul- monary Tuberculosis and later developed carcinoma lung. CASE But symptoms of patient worsened in form that she developed breathing difficulty and loss of appetite.Repeat Xray showed right sided moderate pleural effusion.Ultrasound chest showed moderate pleural effusion on right side and minimal on left side.CTscan with CTguided biopsy was done which showed Mitotic mass in right upper lobe showing mediastinal invasion with moderate degree of right sided pleural effusion ,pleural deposits . Cytology showed High grade malignant spindly cell sarcoma/high grade undifferentiated pleomorphic sarcoma. Antitubercular treatment was continued and Patient was f d t hi h t f t t t f l i CT Scan (Fig :3,4) showed consolidative changes involving right upper lobe with areas of breakdown and soft tissue attenuation within the cavity, with adjacent consolidative changes involving right upper lobe. CT Scan (Fig :3,4) showed consolidative changes involving right upper lobe with areas of breakdown and soft tissue attenuation within the cavity, with adjacent consolidative changes involving right upper lobe. Figure3:CTscan Chest Patient was given broad spectrum antibiotics. Bronchoalveolar lavage was done which had shown Acid fast bacilli positivity.On this basis Antitubercular treatment (4drugs HRZE)was started. But symptoms of patient worsened in form that she developed breathing difficulty and loss of appetite.Repeat Xray showed right sided moderate pleural effusion.Ultrasound chest showed moderate pleural effusion on right side and minimal on left side.CTscan with CTguided biopsy was done which showed Mitotic mass in right upper lobe showing mediastinal invasion with moderate degree of right sided pleural effusion ,pleural deposits . Patient was given broad spectrum antibiotics. Bronchoalveolar lavage was done which had shown Acid fast bacilli positivity.On this basis Antitubercular treatment (4drugs HRZE)was started. But symptoms of patient worsened in form that she developed breathing difficulty and loss of appetite.Repeat Xray showed right sided moderate pleural effusion.Ultrasound chest showed moderate pleural effusion on right side and minimal on left side.CTscan with CTguided biopsy was done which showed Mitotic mass in right upper lobe showing mediastinal invasion with moderate degree of right sided pleural effusion ,pleural deposits . Cytology showed High grade malignant spindly cell sarcoma/high grade undifferentiated pleomorphic sarcoma. Antitubercular treatment was continued and Patient was referred to higher center for treatment of lung carcinoma. JAIM | volume 06 |number 01 | issue 11 | January-June 2017 CASE Chest Xray (Fig:1,2)of patient showed consolidation initially which had progressed to pleural effusion in one month. History: 72 year old female presented with breathlessness and cough for 4 months .She had history of decreased appetite. She is a known case of Diabetes mellitus and hypertension and is on medication for the same. No family history of similar complaints or chronic illness.Past history of hospitalisation in our hospital 2 months back for similar complaints for two weeks.H/o of intake of Antitubercular medication for last two months4 drugs HRZE (DOTS regime). History: 72 year old female presented with breathlessness and cough for 4 months .She had history of decreased appetite. She is a known case of Diabetes mellitus and hypertension and is on medication for the same. No family history of similar complaints or chronic illness.Past history of hospitalisation in our hospital 2 months back for similar complaints for two weeks.H/o of intake of Antitubercular medication for last two months4 drugs HRZE (DOTS regime). Figure1: Chest Xray Physical examination revealed pallor.There was no icterus, cyanosis, clubbing and bony tenderness. Heart rate- 90/min,Respiratory rate 32/min.Respiratory distress present.use of accessory muscles of respiration present. Chest examination:Right side of chest appeared prominent. Breath sounds were decreased on right side of chest in mammary, infraaxillary, infrascapular area Bronchial breath sounds and whispering pectoriloquy were present on right mammary area and axilllary area. Systemic examination was normal. *Corresponding author Dr Neeraj Lata. Senior Resident. Department of Pediatrics. Government Medical College, Amritsar neerajlatavijay@gmail.com On Investigations:Patient had anemia with leucocytosis. Erythrocyte sedimentation rate was raised (64).Liver function page 11 Journal of Advances in Internal Medicine | Case Report Figure2 ChestXray(done after one month) CT Scan (Fig :3,4) showed consolidative changes involving right upper lobe with areas of breakdown and soft tissue attenuation within the cavity, with adjacent consolidative changes involving right upper lobe. Figure3:CTscan Chest Figure2 ChestXray(done after one month) Figure2 ChestXray(done after one month) Figure2 ChestXray(done after one month) Figure4 CT scan Chest Figure4 CT scan Chest Figure4 CT scan Chest Figure4 CT scan Chest Figure4 CT scan Chest Patient was given broad spectrum antibiotics. Bronchoalveolar lavage was done which had shown Acid fast bacilli positivity.On this basis Antitubercular treatment (4drugs HRZE)was started. ABBREVIATIONS HRZE-Isoniazid,Rifampicin,Pyrazinamide,Etambutol DOTS-Directly Observed Treatment Shortterm TB-Tuberculosis HRZE-Isoniazid,Rifampicin,Pyrazinamide,Etambutol DOTS-Directly Observed Treatment Shortterm TB-Tuberculosis 8. Wu CY, Hu HY, Pu CY, Huang N, Shen HC, Li CP, et al. Pulmonary tuberculosis increases the risk of lung cancer: a population-based cohort study. Cancer. 2011;117(3):618-24. CONCLUSION High index of suspicision of coexistence of lung carcinoma and pulmonary tuberculosis should be kept when an old aged patient presents with signs and symptoms of any of these. It has been suggested that inflammation and pulmonary fibrosis caused by tuberculosis can induce genetic damage, which can increase lung carcinoma risk.( 11-13 ) The increased occurrence of Lung carcinoma in patients with tuberculosis can also be related to infection-induced immunosuppression.14    Cancer- induced or chemotherapy-induced immunosuppression can also lead to increased rates of tuberculosis reactivation in patients with solid tumors.15 DISCUSSION: Lung cancer, also known as lung carcinoma,[1] is a malignant lung tumor characterized by uncontrolled cell growth in tissues of the  lung.[2] Worldwide in 2012, lung cancer occurred in 1.8 JAIM | volume 06 |number 01 | issue 11 | January-June 2017 JAIM | volume 06 |number 01 | issue 11 | January-June 2017 page 12 page 12 Mahajan S, et al. Coexistence of Pulmonary tuberculosis| Case Report million people and resulted in 1.6 million deaths.[3] The most common age at diagnosis is 70 years. In 2015, there were an estimated 10.4 million new (incident) TB cases worldwide, of which 5.9 million (56%) were among men, 3.5 million (34%) among women and 1.0 million (10%) among children.4 Our case report showed that Tuberculosis and carcinoma can coexist in lung and requires early institution of treatment.The association between these two diseases is important, since both are quite prevalent and have a major impact on public health. The simultaneous or sequential occurrence of pulmonary tuberculosis and Lung carcinoma in the same patient has been reported in various case series and case-control studies.(5-10) BIBLIOGRAPHY 1. «Lung Carcinoma: Tumors of the Lungs». Merck Manual Professional Edition, Online edition. Retrieved  15 August 2007. 9. Yu YH, Liao CC, Hsu WH, Chen HJ, Liao WC, Muo CH, et al. Increased lung cancer risk among patients with pulmonary tuberculosis: a population cohort study. J Thorac Oncol. 2011;6(1):32-7. 9. Yu YH, Liao CC, Hsu WH, Chen HJ, Liao WC, Muo CH, et al. Increased lung cancer risk among patients with pulmonary tuberculosis: a population cohort study. J Thorac Oncol. 2011;6(1):32-7. 9. 2. 2  “Non-Small Cell Lung Cancer Treatment –Patient Version (PDQ®)”. NCI. May 12, 2015. Retrieved  5 March 2016. 3. World Cancer Report 2014. World Health Organization. 2014. pp. Chapter 5.1. ISBN 92-832-0429-8. 10. Ballaz S, Mulshine JL. The potential contributions of chronic inflammation to lung carcinogenesis.Clin Lung Cancer. 2003;5(1):46- 62. 4. Global tuberculosis report ;2016:WHO 11. Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420(6917):8607. 5. Brenner AV, Wang Z, Kleinerman RA, Wang L, Zhang S, Metayer C, et al. Previous pulmonary diseases and risk of lung cancer in Gansu Province, China. Int J Epidemiol. 2001;30(1):118-24. Dacosta NA, Kinare SG. Association of lung carcinoma and tuberculosis. J Postgrad Med. 1991;37(4):185-9. 12. Engels EA. Inflammation in the development of lung cancer: epidemiological evidence. Expert Rev Anticancer Ther. 2008;8(4):605-15. 13. Rybacka-Chabros B, MaÅ”dziuk S, Berger-Lukasiewicz A, DaÅ”ko-MroziÅ”ska M, Milanowski J. The coexistence of tuberculosis infection and lung cancer in patients treated in pulmonary department of Medical Academy in Lublin during last ten years (1990-2000). Folia Histochem Cytobiol. 2001;39 Suppl 2:73-4. 6. Liang HY, Li XL, Yu XS, Guan P, Yin ZH, He QC, et al. Facts and fiction of the relationship between preexisting tuberculosis and lung cancer risk: a systematic review. Int J Cancer. 2009;125(12):2936-44. 7. Shiels MS, Albanes D, Virtamo J, Engels EA. Increased risk of lung cancer in men with tuberculosis in the alpha- tocopherol, beta-carotene cancer prevention study. Cancer Epidemiol Biomarkers Prev. 2011;20(4):672-8. 14. Kim HR, Hwang SS, Ro YK, Jeon CH, Ha DY, Park SJ, et al. Solid-organ malignancy as a risk factor for tuberculosis. Respirology. 2008;13(3):413-9. JAIM | volume 06 |number 01 | issue 11 | January-June 2017 page 13 page 13
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Disordered breathing in a mouse model of Dravet syndrome
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. CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint Disordered breathing in a mouse model of Dravet syndrome Fu-Shan Kuo, Joseph L. LoTurco, Xinnian Chen, Daniel K. Mulkey Dept. of Physiology and Neurobiology, Univ. of Connecticut, Storrs, CT 06269. Abbreviated Title: loss of SCN1a function disrupts brainstem respiratory control Keywords: SCN1a missense mutation, retrotrapezoid nucleus, brainstem, chemoreception, SUDEP Number of words Abstract: 150 Number of words Main text: 4266 Number of figures: 6 Number of references: 45 Address of Corresponding Author: Daniel K. Mulkey Dept. Physiology and Neurobiology University of Connecticut 75 N. Eagleville Rd. U-3156 Storrs, CT 06269 Email: daniel.mulkey@uconn.edu Phone: 860-486-5700 Fax: 860-486-3303 Disordered breathing in a mouse model of Dravet syndrome Fu-Shan Kuo, Joseph L. LoTurco, Xinnian Chen, Daniel K. Mulkey Shan Kuo, Joseph L. LoTurco, Xinnian Chen, Daniel K. Mulkey Dept. of Physiology and Neurobiology, Univ. of Connecticut, Storrs, CT 06269. Abbreviated Title: loss of SCN1a function disrupts brainstem respiratory control Keywords: SCN1a missense mutation, retrotrapezoid nucleus, brainstem, chemoreception, SUDEP Address of Corresponding Author: Daniel K. Mulkey Dept. Physiology and Neurobiology University of Connecticut 75 N. Eagleville Rd. U-3156 Storrs, CT 06269 Email: daniel.mulkey@uconn.edu Phone: 860-486-5700 Fax: 860-486-3303 1 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint ABSTRACT Dravet syndrome (DS) is a form of epilepsy with a high incidence of sudden unexpected death in epilepsy (SUDEP). Respiratory failure is a leading cause of SUDEP, and DS patients’ frequently exhibit disordered breathing. Despite this, mechanisms underlying respiratory dysfunction in DS are entirely unknown. We found that mice expressing a recurrent SCN1a missense mutation (A1783V) conditionally in inhibitory neurons (SCN1aA1783V/+; mixed C57B/6 background) exhibit spontaneous seizures, die prematurely and present a respiratory phenotype similar to DS patients including hypoventilation, apnea and a diminished ventilatory response to CO2. At the cellular level in the retrotrapezoid nucleus (RTN), we found inhibitory neurons expressing the SCN1a A1783V variant are less excitable, whereas chemosensitive RTN neurons, which are a key source of the CO2/H+-dependent drive to breathe, are hyper-excitable in slices from SCN1aA1783V/+ mice. These results show loss of SCN1a function can disrupt brainstem respiratory control including at the level of the RTN. 2 2 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint Dravet syndrome DS (aka. severe myoclonic epilepsy of infancy) is a severe form of early-onset epilepsy that is resistant to anti-epileptic drugs and has a high incidence of sudden unexpected death in epilepsy (SUDEP) (Kalume 2013; Kearney 2013; Shmuely et al. 2016). The cause of death in DS patients is thought to involve seizure-induced parasympathetic suppression of cardiac activity (Kearney 2013; Kalume et al. 2013; Gataullina and Dulac 2017). However, recent evidence suggests that respiratory dysfunction contributes to SUDEP associated with DS, as patients exhibit breathing problems including hypoventilation and apnea prior to the manifestation of bradycardia, a slower than normal heart rate (Kim et al., 2018). Patients with DS also exhibit a blunted ventilatory response to CO2 (Kim et al., 2018). ABSTRACT 2013), and conditional deletion of SCN1a from forebrain inhibitory neurons results in a DS-like phenotype similar to global SCN1a deletion (Cheah et al. 2012). For these reasons, most studies have used global or inhibitory neuron-specific SCN1a deletions to model DS (Catterall WA. 2012), with few studies focusing on other high-priority genetic risk factors like SCN1a missense mutations, which represent ~40% of DS-associated mutations (Depienne et al., 2009; Parihar and Ganesh 2013). Thus, the extent to which expression of SCN1a loss-of-function mutations recapitulate features of DS remains unclear. Furthermore, despite the lethality associated with SCN1a mutations, nothing is known regarding how loss of SCN1a affects brainstem respiratory centers. this, conventional SCN1a knockout mouse models (in a mixed C57B/6 background) recapitulate characteristic features of DS, including motor problems, seizures and premature death, in a remarkably titratable manner. For example, homozygous SCN1a knockout mice develop ataxia and die at 15 days postnatal, whereas heterozygous SCN1a deficient mice show seizure activity and early mortality starting at 3 weeks of age (Yu et. al., 2006; Ogiwara et al., 2007). The cellular basis for many features of DS including seizures and premature death appears to involve disinhibition, as global deletion of SCN1a suppresses activity of inhibitory but not excitatory neurons in the cortex and hippocampus (Yu et al. 2006; Dutton et al. 2013), and conditional deletion of SCN1a from forebrain inhibitory neurons results in a DS-like phenotype similar to global SCN1a deletion (Cheah et al. 2012). For these reasons, most studies have used global or inhibitory neuron-specific SCN1a deletions to model DS (Catterall WA. 2012), with few studies focusing on other high-priority genetic risk factors like SCN1a missense mutations, which represent ~40% of DS-associated mutations (Depienne et al., 2009; Parihar and Ganesh 2013). Thus, the extent to which expression of SCN1a loss-of-function mutations recapitulate features of DS remains unclear. Furthermore, despite the lethality associated with SCN1a mutations, nothing is known regarding how loss of SCN1a affects brainstem respiratory centers. The main goal of this study was to provide the first detailed characterization of breathing in a SCN1a missense mutation mouse model of DS. We modeled DS by expressing a loss-of- function missense mutation (A1783V) conditionally in inhibitory neurons (referred to as SCN1aA1783V/+ mice). The A1783V variant is a recurrent DS mutation (Marini et al. 2007, Lossin 2009, Klassen et al. 2014) predicted to result in loss of function by increasing Nav1.1 voltage- dependent inactivation. ABSTRACT This finding suggests that respiratory dysfunction, possibly at the level of respiratory chemoreceptors (neurons that regulate breathing in response to changes in tissue CO2/H+), contributes to the pathology of DS. Despite this, mechanisms underlying respiratory dysfunction in DS or epilepsy in general are entirely unknown. Leading hypothesis propose that seizure activity disrupts respiratory control by a feed-forward mechanisms involving spreading depolarization (Aiba and Noebels 2015) or activation of inhibitory subcortical projections to brainstem respiratory centers (Dlouhy et. al., 2015; Lacuey et al., 2017); however, a yet unexplored additional possibility is that epilepsy- associated mutations may directly affect brainstem respiratory centers and serve as a common substrate for both seizure and respiratory dysfunction. Most DS cases (70-95%) are caused by mutations in the SCN1a gene (MIM#182389), which encodes the pore-forming subunit of a voltage-gated Na+ channel (Nav1.1) (Meisler and Kearney 2005; Fujiwara 2006; Catterall et al. 2010; Akiyama et al. 2012). Approximately 700 different SCN1a mutations have been identified in DS patients, the majority of which are missense or frameshift mutations that result in loss of function (Parihar and Ganesh 2013). Consistent with 3 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint this, conventional SCN1a knockout mouse models (in a mixed C57B/6 background) recapitulate characteristic features of DS, including motor problems, seizures and premature death, in a remarkably titratable manner. For example, homozygous SCN1a knockout mice develop ataxia and die at 15 days postnatal, whereas heterozygous SCN1a deficient mice show seizure activity and early mortality starting at 3 weeks of age (Yu et. al., 2006; Ogiwara et al., 2007). The cellular basis for many features of DS including seizures and premature death appears to involve disinhibition, as global deletion of SCN1a suppresses activity of inhibitory but not excitatory neurons in the cortex and hippocampus (Yu et al. 2006; Dutton et al. ABSTRACT Consistent with other DS models (Yu et al. 2006; Kalume et al. 2013; Kim et al. 2018), we found that SCN1aA1783V/+ mice exhibited spontaneous seizure activity and 4 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint premature death starting at ~2 weeks of age, confirming this is a model of SUDEP in DS. At this same developmental time point, SCN1aA1783V/+ mice hypoventilated, exhibited frequent apneas under baseline conditions and showed a reduced ventilatory response to CO2, thus recapitulating the respiratory phenotype exhibited by DS patients (Kim et al. 2018). At the cellular level in a key brainstem respiratory chemoreceptor region known as the retrotrapezoid nucleus (RTN), we found that inhibitory neurons expressing A1783V show less spontaneous activity and a diminished ability to maintain firing during sustained depolarization. This is consistent with the possibility that A1783V increases Nav1.1 channel inactivation. Also consistent with a brainstem disinhibition mechanism, we found that basal activity and CO2/H+-sensitivity of excitatory chemosensitive RTN neurons was enhanced in slices from SCN1aA1783V/+ mice. These results show that RTN chemoreceptor function is altered in this DS model and may contribute to premature death. + mice have spontaneous seizures and die prematurely SCN1aA1783V/+ mice were born in the expected ratios, were viable, and by ~15 days postnatal, were similar in terms of body weight (T46 = 1.62, p = 0.11) and temperature (T26 =0.77, p = 0.44) as their SCN1a+/+ littermates (Figures 1A-C). However, SCN1aA1783V/+ pups showed seizure-like behavior by ~2 weeks of age (Table 1). More specifically, based on the Racine seizure-behavior scoring paradigm, only 22.7% of control mice (N = 22) showed seizure-like behavior, which mainly manifested as head-bobbing (category 1). By contrast, 77.3% of SCN1aA1783V mice (N = 22) showed severe seizure behavior, including forelimb tremor (category 3), rearing alone (category 4) or in conjunction with falling over, and full-body tonic-clonic seizure (category 5). Unlike control animals, several of the mutant mice exhibited behavioral arrest that we consider to be absence seizure-like activity. In conjunction with seizure-like behavior, SCN1aA1783V mice also started dying unexpectedly, reaching 100% lethality by 23 days postnatal (Figure 1D). VGAT+ cells (T172 = 0.142, p = 0.88). We also observed numerous bright fluorescent puncta, which corresponded to SCN1a transcript in the soma of VGAT+ cells and, to a lesser extent, in VGLUT2+ cells in slices from control mice (F3,321 = 24.07, p < 0.0001). In slices from SCN1aA1783V/+ mice, we found a modest reduction in SCN1a transcript in VGAT+ but not VGLUT2+ cells (F3,321 = 24.07, p < 0.05; see Figures 1E-F). This result suggests this mutation may compromise channel expression. In a separate experiment to validate cell-type-specific Cre expression, we crossed VGATcre mice with a Rosa26TdTomato reporter line and found that all tdT- labeled cells expressed VGAT, but not VGLUT2, mRNA (not shown). These results suggest inhibitory neurons from SCN1aA1783V/+ mice express SCN1a transcript, albeit at modestly reduced levels. Therefore, given that heterozygous deletion mutations can give rise to severe forms of DS (Yu et al. 2006, Miller et al. 2014), we expected SCN1aA1783V/+ mice to exhibit a mild epilepsy-like phenotype. Contrary to this expectation, SCN1aA1783V/+ mice exhibited a severe SUDEP-like phenotype. SCN1aA1783V/+ mice were born in the expected ratios, were viable, and by ~15 days postnatal, were similar in terms of body weight (T46 = 1.62, p = 0.11) and temperature (T26 =0.77, p = 0.44) as their SCN1a+/+ littermates (Figures 1A-C). However, SCN1aA1783V/+ pups showed seizure-like behavior by ~2 weeks of age (Table 1). + mice have spontaneous seizures and die prematurely We first sought to determine whether inhibitory neurons from SCN1aA1783V/+ mice express SCN1a channel transcript. We prepared brainstem sections containing the RTN from control and SCN1a A1783V/+ mice (15 days old) for subsequent fluorescent in situ hybridization using probes for 1) SCN1a, which does not distinguish SCN1a channel variants; 2) vesicular GABA transporter (VGAT) to identify GABAergic and glycinergic inhibitory neurons; and 3) vesicular glutamate transporter 2 (VGLUT2) to identify excitatory glutamatergic neurons, including chemo-sensitive RTN neurons. We labeled all cell nuclei with DAPI. Inhibitory VGAT+ cells were present in the RTN region and were in close proximity to excitatory VGLUT2+ neurons (i.e., putative RTN chemoreceptors). Both genotypes showed similar relative distributions of 5 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint VGAT+ cells (T172 = 0.142, p = 0.88). We also observed numerous bright fluorescent puncta, which corresponded to SCN1a transcript in the soma of VGAT+ cells and, to a lesser extent, in VGLUT2+ cells in slices from control mice (F3,321 = 24.07, p < 0.0001). In slices from SCN1aA1783V/+ mice, we found a modest reduction in SCN1a transcript in VGAT+ but not VGLUT2+ cells (F3,321 = 24.07, p < 0.05; see Figures 1E-F). This result suggests this mutation may compromise channel expression. In a separate experiment to validate cell-type-specific Cre expression, we crossed VGATcre mice with a Rosa26TdTomato reporter line and found that all tdT- labeled cells expressed VGAT, but not VGLUT2, mRNA (not shown). These results suggest inhibitory neurons from SCN1aA1783V/+ mice express SCN1a transcript, albeit at modestly reduced levels. Therefore, given that heterozygous deletion mutations can give rise to severe forms of DS (Yu et al. 2006, Miller et al. 2014), we expected SCN1aA1783V/+ mice to exhibit a mild epilepsy-like phenotype. Contrary to this expectation, SCN1aA1783V/+ mice exhibited a severe SUDEP-like phenotype. + mice have spontaneous seizures and die prematurely 2018), we consider the SCN1aA1783V/+ mouse model to be useful for dissecting the mechanisms that underlie respiratory failure in DS. To determine whether SCN1aA1783V/+ mice exhibit abnormal brain activity, we obtained electrocorticogram (ECoG) recordings from control and SCN1aA1783V/+ mice. We allowed mice 12 hours to recover after implanting them with the ECoG head stage. We continuously recorded ECoGs over a two-hour period, between the hours of 9:00 AM – 2:00 PM. Consistent with frequent polyspike activity observed in the ECoG recordings of DS patients (Bender et al., 2012), SCN1aA1783V/+ mice showed spontaneous high-amplitude spike-wave discharges (SWD) that occurred at a frequency of 0.37 ± 0.05/min (T10 = 3.009, p < 0.01) and had an average duration of 12.4 ± 5.5 s (Figs. 2A-B; T10 = 2.268, p < 0.05). Conversely, control animals showed SWD events less frequently (0.13 ± 0.1/min) and with shorter durations (6.7 ± 2.2s) compared to SCN1aA1783V/+ mice (Figure 2A; T9 = 2.4, p > 0.05). Power spectral analysis of SCN1aA1783V/+ SWD events showed increases in both alpha and beta frequency (F 4, 840 = 5.605, p < 0.001). These results suggest SCN1aA1783V/+ mice have frequent, spontaneous seizures and die prematurely (Figure 1D). Because this SUDEP-like phenotype is virtually identical to other DS models, including global and inhibitory neuron-specific SCN1a haplo-insufficient models (Yu et al. 2006, Kalume et al. 2013, Kim et al. 2018), we consider the SCN1aA1783V/+ mouse model to be useful for dissecting the mechanisms that underlie respiratory failure in DS. + mice have spontaneous seizures and die prematurely More specifically, based on the Racine seizure-behavior scoring paradigm, only 22.7% of control mice (N = 22) showed seizure-like behavior, which mainly manifested as head-bobbing (category 1). By contrast, 77.3% of SCN1aA1783V mice (N = 22) showed severe seizure behavior, including forelimb tremor (category 3), rearing alone (category 4) or in conjunction with falling over, and full-body tonic-clonic seizure (category 5). Unlike control animals, several of the mutant mice exhibited behavioral arrest that we consider to be absence seizure-like activity. In conjunction 6 6 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint To determine whether SCN1aA1783V/+ mice exhibit abnormal brain activity, we obtained electrocorticogram (ECoG) recordings from control and SCN1aA1783V/+ mice. We allowed mice 12 hours to recover after implanting them with the ECoG head stage. We continuously recorded ECoGs over a two-hour period, between the hours of 9:00 AM – 2:00 PM. Consistent with frequent polyspike activity observed in the ECoG recordings of DS patients (Bender et al., 2012), SCN1aA1783V/+ mice showed spontaneous high-amplitude spike-wave discharges (SWD) that occurred at a frequency of 0.37 ± 0.05/min (T10 = 3.009, p < 0.01) and had an average duration of 12.4 ± 5.5 s (Figs. 2A-B; T10 = 2.268, p < 0.05). Conversely, control animals showed SWD events less frequently (0.13 ± 0.1/min) and with shorter durations (6.7 ± 2.2s) compared to SCN1aA1783V/+ mice (Figure 2A; T9 = 2.4, p > 0.05). Power spectral analysis of SCN1aA1783V/+ SWD events showed increases in both alpha and beta frequency (F 4, 840 = 5.605, p < 0.001). These results suggest SCN1aA1783V/+ mice have frequent, spontaneous seizures and die prematurely (Figure 1D). Because this SUDEP-like phenotype is virtually identical to other DS models, including global and inhibitory neuron-specific SCN1a haplo-insufficient models (Yu et al. 2006, Kalume et al. 2013, Kim et al. ventilatory response Recent evidence (Kim et al., 2018) showed that DS patients have post-ictal respiratory abnormalities, including hypoventilation, apnea and impaired CO2 chemoreception. These symptoms can last for several hours after seizure, which indicates that respiratory problems contribute to SUDEP in DS. Therefore, to determine whether SCN1aA1783V mice exhibit 7 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint respiratory problems, we used a whole-body plethysmography to measure baseline breathing and the ventilatory response to CO2 in 15-day-old control and SCN1aA1783V mice. We found that compared to control mice, SCN1aA1783V mice showed a diminished respiratory output under room air conditions. Specifically, SCN1aA1783V exhibited suppressed frequency (246 ± 15.3 bpm for controls compared to 199.5 ± 8.4 bpm for SCN1aA1783V/+; T25 = 2.665; p < 0.05); tidal volume (13.8 ± 2.0 µl/g for controls compared to 7.3 ± 1.7 µl/g for SCN1aA1783V/+, T37 = 2.351, p < 0.05); and minute ventilation (3.4 ± 0.5 µl/min/g for controls compared to 1.8 ± 0.4 µl/min/g for SCN1aA1783V/+; T37 = 2.173, p < 0.05; see Figures 3A-D for all). Although both genotypes exhibited apneic events at similar frequencies (0.23 ± 0.1/min for control and 0.11 ± 0.04/min for SCN1aA1783V/+;; p = 0.6), the duration of these events were longer in SCN1aA1783V/+ mice (Figure 3E; 1,104 ± 58.6 ms for controls versus 1,350 ± 99.2 ms SCN1aA1783V/+;; T51 = 2.135; p < 0.05). We also found that SCN1aA1783V/+ mice had a diminished capacity to increase respiratory frequency in response to graded increases in CO2 (Figure 3F). Specifically, respiratory frequency in 7% CO2 (balance O2) was higher in controls (363.1 ± 7.7 bpm; N = 22) versus SCN1aA1783V/+ (300.7 ± 17.4 bpm; N = 17; F1,37 = 5.69, p < 0.05). ventilatory response Although tidal volume responses to CO2/H+ are similar between genotypes (p = 0.47), total respiratory output, as measured by minute ventilation— the product of respiratory frequency and tidal volume—was diminished in SCN1aA1783V/+ mice compared to controls (F3,111 = 3.167, p < 0.05; Figures 3G-H). Specifically, increasing inspired CO2 from 0% to 3% increased minute ventilation in control mice by 3.3 ± 0.5 µl/min/g (p < 0.0001). These same conditions, however, led to a much smaller and non-significant increase in minute ventilation among SCN1a A1783V/+ mice (increase of 1.5 ± 0.5 µl/min/g; p = 0.07). These results show that SCN1aA1783V/+ mice exhibit a respiratory phenotype similar to that observed in DS patients, and further supports the possibility that respiratory problems may contribute to mortality in this DS model. respiratory problems, we used a whole-body plethysmography to measure baseline breathing and the ventilatory response to CO2 in 15-day-old control and SCN1aA1783V mice. We found that compared to control mice, SCN1aA1783V mice showed a diminished respiratory output under room air conditions. Specifically, SCN1aA1783V exhibited suppressed frequency (246 ± 15.3 bpm for controls compared to 199.5 ± 8.4 bpm for SCN1aA1783V/+; T25 = 2.665; p < 0.05); tidal volume (13.8 ± 2.0 µl/g for controls compared to 7.3 ± 1.7 µl/g for SCN1aA1783V/+, T37 = 2.351, p < 0.05); and minute ventilation (3.4 ± 0.5 µl/min/g for controls compared to 1.8 ± 0.4 µl/min/g for SCN1aA1783V/+; T37 = 2.173, p < 0.05; see Figures 3A-D for all). Although both genotypes exhibited apneic events at similar frequencies (0.23 ± 0.1/min for control and 0.11 ± 0.04/min for SCN1aA1783V/+;; p = 0.6), the duration of these events were longer in SCN1aA1783V/+ mice (Figure 3E; 1,104 ± 58.6 ms for controls versus 1,350 ± 99.2 ms SCN1aA1783V/+;; T51 = 2.135; p < 0.05). We also found that SCN1aA1783V/+ mice had a diminished capacity to increase respiratory frequency in response to graded increases in CO2 (Figure 3F). Specifically, respiratory frequency in 7% CO2 (balance O2) was higher in controls (363.1 ± 7.7 bpm; N = 22) versus SCN1aA1783V/+ (300.7 ± 17.4 bpm; N = 17; F1,37 = 5.69, p < 0.05). Although tidal volume responses to CO2/H+ are similar between genotypes (p = 0.47), total respiratory output, as measured by minute ventilation— the product of respiratory frequency and tidal volume—was diminished in SCN1aA1783V/+ mice compared to controls (F3,111 = 3.167, p < 0.05; Figures 3G-H). Disinhibition and altered RTN chemoreception may underlie breathing problems in SCN1a mice The RTN is an important respiratory center and disrupting CO2/H+-sensitive cells in this region results in a respiratory phenotype similar to SCN1aA1783V/+ mice (Figure 3). Evidence also suggests that inhibitory neurons in the RTN region contribute to respiratory drive (Ott et al. 2011). We therefore sought to determine whether loss of SCN1a function in inhibitory neurons decreases inhibitory neuron activity and disinhibits excitatory, chemosensitive, neurons. To facilitate the identification of inhibitory neurons, we crossed VGATcre mice with the Rosa26TdTomato reporter line. We crossed the resulting offspring with floxed-stop SCN1aA1783V mice. VGAT+ cells that undergo this recombination express tdT and the A1783V variant (VGATtdT/+:SCN1aA1783V/+). Consistent with other SCN1a knockout (Tai et al. 2014) or missense knockin (Ogiwara et al. 2007, Mashimo et al. 2010, Hedrich et al. 2014) DS models, the loss of SCN1a function in inhibitory neurons suppressed inhibitory neural activity. We performed whole-cell current clamp recordings of inhibitory neurons in the RTN region in VGATtdT/+ slices, which yielded the following results: the inhibitory neurons of SCN1aA1783V/+ mice showed lower basal activity than those of controls (14.39 ± 1.5 Hz for controls vs. 9.902 ± 0.64 Hz for SCN1aA1783V/+; T60 = 2.97, p < 0.01; Figures 4A-B). Furthermore, SCN1aA1783V/+ inhibitory neurons fired fewer action potentials in response to depolarizing current steps (0-300 pA; ∆ 20 pA) from a holding potential of -80 mV. This activity deficit became more pronounced during large (200-300 pA) sustained (1,000 ms) current injections where inhibitory neurons expressing SCN1aA1783V/+ showed pronounced spike amplitude and frequency decrement (Figures 4A and D). That is, the number of spikes elicited by a +300 pA current step (1,000 ms) was 53.7 ± 11 for controls (N = 13) compared to 13.9 ± 6.4 for SCN1aA1783V/+ (N = 20; F15,465 = The RTN is an important respiratory center and disrupting CO2/H+-sensitive cells in this region results in a respiratory phenotype similar to SCN1aA1783V/+ mice (Figure 3). Evidence also suggests that inhibitory neurons in the RTN region contribute to respiratory drive (Ott et al. 2011). We therefore sought to determine whether loss of SCN1a function in inhibitory neurons decreases inhibitory neuron activity and disinhibits excitatory, chemosensitive, neurons. To facilitate the identification of inhibitory neurons, we crossed VGATcre mice with the Rosa26TdTomato reporter line. We crossed the resulting offspring with floxed-stop SCN1aA1783V mice. VGAT+ cells that undergo this recombination express tdT and the A1783V variant (VGATtdT/+:SCN1aA1783V/+). ventilatory response Specifically, increasing inspired CO2 from 0% to 3% increased minute ventilation in control mice by 3.3 ± 0.5 µl/min/g (p < 0.0001). These same conditions, however, led to a much smaller and non-significant increase in minute ventilation among SCN1a A1783V/+ mice (increase of 1.5 ± 0.5 µl/min/g; p = 0.07). These results show that SCN1aA1783V/+ mice exhibit a respiratory 8 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint Disinhibition and altered RTN chemoreception may underlie breathing problems in Consistent with other SCN1a knockout (Tai et al. 2014) or missense knockin (Ogiwara et al. 2007, Mashimo et al. 2010, Hedrich et al. 2014) DS models, the loss of SCN1a function in inhibitory neurons suppressed inhibitory neural activity. We performed whole-cell current clamp recordings of inhibitory neurons in the RTN region in VGATtdT/+ slices, which yielded the following results: the inhibitory neurons of SCN1aA1783V/+ mice showed lower basal activity than those of controls (14.39 ± 1.5 Hz for controls vs. 9.902 ± 0.64 Hz for SCN1aA1783V/+; T60 = 2.97, p < 0.01; Figures 4A-B). Furthermore, SCN1aA1783V/+ inhibitory neurons fired fewer action potentials in response to depolarizing current steps (0-300 pA; ∆ 20 pA) from a holding potential of -80 mV. This activity deficit became more pronounced during large (200-300 pA) sustained (1,000 ms) current injections where inhibitory neurons expressing SCN1aA1783V/+ showed pronounced spike amplitude and frequency decrement (Figures 4A and D). That is, the number of spikes elicited by a +300 pA current step (1,000 ms) was 53.7 ± 11 for controls (N = 13) compared to 13.9 ± 6.4 for SCN1aA1783V/+ (N = 20; F15,465 = 9 9 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint 9.536; p < 0.0001). We also found that inhibitory neurons from each genotype had similar input resistance (517.6 ± 82.2 MΩ for control vs. 519.2±38.9 MΩ for SCN1aA1783V/+; T25 = 0.02; p = 0.3; Figure 4C). These results suggest that expression of SCN1aA1783V/+ in brainstem inhibitory neurons reduces spontaneous activity and the neuron’s ability to respond to a wide range of excitatory inputs. The A1783V mutation is located in the S6 segment of domain 4 (Marini, Mei et al. 2007, Lossin 2009), a region thought to regulate voltage-dependent inactivation (Catterall WA 2000). Disinhibition and altered RTN chemoreception may underlie breathing problems in Given that inhibitory neurons that express A1783V show reduced excitability, we hypothesized that the SCN1a A1783V variant results in loss of function by causing Nav1.1 channels to inactivate at more negative voltages. Consistent with this hypothesis, when examining spontaneous action potentials (as measured under resting conditions with a 0 pA holding current) in inhibitory neurons in slices of SCN1aA1783V/+ and control mice, the latter showed a higher amplitude (73.5 ± 1.9 mV) than SCN1aA1783V/+ (61.9 ± 2.6 mV; F1,95 = 9.931, p < 0.001). The maximum rate of depolarization was higher for controls (134.4 ± 5.0 mV/mS) compared to SCN1aA1783V/+ (89.4 ± 5.3 mV/mS; F1,96 = 35.2, p < 0.0001; see Figures 5A, D-F). Action potential threshold was also higher in inhibitory neurons in slices from SCN1aA1783V/+ (-29.2 ± 0.9 mV) compared to controls (-32.4 ± 0.6 mV; F1,95 = 7.403, p < 0.05; Figures 5A, F). Next, we characterized the properties of the first action potential elicited after holding cells at potentials that either remove or enhance Nav1.1 channel inactivation. We found that differences in action potential waveform properties between genotypes were minimized when we held cells at a negative voltage to remove Na+ channel inactivation. For example, holding inhibitory neurons expressing SCN1aA173V/+ at a negative pre-potential by injecting a hyperpolarizing 10 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint current (-100 pA; 1,000 ms) increased action potential amplitude (78.01 ± 2.0 mV SCN1aA1783V/+; F1, 95 = 9.931, p < 0.0001) to an amount similar to spikes from control cells (81.05 ± 1.2 mV control; p = 0.83) (Figures 5B, E). Disinhibition and altered RTN chemoreception may underlie breathing problems in It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint considered neurons that were spontaneously active in 5% CO2 and responded to 10% CO2 with at least a 1.0 Hz increase in firing rate to be chemosensitive. Chemosensitive RTN neurons also have been shown to express the transcription factor Phox2b; therefore, at the end of each experiment we filled all recorded cells with Lucifer yellow for later immunohistochemical confirmation of Phox2b expression. Chemosensitive RTN neurons in slices from control mice had an average basal activity of 1.3 ± 0.4 Hz under control conditions (5% CO2; pH 7.3). These cells were strongly inhibited by decreasing CO2 to 3% (pHo = 7.6) (1.02 ± 0.3 Hz) and showed a linear firing increase in response to 7% (pHo = 7.2) (2.4 ± 0.5 Hz) and 10% CO2 (pHo = 7.0) (2.8 ± 0.4 Hz) (Figures 6A, B-C). This CO2 response profile is consistent with type I chemoreceptors (pH50= 7.3), which were described previously in a Phox2b mouse reporter line (Lazarenko et al. 2009). Consistent with our hypothesis, chemosensitive RTN neurons in slices from SCN1aA1783V/+ mice were more active under control conditions (5% CO2) (2.4 ± 0.35 Hz) (Figure 6C) (T21=2.223, p < 0.05) and showed an enhanced firing response to high CO2/H+ (Figure 6D) (slope: 0.3 ± 0.01 control vs. 0.37 ± 0.01, F1,4 =8.04, p < 0.05). These results show that loss of SCN1a function in inhibitory neurons disrupts activity of RTN chemoreceptors. Disinhibition and altered RTN chemoreception may underlie breathing problems in Under these conditions, the maximum rate of depolarization also increased 140.3 ± 5.441 mV/ms (F 1, 96 = 35.21, p < 0.0001) (Figures 5B, D); this rate was similar to that measured in spontaneous spikes from control animals (p > 0.99) but slower than spikes from control cells following a negative pre-potential (166.6 ± 7.3 mV/mS, F1,96 = 35.21, p < 0.05). Holding inhibitory neurons expressing SCN1aA1783V/+ at a negative pre-potential also lowered the threshold for spike initiation (-35.68 ± 0.7 mV SCN1aA1783V/+; F1, 95 = 7.403, p<0.001) to a level similar to control cells (-37.2 ± 1.2 mV control; F1, 95 = 7.403, p = 0.06) (Figures 5B, F). We also found that delivering a +180 pA current for 1,000 ms to enhance Na+ channel inactivation in control cells resulted in similar action potential amplitude (F1, 95 = 9.931, p > 0.99), rate of depolarization (F 1, 96 = 35.21, p = 0.58) and spike threshold (F1, 95 = 7.403, p = 0.97) as spikes measured in inhibitory neurons from SCN1aA1783V/+ slices under resting conditions (holding current = 0 pA) (Figures 5C, D-F). Together, these results are consistent with the possibility that the SCN1a A1783V mutation results in loss of function due to enhanced Nav1.1 inactivation. Previous evidence suggests that inhibitory neurons in the RTN region regulate activity of chemosensitive neurons (Ott et al. 2011). Because expression of SCN1aA1783V suppresses activity of inhibitory neurons in the RTN region (Figures 4 and 5), we predict that loss of inhibitory tone would enhance basal activity and CO2/H+ sensitivity of excitatory chemosensitive neurons. To test this, we characterized the firing activity of chemosensitive RTN neurons in slices from each genotype during exposure to CO2 levels ranging from 3 to 10%. We initially identified chemosensitive RTN neurons in each genotype by their firing response to CO2. We 11 11 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. DISCUSSION Epilepsy patients have a 40-fold higher mortality rate than the general population (Dlouhy et al. 2016). The most common cause of death for this patient population is SUDEP, a leading cause of which is respiratory failure (Surges et al. 2009, Ryvlin et al. 2013, Kennedy and Seyal 2015, Dlouhy et al. 2016). However, the mechanisms underlying respiratory dysfunction in epilepsy and SUDEP are largely unknown. This is particularly true in the context of DS, where patients have an exceedingly high mortality rate and commonly exhibit life-threatening respiratory problems (Kim et al. 2018), yet nothing is known regarding how loss of SCN1a function impacts 12 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint brainstem respiratory centers. The results presented here address this knowledge gap by showing that expression of the recurrent DS-associated SCN1a variant A1783V in inhibitory neurons results in seizures and pre-mature death (Figures 1D, 2). Moreover, this mouse model presents with a respiratory phenotype strikingly similar to that exhibited by DS patients. Perhaps not surprising, we found that loss of SCN1a function in inhibitory neurons in the RTN diminished activity in a cell-autonomous manner but, importantly, also enhanced baseline activity and CO2/H+ sensitivity of excitatory chemosensitive neurons. These results suggest that disruption of SCN1a in inhibitory neurons can impact brainstem respiratory centers and contribute to pathological features of DS including disordered breathing associated with SUDEP By ~ 2 weeks of age, SCN1aA1783V mice exhibited a respiratory phenotype similar to DS patients including hypoventilation, increased apneas and diminished ventilatory response to CO2/H+. These breathing problems occurred in conjunction with a marked increase in mortality, thus correlatively supporting the possibility that respiratory failure contributes to premature death in DS. DISCUSSION Although the mechanisms contributing to respiratory dysfunction in DS are unknown, previous work showed that loss of SCN1a from inhibitory neurons in the forebrain, but not the brainstem where respiratory control centers are located, resulted in premature death (Cheah et al. 2012). These results are consistent with the possibly that respiratory dysfunction is a secondary consequence of cortical seizure activity propagating to and disrupting brainstem function. There are numerous direct and indirect projections from the cortex to brainstem respiratory centers (Shea 1996) that may serve as the anatomical substrate for seizure-induced respiratory dysfunction. For example, recent work in humans showed that apnea and arterial oxygen desaturation occurred when cortical seizure activity spread to the amygdala (Dlouhy et al. 2015) 13 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint and presumably activated descending inhibitory projections to brainstem respiratory centers. However, SUDEP can also occur in epilepsy patients in the absence of an overt seizure or outside the peri-ictal period (Lhatoo and Shorvon 1998), suggesting that factors other than acute seizure predispose individuals to SUDEP. For example, it is possible that repeated bombardment of brainstem respiratory centers by frequent cortical seizure events alters cellular or neural network function, leading to progressive respiratory disruption and increased SUDEP propensity. Consistent with this possibility, patients with temporal lobe epilepsy (a common type of focal epilepsy) show widespread alterations in neural network activity including at the level of the brainstem (Englot et al. 2018). However, it remains unclear whether elements of respiratory control are compromised by repeated seizure activity in a similar manner. Our results show that SCN1a transcript is highly expressed by brainstem inhibitory neurons and to a lesser extent by glutamatergic neurons (Figures 1E-F); therefore, loss of SCN1a function is likely to directly impact brainstem inhibitory neurons independent of descending seizure activity. DISCUSSION Consistent with this possibility and analogous to cortical inhibitory neurons in SCN1a-/+ knockout models (Cheah et al. 2012) and induced pluripotent stem cells derived from DS patients with an SCN1a truncation mutation (Higurashi et al. 2013), we found that inhibitory neurons in the RTN region expressed the SCN1a A1783V variant produced fewer action potentials in response to sustained depolarizing current injection and were more prone to depolarization block compared to inhibitory neurons from control mice (Figures 4A-D). These results suggest that loss of SCN1a might suppress inhibitory tone in brainstem respiratory centers including the RTN where inhibitory neurons appear to interact with and regulate the activity of excitatory chemosensitive neurons (Ott et al. 2011). 14 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint We confirmed this possibility at the cellular level by showing that baseline activity and CO2/H+- dependent output of RTN chemoreceptors in slices from SCN1aA1783V/+ mice were enhanced compared to RTN chemoreceptors in slices from control mice, thus demonstrating that RTN chemoreceptor function is altered in this DS model. However, because chemosensitive RTN neurons provide an excitatory drive to breathe (Guyenet et al. 2016), we would predict that disinhibition of these neurons would potentiate rather than suppress respiratory function as observed in DS patients (Kim et al. 2018) and SCN1aA1783V/+ mice (Figure 3). Therefore, it is likely that other respiratory elements also contribute to the observed respiratory phenotype. In particular, inhibitory signaling within the pre-Bötzinger complex (pre-BötC) – a brainstem region downstream of the RTN that regulates inspiratory rhythm – is required for rapid breathing; Studies have reported that disrupting inhibitory neuromodulation within this region prolongs the post-burst refractory period exhibited by pre-BötC inspiratory neurons, resulting in reduced respiratory frequency (Baertsch et al. 2018). DISCUSSION Therefore, it is reasonable to speculate that global loss of SCN1a function could overactivate pre-BötC inspiratory neurons and increase the inspiratory burst refractory period, thus slowing respiratory frequency. Furthermore, as chemosensitive RTN neurons send excitatory glutamatergic projections directly to inspiratory pre-BötC neurons, disinhibition at the level of the RTN would likely further compromise inspiratory output by the pre-BötC. Despite the prevalence of SCN1a missense mutations in DS (Parihar and Ganesh 2013), few studies have characterized the pathophysiology associated with specific mutant alleles. This is particularly important for the development of patient-directed therapies because the aberrant products of SCN1a missense mutations are potentially expressed, thus representing a novel therapeutic target that is absent from haploinsufficient models of DS. Here, we show that 15 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint expression of a DS-associated missense mutation (A1783V) in inhibitory neurons results in seizures and premature death on a similar time scale as haploinsufficient DS models (Caterall WA 2012). Inhibitory neurons from SCN1aA1783V/+ mice showed a modest reduction in channel transcript that may contribute to loss of inhibitory tone; however, the repetitive firing characteristics of SCN1aA1783V/+-expressing neurons was more consistent with loss of function due to increased Nav1.1 channel inactivation. For example, genotype differences in the action potential amplitude and rate of depolarization were diminished under experimental conditions designed to remove Na+ channel inactivation. Therefore, an effective treatment for SCN1a A1783V/+ -associated pathology could be to selectively potentiate Nav1.1 channel activity by slowing voltage-dependent inactivation. This approach, using a spider venom called heteroscodratoxin-1 (Hm1a), has been show to decrease seizures and mortality in an SCN1a haploinsufficient model of DS, albeit at a concentration well above the Hm1a EC50 of Nav1.1 channels (Richards et al., 2018). DISCUSSION CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint ACKNOWLEDGEMENTS We thank Drs. Ana Mingorance (Chief Development Officer of the Loulou Foundation) and Anastasios Tzingounis (Dept. Physiology and Neurobiology, Univ. Connecticut) for their constructive suggestions regarding this project. This work was supported by funds from the National Institutes of Health Grants HL104101 (DKM), HL137094 (DKM) and NS104999 (JLL). Additional funds were also provided by the Dravet Foundation Grant AG180243 (DKM) and American Epilepsy Society (F-SK). AUTHOR CONTRIBUTIONS F-SK: experimental design; collection and analysis of data; revising the manuscript; final approval of the manuscript. JJL: experimental design; revising the manuscript; final approval of the manuscript. JJL: experimental design; revising the manuscript; final approval of the manuscript. JJL: experimental design; revising the manuscript; final approval of the manuscript. XC: data analysis; revising the manuscript, final approval of the manuscript. DKM: experimental design; data analysis; drafting the manuscript; revising the manuscript, final approval of the manuscript. DKM: experimental design; data analysis; drafting the manuscript; revising the manuscript, final approval of the manuscript. DISCUSSION If the potency of Hm1a depends on channel availability, then we predict that Hm1a would rescue the function of inhibitory neurons expressing A1783V mutant channels more effectively than neurons expressing only one functional SCN1a allele. However, limitations associated with Hm1a, including poor blood-brain barrier permeability and a short half-life, preclude testing this possibility in a clinically relevant manner at this time. expression of a DS-associated missense mutation (A1783V) in inhibitory neurons results in seizures and premature death on a similar time scale as haploinsufficient DS models (Caterall WA 2012). Inhibitory neurons from SCN1aA1783V/+ mice showed a modest reduction in channel transcript that may contribute to loss of inhibitory tone; however, the repetitive firing characteristics of SCN1aA1783V/+-expressing neurons was more consistent with loss of function due to increased Nav1.1 channel inactivation. For example, genotype differences in the action potential amplitude and rate of depolarization were diminished under experimental conditions designed to remove Na+ channel inactivation. Therefore, an effective treatment for SCN1a A1783V/+ -associated pathology could be to selectively potentiate Nav1.1 channel activity by slowing voltage-dependent inactivation. This approach, using a spider venom called heteroscodratoxin-1 (Hm1a), has been show to decrease seizures and mortality in an SCN1a haploinsufficient model of DS, albeit at a concentration well above the Hm1a EC50 of Nav1.1 channels (Richards et al., 2018). If the potency of Hm1a depends on channel availability, then we predict that Hm1a would rescue the function of inhibitory neurons expressing A1783V mutant channels more effectively than neurons expressing only one functional SCN1a allele. However, limitations associated with Hm1a, including poor blood-brain barrier permeability and a short half-life, preclude testing this possibility in a clinically relevant manner at this time. In sum, our results show that expression of SCN1aA1783V in inhibitory neurons mirror clinical features of DS including spontaneous seizures and respiratory dysfunction. At the cellular level, brainstem inhibitory neurons in the RTN of slices from SCN1aA1783V are less excitable whereas glutamatergic chemosensitive neurons are more excitable. Thus, our findings indicate that RTN chemoreceptors are a potential substrate for respiratory dysfunction in DS. 16 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . DECLARATION OF INTERESTS DECLARATION OF INTERESTS The authors declare no competing interests 17 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint Dlouhy, B. J., B. K. Gehlbach, C. J. Kreple, H. Kawasaki, H. Oya, C. Buzza, M. A. Granner, M. J. Welsh, M. A. Howard, J. A. Wemmie and G. B. Richerson (2015). 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It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint FIGURE LEGENDS Figure 1. Conditional expression of SCN1aA1783V/+ in inhibitory neurons results in premature death. A-C, SCN1aA1783V/+ mice did not show any obvious differences gross morphology (A) body weight (A) or temperature (C) compared to age-matched litter mate control mice. D, survival curve shows that control mice (n = 57) survive to adulthood (30 days postnatal) while SCN1aA1783V/+ mice (n = 41) die prematurely starting at 9 days postnatal and reaching 100% lethality by 25 days (χ2 = 63.9, p < 0.0001). E-F, fluorescent in situ hybridization was performed to characterize expression of SCN1a transcript in inhibitory (VGAT+) and glutamatergic (VGLUT+) neurons in the RTN region in brainstem sections from control and SCN1aA1783V/+ mice. E, brainstem sections from control and SCN1aA1783V/+ mice containing the RTN show SCN1a labeling (green puncta) of both VGAT+ and VGLUT neurons. F, summary data show SCN1a transcript expression (normalized to cell size) in VGAT+ and VGLUT+ RTN neurons from each genotype; channel transcript was reduced in VGAT+ cells from SCN1aA1783V/+ mice (0.43 ± 0.7 mRNA/area, n = 94 cells) compared to control (0.73 ± 0.9 mRNA/area, n = 82 cells) (p < 0.05), whereas VGLUT+ cells showed low channel transcript across both genotypes. These results were compared using a two-way ANOVA and Sidak multiple comparison test. *, p < 0.05; ***p < 0.001; ****p < 0.0001. REFERENCES CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint Parihar, R. and S. Ganesh (2013). The SCN1A gene variants and epileptic encephalopathies. J Hum Genet 58, 573-580. Ryvlin, P., L. Nashef, S. D. Lhatoo, L. M. Bateman, J. Bird, A. Bleasel, P. Boon, A. Crespel, B. A. Dworetzky, H. Hogenhaven, H. Lerche, L. Maillard, M. P. Malter, C. Marchal, J. M. Murthy, M. Nitsche, E. Pataraia, T. Rabben, S. Rheims, B. Sadzot, A. Schulze-Bonhage, M. Seyal, E. L. So, M. Spitz, A. Szucs, M. Tan, J. X. Tao and T. Tomson (2013). Incidence and mechanisms of cardiorespiratory arrests in epilepsy monitoring units (MORTEMUS): a retrospective study. Lancet Neurol 12, 966-977. Shea, S. A. (1996). Behavioural and arousal-related influences on breathing in humans. Exp Physiol 81, 1-26. Shmuely, S., S. M. Sisodiya, W. B. Gunning, J. W. Sander and R. D. Thijs (2016). Mortality in Dravet syndrome: A review. Epilepsy Behav 64, 69-74. Surges, R., R. D. Thijs, H. L. Tan and J. W. Sander (2009). Sudden unexpected death in epilepsy: risk factors and potential pathomechanisms. Nat Rev Neurol 5, 492-504. Tadel F, Baillet S, Mosher JC, Pantazis D, Leahy RM (2011). Brainstorm: a user-friendly application for MEG/EEG analysis. Comput Intell Neurosci. 2011, 879716. Tai, C., Y. Abe, R. E. Westenbroek, T. Scheuer and W. A. Catterall (2014). Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome. Proc Natl Acad Sci U S A 111, E3139-3148. Yu, F. H., M. Mantegazza, R. E. Westenbroek, C. A. Robbins, F. Kalume, K. A. Burton, W. J. Spain, G. S. McKnight, T. Scheuer and W. A. Catterall (2006). Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci 9, 1142-1149. 21 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . Figure 2. SCN1aA1783V/+ activity show that SCN1a A1783V/+ mice exhibit frequent spontaneous burst of high amplitude spike-wave discharges (SWD). The arrow identifies a typical seizure-like SWD event that was analyzed further by power spectral analysis in panel D. B, summary data show that seizure-like SWD events occurred more frequently (control 0.13 ± 0.1 events/2 hr, n = 6; SCN1a A1783V/+ 0.37 ± 0.05 events/2 hr, n = 6; T10=3.009, p < 0.01 ) and lasted for a longer duration (control 6.7 22 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint ± 2.2 ms, n = 6; SCN1a A1783V/+ 12.4 ± 5.5 ms, n = 6, T10=2.268, p < 0.05) in SCN1a A1783V/+ mice compared to control animals. Ci, representative power spectrum density (PSD) plots of SWD events show typical strong activity in the theta-, alpha and beta frequency range in SCN1aA1783V/+ but not control mice. Cii-Ciii, summary data (normalized to the maximum value at each event) show PSD peak (Cii) and PSD area under the curve (Ciii) of each frequency range for each genotype. Note that SWD events measured in SCN1aA1783V/+ mice show increased activity in the theta, alpha and beta range but no differences in the delta or gamma band compared to SWD events measured in control mice. Di-iii, SWD event recorded from a SCN1aA1783V/+ mouse (arrow in panel A) plotted on an expanded time scale (Di) and corresponding time frequency distribution (Dii) and deconstructed spectrum into its various frequency domains (Diii). These results were compared using a two-way ANOVA and the Sidak multiple comparison test. *, p < 0.05; **, p< 0.01; ***p < 0.001. ± 2.2 ms, n = 6; SCN1a A1783V/+ 12.4 ± 5.5 ms, n = 6, T10=2.268, p < 0.05) in SCN1a A1783V/+ mice compared to control animals. Ci, representative power spectrum density (PSD) plots of SWD events show typical strong activity in the theta-, alpha and beta frequency range in SCN1aA1783V/+ but not control mice. Cii-Ciii, summary data (normalized to the maximum value at each event) show PSD peak (Cii) and PSD area under the curve (Ciii) of each frequency range for each genotype. Figure 2. SCN1aA1783V/+ Note that SWD events measured in SCN1aA1783V/+ mice show increased activity in the theta, alpha and beta range but no differences in the delta or gamma band compared to SWD events measured in control mice. Di-iii, SWD event recorded from a SCN1aA1783V/+ mouse (arrow in panel A) plotted on an expanded time scale (Di) and corresponding time frequency distribution (Dii) and deconstructed spectrum into its various frequency domains (Diii). These results were compared using a two-way ANOVA and the Sidak multiple comparison test. *, p < 0.05; **, p< 0.01; ***p < 0.001. Figure 3. SCN1aA1783V/+ mice show reduced respiratory output under control conditions and during exposure to high CO2. A, traces of respiratory activity from a control and SCN1aA1783V/+ mouse during exposure to room air, 100% O2 and 3-7% CO2 (balance O2). B-D, summary data (n=22 control; n=17 SCN1aA1783V/+) show respiratory frequency (B), tidal volume (C) and minute ventilation (D) are reduced in SCN1AA1783V/+ mice compared to control under room air conditions. E, traces of respiratory activity (left) and summary data (right) show that under room air conditions both control and SCN1aA1783V/+ mice exhibit periods of apnea; the frequency of these events were similar between genotypes, however, they lasted for a longer duration in SCN1aA1783V/+ mice compared to control. F-H, summary data shows the respiratory frequency (F), tidal volume (G) and minute ventilation response of control and SCN1aA1783V/+ mice to graded increases in CO2 (balance O2). SCN1aA1783V/+ mice showed a blunted respiratory CN1aA1783V/+ mice show reduced respiratory output under control conditions 23 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint frequency to 5% and 7% CO2 which resulted in a diminished CO2/H+-dependent increase in minute ventilation. These results were compared using either unpaired t test (panels B-E) or two-way ANOVA followed by the Holm-Sidak multiple comparison test (panels F-H). *, difference between means p < 0.05, #, different interaction factor, p < 0.05. Figure 4. Brainstem inhibitory neurons in slices from SCN1aA1783V/+ show diminished basal activity and repetitive firing behavior during sustained depolarization. A, segments of membrane potential from inhibitory neurons in the RTN region in slices from control and SCN1aA1783V/+ mice during depolarizing current injections (40 to 220 pA; 1 s duration) from a membrane potential of –80 mV. B, summary data shows inhibitory neurons in slices from SCN1aA1783V/+ mice (n=36) are less active under resting conditions (0 pA holding current) compared to control cells (n = 26 cells). C, summary data and representative voltage responses to a -60 pA current injection show that inhibitory neurons from each genotype had similar input resistance. D, input-output relationship show that inhibitory neurons from SCN1aA1783V/+ mice generate fewer action potentials in response to moderate depolarizing current injections (1 s duration) and at more positive steps go into depolarizing block. Results were compared using t- test (B-C) and two-way ANOVA and Sidak multiple comparison test (D). *, p < 0.05; **, p< 0.01; ***, p < 0.001. Figure 5. The SCN1a A1783V missense mutation appears to result in loss of channel function by increased voltage-dependent inactivation. A, average spontaneous action (control n= 24 spikes, SCN1aA1783V/+ n= 29 spikes (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and SCN1aA1783V/+ mice (Ai) and corresponding phase plot (Aii) (dV/dt; Y-axis vs mV; X-axis) of the traces in panel Ai show Figure 5. The SCN1a A1783V missense mutation appears to result in loss of channel function 24 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint that cells expressing SCN1aA1783V/+ depolarize slower compared to control cells. B, average first action potential following a hyperpolarizing pre-potential (-100 pA; 1 s) (control n= 13 spikes, SCN1aA1783V/+ n= 16 spikes) (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and SCN1aA1783V/+ mice (Bi) and corresponding phase plot (Bii) of traces in panel Bi show that holding cells at a negative pre- potential to remove sodium channel inactivation improved the depolarization kinetics of subsequent spikes. C, average first action potential following a depolarizing pre-potential (+180 pA; 1 s) (control n= 9 spikes, SCN1aA1783V/+ n= 11 spikes (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and SCN1aA1783V/+ mice (Ci) and corresponding phase plot (Cii) of traces in panel Ci show that holding cells at a depolarized pre-potential to increase sodium channel inactivation diminished genotype differences in action potential kinetics. D-F, summary data showing the maximum rate of depolarization (D), action potential amplitude (E) and action potential threshold (F) of spontaneous action potentials and first spikes following positive or negative pre-potentials recorded in slices from control and SCN1aA1783V/+ mice. Results were compared by two-way ANOVA and the Sidak multiple comparison test.. *, p < 0.05; **, p< 0.01; ***, p < 0.001; ****, p < 0.0001. that cells expressing SCN1aA1783V/+ depolarize slower compared to control cells. Figure 5. The SCN1a A1783V missense mutation appears to result in loss of channel function B, average first action potential following a hyperpolarizing pre-potential (-100 pA; 1 s) (control n= 13 spikes, SCN1aA1783V/+ n= 16 spikes) (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and SCN1aA1783V/+ mice (Bi) and corresponding phase plot (Bii) of traces in panel Bi show that holding cells at a negative pre- potential to remove sodium channel inactivation improved the depolarization kinetics of subsequent spikes. C, average first action potential following a depolarizing pre-potential (+180 pA; 1 s) (control n= 9 spikes, SCN1aA1783V/+ n= 11 spikes (top) and first time derivative of average action potentials (bottom) recorded from inhibitory neurons in slices from control and SCN1aA1783V/+ mice (Ci) and corresponding phase plot (Cii) of traces in panel Ci show that holding cells at a depolarized pre-potential to increase sodium channel inactivation diminished genotype differences in action potential kinetics. D-F, summary data showing the maximum rate of depolarization (D), action potential amplitude (E) and action potential threshold (F) of spontaneous action potentials and first spikes following positive or negative pre-potentials recorded in slices from control and SCN1aA1783V/+ mice. Results were compared by two-way ANOVA and the Sidak multiple comparison test.. *, p < 0.05; **, p< 0.01; ***, p < 0.001; ****, p < 0 0001 Figure 6. Chemosensitive RTN neurons in slices from SCN1aA1783V/+ mice are hyper- excitable. A, firing rate traces from chemosensitive neurons in slices from control (top) and SCN1aA1783V/+ mice (bottom) show that neurons from both genotypes respond to changes in CO2/H+; RTN neurons are spontaneously active under control conditions (5% CO2; pHo 7.3) and respond to 7% CO2 (pHo 7.2) and 10% CO2 (pHo 7.0) with a linear increase in activity, whereas exposure to 3% CO2 (pHo 7.6) decreases neural activity. However, basal activity and CO2/H+- 25 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint dependent output of RTN chemoreceptors from SCN1aA1783V/+ tissue is enhanced compared to control cells. B, double-immunolabeling shows that a Lucifer Yellow-filled CO2/H+-sensitive RTN neuron (green) is immunoreactive for phox2b (magenta), the merged image is shown to the right. We confirmed that all CO2/H+-sensitive neurons (control n= 12; SCN1aA1783V/+ n = 11) included in this study were phox2b-positive. C-D, summary data shows that RTN chemoreceptors in slices from SCN1aA1783V/+ mice have higher basal activity (C) and enhanced CO2/H+-dependent output between 3-10% CO2 (D). Results were compared by t-test (C) or ANCOVA test (D). *, p < 0.05. TABLES Table 1: Behavioral Assessment of Seizure activity Racine Score 1 2 3 4 5 Behavioral Arrest Control 5 0 0 0 0 0 SCN1aA1783V/+ 3 3 6 2 3 5 Table 2: Probes used for FISH Gene name Probe Cat no. Target region Scn1a 434181 1624 - 2967 SLC32A1 319191-C2 894 - 2037 Slc17a6 319171-C2 1986 - 2998 TABLES Table 1: Behavioral Assessment of Seizure activity Racine Score 1 2 3 4 5 Behavioral Arrest Control 5 0 0 0 0 0 SCN1aA1783V/+ 3 3 6 2 3 5 Table 2: Probes used for FISH Gene name Probe Cat no. SCN1a A1783V/+ m All animal use was in accordance with guidelines approved by the University of Connecticut Institutional Animal Care and Use Committee. SCN1a A1783V /+ mice were generated by crossing offspring of VGAT-ires-cre (JAX no. 016962) and tdTomato mice (JAX no. 007914) with mice carrying cre-driven heterozygous SCN1a A1783V mutation (Jax no. 026133) to introduce the SCN1a variant A1783V conditionally in inhibitory neurons. Experimental animals express both the reporter and the SCN1aA1783V/+ mutation (SCN1a A1783V/+ mice), whereas litter mate control animals were those that express the tdTomato reporter under the VGAT promotor. Aged matched mice from both genotypes and sexes were used for all experiments included in this study. Figure 6. Chemosensitive RTN neurons in slices from SCN1aA1783V/+ mice are hyper- Target region Scn1a 434181 1624 - 2967 SLC32A1 319191-C2 894 - 2037 Slc17a6 319171-C2 1986 - 2998 TABLES Table 1: Behavioral Assessment of Seizure activity Racine Score 1 2 3 4 5 Behavioral Arrest Control 5 0 0 0 0 0 SCN1aA1783V/+ 3 3 6 2 3 5 Table 2: Probes used for FISH Gene name Probe Cat no. Target region Scn1a 434181 1624 - 2967 SLC32A1 319191-C2 894 - 2037 Slc17a6 319171-C2 1986 - 2998 26 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint Fluorescent in situ hybridization (FISH) To prepare fresh frozen slice, postnatal week 2 mice of both genotypes were anesthetized with isoflurane, decapitated, and brainstem tissues were rapidly frozen with dry ice and embedded with OCT compound. Brainstem slices (14um thick) containing the retrotrapezoid nucleus (RTN) were crysectioned and collected onto SuperFrost Plus microscope slides. Slices were fixed with 4% paraformaldehyde and dehydrated with 50%,70% and 100% ethanol. FISH were processed with the instruction of RNAscope® Multiplex Fluorescent Assay (ACD, 320850), the probes used in our study were designed and validated by ACD (Table 2). Unrestrained whole-body plethysmography Respiratory activity was measured using a whole-body plethysmograph system (Data Scientific International; DSI), utilizing animal chamber (600 mL volume) maintained at room temperature 27 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint and ventilated with air (1.3 L/min) using a small animal bias flow generator. Fifteen day old mice (~7 g) were individually placed into a chamber and allowed 2 hour to acclimate prior to the start of an experiment. Respiratory activity was recorded using Ponemah 5.20 software (DSI) for a period of 15 minutes in room air followed by exposure to graded increases in CO2 from 0% to 7% CO2 (balance O2). Body temperature was measured before and after each experiment and although body temperature tended to drop ~ 1 ◦C by the end of an experiment, there were no genotype difference in the degree of cooling (p = 0.37). Parameters of interests include respiratory frequency (FR, breaths per minute), tidal volume (VT, measured in mL; normalized to body weight and corrected to account for chamber and animal temperature, humidity, and atmospheric pressure), and minute ventilation (VE, mL/min/g). A 20 second period of relative quiescence after 2 minutes of exposure to each condition was selected for analysis. Spontaneous apneic events, conservatively defined as 3 or more missed breaths not preceded by a sigh or augmented breath, were analyzed off-line. All experiments were performed between 9 a.m. and 6 p.m. to minimize potential circadian effects. Acute slice preparation and in vitro electrophysiology Slices containing the RTN were prepared as previously described (Mulkey et al., 20007). In short, rats were anesthetized by administration of ketamine (375 mg/kg, I.P.) and xylazine (25 mg/kg; I.P.) and rapidly decapitated; brainstems were removed and transverse brain stem slices (300 µm) were cut using a microslicer (DSK 1500E; Dosaka) in ice-cold substituted Ringer solution containing the following (in mM): 260 sucrose, 3 KCl, 5 MgCl2, 1 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, and 1 kynurenic acid. Slices were incubated for 30 min at 37°C and subsequently at room temperature in a normal Ringer’s solution containing (in mM): 28 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint 130 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. Both substituted and normal Ringer’s solutions were bubbled with 95% O2 and 5% CO2 (pH=7.30). 130 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. Both substituted and normal Ringer’s solutions were bubbled with 95% O2 and 5% CO2 (pH=7.30). Individual slices containing the RTN were transferred to a recording chamber mounted on a fixed-stage microscope (Olympus BX5.1WI) and perfused continuously (~2 ml/min) with a bath solution containing (in mM): 140 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose (equilibrated with 5% CO2; pH=7.3). All recordings were made with an Axopatch 200B patch- clamp amplifier, digitized with a Digidata 1322A A/D converter, and recorded using pCLAMP 10.0 software (Molecular Devices, Sunnyvale, CA). Acute slice preparation and in vitro electrophysiology Recordings were obtained at room temperature (~22° C) with patch electrodes pulled from borosilicate glass capillaries (Harvard Apparatus, Molliston, MA) on a two-stage puller (P-97; Sutter Instrument, Novato, CA) to a DC resistance of 5–7 MΩ when filled with a pipette solution containing the following (in mM): 125 K-gluconate, 10 HEPES, 4 Mg-ATP, 3 Na-GTP, 1 EGTA, 10 Na-phosphocreatine (uM), 0.2% Lucifer yellow (pH 7.30). Electrode tips were coated with Sylgard 184 (Dow Corning, Midland, MI). The firing response of chemosensitive RTN neurons to CO2 (3-10% CO2) was assessed in the cell-attached voltage-clamp configuration (seal resistance > 1 GΩ) with holding potential matched to the resting membrane potential (Vhold = −60 mV) and with no current generated by the amplifier (Iamp = 0 pA). Firing rate histograms were generated by integrating action potential discharge in 10 to 20-second bins using Spike 5.0 software (Cambridge Electronic Design, CED, Cambridge, U.K.). We confirmed that all chemosensitive RTN neurons included in this study were immunoreactive for the transcription factor Phox2b. 29 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint To characterize action potential properties and repetitive firing behavior of inhibitory neurons, we made whole-cell current-clamp recordings from fluorescently labeled neurons located in the region of the RTN in slices from VGAT:TdTomato mice. Repetitive firing responses to 1 second depolarizing current steps from 0 to 300 pA (∆ 20 pA increments) were characterized from an initial holding potential of -80 mV. Action potential amplitude, threshold (dV/dT > 10mV/mS) and the maximum rate of depolarization obtained from the peak of the first time derivative of the action potential were characterized for spontaneous spikes measured under resting conditions (holding current = 0 pA) and for the first spike elicited after delivering a positive (+200 pA) or negative (-100 pA) 1 second current step. Acute slice preparation and in vitro electrophysiology All whole-cell recordings had an access resistance (Ra) < 20 MΩ, recordings were discarded if Ra varied >10% during an experiment. A liquid junction potential of -14 mV was accounted for during each experiment. Immunofluorescence staining Slices were fixed in 4% PFA over night after recording, and blocked with 5% normal horse serum in 1X PBS with 2.5% triton for 1 hr. Slices were incubated in goat anti-phox2b antibody (R&D, AF4940) and rabbit anti-lucifer yellow antibodies (Invitrogen, A-5750) mixed in blocking solution under 4℃ overnight. After washing the primary antibody a secondary antibody was applied for 2 hrs followed by an additional wash and mounting with ProLong® Gold Antifade Reagent (Invitrogen, P36934). Slices were imaged using a Leica SP8 confocal microscope (40x/1.3 HC oil objective) to identify cells that co-express Alex Fluro 647 for phox2b and Alex Fluro 488 for lucifer yellow. Electrocorticography recording 30 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint Subdural EcoG electrodes were implanted in 15 day old control and SCN1aA1783V/+ mouse pups. To minimize damage we used stainless steel wire electrodes (diameter = 0.003 in) (A-M system, 790900) inserted just under the skull for a length of 2 mm into each hemisphere near the fontal cortex. A reference wire electrode was placed in the posterior cortex. Each electrode was connected to a Mill-MAX miniature socket (digikey, ED11265-ND) and secured to the skull with super glue. Differential voltage signals were amplified 1000× with a DAM-50 differential amplifier (1 Hz low filter, 10 kHz high filter), digitized at 5 kHz and recorded using Sirenia Software (Pinnacle technology). Mice were allowed to 12 hours to recover from surgery before recording EcoG activity for a period of 2 hours. We also video recorded all experiments to correlate animal behavior with EcoG recordings. Only spike wave discharge (SWD) activity that occurred in conjunction with observable seizure behavior was included in the analysis. Any data including movement artifacts were excluded from analysis. The same criteria for seizure events were used for both mutant mice and control group. The full duration of each seizure event was segmented and then down sampled from 600 Hz to 100 Hz to focus on the frequency range of interest (0-50 Hz) prior to performing the power spectral analysis in Matlab (MathWorks). Frequency ranges of EcoG signals are defined as follows: delta, δ (1–5 Hz), theta, Ө (6–8 Hz), alpha, α (9–16 Hz), beta, β (17–36 Hz), and gamma, γ (37–50 Hz). Frequency analysis results were normalized to the maximum frequency amplitude at each event. For each frequency range, maximal amplitude and area under each frequency range were calculated to report the spectral power. Seizure behavior Scoring We video monitored mice for 1 hour after placing them individually in a cage and giving them access to food and water ad lib. Seizure behavior during this time was evaluated using the Racine scoring system as follows: score 1, mouth and facial movements; score 2, head nodding; score 3, forelimb clonus; score 4, rearing with limb clonus; score 5, full body clonus, rearing and falling. Electrocorticography recording To show the time- varying frequency distribution, time frequency analysis using Hilbert and Morlet transformations were also performed using Brainstorm 3.0 (Tadel et al. 2011). 31 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint Statistical Analysis Data are reported as mean ± SE. All statistical analysis was performed using Prism 7 (GraphPad Software, Inc., La Jolla, CA). Data were normally distributed (Shapiro-Wilk normality test) and comparisons were made using t-test, Chi Square test, one-way or two-way ANOVA followed by multiple comparison tests as appropriate. Relevant values used for statistical analysis are included in the results section. 32 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint 0 12 body weight (g) 0 45 body temp (°C) Control Figure 1 A1783V/+ SCN1a control A1783V/+ SCN1a A B C D E F con mutant 0 2.0 Scn1A mRNA/ Area VGAT VGLUT ******** (4) (3) (3) (4) VGAT VGAT VGLUT VGLUT SCN1A DAPI 5uM n.s. n.s. n.s. con A1783V/+ SCN1a postnatal days 0 15 30 7 100 alive (%) control A1783V/+ SCN1a # animals D postnatal days 0 15 30 7 100 alive (%) control A1783V/+ SCN1a 0 12 body weight (g) 0 45 body temp (°C) A B C D con mutant n.s. n.s. con A1783V/+ SCN1a postnatal days 0 15 30 7 100 alive (%) control A1783V/+ SCN1a D A 1 B C 0 12 body weight (g) Control Figure 1 A1783V/+ SCN1a control A1783V/+ SCN1a E F 0 2.0 Scn1A mRNA/ Area VGAT VGLUT ******** (4) (3) (3) (4) VGAT VGAT VGLUT VGLUT SCN1A DAPI 5uM n.s. con SCN1a # animals control A1783V/+ SCN1a F 0 2.0 Scn1A mRNA/ Area VGAT VGLUT ******** (4) (3) (3) (4) n.s. # animals F Control A1783V/+ SCN1a E VGAT VGAT VGLUT VGLUT SCN1A DAPI 5uM E Figure 1 . 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Statistical Analysis ; https://doi.org/10.1101/470609 doi: bioRxiv preprint 200 uV 5 min Control 40 0 25 0 Frequency (Hz) 5 0 power spectrum Hilbert transform 0 10 20 30 40 50 Time (s) Figure 2 200 μV A Di Dii Diii β 2 γ 1 γ α θ δ A1783V/+ SCN1a SWD event 1.0 0 event/2hr SWD freq 25 0 mS SWD duration PSD peak PSD area *** ** ** 1 0 ** ** θ α β θ α β 1 0 Bi Bii Ci Cii Ciii Power 0 1.0 PSD 10 20 30 40 50 Control A1783V/+ SCN1a (Hz) A1783V/+ SCN1a * * con A1783V/+ SCN1a 2 2 (10 uV /Hz) 4 2 (10 uV /Hz) 200 uV 5 min Control 40 0 25 0 Frequency (Hz) power spectrum 200 μV A Di Dii A1783V/+ SCN1a SWD event 1.0 0 event/2hr SWD freq 25 0 mS SWD duration PSD peak PSD area *** ** ** 1 0 ** ** θ α β θ α β 1 0 Bi Bii Cii Ciii A1783V/+ SCN1a * * A1783V/ 2 2 (10 uV /Hz) 200 uV 5 min Control Frequency A 1.0 0 event/2hr SWD freq 25 0 mS SWD duration PSD peak PSD area *** ** ** 1 0 ** ** θ α β θ α β 1 0 Bi Bii Cii Ciii A1783V/+ SCN1a * * con A1783V/+ SCN1a 40 0 25 0 Frequency (Hz) power spectrum 200 μV Di Dii A1783V/+ SCN1a SWD event 2 2 (10 uV /Hz) A A1783V/+ SCN1a SWD event Di 200 uV Bi 25 0 2 2 (10 uV /Hz) Frequency 5 0 Hilbert transform 0 10 20 30 40 50 Time (s) Diii β 2 γ 1 γ α θ δ 4 2 (10 uV /Hz) Ci Power 0 1.0 PSD 10 20 30 40 50 Control A1783V/+ SCN1a (Hz) con 83 / SCN1a . 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Statistical Analysis ; https://doi.org/10.1101/470609 doi: bioRxiv preprint control 1S 0.1ml/s p Air 100% O2 3% CO2 5% CO2 7% CO2 0 * 20 0 300 10 0 400 0 40 0 15 0 * * * 3 0 apnea duration in air (s) * 2 s 0.2 ml/S Figure 3 freq (breaths/min) freq in air (breaths/min) 0 tidal vol in air (μl/g) 0 0 3 5 7 0 3 5 7 0 3 5 7 tidal vol (μl/g) A B C D E F G H in air (μl/g/min) VE (μl/g/min) VE % CO2 % CO2 % CO2 apnea apnea control A1783V/+ SCN1a * A1783V/+ SCN1a # 0 * 20 0 300 10 * freq in air (breaths/min) 0 tidal vol in air (μl/g) 0 B C D in air (μl/g/min) VE * 0 * 20 0 300 10 * 3 0 apnea duration in air (s) * 2 s 0.2 ml/S freq in air (breaths/min) 0 tidal vol in air (μl/g) 0 B C D E in air (μl/g/min) VE apnea apnea control A1783V/+ SCN1a * D 3 B A 3 0 apnea duration in air (s) * 2 s 0.2 ml/S E apnea apnea control A1783V/+ SCN1a 0 400 0 40 0 15 0 * * 2 s freq (breaths/min) 0 3 5 7 0 3 5 7 0 3 5 7 tidal vol (μl/g) F G H (μl/g/min) VE % CO2 % CO2 % CO2 # 1 H 4 F . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint 200 mS 20 mV 0 (GΩ) 30 0 Basal activity (Hz) * 40 pA Action potential Number 0 80 p <0.0001 1 Figure 4 A control depol. Statistical Analysis block B input resistance 200 mS 20 mV -60 pA con 0 60 180 240 300 120 con * * * * * * C D A1783V/+ SCN1a A1783V/+ SCN1a A1783V/+ SCN1a -80 mV 80 pA 100 pA 180 pA 220 pA 200 S 20 mV A control A A 200 mS 20 mV 40 pA A control depol. block A1783V/+ SCN1a -80 mV 80 pA 100 pA 180 pA 220 pA 0 (GΩ) 30 0 Basal activity (Hz) * Action potential Number 0 80 p <0.0001 1 Figure 4 B input resistance 200 mS 20 mV -60 pA con 0 60 180 240 300 120 con * * * * * * C D A1783V/+ SCN1a A1783V/+ SCN1a 0 (GΩ) 30 0 Basal activity (Hz) * Action potential Number 0 80 1 B input resistance 200 mS 20 mV -60 pA con C D A1783V/+ SCN1a Action potential Number 0 80 p <0.0001 S 0 60 180 240 300 120 con * * * * * * D A1783V/+ SCN1a B . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint 250 0 dV/dT (mV/mS) -60 60 mV 0 150 100 0 -120 dV/dT (mV/mS) Spontaneous Action Potential Figure 5 Ai **** 20 mV 50 dV/dT 2 mS D Aii 20 mV 2 mS 50 dV/dT 150 0 mV -80 60 mV 0 150 0 -120 dV/dT (mV/mS) Negative Pre-potential Bi Ci E Bii Positive Pre-potential 2 mS 50 dV/dT 20 mV F -80 60 mV 0 150 0 -120 dV/dT (mV/mS) Cii * ** n.s. 0 -50 mV * n.s. **** spontaneous negative pre-potential positive pre-potential control A1783V/+ SCN1a spike amplitude max. rate of depol. spike threshold * n.s. n.s. Statistical Analysis 250 0 dV/dT (mV/mS) -60 60 mV 0 150 100 0 -120 dV/dT (mV/mS) Spontaneous Action Potential Ai **** 20 mV 50 dV/dT 2 mS D Aii Bi E Bii * * spontaneous negative pre-potential positive pre-potential control A1783V/+ SCN1a max. rate of depol. D Ai Bi E 150 0 mV -80 60 mV 0 150 0 -120 dV/dT (mV/mS) ntial E Bii ** n.s. n.s. **** spike amplitude Ci Positive Pre-potential Cii Figure 5 Ci Positive Pre-potential 2 mS 50 dV/dT 20 mV F -80 60 mV 0 150 0 -120 dV/dT (mV/mS) Cii 0 -50 mV spike threshold * n.s. n.s. ntial F -80 60 mV 0 150 0 -120 dV/dT (mV/mS) Cii 0 -50 mV spike threshold * n.s. n.s. F Figure 5 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted November 14, 2018. ; https://doi.org/10.1101/470609 doi: ioRxiv preprint control 0 Firing Rate (Hz) 8 2 0 Firing Rate (Hz) 2 5 min 5 min 6 0 m = 0.37* m = 0.30 + CO /H -sensitivity 2 Lucifer Yellow Phox2b merge 4 0 Firing Rate (Hz) * 50 μm Figure 6 B A 5 10 3 7 %CO2 5 10 3 7 %CO2 ventral surface baseline 3 5 7 10 C D Firing Rate (Hz) A1783V/+ SCN1a % CO2 con A1783V/+ SCN1a control 0 Firing Rate (Hz) 2 5 min B A 5 10 3 7 %CO2 Lucifer Yellow Phox2b merge 50 μm B ventral surface B A control 50 μm C Firing Rate (Hz)
https://openalex.org/W4313512603
https://papiro.unizar.es/ojs/index.php/tropelias/article/download/5543/4548
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Placeres de la autoescisión: la abyección y el masoquismo en "La vida secreta de Salvador Dalí"
Tropelías/Tropelías
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cc-by
10,512
li d r~ j", i: 1: PLACERES DE LA AUTOESCISIÓN: LA ABYECCiÓN Y EL MASOQUISMO EN LA VIDA SECRETA DE SALVADOR DALÍ i David Vilascca Universitat Autònoma de Rarcelona I I i; !, " Cada hora era una nueva victoria del "ego" sobre la muerte. Por un lado, a mi alrededor sólo veía continuos compromisos con la muerte. i Yo, ni hablar! Con ]a muerte no pactaría nunca (Dalí, 1949, 152), La sexualidad sería lo que es intolerab]e al sujeto estructurado (13ersani, ] 9R6, 3R). li i: 1\ f1 !i Incansable constructor, el excluido es, en pocas palabras, un extraviado. Va en viaje, durante la noche, cuyo final se aleja cada vez más. Tiene nociÓn del peligro, un '1 ~j !; ! de la pérdida que el pseudo-objeto que lo atrae representa para él, pero no puede evitar arriesgarse en el momento mismo en que se divide. Y cuanto más se extravía, más se salva (Kristeva, El "exasperado 19R2, R). hiperindividua]ismo" de Salvador Oalí, su "furiosa afirmación de (...) 1949, 1I R; 152), va estrechamente ligado e imperialista" (Dalí, [una I personalidad creciente en la autobiografía del artista, La vida secreta de Salvador Dalí (1942)1, a un instinto igualmente endémico y furioso hacia el dominio de su propio yo, de la gente que le rodea y de las situaciones en que se envuelve. "Cuando llegué al tejado sentí volvenne único otra vez", es- 1.- La primera ediciÓn de '{'he Sec,.el L((e "(S(/{v(ul,,,. f)(/Ií by S(/ll'llcl",. [)(/Iíaparcce en Nueva York en 1942. La ediciÓn que utilizo es la que publica Vision (Londres, I ()4<)). Todas las traducciolles que aparecen en el artículo SOIl mías a no ser que se indique lo contrario. Tropelías, n." 4. 193 1<)<)3 !I i , . I! !! i; tI '1 ~! !~ ~ .\ ì!->'i'q! 11' 1', I DA VID VILASECA cribe Dalí de su ya conocida fascinación (tanto ]iteral como metafórica) por estar siempre "en la cumbre": "Todo el panorama hasta la bahía de Rosas parecía o/JedecCI'lIIe y depender de mi mirada" (] 949,72; ]a cursiva es mía). La compulsiva necesidad de controlar el exterior y las afirmaciones megalómanas de independencia y unidad subjetiva se implican mutuamente en las memorias de Salvador Dalí. En ambos casos, el dominio es obtenido por el narrador por medio visual o escopofílico (como en la cita anterior), por medio de una introyección oral (comiéndose literal o lïgurativamente el objeto externo), o bien desplegando estratagemas de un marcado carácter sádico. En esencia, se diría que ]a función de una subjetividad unificada y totalizadora (Dalí como "amo" de su vida y de su yo) solamente aparece, en las memorias, como resultado de ]a alegre complacencia con que Dalí controla e] extcrior, y a través de su consiguiente identificación narcisista con aquella ilusión de control. I.a pida secrela.., narra la historia de Dalí dándose a luz a sí mismo, El libro es ]a historia de una (auto)creación, cuya efectividad depende en gran medida de] modo en que Dalí se a lo que no quiere ser o a lo que no quiere considerar como parte de sí mismo (del modo, pues, en que el narrador lucha por imponer demarcaciones estrictas que le permitan distinguir su sujeto de los objetos, su ser de su no-ser, su interior de su exterior), posiciona respecto La descripción de] mecanismo de la abyección, que encontramos en POllpoirs de Kristeva (]9~()), reviste aquí un particular interés, Según Kristeva, lo "abyecto" es una imagen a la vez terrorífica y asquerosa, imagen que representa para el indivi/'/101'1'1'111' de Julia duo todo lo que tiene que ser apartado y excluido de sí para preservar la pureza narcisista de sus identificaciones, así como la ilusión de su coherencia individual. Sin embargo, como en seguida veremos, dado que el mecanismo de la abyección, para Kristeva, conlleva en último término el rechazo y la exclusión del propio sujeto, tal mecanismo constituye una función ambivalente dirigida tanto hacia el establecimiento de la identidad "propia y limpia"2 de uno mismo, como, inversamente, hacia el trastorno de la propia organización psíquica, Aquí, la teoría de la abyección de Kristeva se brinda a una interesante comparación con el concepto de masoquismo que expresa Leo Bersani tanto en The Frelldial1 ßody (1986) como, más recientemente, en JÏ1e CII/ll/re (~r RedellljJliol1 (] 990), Como apunta Kristeva, el mecanismo de ]a abyección se extravía en e] mismo momento en que divide al sujeto, dando lugar no a una sino a un número indefinido de posiciones fantasmáticas desde las cuales el dominio o control (de uno mismo y del exterior) no representa más que un (auto)engaíïo provisional y sielllpre ya condenado al fracaso, Sin embargo, siguiendo a Bersani, intentaré mostrar cómo, en Dalí, no cabe únieamentc explicarse la abyección como el intento (tan reiterado como imposible) de establecer la propia identidad en oposición estable y esencial a algo que se le ha vuelto terrorífica mente cercano; por el contrario, ésta representa, en primer lugar, una fuente de goce masoquista para el sujeto, lIna alteración momentánea de la organi- El adjetivo francés 1'1'01'1'1', que Kristeva utiliza en la expresión ('orps p1'Opl'C, ha mantenido el sentido del 2.originallatin /JI'o/Jl'ills ("lo de uno, lo característico") )' ha adquirido también otro nuevo: "limpio", La polise- mia de la palabra juega en favor del argumento de Kristeva. Siguiendo la sugerencia del traductor de POlIl'oil's dc I'hol'l'clIl' al inglés, Leon S. Roudiez, propongo "el cnerpo propio)' limpio" como la solución más cercana al original, sacrilÏcando la elegancia a la claridad conceptual (vean la "Translator's Note" en Krisleva, 19H2, viii), 194 :'j.~ PLACERES DE LA AUTOESCISIÓN zación subjetiva que le sitúa cn el umbral arcaico tanto dcl nacimiento como dc su propia muerte. 1. "Superior a lo que yo podía resistir": la abyecci6n y el impulso de control La Icctura lacaniana de la narración de 1 Ienry James (}fm V/fe/fa de f/ferCa, quc emprende Soshana Fclman en un artículo ya clásico, pone en evidcncia el hecho de que controlar (lIwsterillg) cl significado implica no sólo cOll1prender sino también, paradójicamentc, negarse a cOll1prender, convertir algunas áreas en positivamente invisibles, excluir del significado precisamcntc lo que uno no quiere_ver: "Ver es marcar un /íll1ife más allá dcl cual se impide la visión", Felman cseribe. "El rígido cierre del abrazo violento que implica el acto (el 'agarro') del entendimiento va ligado, sin duda, a la violencia que se requiere para imponcr un /íll1ite más allá del cual nuestros ojos sc dcben cerrar" (relman, 1977, 66; la cursiva es del original). l' :1 .' .' 1: '! i ,o' !1 i: ~ : " El mecanismo de la abyceeión prescnta muchos puntos de contacto con estc acto de ceguera volLlI1taria que cualquier intento de establecer el significado nccesariamcnte exigc. Se- gún Kristeva, la abyceción describc el mecanismo por el cual el sujeto, a fin de mantcner el control sobrc sí mismo y su exterior, ticnc que rechazar con violencia (y, en estc scntido, ncgarse a "vcr") ciertos "no-objetos", clcmentos quc son percibidos por el individuo como par- ticularmente asqucrosos y horripilantes. Antcs de adentrarmc cn una explicación tcórica del concepto de abyección en Kristeva, quisicra referirme a dos escenas dc La vida seC/'eWoo. cn las quc la representación de elcmcntos nauseabundos adquiere un carácter más relevantc. I.a primera esccna que me propongo analizar, calificada por Dalí como "una de las más tcrroríficas que pucblan mi mcmoria" (Dalí, 1949,93), tienc lugar durantc la infancia del pintor, y gira alrcdedor dc uno de los animales que formaba parte de su pceuliar colccción zooló- i: ,. gica durante su estancia en casa dc la familia Pitxot3. Se trata dc un gran crizo encontrado lIluerto detrás dc un montón de ladrillos, cn avanzado estado dc putrefacción. Escribc Dalí: El gran erizo, quc me había sido imposible encontrar durante más de una scmana (oo.) apareció de repente cn un rincón del gallinero bajo un montón de ladrillos y ortigas. Estaba muerto. Llcno de repulsa, mc acerqué a él. La gruesa piel de su cspalda, cubicrta de púas, se agitaba con el incesantc ir y venir dc una frenética masa de gusanos que sc rcmovían. Ccrca de la cabeza, csta pululación era tan intensa quc uno habría dicho que un verdadcro volcán interno de putrefacción iba a explotar en i i 1: :j , , t;r~ _1 i; cualquier momento a través de aquclla piel rota por cl horror dc la muertc cn una irrupción inminentc de ignominia fÏnal (Dalí, 1949,94). El texto pone un gran énfasis en cl angustioso cfccto que esta visión tienc cn el narrador. La rcacción de Dalí ante el erizo se describe con voluptuoso dctalle. Las rcferencias a su dcbilidad y a sus temblorcs dc pánico van ligadas a una incquívoca fascinación y placcr fí- !~ sico. Como afirmaría Leo RersailÎ, el gocc (masoquista) de Dalí va ligado, aquí, al trastorno de la organización psíquica que csta asquerosa visión le produce. Escribe: 1-1 3.- Los I'it.~ot eran una familia de artistas que residían en el Molí de la Torre, una casa de campo situada a unas dos horas de Figueras. La estancia de Dalí con ellos. que Meryle Secrest sitúa alrededor de 1910 (Seerest. l <JHó, 33), tu\'o una gran influencia cn la etapa inicial de la carrera del pintor. 195 :~~ DA VID VILASECA Un ligero temblor y una gran debilidad se ampararon de mis piernas, y unos deque ascendían verticalmente por mi espalda se abrían como un abanico al llegar a mi cogote, de donde caían otra vez esparcicndo sus ramas por todo licados escalofríos ~: mi cuerpo, como una verdadera explosi6n de fuegos artificiales en una fiesta de apoInvolunlariamenle, me acerqué aún más a esta bola infecta que leosis de mi terror. continuaba atrayéndome con una repugnante fascinaci6n. Tenía que darle una buena mirada (Dalí, 1949, 94). Sin embargo, después de esta primera fase de observación, nuestro narrador, habiendo dado la vuclta al animal y habiendo descubierto el espectáculo aún más asqucroso de su barriga putrefacta, está al borde del dcsmayo. Seguir mirando se vuelve ahora demasiado penoso para él, y Dalí tiene que alejarse. "Esta vez, era superior a lo que yo podía resistir", es- cribe: !;! ", i', ;~ '" l " ,.~ Agité este montón erizado de pesadilla con una intensidad tan aterradora y una voluptuosidad tan morbosa que por un momento pensé que iba a desmayarme. Especialmente cuando (...) llevado por la curiosidad, di finalmente la vuelta al erizo. Entre sus cuatro rígidas patas, vi una masa de gusanos agitados, grandes como mi puño, que rezumaban de manera abominable después de haber reventado y atravesado la fina y violácea membrana del vientre que hasta entonces los había mantenido en una 1: 1: 1;: :~; mezcla compacta, impaciente y devoradora. Huí de aquel lugar (...) Esta vez, era superior a lo que yo podía resistir (Dalí, 1949, (5). Estrechamente relacionada con ésta, la segunda escena que quiero traer a colación aquí tiene lugar mucho más tarde, durante una de las estancias de Dalí en París cuando se rodaba UII perro alldalllz4. En su habitaci6n de hotel, en el momento que va a levantarse, Dalí se ha dado cuenta de que un insecto que había visto en el techo la noche anterior ya no se encuentra allí. Dalí sacude las sábanas y mira por todo su cuerpo, temiendo de repente que el animal (una garrapata) pueda haberle caído encima durante la noche. Efectivamente, lo descubre con la ayuda de un espejo, pegado a su espalda. Presa del asco y del horror, Dalí intenta de- senganchar la garrapata, pero se encuentra demasiado bien adherida a su piel y ofrece una resistencia increíble. El narrador nos describe su angustia y sus esfuerzos por desprenderse del parásito con su característica minuciosidad: Cerré los ojos y apreté los dientes dispuesto a soportar cualquier cosa con tal de poder librarme de aquella diminuta pesadilla que me estaba paralizando. Cogí la garrapata entre el pulgar y el índice y pcllizqué el punto donde se juntaba a mi piel con las afiladas pinzas de mis uñas (...) La garrapata estaba tan sólidamente pegada a mí, que no conseguí desprenderla ni una pizca. Era como si estuviera formada por mi propia carne, como si ya constituyera una parte inherente e inseparable de mi propio cuerpo; como si, de repente, en vez de un insecto se hubiera vuelto el germen terrorífieo de un pequeño embri6n de hermano siarnés que estuviera creciendo en mi espalda, como la más apocalíptica e infernal de las enfermedades (Dalí, 1949,214). película cn dos scmanas durantc la primavcra dc 1929. Un j!l'I'ro (//Il/II/nl sc proycctÓ cn 4.- Bniincl rodÓ la público cn junio dclmismo aiio (vcr Sánchcl. Vidal, 19HR, 355). 19ó ;~ðl 11 PLACERES DE LA AUTOESCISIÓN ~i La escena adquiere tintes verdaderamente frenéticos, y acaba convirtiéndose en una "orgía de sangre" cuando Dalí, "con una brutalidad proporcional (...) a Isul terror" (1949, 214), se dispone a usar una hoja de afeitar para separar el parásito de la piel. Al borde del desmayo, las paredes de la habitación salpicadas por completo de sangre, Dalí se hace con el " timbre de recepción y es finalmente asistido por el encargado del hotel y un doctor, quienes le revelan la sorprendente verdad del suceso. El episodio no fue más que el resultado de una treta que le jugó a Dalí su imaginación hipocondríaca y sobreexcitada. Lo que había tomado 1; por una garrapata era en realidad una pequeña marca de nacimiento, una peca. Luego en vez de un cuerpo extraño, Dalí había estado intentando arrancarse un pedazo de su propia carne. l' i! I A pesar de su brevedad, los pasajes citados nos dan una idea precisa de la naturaleza del asco y el horror en La vida secreta... Tales sentimientos aparecen relacionados, en Dalí, :J 1: 11 con lo pegajoso y lo que carece de contornos, con lo que literal o metafóricamente amenaza los límites y oposiciones que constituyen la base de su subjetividad. i: En la primera escena, por ejemplo, lo que provoca el mareo del narrador no es solamente la visión del erizo muerto (el cuerpo del animal por sí mismo) sino, más específicamente, esa "frenética masa de gusanos que se removían" saliendo de él. Luego parece que no es sólo la muerte lo que provoca la repulsa en Dalí, sino el hecho de que la muerte pueda en- i' gendrar una forma de vida, el hecho, precisamente, de que muerte y vida ya no sean opuestas ni estén e1aramente distinguidas la una de la otra ante la visión del erizo. De manera similar, el objeto del terror de Dalí, en el segundo episodio, es una garrapata sintomátieamente pereibida como el "germen terrorífico (...) de un hermano siamés (...) creciendo en mi espalda". Lo que produce "la más apocalíptica e infernal de las enfermedades", aquí, no es el parásito (o no sólo el parásito) sino el hecho de que, adherido a la piel, amenace y contamine la pureza y singularidad individual de Dalí. El animal pone en duda los contornos del sujeto y, como un hermano siamés, socava simbólicamente la distinción entre oriRinal y doble (lo interno y lo externo, lo "propio" y lo "impropio") donde descansa y se funda su identidad, su existencia como sujeto único, individual y presente a sí mismo. Así, sea delante del erizo o de la garrapata, las reacciones de Dalí siguen un modelo muy similar: primero se siente atraído hacia lo repugnante y asqueroso, y la debilidad física y anímica que acompaña a la visión le producen un inequívoco placer voluptuoso; luego, justo antes de per- der el conocimiento o de desmayarse, por medio de la huida o de arrancar literalmente de sí el cuerpo extraño, Dalí se desprende de aquello cuya visión causa su anulación. Lo excluye de sí, lo abyecta. Caracterizado por su falta de forma, por su inconsistencia y ambigÜedad, lo abyecto representa, según Kristeva, algo que el sujeto, como entidad simbólica y lingÜística, tiene que rechazar para poder existir: "No es la falta de limpieza o de salud la causante de la abyec- ción, sino lo que altera la identidad, los sistemas, los órdenes. Lo que no respeta fronteras, posiciones, reglas. Lo que está en medio, lo ambiguo, lo compuesto" (Kristeva, 19R2, 4). Para Freud, lo misterioso o extraño (das Unheill1liche) no es, en realidad, "nada nuevo o ajeno, sino algo familiar y bien arraigado en la mente que se ha vuelto extraño a ella sólo a través del proceso de la represión" (Freud, 19 I 9, 24 1). En una línea similar, lo abyecto de Kristeva es lo que le "recuerda" al sujeto sus primeros intentos por demarcarse respecto de la entidad maternal, atrayéndole de nuevo (y de ahí su carácter terrorífico) hacia un estadio de preidentidad en el cual el "yo" se encuentra todavía debatiéndose por establecerse autónomamente a través del lengu,~ie. En el límite que separa las oposiciones fundamentales entre ! ' ! !: 197 , . I ~'i3r DA VID VILASECA ~.~ "vida" y "muertc", lo "intcrior" y lo "extcrior", cl "yo" y lo "otro", lo abyccto existc antcs dc sido mutuamcntc cstablccidos por mcdio dc la difcrcncialjuc cl sujcto y sus objetos hayan lingÜística, ción crcando así una amcnazantc "falta dc scntido", un algo quc propiamcnte no cs nada, un no-objcto (1982,2)5. quc, en palabras dc Kristeva, "si lo rcconozco, mc aniquila" Según Kristcva, lo quc nos rcpugna cs "lo quc dcsobedece las rcglas dc clasificación espccíficas dc un sistema simbólico dado" (19R2, 92). En prcscncia de un objcto quc .I'ignifïcaJ'({ realmente lo mucrto (cl cucrpo inmóvil del erizo, por cjcmplo), Dalí hubicra entcndido y sc hubicra rcsignado. Pcro csa "masa oscura y rcpugnantc" (Dalí, 1949, 95) de gusanos 'piel dcl animal no .I'ign(jh'(/// algo mucrlo. Al contrario, la quc han reventado y atravcsado la visión del conjunto representa un ambiguo término mcdio entrc vida y mucrtc, un "objcto" mal definido, un no-objcto quc, cn consccucncia, impidc al sujcto cstablcccrse propiamcntc en sus dcmarcacioncs 1:; \)1 lingiiísticas (dcmarcacioncs basadas, como sabcmos dcsdc Saussurc, en un sistcma dc oposicioncs binarias cxcluycntcs). Ese "objcto", pucs, cnfrcnta a Dalí con el vacío y la ncgatividad quc tienc quc rechazar para dcmarcar un espacio cn el quc las oposicioncs dcl lenguaje puedan cxistir (aunquc cl vacío y la ncgatividad cstén sielllpre ya c il/el'ilah/elllel/le instalados cn él). Es el "horror en .;i nosotros mismos" (Kristcva, 19R2, 53), una "otra parte" que dc repcnte dcsposec a Dalí tanto dc sí mismo como también, y cn el mismo movimicnto, de sus objctos. En estc scntido, es sintomático quc tanto cl crizo como la garrapata sitúen a Dalí al borde mismo dcl dcsmayo, dc su (momcntánca) desaparición como sujcto. Dc repcnte, sin un cspacio simbolizado dondc podcr cxistir, Dalí "pcnsl ó J quc iba a dcsmayarl se 1" (Dalí, 1949, (5). La abyccción (el huir, el arrancarsc cl parásito o el ccrrar los ojos antc la visión dcl crizo) reprcscntan, así, el intcnto dc Dalí de permancccr dcntro dellcnguajc, una protccción en contra dcl derrumba- miento total dcl sistcma al cual dcbc su propia existcncia subjetiva. Prcstcmos una atcnción más dctallada a estc aspccto. Dentro de la historia pcrsonal del sujcto, Kristcva localiza la abyccción en un cstadio de preidcntidad, quc coincide con la llamada "rcprcsión primaria" (Kristcva, 1982, 10-11). En cstc cstadio, aunque el sujcto todavía no csté dcfinido (y pucs sus "objctos" tampoco cxistan propiamcntc), el futuro "yo" cs ya ca- paz de scpararsc y dcmarcarsc por opo.l'iciÚI/. Según Kristcva, estc posicionamicnto no consistc tanto cn una oposiciÚn a lo quc el sujeto l/O es (el "objcto", distinciÚn quc sólo llcgará más tarde, como rcsultado dc la "rcprcsiÖn sccundaria") sino contra aqucl "otro lado" dcl límitc cn cl cual la oposiciÚn quc cxistirá entrc sujcto y objeto no podría tencr lugar. Kristcva 1: apunta: "Incluso antcs dc ser, (oo.) 'yo' no soy pero sí separo, rechazo, a/Jyeclo" (Kristcva, 19R2, 13; la cursi va cs dcl original). Lo abyccto, pucs, cnfrcnta al sujeto con cl vacío y la ncgatividad contra y cn basc a los cuales su subjctividad fuc fundada. Para conscrvar la purcza ilusoria dc su idcntidad, cl sujcto dcbc rcchazar las imágcncs quc filtran la arcaica cconomía dc la separación cntrc los cspaeios de su mundo simbolizado. Pcro cxistc un importantc impcdimento. Dado quc lo ab- particular cn rclaci(,n con los análisis dc Krisleva dc la diferencia scxnal, 5.- Para la noci(,n dc lo (/I',reclo, cn recomicndo la colecci6n de ensayos cditados por Jolm r1ctcher y Andrc\\' Benjalllin Abjeclirm. MI'i(/l/cllOli(/ (1990); consullcn también el libro de John Leclllc's ./(/Ii(/ Kri.l'/('\'(/ (//III I.ol'/': 'Ihe \llorA: 01./1I1i(/ Kri.\'lel'(/ (1990), 157-19R. 198 i1.m ....>)! I'L^CERES DE L^ ^lJTOESCISIÓN yecto, según Kristeva, prcc.\'i.l'IC a la propia oposición entre sujeto y objeto (luego, propiamente hablando, no constituye un "objcto" para "mí"), el acto dc expcler y rechazar "eso" es inseparable del de expelerme y rechazarme a f: " "mí" mismo. Volviendo a los ejemplos anteriores, para poder establecer su "limpieza y propiedad" como individuo, Dalí tiene que posicionarse en contra de la negatividad (tiene que localizarla, situarla como exterior a sí mismo). Sin embargo, como ejemplifica el episodio de la garrapata/peca tanto en el plano simbólico como en el litcral, éste es un trabajo sin resolución posiblc, ya que el sujeto se corta a .I'í lIIi.l'1II0, se arranca a sí lIIislIIO, se abyecta a .I'í mislllo en el mismo movimiento por el cual intentaba establecer su identidad y su propio territorio. El acto de rechazo de lo abyecto (de lo exterior, de lo ajcno a mí) conlleva, pues, el rechazo del propio sujeto que lucha por excluirlo. La cita de Dalí lo ejemplifica perfecta- ti mente: l' ,1 1: '1 !: !i Me hice con una hoja de afeitar, mantuve la garrapata estrechamente aprisionada entre las uñas y empecé a cortar el intersticio entre la garrapata y la piel, que ofrecía una increíble resistencia. Pero, en mi desazón, corté, corté y corté, cegado por i! la sangre que empezaba ya a chorrear. La garrapata cedió finalmente y, medio desmayado, caí al suelo bañado en mi propia sangre (Dalí, 1949,2 (4). 1, * ij !: !: ::: Los episodios analizados de I.a vida .I'cer('(a... nos permiten observar cómo el acto de establecer control subjetivo va a menudo de la mano de un impulso hacia el trastorno de la propia organización psíquica. Por un lado, el mecanismo de la abyección yerra en el mismo instante en lJue divide al sujeto, ya que genera no una, sino una secuencia sin fin de posiciones desde las cuales el control sobre lmo mismo se vuelve una ilusión provisional, sielll/J/(! ya perdida y condenada al fracaso (ilusión de propiedad, de totalidad y de coherencia). De aquí que la abyección tienda hacia la dispersión de las unidades, al desmantelamiento de las estructuras y, en último término, hacia la anulación del propio sujeto abyectante. Por otro lado, la finalidad última de la abyección vendría a ser un sujeto en una completa identificación narcisista consigo mismo (en el felicísimo sueño de una jOllis.I'allec solipsística), lo que conllevaría también la desaparición del sujeto en tanto lJue entidad deseante. Sin embargo, Dalí está lejos de este hipotético movimiento final. "Divisor de territorios, de lenguajes y obras", Kristeva apunta, en palabras que pueden ser aplicadas al propio narrador de J.a vida .I'cercla..., "el excluido nunca termina de demarcar un universo cuyos fluidos confines -ya que están constituidos por un no-objeto, lo abyecto- cuestionan constantemente su solidez y lo impulsan a empezar de nuevo" (Kristeva, 1982, 9). Situacionalista, siempre cuestionándose por su posición (i,dÓllde estoy yo?) más que por su ser (i//lliéll soy yo?), Dalí lucha por encontrar una posición desde donde separar(se), excluir(se) y controlar(se). ^byecta y, al hacerlo, se pierde a sí mismo y debe volver a empezar. Como reza lll10 de los epígrafes a este artículo, Dalí comparte la suerte del "extraviado": el final de su viaje se aleja continuamente, y "cuanto más se extravía, más se salva" (Kristeva, 1982,9). " J~ 2. La ahyección y la cuestión de hl madre arcaica Antes de introducirme en la cuestión del masoquismo, quisiera referirme a un aspecto de gran importancia tanto para el concepto de la abyección en Kristeva corno para las memorias de Dalí: la cuestión de la madre arcaica. i ì! 199 I :1 fJ ; ~ \k'~ ;11 ;" t~ ;~ e DA VID VILASECA !~ Uno de los aspectos más complejos del trabajo de Julia Kristeva es la relación existente entre lo abyccto y lo materno. Relacionada muy estrechamcnte con el concepto dc cOJ'([ o rcccptáculo (que Kristcva toma prestado de Platón), la madre arcaica representa, como Noreen O'Connor apunta en un artículo reciente, "el lugar donde el sujeto es generado y negado al mismo tiempo, donde la unidad del sujeto es fragmcntada por los cambios y estadios que lo produjeron" (O'Connor, 1990, 46). De modo similar a lo abyecto, la madre arcaica representa lo que constantemcntc amenaza la ilusión de indcpcndencia y presencia a sí mismo del sujeto, lo quc le "recuerda" su estadio de indiferenciación presimbólica. Como apunta otra o' " !: !~ " de Kristeva, Elisabeth Grosz, la madre arcaica "define el lugar de una dcuda de vida y de existencia impagables, inefables, deuda que el sujeto (y la cultura) deben al cuerpo materno" (Grosz, 1990, (2). de las comentaristas Sin embargo, según Kristeva, la madre arcaica (como revela el discurso psicoanalítico) también reprcsenta el deseo del sujeto por volver a un punto de origen, origen que es "resistente al significado, innombrable" (O'Connor, 1990, 44). Lo materno es, pues, tanto un estadio presimbólico del cual el sujeto debe sustraerse para establecer su propia posición lingÜística en oposición a lo que pasará a ser otro (su madre), como el impulso final del deseo nostálgico del sujeto por volver a un hipotético punto de "origen". Sin embargo, como su- :1 giere .Iane Gallop, esta nostalgia va más allá del lIostos, "más allá del instinto de retorno" (Gallop, 19R5, 151), ya que, por efecto de la represión primaria, ese "lugar" añorado por el sujeto se encuentra estrueturalmente exeluido. Es un lugar donde él nunca ha estado propiamente, y que, por consiguiente, marca su nostalgia como constitutivamente imposible de sa- tisfacer. I.os conceptos de la madre (arcaica), de la represión primaria y de lo abyecto se encuentran, pues, estrechamente relacionados en Kristeva. "I,uego -afirma esta autora- lo abyecto sería el 'objeto' de la represión primaria" (Kristeva, 19R2, 12). Estas consideraciones explícito a la coJ'([, no serían necesarias si La vida secreta... no contuviese un tributo al receptáculo primario de la entidad materna. En el capítulo llamado iI i:, , , ' o' :: "Memorias intrauterinas", Dalí alaba los placeres del "paraíso perdido" (la frase es suya) de su existencia dentro del útero materno, lugar que afirma recordar "como si fuera ayer". Es- cribe Dalí: Sí, me acuerdo de ese período como si fuera ayer. Por esta razón propongo empezar el libro de mi vida secreta con este comienzo real y auténtico, a saber con estas memorias, tan raras y líquidas, que he conservado de mi vida intra-uterina I , ! (Dalí, 11: 1949,26). Más arriba he estudiado dos ejemplos, entre muchos otros que contiene la autobiografía daliniana, de imágenes y objetos que producen asco y horror a Dalí. Tales imágenes se relacionan con elementos blandos y pegajosos, con lo que no tiene contornos bien definidos, lo que amenaza con invadir nuestro espacio y volverse indistinguible de nosotros mismos. Ii i ! 1: Ahora bien, si leemos atentamente, nos damos cuenta de que similares connotaciones de liquidez, falta de contornos, viscosidad, etc., se usan también, en l.a vida secreta..., para caracterizar el período embrionario dc Dalí en el interior de la matriz. Ésta es la descripción de aquel "lugar": ! ! Realmente, si me preguntan cómo se cstaba "allí dentro", debo contestar inmediatamente, "era divino, era el paraíso". (,Pero, cómo era este paraíso? (...) Déjenme empezar con una descripción general: el paraíso intrauterino era del color del in- i 01 200 I-! -,", ~. -1 t""i ï ~~ PLACERES DE LA AUTOESCISIÓN l' fierno, es decir, rojo, naranja, amarillo y azulado, del color de las llamas, del fuego; por encima de todo, era h!alldo, ill/11!Ívil, tihio, simétrico, dohle, pegqjoso (Dalí, 1949,27; la cursiva es mía). a la viscosidad del interior materno de la de aquella del del vientre erizo es que esta última ha pasado a ser un inquietante símmasa de gusanos bolo de negatividad y de muerte. Las memorias de Dalí van del interior del cuerpo materno Lo Único que, en Dalí, diferencia i: del paraíso líquido del Útero a la no menos líquida, pero ahora inconsistencia de los cadáveres, los gusanos y las abyectas garrapatas. Kristeva se pregunta: "i.l'or qué vericuctos se asocia el interior de la madre con la putrefacción'!" (l9X2, 10 1) ~Por qué vericuetos, podcmos preguntar a Dalí, la co/'{/ maternal ha pasado a al cuerpo cn descomposición, repugnante, convertirse en lo abyecto? La evocaciÓn del cuerpo materno, en el capítulo dedicado a las memorias intrauterinas, induce cn Dalí la imagen de su nacimiento como un acto de expulsiÓn violento y terrible por el cual se arrancÓ a sí mismo de la materia del interior materno. Como apunta Elisabeth Grosz, lo materno, como algo quc permanecc fuera de toda simbolización, "tiene que ser reprimido o sublimado para quc sea posible para el niño obtener la unidad, la estabilidad, la identidad y una posiciÓn desde la cual poder hablar" (1990, \0\). En la Última sección de este artículo estudiaré un ejemplo concreto en que esta sublimaciÓnlrepresión de lo materno se lleva a cabo en Dalí. .:. ::: 1: ::: Hasta aquí hc analizado el modo en quc la voluntad de dominio y de control (tanto de sí mismo como del exterior) se relaciona en Dalí con la noción de lo abyccto de Kristeva. Sin embargo, aunque cxtremadamente productiva en su modelo gcneral, la nociÓn dcl mecanismo de la abyección sólo puede dar cuenta de la vocaciÓn abyectante de Dalí de modo parcial. Informado por ideas como la del "extraviado" (que sugieren tanto una compulsión ciega establecer la propia identidad en términos de pureza y negación de lo "otro", como la im- a posibilidad última de conseguir tal cosa), el argumento de Kristeva ignora un factor que es de crucial importancia en los usos dalinianos de la subjetividad: el placer del sujeto. No tiene en cuenta el hecho de que la anulación momentánea de la organizaciÓn psíquica (favorecida por la abyección) puede constituir también, y en Dalí quizás antes que cualquier otra cosa, una experiencia gozosa de carácter inequívocamente masoquista, La nociÓn del masoquismo del teórico nortcamericano Leo l3ersani nos ofrece, en este sentido, una muy rica contribuciÓn. SegÚn Bersani, el deseo del sujeto no tendería hacia su propia extinciÓn en la satisfacción; por el contrario, el único objeto del placer libidinal lo constituiría, segÚn él, la necesidad de repetir y aumentar el trastorno placentero del equilibrio psíquico (una necesidad que se describe como una "sublimación original", y que' la sexualidad mantendría y reproduciría incesantemente). Bersani cscribe: "I.a tensión dc la sexualidad entre lo placentero y lo no placentero -el dolor de una excitación autoanulante- tiende a ser mantenido, reproducido e ineluso aumentado" (1990, 37). En las páginas que siguen, quisiera argumentar qpe las abyecciones de Dalí en La vida secreta... no pueden scr entendidas meramente como el resultado de un repetido fracaso al intentar establecer su propio yo como una entidad "propia y limpia", plenamente unificada y presente a sí misma. Por el contrario, la abyecciÓn conlleva en Dalí una desestabilización ra- 201 '~$ 1: :; i: i: DA VID VILASECA dical de tal economía, una afirmación exultante de goce masoquista como destrucción de la coherencia subjeti va y como muerte del Yo. 3. El masoquismo y la abyección 1, " 1: l.; los "Tres cnsayos sobrc la tcoría scxual" (1905) de Freud po- Como apunta Bcrsani, drían apuntar hacia la idea de que la excitación scxual ticnc lugar cuando es excedido el nivelnormal de sensacioncs del cuerpo, y cuando la organización dcl yo cs momentáneamente í trastornada por estímulos que van "más allá" dc lo que rcsulta compatible con la organizaeión psíquiea. Bersani cscribe: "Freud simultáneamente apunta hacia y sc retrae de una definición de excitación scxual como algo similar a un trastorno dcl cquilibrio psíquico disfrutado dc modo masoquista. Por lo mcnos cn la mancra cn quc se eonstituye, pucdc scr una tautología por clmasoquismo" (Bcrsani, 1990, 37)C'. la sexualidad El masoquismo no constituye sólo la característica principal de la sexualidad humana, según Bersani, sino que se halla también en el mismo origen de la historia del sujeto. Para Bersani, "el sujeto humano es originalmente escindido ell la sexualidad" (1990, 36); para este autor, la primcra organización del yo tiene lugar simultáneamcntc a la sublimación dcl autoerotismo, sublimación que no implica ninguna idea "redentora" dc una tendeneia "más elevada", sino mcramcnte la desviación del instinto sexual desdc una aetividad fijada en un objeto (tal como chupar del pecho de la madre) hacia otra en la cual la única fucnte de placer (Bcrsani, 1990, 35-37). Según Ikrde una organización psíquica conllcva incvitablemente la expe- scría la propia experieneia masoquista de la autocscisión sani, la primera expericncia ricncia de la ruptura; cl yo nacc como una totalidad ya cscindida, como una agencia "sedueida hacia el ser" (la locución cs dc Bcrsani) por la propia pcrspectiva de ser escindida (1990, 37)7. Además, y cn cstc punto converge el masoquismo con el deseo de control, la tcndencia hacia el desmantelamicnto de la organización psíquica también está prcscntc cn los movimientos instintivos dcl sujeto. Bersani apunta que, respecto de las relacioncs dcl organismo con objetos, los instintos del yo y los instintos sexuales tienden hacia un fin común: "la climinación de cualquicr cosa extcrna al organismo" (1986, 87). Al mcnos cn cl cstadio pregenital de la sexualidad infantil, la incorporación dcl objcto parccc inhcrcntc al dcseo sexual: "El amor en la fase oral -escribe Bersani- conlleva la abolición dc la cxistcncia scparada del objeto. En el estadio dc organización sadístico-anal, el afán por el objcto aparcce bajo la forma de una necesidad de control" (Bersani, 1986, 87). !~ i 1': -, ! Sin embargo, dado que la incorporación de un objeto amado conlleva inevitablemcntc una cierta "invasión", una desestabilización del ego por estímulos proeedentes del objeto, la necesidad dc incorporar y dominar elementos externos puede ser eoncebida como una forma Por otro lado, un control absoluto tanto del objeto externo como dc uno de masoquislllo. '/11(' F/"('lIdia1/ /lod.l' (1 <)86). 32-3<): también, su artículo "Repre6.- Vean también el libro anterior de Bersani sentation anu its Discontents" (1 ')H t), 5,,7. el sufrimiento ext1Ítico dc un 7.- Bersani escribe: "El ueseo masoquista primario tcndería meramcntc a repctir <'/)/"(111/('11/('1/1. La primcra totalidad psíquica estaría constituida por un dcseo de cscindir la totalidad. U puro illf(>r(,1/Ó" ego, ('11 Sil o/"ig(,lI, 1/0 serí" II/(ís Ij/(e 11110 "p"sio//(/(/a pio d('.\'1//(/IIIe!(//lIi('lIlo" (Bcrsani, I <)<)(), 3H. El énfasis es mío). 1I('c('.\'il"do po/" e!/)/a('('/" "lIlicip"do de Sil pro- I! i J 1, i: le! 1: 202 1'; Ij l ~ k.;~ '" [1..j " ....' -.::....;. j:' ;!-r-2: 1; PLACERES DE LA AUTOESCISIÓN mismo implicaría el fin de todo requerimiento, "el fin del movimiento, una quietud no-de1977, X6), lo que equivaldría también a la seante como la de la materia inorgánica" (Bersani, anulación masoquista del sujeto deseante. En base a esta argumentación, Leo l3ersani llega a un total replanteamiento de los conceptos de sadismo, masoquismo y narcisismo, Para este autor, el masoquismo ya no es se- ,. cundario en relacilÍn al sadismo, como Freud había sostenidox. Por el contrario, el masoquismo está en la base tanto del odio al objeto externo que caracteriza al sadismo como de la i: voluntad de dominio y de poder que acompaña al narcisismo. Un denso párrafo resume la relacilÍn existente, según Bersani, entre estos tres conceptos: ,1 1, .. i! Si el odio de un objeto externo, en virtud de su misma intensidad, constituye un H fenlÍmeno sexualizante, IlÍgicamente debería ser descrito como una forma de sadi,\ï/IO, Si además este odio, al romper los límites entre el yo y el mundo, satisface del yo les decir, sus deseos de poder y de con- los "viejos deseos de omnipotencia" trol J. entonces debería ser tratado también como una forma de narcisisll/o. Por último, si la sexualidad es por definición algo excesivo, una escisión psíquica debida a , . , la brecha existente entre el nivel de estimulacilÍn a que se somete al yo y su capacidad estructuradora, entonces el odio de objetos externos por parte del yo, su invasión por los estímulos provenientes de esos objetos, y su necesidad de incorporar objetos amados, puede ser identificada también con eln/((.\OCfuisll/o (Bersani, 19X6, XX-X9). Quisiera apuntar específicamente a la relacilÍn entre masoquismo y abyeccilÍn. El paralelismo entre ambos conceptos se puede trazar a distintos niveles. Desde un punto de vista onlogcnélico (referente a la historia del sujeto), lo que 13ersani llama masoquismo se puede interpretar como una represión primaria (aunque, de "primaria", nada) por la cual el yo-queva-a-ser, al mismo tiempo que sublima su autoerotismo, se eseinde o abyecta a sí mismo de la entidad maternal en un intento de establecer su propia independencia y unidad respecto a li ella. Por otro lado, desde un punto de vista sincrÓnico, el mecanismo de la abyección tiene un carácter marcadamente masoquista, ya que induce a la escisión y a la desorganización psíquica de la misma identidad subjetiva que está intentando establecerse por oposiciÓn a esos (no- lobjetos. i'I , r~ h.; En los dos episodios de l.a pida sec/'eW... que cité en la secciÓn anterior, por ejemplo, pudimos observar que, antes de que el rechazo del animal abyccto sc produjera, sc detectaba una incquívoca atraceilÍn r: fascinación por lo tcrrorífico y asqueroso por parte del narrador. "Voluptuosidad morbosa", "fascinacilÍn repugnantc" (Dalí, 1949, 94), una asquerosidad que o !,,"j , , "era imposible no mirar constantemente" (Dalí, 1949, 3(5) son expresiones que encontramos menudo en el texto daliniano, y que registran elocuentemente el goce masoquista que, para a Dalí, acompaña a la percepcilÍn de lo abyecto. En relacilÍn con este aspecto, no es casualidad, sin duda, que la abyeccilÍn tenga lugar justo en el momento anterior a que se produzca el l' desmayo, momento en que el yo de Dalí quedaría (aunque momentáneamente) anulado por completo, por lo que la escisiÓn subjetiva dejaría de ser propiamente una experiencia placentera para "él". Vcan Frcud (1915, 127-12H). Para una crítica dc la nociÓn frcudiana dc masoquismo como "sadismo vuclto en contra del propio sujeto", vean el libro dc Gillcs J)clcuzc Ma.l'o"l1i.l'1II (1()H9), 106 Y ss. H,- 203 I 1: , ! ; : e, , , ~~~ '%t. ~, ~:~ 1 ~ ~::. DA VID VILASECA ;'J : En la última sección de este artículo me propongo estudiar la relación existente entre el instinto de control y el masoquismo en I.a vida secreta... Basándome en el episodio de la re- , , ' , . lación adolescente de Dalí con una novia de Figueras (Dalí, 1949, 142-153), observaré el modo en que la introyeeción del objeto amado (a quien consideraré como una eontrafigma de la madre arcaica) se relaciona en la autobiografía con el instinto de control, así como con tendencias de carácter sadomasoquista dirigidas, por parte del protagonista, a separar su identidad "limpia y propia" de la de ese objeto amad%diado. Sin embargo, teniendo en , , , . cuenta la discusión anterior sobre el masoquismo, mi análisis subrayaní no tanto la voluntad de Dalí de establecer su propia identidad por medio de maltratar y reb,~iar sádicamente a su amante, sino la manera en que (en su juego de identificaciones fantasmáticas cambiantes) Dalí inflige activamente dolor al objeto mientras que, por otro lado, lo disfruta él pasivamente como una experieneia de trastorno y escisión psíquicos. , , I 4. El (sado)masoquismo de Dalí: la historia de la amante que duró cinco años Dmante el período previo a sus estudios en la Academia de San Fernando de Madrid, el narrador de la autobiografía nos cuenta que entra en una relación de eineo años con una chica de Figueras, cuyo nombre no se explicita. Dalí y la joven se conocen estando en un atraídos el uno por el otro. Abandonan la clase y corren hacia los campos de trigo, donde se besan y Dalí palpa los pechos de ella, "meneándose bajo la blusa como un pescado atrapado en sus manos" (Dalí, 1949, 143). cmso de filosofía y se sienten inmediatamente Desde el principio se establece entre ellos una relación de amo a esclava, relación en la cual Dalí obtiene placer por mcdio de humillar y rebajar a la chica tanto moral como físicamente. Dalí escribe: "Una calma completa poseía mi mente. Llevaba a cabo mis planes hasta el más ínfimo detalle, con una frialdad tan caleuladora que sentía helárseme mi propia alma" (1949, 143). Dalí es el amo y está en control de la situaciÖn. Sabiendo que quiere marcharse a Madrid para estudiar, calcula de antemano cuánto tiempo va a durar la relaciÖn. Cuando la chica le pregunta si querrá salir con ella al día siguiente, Dalí contesta: "j Mañana por la tarde, sí, y durante otros cinco años, pero ni un día más!". Y luego explica cínicamente: "Tenía mi plan -jera mi plan quinquenal!-" (1949,143). Además, Dalí disfruta diciendo a la ehica que no la ama: '''jNo te amo!' le dije. 'Y nunca amaré a ninguna mujer'" (1949, 143). Obtiene poder sobre ella haciéndola sentir inferior, y manteniendo el amor de ella hacia él insatisfecho. En lo que constituye uno de los episodios más obviamente sexistas de la autobiografía, Dalí.es- cribe: Dmante este noviazgo de einco años puse en juego todos los resortes de mi perversidad sentimental. Conseguí erear en ella una necesidad tan grande de mí (...) que veía crecer mi influencia día tras día (...) El sentimiento de inferioridad (...) creÖ en su mente una insatisfacción tal (...) que no pudi"endoobtener de mí, en lo tocante a la pasión, más de lo que le había mostrado en aquella ocasión -más bien todo lo contrario (pues la simulaeión de frialdad era una de mis armas más formidables)- su amor, prodigaba, contribuyó indudablemente a mantener aquel estado de creciente tensiÖn amorosa (...) más y más vulnerable a las crisis materiales del crimen, del suicidio o del ataque de nervios (Dalí, 1949, 144). que constantemente 204 r; ''i!i;"", PI,ACERES DE LA AlJTOESCISIÖN Una y otra vez, Dalí exige que la chica lleve a cabo actos y sacrificios para él, talcs como simular que estä muerta o descubrir sus pechos. "Muéstramelos", Dalí le ordena a menudo (1949, 151). Cuando Dalí se marcha a pasar el verano a Cadaqués, contesta a las devotas cartas de ella sólo muy de vez en cuando, y "siempre con una espina de veneno que sabía no podría dejar de envenenarla y hacerla volver amarilla como la cera" (1949, l4ó). Por si esto fuera poco, Dalí disfruta manipulando los sentimientos de la chica, aisländola de sus amigos y convirtiéndola en totalmente dependiente de él: ; 1'; Por eneima de todo, no quería que tuviera ningún amigo (oo.) Tan pronto sabía a alguien, o si me hablaba de alguien de modo favorable, inme- que había conocido diatamente intentaba mostrar desaprobación, arruinar y aniquilar a esa persona en su mente, cosa quc siempre conseguía (oo.) Exigía la sumisilÍn de sus sentimientos al pie de la letra, y eada infracción de mi inquisición sentimental implacable tenía que pagarse con sus amargas lágrimas. Un tono desdeÍioso dirigido a ella, soltado como quien no quiere la cosa en una conversación casual, era suficiente para hacer quc se a punto de morir. Ya no tenía esperanzas dc que yo pudiera amarla, pero se sintiera aferraba a mi estima como una mujcr ahogändose (Dalí, 1949, 150-151). Por medio de infligir tácticas sádieas a su víctima femcnina, Dalí puede identificarse provisionalmente con una ilusión de control tanto sobre la situaeilÍn como sobre sí mismo: "i,Cómo había podido, en tan poco tiempo, volver a tener dominio sobre mí mismo otra vez'!", Dalí se pregunta (1949, 143). En este sentido, Dalí opone explícitamente la situación según él, características totala una de verdadero amor, que conllevaría, en que se encuentra mente opuestas: fusilÍn con cI objeto amado, la disolución del yo y la imposibilidad de arbitrar sobre sus propias acciones. Dalí escribe: Al no amarla, mantenía intaeta mi soledad (oo.) Sabía que amar (oo.) era algo totalmente distinto, algo que exigía la aniquilación del yo en una confusión omnipotente de todos los sentimientos, donde todo discernimiento consciente, toda decisilÍn metodollÍgica sobre las acciones amenazaba perpetuamente con arruinarse de la manera más paradójicamente imprevisible (Dalí, 1949, 145). Sin embargo, llegados a este punto, empezamos a damos cuenta de lo que pueda estar ocurriendo realmente en este episodio. Sabemos bien cuánto aterrorizan a Dalí las imágenes .~ y las figuras que, de uno u otro modo, amenazan la estabilidad de su identidad, mostrándole la negatividad y la muerte en base y en oposición a las cuales se construye su subjetividad en el lenguaje. .~ j Además, como apunté más arriba, la ausencia o vacío que representa la madre arcaica seducen al sujeto con una alucinación de su propia aniquilación, constituyendo así, para él, el origen (estructuralmente excluido) de un instinto de retorno que es eonstitutivamente imposible de satisfacer (un retorno donde, propiamente, el "yo" no ha estado nunca). i. Pues bien, sostengo que la amante de Figueras representa para Dalí una contra figura de la madre (arcaica). De modo inconsciente, Dalí ha desplazado en su novia todo su terror y su odio hacia la figura materna, lo que le permite, a nivel consciente, mantener intacto su amor l' hacia la madre "real". Naturalmente, Dalí no ama a la chica, incluso la puede despreciar y odiar; slÍlo así puede desvincularse de todo lo que ella representa, y asegurar, por consi- i' guiente, tanto la "pureza" de su amor consciente hacia la madre como la ilusión de su propio yo uni ficado e independiente. i. I I~ 205 1: !'( i: ,. ~l ~ ~ DA VID VILASECA " . , ~ Varios elementos de] episodio apuntan hacia una equivalencia simbólica subyacente entre la amante y la madre de Dalí. Físicamente, la chica es "una criatura muy bella", de cabe145), adjetivos que concuerdan con las cualidades idealizadas que su madre sólo unas páginas más adelante. Ésta, la madre, tenía un "alma llos rubios (Dalí, 1949, Dalí atribuye a santa", "yo la adoraba", Dalí escribe (1949, 152). Por otro lado, el interés erótico de Dalí en la chica está casi exclusivamente dirigido a sus pechos, la parte del cuerpo femenino que más puede recordarnos a la relación entre madre e hijo. I.os pechos de la chica se describen con un detalle feticbista que apunta inequívocamente a la función nutritiva: "Eran incompa- rablemente bonitos y blancos; sus pezones eran exactamente como frambuesas" (1949, 151). :j '1 '. Sin embargo, el momento en que la equivalencia simbólica entre la novia y la madre re- sulta ya evidente llega cuando la muerte metafórica de la chica (después de que Dalí la haya rechazado, transcurridos los cinco años) se hace coincidir con la muerte real de la progeni- tora al final del capítulo. En uno de sus paseos por Cadaqués, Dalí tiene una premonición de la muerte de su amante: "Un presentimiento cruzó mi cerebro como un rayo: (,se habrá muerto?", Dalí se li pregunta. Pero es un pensamiento irreal, pronto refutado por ]a llegada de otra de sus cartas. !: Dalí escribe: Me asaltó un sudor frío, que no me dejó hasta que llegué a casa, donde me estaba esperando una carta de mi amante, que coneluía diciendo: "Estoy engordando, y todo el mundo dice que tengo muy buen aspecto. Pero a mi sólo me interesa lo que tú pensarás de mí (Oo.) Mil besos, y otra vez, nunca te podría olvidar, etc., etc., (Oo.) i La muy idiota' (Dalí, 1949, 14R). Un poco más adelante tiene lugar ]a muerte real de la madre de Dalí. Al contrario de la idiotez de la amante, la madre se representa en términos muy idealizados y sublimados. Es sintomático que la madre de Dalí (que está casi totalmente ausente en las páginas anteriores de la autobiografía) tenga que aparecer por primera vez en el momento de su muerte, que tenga que sernos presentada y hacerse parte de las memorias sólo a través de su idealización ((l/sencia, a través de una sublimación que requiere necesariamente su desaparición en tanto que presencia. Dalí alaba el "alma santa" de su madre, pero sólo a expensas de rechazarla en el cuerpo de la amante. Sólo después de matar y abyectar el peligro que el cuerpo materno representa (en la figura de la amante) puede empezar sin ningún peligro el proceso COl1lO de idealización de la madre real. Dalí escribe: i~ : I l' I! i. !! ; l' Sobrevino la muerte de mi madre', y éste fue el golpe más grande que había recibido en toda mi vida. La adoraba; su imagen se me aparecía como única. Sabía que los valores morales de su alma santa estaban muy por encima de todo lo humano, y no me podía resignar a la pérdida de un ser con quien contaba para hacer invisibles las máculas de mi alma (Oo.) Ella me adoraba con un amor tan íntegro y tan orgulloso La muerte de mi madre me pareció como una afrenta del destino (Dalí, 1949, 152-153). (oo.) Como ya apunté basándome en E. Grosz, el cuerpo materno tiene que ser o bien reprimido, o sublimado por el sujeto (Grosz, 1990, 10 1). Freud confirma esta teoría en su artículo "Sobre ]a más generalizada degradación en la vida amorosa" (1912), donde se apunta que el sujeto a menudo degrada el objeto sexual a fin de sobreponerse 206 a las ansiedades de castra- :; ~'Q PLACERES DE LA AUTOESCISIÓN fijaciÓn inconsciente de la libido en la figura materna~. Esto equidegradar una representacicín inconsciente de la madre para poder sal varia reprimir y cicín producidas por una vale conscientemente, a sobrevalorar su "alma" mientras se reprime s<Ídicamente su realidad corporal, a darle vida mientras se mata y abyecta su amenazante "maternidad". a Rechazando y degradando a la madre el/ su novia (abyectando la posibilidad de la madre arcaica en él), Dalí puede identificarse con una ilusión de control tanto sobre la situación en que se encuentra como sobre sí mismo. Sin embargo, si Icemos atentamente, el episodio de la novia de Figueras nos da pistas suficientes, si no de que lo que quiz<Ís esté ocurriendo lo contrario, sí al menos de que el sistema de identificaciones del cual Dalí extrae su jlli.l'.I'al/ce en relacicín a la chica es mucho m<Ís complejo y fluido de lo que hasta aquí ha quedado dicho. No es sólo sádico, sino masoquista también, no sólo encaminado a establecer un espejismo de coherencia y autonomía subjetivas sino también, paradcíjicamente sea precisamente y en un mismo impulso, hacia la desestabilización radical de tal instancia. En su conocido artículo "Pulsiones y destinos de pulsicín" (1915), rreud subraya el papel de las identificaciones que desarrollan, en el plano de la fantasía, los pacientes s<Ídicos y/o masoquistas respecto a la otra persona en el intercambio erótico. Por un lado, rreud apunta que, en el caso del masoquismo, el yo en el papel pasivo "se sitÚa también, en su imaginacicín, en el otro papel, que ha sido tomado por el sujeto extraño" (freud, 1915, l2S). Por otro lado, en el sadismo, "mientras el dolor se inflige a otras pcrsonas, el sujeto lo disfruta de modo masoquista a través de una identificación con el objeto sufriente" (rreud, 1915, 12lJ). La fluidez y la inestabilidad radicales que, según rreud, caracterizan la posición del sujeto en relaciÓn al objeto amad%diado en el intercambio sadomasoquista, se hace también patente en el episodio de las memorias de Oalí que estamos estudiando. Por un lado, tal inestabilidad en el plano identificativo se transluce como efecto paradÓjico del exceso retcírico con que el episodio parece postular lo exactamente opuesto. Me explico: la singularidad de Oalí en oposición a la chica es afirmada tan repetidamente, y de modo tan arrogante y sin fisuras, que el texto acaba por inducir a la subversicín de la propia economía binaria en la cual se apoya la diferencia entre ambos personajes. Uno de esos momentos de exceso retórico tiene lugar en el siguientc pasaje, donde el uso de los pronombres por parte de Dalí se hace tan confuso que rcsulta difícil ya poder distinguir con nitidez entre él y la chica: Pero la piedra angular de esta cúpula de tortura moral quc estaba construyendo conocimiento, totalmente compartido por los dos, de que yo no la quería. Ciertamente yo sabía y e/la sabía que yo no la quería; yo sabía que ella sabía que yo no la quería; e/la sabía que yo sabía que e/la sabía que yo no la quería (...) (Oo.) era, sin duda, el (Dalí, llJ49, 145. El énfasis es mío). Es sintomático que la "piedra angular", el nudo central del intercambio sadomasoquista entrc Dalí y su novia produzca precisamcnte el desdibujamiento de los límites que diferencian el uno de la otra, mostrando así el car<Ícter intercambiable que define las posiciones de ambos (tanto a nivel sintáctico como en el de la fantasía) por lo que se refiere a quién est<Í haciendo qué a quién, quién es el sujeto y quién el receptor del amor, del acto de sadismo, del placer y del dolor. Mostrando la recursividad potencialmente infinita del lenguaje (yo sabía que e/la sabía que yo sabía que e/la sabía...), la cualidad intercambiable que dcrine la po- 9.- Vean cl artículo dc Freud "Sobre la lll1Ís gcncralizada dcgradaciÓn cn la vida alllorosa" (1912), I g 1-1 lO. 207 i.!?: DA VID VILASECA sición dcl hahlante "yo", rcspecto del apelado "tú", Dalí socava, cn cl plano dc la escritura, la oposición entre el yo y el otro (sujcto activo y rcceptor paciente, amo y csclavo) quc cl contenido manificsto del cpisodio sosticnc dc modo tan clocuente y reiterativo. Así. qucda subrayado quc son contradictorios y múltiples (y no uno c invariablc) los roles e idcntidades quc, a nivcl fantasnuítico, dan cuenta de la implicaciÖn dc Dalí en la cscena sadomasoquista. l' I l' 1: !! " Hay otro aspccto sobre cl cual quisiera llamar la atcnción, ya que muestra la fluidcz de las localizaciones dc Dalí de manera aún más cxplícita. Me reficro a la incspcrada apariciÖn cn cstc cpisodio del mito de San Sebastián, santo cuyo martirio ticnc notorias connotacioncs sadomasoquistas, y a quicn Dalí sc refierc a menudo tanto en sus mcmorias como cn otras lA 1: !: j partes de su corpus cscrito y pictórico1ll. La mcnciÖn tienc lugar justo después de los ya citados comentarios dc Dalí sobre lo que él eonsidcra cl amor "real" (como "confusión omnipotcntc de todos los scntimicntos" y "aniquilaci(m del yo"), sentimicnto que se oponc a la conciencia dc estar cn control y dominio absolutos, característica dc su rclación con la prcscntc amantc. Ilustra el pasaje, cn la edición un andrógino dibujo del propio Dalí como San Sebastián, atado bol con varias flechas clavadas en su cucrpo desnudo. Escribe Dalí: de la autobiografía, a un ár- Aquí, por cl contrario, mi chica sc convirtió en el blanco constante dc mis prucbas dc habilidad, quc yo sabía que mc iban a "scrvir" más tarde. Me daba perfccta cucnta de que cl amor cs rccibir la flecha, no dispararla; y probaba sobre su carnc aqucl San Sebastián quc yo llevaba cn mi propia piel en cstado latcntc, y que hubiera qucrido lanzar afuera como la serpiente hace la muda (Dalí, 1949, 145). El pasaje mcrccc scr comentado con cicrto dctallc. Por un lado, a lo largo dc este episodio, Dalí se ha idcntificado con el amo cn cl intcrcambio sadomasoquista: él ha sido quien cstaba en la posiciÖn dc control, cl que no amaba; él era quicn lanzaba las flechas a un escindido San Sebastián que, por consiguiente, no podía ser nadie más que su víctima fcmenina. Sin embargo, por otro lado, éstc cra un San Sebastián "que I él] llevaba en Isu] propia picl cn estado latente", San Sebastián para el cual la víctima dc Dalí era sólo "cl blanco constantc dc [susJ prucbas dc habilidad", prucbas que sabía "Ile] iban a 'scrvir' la élJmás tarde". Luego, la rcfcrcncia a San Scbastián (y la identificación cxplícita dcl pintor con él cn cl dibujo adya- ccntc) apunta hacia una imagen fantasmática de Dalí viéndose a sí mismo como el objcto dcl dolor quc inflige a la chica; viéndosc como cl objeto de las hiricntcs flechas del amor, y no como el dcspiadado tirador al arcoll. 1': i: 1: li ji 1: ! i. i ! I! !\ i, i: 1: 1; li !! 10.- El mito de San Sebastián constituyc un tema recurrente en los primeros escritos de Dalí. En 1927 publicÚ un poema con el nombre del santo en la revista I:Alllic de les Arl.\'. y también aparece a menudo en su correspondencia con García Lorca, como ha subrayado Rafael Santos Torroella en su artículo "Sant Sebastià i el mi te daliniÜ" (1990), San Sebastián se convirtiÚ en una referencia comÚn entre ambos artistas, con la cual los i1 !i 1: " , dos se podían identificar indistintamente, "En mi San Sebastián eres tÚ (..) mucho y veces me parece que jA ver si resulta que San Sebastián eres tÚ!". escribe Dalí a Lorca en 1926; y luego apunta. idenlilïte recuerdo a eándose ahora él mismo con el santo: "Pero por 'Ihora déjame que use su nombre para firmar. Un gran abrazo de tu San Sebastián" (Dalí, I 97R, 44). 11.- En el pnílogo a su novela Hiddell Faces (1944), Dalí hace un lÚcido comentario que apunta directamente a la movilidad de posiciones subjetivas que. segÚn vengo defendiendo. caracterizan al individuo en el intercambio sadomasoquista. Dalí define el "cledanismo" (nomhre derivado del de la protagonista de la novela) como la síntesis entre sadismo y masoquismo. síntesis que consiste en el placer de identificarse con el dolor inlligido al objeto. Escribe Oalí: "El sadismo puede ser definido como el placer experimentado a través dcl ' jl I I! , ' l' j-I l' '1 11 1: 208 li 1; 1! l' liJ -, ,,' l" ~~ ~ PLACERES DE LA AUTOESCISlc)N A través de su identificación con el ohjeto, Dalí disfruta de modo masoquista el dolor que (sádicamente) inflige a su víctima. El episodio sugiere, pues, de acuerdo con Leo ßersani, que el sadismo de Dalí (así como su instinto de control y su vocación ahyectante) no son primarios en relación con sus tendencias masoquistas. Por el contrario, el masoquismo es el San Sebastián que él lleva "dentro de sí mismo", la separación primaria, la escisión de su unidad suhjetiva que establcce tanto la imposibilidad de un origen absoluto de su yo estruc- turado como el fascinante y último abyecto que lo seduce hacia su propia anulación subjetiva. :+: .:. .:. En cl presente artículo he querido mostrar cómo cl instinto de control y poder de Dalí entra en relación con el mecanismo de la abyección y con las tendencias masoquistas que varios teóricos contemporáneos establecen como características de la subjetividad y de la sexualidad humanas. Habiendo analizado la variedad de identificaciones por medio de las cuales Dalí se define en oposición no sólo a lo terrorífico y asqueroso, sino también a su novia de Figueras ("no-objetos" que, en último término, ocupan el lugar de la madre arcaica), he sostenido que la tendencia a la abyección no se puede explicar solamente como un intento repetido de establecer la limpieza y propiedad del sujeto. Por el contrario, siendo a la vez esclavo y amo (tanto el sujeto como el receptor de la violencia), en la multiplicidad de sus posiciones y fantasías de autoeseisión, Dalí nos muestra que está siempre dispuesto a recomen- zar sus representaciones experimentales de quien, por quien y por medio de que su organización psíquica ha sido escindida. Un San Sebastián (un extraviado, pero vocacional), la abyección ha mostrado a Dalí el placer masoquista de su propia escisión: una visión de un lugar donde él nunca estuvo, donde él propiamente nunca podría volver a estar. Referencias bibliográficas BERSANI, Leo, (l9gG), The Frelldiall Body: PsycllOallalysis (lIld Art Nueva York, Colum, bia LJ ni versity I'r~ss. - (1990), n/e Cllltllre (~f'Redelllptioll, Londres, Harvard University Press. I i DALÍ, Salvador, (1949), The Secret L(f'e qf' 5ìalvador Datí by Salvador Dalí (trad. I [aakon M. Chevalier), Londres, Vision [trad. española: La vida secreta de Salw/dor /)alí (trad. José Martínez), Barcelona, Dasa, 19911. - - (1952), Rostros oCllllOs, Barcelona, Caral!. (19n), Salvador /)alí escribe a Federico Garda Lorca (ed. Rafael Santos Tormclla), Madrid, Ministerio de Cultura. DELEUZE, Gilles, (19g9), !vfasochi.\ïll (trad. Jean McNeil), Nueva York, Zone ßooks [del originall'réselltatioll de Sacher !vfasoch, París, Minuit, 19671. dolor infligido al objeto; el masoquismo, eomo el placer producido a través del dolor intlingido por el objeto. El cledanismo es el plocer y el dolor subli/l/odos Iwr //1/(/ o!>solll1o identificoción trascendente con el objeto" (Dalí, 1952, 10; la cursiva es mía). 209 'W-o. DA VID VILASECA FELMAN, Shoshana, (1977), "TlIrning the Screw of Interpretation", 55-56 (VI), 94-207. Yale French Stl/dies, FREUD, Sigmllnd, (1905), "Three Essays on the Theory of Sexuality", The Standard Edi- :"1 \) tion 4the Complete PsycllOlogical Works (~f Sigml/nd Freud (trad. James Strachey), Hogarlh Press, 195/ì-1974; VII, 125-245 [trad. española: "Tres ensayos soLondres, bre la teoría sexual", Obras completas de Sigmul/d Frel/d (trad. José L. Etcheverry), Buenos Aires, Amorrortu, 1976; XXIV, 109-2241. (1912), "On the Universal Tendeney lO Debasemenl in lhe Sphere of Love", S.E., XI, 177-190 [trad. española: "Sobre la más generalizada degradacicín en la vida amorosa", O.c., XI, 169-1!ì41. - I " I i~ :. " " (1915), "Instincts and their Vicissitudes", - SE, XIV, 109-140 [trad. española: "Pulsiones y destinos de pulsión", O.C., XIV, 105-1341. (1919), "The Uncanny", - H i S,F;., XVIII, 217-256 [trad. española: "Lo ominoso", O.c., XVII, !: 215-252). GROSZ, Elisabeth, (1990), "The Body 01' Significmion", en FLETCHER, John y BEN1AMIN, Andrcw (eds.), Abjection, Melancholia and LOl'e: 'Ihe Work (~f.ll/lia Kristel'a, Londres, Routledge, \990, /ì0-1 03. 1- i: :' KRISTEV A, Julia, (1982), I'mvers (~f 1I0rror: An Essay 011 A/~iectic!/l (lrad. Leon S, Roudiez), Nueva York, Columbia University Press [del original Pouvoir.\' de I'horrel/r, París, Seuil, 19/ì01. LECHTE, 101111, (1990), .Iulia Kristeva, Londres, Routledge, O'CONNOR, Noreen, (1990), "The An-Arche of Psychotherapy", en FLETCHER, John y BENJAivlIN, Andrew (eds.), AI~iection, lvlelancholia al/(Il_ove: n/e Work (~f .Iulia Kristeva, Londres, Routledge, 42-52, SÁNCI-IEZ- VIDAL, Agustín, (1988), l/mIl/el, Lorca, Dalí: El enigma sin .fIn, Barcelona, ,i: ,- l' I i. l' i: l. !j Planeta. SANTOS TORROELLA, Rafael, (1990), "Sant Scbastià i el mite daliniÙ", en FANES, Félix (ed,), :' Dalí escriptor, Barcelona, [<undacicí Caixa de Pensions, 33-47. SECREST, ivlcryle, (19/ì6), Salvador l)alí: the Surrealist .les ter, Londres, Paladin Books, rl ~ : : 1, f..i !i I~ :: r-l i 210 : \ I \j 1; b{ ~.,j f'!i , . 'i.#
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Microbial communities in sediment from <i>Zostera marina</i> patches, but not the <i>Z. marina</i> leaf or root microbiomes, vary in relation to distance from patch edge
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How to cite this article Ettinger et al. (2017), Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge . PeerJ 5:e3246; DOI 10.7717/peerj.3246 Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge Cassandra L. Ettinger1, Sofie E. Voerman2,3, Jenna M. Lang1,4, John J. Stachowicz5 and Jonathan A. Eisen1,5,6 1 Genome Center, University of California, Davis, CA, United States 2 Climate Change Cluster, University of Technology Sydney, Sydney, Australia 3 School of Life Sciences, University of Technology Sydney, Sydney, Australia 4 Trace Genomics, San Francisco, CA, United States 5 Department of Evolution and Ecology, University of California, Davis, CA, United States 6 Department of Medical Microbiology and Immunology, University of California, Davis, Davis, CA, United States 1 Genome Center, University of California, Davis, CA, United States 2 Climate Change Cluster, University of Technology Sydney, Sydney, Australia 3 School of Life Sciences, University of Technology Sydney, Sydney, Australia 4 Trace Genomics, San Francisco, CA, United States , , , 5 Department of Evolution and Ecology, University of California, Davis, CA, United States 6 Department of Medical Microbiology and Immunology, University of California, Davis, Davis, CA, United States INTRODUCTION The seagrass, Zostera marina, is a foundation species in protected bays and estuaries throughout the temperate northern hemisphere. Seagrasses are fully submerged marine angiosperms and are a paraphyletic group comprised of three lineages in the order Alismatales that convergently adapted to the marine environment between 70 and 100 million years ago (Les, Cleland & Waycott, 1997). There are only approximately 60 species of seagrass compared to the 250,000 species of terrestrial angiosperms, a testament to the strict selective pressure posed by re-entry to the marine environment (Orth et al., 2006). Seagrass patches serve as habitat and nursery grounds for many marine species, play key roles in nutrient cycling and carbon sequestration, and serve to protect the coastline from erosion (Williams & Heck Jr, 2001). Z. marina populations, like those of many seagrass species, are negatively affected by climate change, pollution and habitat destruction and so far, restoration efforts have been costly and ineffective (Orth et al., 2006). As a result, Z. marina is vulnerable to habitat fragmentation and loss. The work described here was originally focused on a phenomenon known as ‘‘edge effects’’ in which the border between habitats is intermediate in abiotic conditions from the center of either adjacent habitat and thus the biotic composition of the border habitat, or edge, may differ from that of interior, intact habitat. Edges often support a mixture of organisms from two adjacent habitats (Fox et al., 1997; Davies-Colley, Payne & Elswijk, 2000), but may be abiotically unsuitable for species found in the center of either habitat. Increased predation and invasion by non-native species can also be a common feature of edges (Paton, 1994; Fox et al., 1997; Harrison & Bruna, 1999; Flaspohler, Temple & Rosenfield, 2001). Prior work on seagrasses have shown edge effects on species abundances (Smith et al., 2008; Smith et al., 2011; Tanner, 2005) and abiotic conditions such as turbulence (Granata et al., 2001; Folkard, 2005), carbon stocks (Ricart et al., 2015) and organic matter deposition (Duarte & Sand-Jensen, 1990; Cebrián et al., 2000). Here we investigate whether such edge effects are evident in the microbiota found in, on and near Z. marina plants. Our interest in the microbiota for this study is driven by our overarching goal of developing Z. marina as a model for studies of microbial communities associated with marine plants. ABSTRACT Background. Zostera marina (also known as eelgrass) is a foundation species in coastal and marine ecosystems worldwide and is a model for studies of seagrasses (a paraphyletic group in the order Alismatales) that include all the known fully submerged marine angiosperms. In recent years, there has been a growing appreciation of the potential importance of the microbial communities (i.e., microbiomes) associated with various plant species. Here we report a study of variation in Z. marina microbiomes from a field site in Bodega Bay, CA. Methods. We characterized and then compared the microbial communities of root, leaf and sediment samples (using 16S ribosomal RNA gene PCR and sequencing) and associated environmental parameters from the inside, edge and outside of a single subtidal Z. marina patch. Multiple comparative approaches were used to examine associations between microbiome features (e.g., diversity, taxonomic composition) and environmental parameters and to compare sample types and sites. Submitted 4 January 2017 Accepted 29 March 2017 Published 27 April 2017 Corresponding author Jonathan A. Eisen, jaeisen@ucdavis.edu, jonathan.eisen@gmail.com Academic editor Gerrit Beemster Additional Information and Declarations can be found on page 16 DOI 10.7717/peerj.3246 Copyright 2017 Ettinger et al. Distributed under Creative Commons CC-BY 4 0 Submitted 4 January 2017 Accepted 29 March 2017 Published 27 April 2017 Corresponding author Jonathan A. Eisen, jaeisen@ucdavis.edu, jonathan.eisen@gmail.com Academic editor Gerrit Beemster Additional Information and Declarations can be found on page 16 DOI 10.7717/peerj.3246 Copyright 2017 Ettinger et al. Distributed under Creative Commons CC-BY 4.0 Results. Microbial communities differed significantly between sample types (root, leaf and sediment) and in sediments from different sites (inside, edge, outside). Carbon:Nitrogen ratio and eelgrass density were both significantly correlated to sediment community composition. Enrichment of certain taxonomic groups in each sample type was detected and analyzed in regard to possible functional implications (especially regarding sulfur metabolism). Discussion. Our results are mostly consistent with prior work on seagrass associated microbiomes with a few differences and additional findings. From a functional point of view, the most significant finding is that many of the taxa that differ significantly between sample types and sites are closely related to ones commonly associated with various aspects of sulfur and nitrogen metabolism. Though not a traditional model organism, we believe that Z. marina can become a model for studies of marine plant- microbiome interactions. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Subjects Marine Biology, Microbiology, Plant Science Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 INTRODUCTION Recently a few culture- independent surveys of seagrass microbiomes have been published and these provide good initial reference points for our work here (Jiang et al., 2015; Sun et al., 2015; Cúcio et al., 2016; Mejia et al., 2016). Although, these studies have similar big picture findings, there are small differences in microbiome composition between them and thus further teasing apart of the factors that shape seagrass microbiomes is necessary and important work. In this study, we characterized and then compared the epiphytic and rhizospheric bacterial communities of eelgrass using root, leaf and sediment samples obtained from the inside, edge and outside of a single subtidal Z. marina patch. We focused on characterizing the bacterial and archaeal members of the microbiome in each of these samples using high throughput sequencing of 16S ribosomal gene PCR libraries. We focus in particular on the following questions: What is the general taxonomic composition of the Z. marina microbiome? Are there changes in sediment microbial community composition or in biodiversity at the patch edge and, if so, what factors are driving observed differences, environmental abiotic factors or presence/absence of Z. marina? This analysis reveals multiple novel insights into the general structure of the Z. marina microbiome and lays the groundwork for further studies. INTRODUCTION Although we speculate that plant microbe interactions are important for seagrasses, little is known about how the roles of microbial communities associated with marine plants may affect plant health and what abiotic and biotic factors affect the composition of these communities. Terrestrial plants, like Arabidopsis (Lundberg et al., 2012), corn (Peiffer et al., 2013; Bouffaud et al., 2014), rice (Peiffer et al., 2013; Edwards et al., 2015) and poplar (Beckers et al., 2016) have been shown to have distinct microbial communities on the inside (endophytes) and on the surface (epiphytes) of plant leaves and roots, as well as in the surrounding soil or sediment (rhizosphere) (Lundberg et al., 2012). 2/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 These communities can vary across different stages of plant development (Chaparro, Badri & Vivanco, 2014) and with local environmental conditions. In terrestrial systems the main drivers of plant associated microbial community composition are considered to be environmental factors, like soil particle size, pH and moisture content, as well as host plant species (Aleklett et al., 2015; Lakshmanan, 2015). Thus, examining eelgrass microbiota across a known environmental gradient from the center to the outside of a patch has the potential to provide insights into factors that shape the eelgrass microbiome, the full community of microorganisms associated with eelgrass. Recently a few culture- independent surveys of seagrass microbiomes have been published and these provide good initial reference points for our work here (Jiang et al., 2015; Sun et al., 2015; Cúcio et al., 2016; Mejia et al., 2016). Although, these studies have similar big picture findings, there are small differences in microbiome composition between them and thus further teasing apart These communities can vary across different stages of plant development (Chaparro, Badri & Vivanco, 2014) and with local environmental conditions. In terrestrial systems the main drivers of plant associated microbial community composition are considered to be environmental factors, like soil particle size, pH and moisture content, as well as host plant species (Aleklett et al., 2015; Lakshmanan, 2015). Thus, examining eelgrass microbiota across a known environmental gradient from the center to the outside of a patch has the potential to provide insights into factors that shape the eelgrass microbiome, the full community of microorganisms associated with eelgrass. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Molecular methods DNA was extracted from leaf (n = 8), root (n = 8) and both shallow (n = 12) and deep sediment (n = 12) samples as well as from a kit control (n = 1) with the PowerSoil DNA Isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. For the DNA extractions, root and leaf tissues were placed directly into PowerBead tubes from the freezer without grinding. Microbial 16S rRNA genes were amplified using a two-step protocol targeting the V4 region using the ‘‘universal’’ 515F and 806R primers (Caporaso et al., 2012). The primer set was modified to include Illumina adapters and barcode sequences using a dual indexing approach as in Lang, Eisen & Zivkovic (2014). The 41 samples described in this paper were multiplexed with 103 samples from other experiments, for a total of 144 samples on the run. Libraries were sequenced by the UC Davis Genome Center Core Facilities on an Illumina MiSeq (Illumina, Inc., San Diego, CA, USA) to generate 250 bp paired-end reads. Sample collection We collected leaf, root and sediment samples for microbiome analysis from 0.25 m2 quadrats (n = 4) located in the interior (2.5 m from the edge), on the edge (but within the eelgrass habitat) and outside (2.5 m from the edge) of a single shallow subtidal eelgrass patch in Bodega Bay, CA (GPS: 38.319435, −123.053838) during the summer of 2013. Quadrats were positioned 2.5 m from each other parallel to the patch’ edge. Samples were collected during low tide (±0.5 m water depth) at night (11 PM). For quadrats located at the center or edge of the eelgrass patch, one eelgrass shoot was sampled and directly separated into root and shoot tissue. The root tissue consisted of one entire root bundle sampled, the leaf tissue consisted of a clipped leaf of ± 3 cm in length positioned at about half way along the shoot length (±20 cm from the base). For each quadrat, sediment samples were collected at two sediment depths, 0.5 cm or less and 3 cm deep, from randomly selected locations within the quadrats. Microbial samples were directly stored on ice and transported to the laboratory within one hour where samples were frozen at −20 ◦C until further analysis. Environmental data and the samples used for microbiome analysis were collected simultaneously. For each quadrat, eelgrass density was estimated by direct count. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 3/25 Temperature, pH, salinity and dissolved oxygen were measured at 20 cm above the sediment with a YSI 556 handheld multimeter (YSI Inc., Yellow Springs, OH, USA), at a similar height as the shoot tissue was sampled. Sediment chemical and physical properties were assessed by separately coring the top 4 cm of sediment (10 cm diameter, taken twice within a quadrat and combined for analysis), to correspond with the sediment layer most influenced by the eelgrass roots. Sediment was dried (three days at 40 ◦C), mixed, sieved (sieve sizes: 710, 500, 355, 250, 180, 90 and 30 µm) and particle size fractions were weighed to investigate particle size distribution. A portion of the mixed sediment samples (±50 g) was separately analyzed for total organic carbon (TOC), total inorganic carbon (TIC) and Carbon:Nitrogen (C:N) ratio by the UC Davis Analytical Laboratory. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Sequence processing A custom in-house script was used to demultiplex, quality check and merge paired reads (https://github.com/gjospin/scripts/blob/master/Demul_trim_prep.pl). The resulting sequences were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) v. 1.9.0 workflow (Caporaso et al., 2010). For a detailed walkthrough of the following analysis using QIIME, see the IPython notebook (http://nbviewer.jupyter.org/gist/casett/86da7fc8749d27574f183498df65134a). The sequencing run for this project included samples from other projects. In total, for the entire run, 14,163,470 reads passed quality filtering (Q20). Of these reads, 4,573,318 were associated with the 41 samples for this project. Of the 4,573,318 reads for this project, 4,212,549 merged successfully (92.11%). The sample with the lowest number of sequences after merging was the negative control with 444 sequences, the next lowest sample, BB039, had 22,897 sequences, approximately a fifty-fold increase. The most abundant sequence in the negative control was chloroplast DNA, and thus, we conclude that these 444 sequences were likely the result of contamination from other samples during sequencing or molecular analyses. We considered removing shared operational taxonomic units (OTUs) or 100 percent identical DNA sequences between the negative control and our environmental samples, but determined both of these actions to be too stringent on the dataset when Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 4/25 taking into account the abundance of the OTU’s/DNA sequences in the samples and the number of reads in the negative control. Instead the negative control was simply removed from downstream analysis. A total of 4,976 chimeras were identified using USEARCH v. 6.1 and were filtered out. The remaining sequences were clustered using the open reference approach into OTUs at 97 percent similarity using UCLUST (Edgar, 2010). Taxonomy was assigned using the assign_taxonomy.py QIIME script with the GreenGenes database (v.13_8) (DeSantis et al., 2006) using UCLUST. Further filtering was performed using the QIIME scripts, filter_taxa_from_otu_table.py and filter_otus_from_otu_table.py, to remove chloroplast DNA, mitochondrial DNA and singletons. Reads classified as ‘‘Unassigned’’ at the domain level were also removed from downstream analysis. After these filtering steps, the lowest number of sequences in a sample dropped to 3,277. This reduction in the number of sequence reads can be largely attributed to the removal of Z. marina chloroplast DNA from the leaf and root samples. To aid in statistical comparison between different sample types (leaf, root, sediment), we subset our 16S rDNA sequences to a minimum sequence count of 3,277 to retain the maximum number of samples. Sequence processing However, when comparing only sediment samples, the 16S rDNA sequences were randomly subset to 20,000 sequences using the single_rarefaction.py QIIME script. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Data visualization and statistical analyses Data visualization was performed exclusively in R and statistical analyses were performed using a combination of QIIME scripts and R (R Core Team, 2016). For analysis done in R, the rarefied OTU tables were converted to json format and exported for analysis using the ggplot2 (Wickham, 2009), vegan (Dixon, 2003) and phyloseq (McMurdie & Holmes, 2013) packages. Initial analysis indicated no significant differences between the microbial communities associated with shallow (0.5 cm or less) and deep (3 cm) sediment samples, thus sediment depth was not considered further here. We describe the different types of analyses below. • Intra-sample (alpha) diversity. We were interested in if significant differences existed between the intra-sample (alpha) diversities (richness, evenness) of the microbial communities associated with different sample types (leaf, root, sediment) and different sediment locations (inside, edge, outside). We calculated the following diversity metrics: Chao1 (Chao, 1984), Observed OTUs, Shannon (Shannon & Weaver, 1949) and Simpson Indices (Simpson, 1949) in R. To determine if there were significant differences between the alpha diversities of different sample types and different sediment locations, we first performed Kruskal–Wallis tests. We then implemented Bonferroni corrected post-hoc Dunn tests to identify which pairwise comparisons were driving differences. • Inter-sample (beta) diversity. We assessed the inter-sample (beta) diversities of the microbial communities associated with different sample groupings (sample type, location, etc) and if there were any significant correlations between environmental variables and community dissimilarity. We used both Unifrac (weighted and unweighted) (Lozupone et al., 2007; Hamady, Lozupone & Knight, 2010) and Bray–Curtis (Bray Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 5/25 5/25 & Curtis, 1957) dissimilarities calculated in R using phyloseq. These dissimilarities were then plotted using principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) methods. Multiple tests were then performed on these beta-diversity results. To test for significant differences in centroids between different sample groupings (sample type, location, etc.) PERMANOVA tests were performed using the adonis function from the vegan package in R with 9,999 permutations (Anderson, 2001). PERMANOVA tests can be sensitive to differences in dispersion when using abundance-based distance matrices (Warton, Wright & Wang, 2012), but are more robust than other tests, especially for balanced designs (Anderson & Walsh, 2013). To test for differences in mean dispersions between different groupings, the betadisper and permutest functions from the vegan package in R were used with 999 permutations. Data visualization and statistical analyses To test for correlations between the Bray Curtis dissimilarities of our samples and the environmental factors (C:N ratio, pH, etc) measured, euclidean distances were calculated in R using vegan and Mantel tests were performed using 9,999 permutations. The supervised_learning.py QIIME script was used to see if a random forest classifier could differentiate between sample type or sediment location using leave-one-out cross validation and 1,000 trees. • Taxonomic variation. To determine if the mean relative abundance of taxonomic orders varied significantly between different sample types and sediment locations, we first used the summarize_taxa.py QIIME script to remove rare OTUs (less than one percent of total abundance) and to collapse OTUs at the Order level. We then used the group_significance.py QIIME script on the resulting OTU table to test for differences using Bonferroni corrected Kruskal–Wallis tests with 1000 permutations. We removed the rare OTUs, as suggested in the documentation for the groups_significance.py QIIME script, to avoid spurious significance from very low abundance OTUS, to simplify analyses and to focus on abundant organisms and overall patterns. • Environmental variation. To determine if environmental factors varied significantly between different locations in the eelgrass patch (inside, edge, outside), ANOVA tests were performed in R for each factor. The post-hoc Tukey’s Honest Significant Difference (HSD) test was performed in R for factors found significantly different by the ANOVA (Tukey, 1953; Kramer, 1956; Kramer, 1957). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Diversity metrics I: intra-sample variation between sample types and locations Diversity metrics I: intra-sample variation between sample types and locations Alpha diversity is greater in the sediment than in the leaves and roots (p < 0.001) for a variety of metrics including observed number of OTUs, Chao1, Shannon and Simpson (Fig. 1). However, there is no difference in alpha diversity between leaf and root samples (p > 0.05) (Table S1 ). This is not altogether unexpected as in terrestrial systems soil has been observed to have increased diversity compared to host associated sample types (Edwards et al., 2015). There is conflict between the diversity metrics when determining if the intra-sample diversity of sediment at different locations (inside, edge, outside) varies Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 6/25 Figure 1 Alpha diversity across samples. Four alpha diversity metrics, observed number of OTUs, Chao1, the Shannon and Simpson diversity indices, are shown as boxplots for (A) different sample types (leaf, root, sediment) and for (B) sediment from different locations (inside, edge, outside). Figure 1 Alpha diversity across samples. Four alpha diversity metrics, observed number of OTUs, Chao1, the Shannon and Simpson diversity indices, are shown as boxplots for (A) different sample types (leaf, root, sediment) and for (B) sediment from different locations (inside, edge, outside). (Table S2). Two of the metrics, observed number of OTUs and Chao1, indicate greater diversity outside compared to inside the patch (p < 0.05). The non-significant metrics, the Shannon and Simpson indices, account for both richness and evenness and are less sensitive to rare taxa than richness only metrics (Bent & Forney, 2008). Thus, one possible explanation for the difference in diversity between the inside and outside sediment is an increased number of rare taxa in sediment from outside the patch. No significant differences were found between the alpha diversity of leaves and roots between the inside and edge of the eelgrass patch. Diversity metrics II: inter-sample variation between sample types and locations Distinct microbial communities were detected in association with Z. marina leaves, roots and sediment (Fig. 2). PERMANOVA tests performed on three different beta diversity metrics, weighted UniFrac, unweighted UniFrac and Bray–Curtis Dissimilarity, found these communities to be significantly different from each other with p = 0.0001 (Table 1). Root and leaf associated communities were found to have more with-in group variance, or dispersion, than sediment communities (p = 0.001), which could indicate that stabilizing Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 7/25 Figure 2 Principal coordinates analysis (PCoA) of microbial communities based on weighted Unifrac distances. Samples are colored by sample type (leaf, root, sediment) with different shapes for location (in- side, edge, outside). Figure 2 Principal coordinates analysis (PCoA) of microbial communities based on weighted Unifrac distances. Samples are colored by sample type (leaf, root, sediment) with different shapes for location (in- side, edge, outside). Table 1 Permanova results. Comparing microbial community composition between different sample types (leaf, root, sediment) and locations (inside, edge, outside) using multiple beta diversity metrics. Category Diversity metric Pseudo-F R2 P (perm) Location Weighted UniFrac 2.22 0.107 0.0213 Unweighted UniFrac 1.91 0.0938 0.0043 Bray Curtis 2.82 0.133 0.0009 Sample type Weighted UniFrac 13.75 0.426 0.0001 Unweighted UniFrac 6.16 0.249 0.0001 Bray Curtis 9.53 0.34 0.0001 LocXType Weighted UniFrac 1.98 0.0541 0.0426 Unweighted UniFrac 1.19 0.0455 0.1586 Bray Curtis 1.482 0.0458 0.0795 selection is acting on these sediment communities. Random forest analysis further validated selection is acting on these sediment communities. Random forest analysis further validated the observed differences between leaves, roots and sediment microbial communities (Table S3). The classifier had an estimated error of 5% (versus a baseline error of 40%) and correctly identified all leaf samples (n = 8) and all sediment samples (n = 24). The classifier did misclassify two of the root samples (n = 8) as leaves, but this is not unexpected as these two samples also appear to cluster more closely with the leaf samples when visualized using Principal Coordinates Analysis (PCoA) (Fig. 2). The reason that these root samples cluster more closely with the leaf samples may be due to which root bundles were sampled; preliminary results indicate that the microbiota associated with the roots can vary depending on the proximity of the root to the base of the leaf, with roots closer to the base appearing more ‘‘leaf-like’’ (HE Holland-Moritz et al., 2017, unpublished data). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Diversity metrics II: inter-sample variation between sample types and locations Figure 4 Average relative abundance of taxonomic groups associated with each sample type (leaf, root, sediment). OTUs are shown grouped by taxonomic order and colored by taxonomic class. Only orders with a mean abundance of at least one percent are shown here. The bars represent the standard error of the mean. Random forest analysis confirmed differences between sediment microbial communities taken from the inside of the patch, the edge and unvegetated sediment from outside the patch (Table S4). The classifier had an estimated error of 12.5% (versus a baseline error of 66.7%) and correctly identified all of the unvegetated sediment (n = 8). The classifier did mistakenly classify one sample from the edge (n = 8) as being from the inside of the patch and two samples from the inside of the patch (n = 8) as being from the edge. In Fig. 3, there is some overlap in the clustering of sediment from the inside and edges of patches which might account for these misclassifications. Diversity metrics II: inter-sample variation between sample types and locations Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 8/25 Figure 3 Principal coordinates analysis (PCoA) of microbial communities in sediment based on weighted Unifrac distances. Samples are colored by location (inside, edge, outside). Figure 3 Principal coordinates analysis (PCoA) of microbial communities in sediment based on weighted Unifrac distances. Samples are colored by location (inside, edge, outside). Table 2 Sediment PERMANOVA results. Comparing sediment microbial community composition be- tween different locations (inside, edge, outside) and eelgrass densities using multiple beta diversity met- rics. Table 2 Sediment PERMANOVA results. Comparing sediment microbial community composition be- tween different locations (inside, edge, outside) and eelgrass densities using multiple beta diversity met- rics Table 2 Sediment PERMANOVA results. Comparing sediment microbial community composition be- tween different locations (inside, edge, outside) and eelgrass densities using multiple beta diversity met- rics. Category Diversity metric Pseudo-F R2 P (perm) Location Weighted UniFrac 8.69 0.453 0.0001 Unweighted UniFrac 2.92 0.217 0.0001 Bray Curtis 8.01 0.433 0.0001 Density Weighted UniFrac 2.81 0.551 0.0002 Unweighted UniFrac 1.51 0.398 0.0001 Bray Curtis 2.86 0.555 0.0001 To determine if there was a difference in community composition at the eelgrass patch edge relative to the inside or outside of the patch, beta diversity metrics were calculated for the sediment microbial communities. As can be seen in Fig. 3, these diversity metrics show the communities clustering by sampling location (inside, edge, outside). PERMANOVA tests indicate that these clusters are significantly different between locations (p = 0.0001) and also for eelgrass shoot densities (p < 0.0002) (Table 2). However, leaf and root microbial communities do not differ significantly based on sampling location, possibly indicating that these plant tissue associated communities are more stable than the sediment communities in regards to location. Whereas sediment communities, although distinct when associated with eelgrass, may be under less selection from the host plant. One possible explanation for the correlation between the sediment communities and eelgrass shoot density may be the release of exudates and oxygen by the roots of the eelgrass, which would increase in concentration with eelgrass density. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 9/25 Figure 4 Average relative abundance of taxonomic groups associated with each sample type (leaf, root, sediment). OTUs are shown grouped by taxonomic order and colored by taxonomic class. Only orders with a mean abundance of at least one percent are shown here. The bars represent the standard error of the mean. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Major patterns in community composition of the leaves, roots and rhizosphere sediment The analysis of diversity metrics presented above shows that there are distinct communities associated with leaves and roots, and these both differ from the sediment, whereas location effects are weaker. We therefore examined in more detail the taxonomic composition and possible functional roles of the microbes on Z. marina leaves, roots and rhizosphere sediment (sediment from the inside and edge of the eelgrass patch). We summarize our findings regarding this here. Figure 4 shows the average relative abundance of different orders of bacteria for leaves, roots and sediment. On leaves, the most abundant orders were Clostridiales, Bacteroidales, Rhodobacterales, Flavobacterales, Saprospirales, Thiotrichales and Unidentified Gammaproteobacteria. On roots, the most abundant orders were Campylobacterales, Bacteroidales, Clostridiales, Desulfobacterales, Flavobacteriales and Desulfuromonadales. In the rhizosphere sediment, the most abundant orders were Bacteroidales, Flavobacteriales, Desulfobacterales, Thiotrichales, Clostridiales and Alteromonadales. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 10/25 We also examined the overall patterns in our results at the class level (Table S5). For leaves, the most abundant class of epiphytes observed was Gammaproteobacteria (20.5 ± 7.3%). Other abundant classes included Clostridia (16.5 ± 12%), Bacteriodia (12.6 ± 8.6%), Alphaproteobacteria (11.4 ± 8.5%), Flavobacteria (7.8 ± 4.7%) and Saprospirae (7.7 ± 4.2%). For roots, the dominant class associated with the roots were Epsilonproteobacteria (17.9 ±15.5%). Other abundant classes observed on the roots include Deltaproteobacteria (13.4 ± 11.3%), Bacteriodia (12.8 ± 8.8%), Gammaproteobacteria (12.6 ± 11%), Clostridia (8 ± 8.4%), Flavobacteriia (6.1 ± 6.1%) and Alphaproteobacteria (4.8 ± 6.3%). In the rhizosphere sediment, the dominant class was Gammaproteobacteria (18.2 ± 3.4%), as it was on the leaves. Other abundant classes found in the rhizosphere sediment include Deltaproteobacteria (14.9 ± 2.6%), Bacteriodia (13.3 ± 2.3%), Flavobacteriia (9.3 ± 3.4%), Clostridia (5.1 ± 3.3%) and Anaerolineae (3.9 ± 1.6%). The summary results above allow a comparison to findings from a recent study on the rhizosphere sediment microbiomes of three seagrass species, including Z. marina, Cúcio et al., 2016. We chose to focus our comparison on the Cúcio et al. study because it is one of the more comprehensive culture independent studies of seagrasses. Overall, there are general similarities and differences when comparing the class-level patterns between the studies. The authors reported that the most abundant classes were Gammaproteobacteria (32– 38% depending on the species sampled), Deltaproteobacteria (23–26%), and Bacteroidia (6–7%). Major patterns in community composition of the leaves, roots and rhizosphere sediment These were the three most abundant classes in our sediment samples as well, but at different relative abundances (see above). These differences could be due to true differences in microbiomes in the sediments sampled, or due to the use of different primer sets, extraction methods, and sample collection strategies (among many other differences). When examined at higher taxonomic ranks, the microbiome of the leaves of Z. marina shares some similarities with the microbiomes of various marine algae (e.g., kelp and seaweeds), with Gammaproteobacteria being the most abundant class in both cases (Hollants et al., 2013). However, these similarities are not seen at lower ranks (e.g., order, family, genus). This finding is similar to what has been observed between different marine algal microbiomes, with similarities observed at higher, but not lower taxonomic levels (Hollants et al., 2013; Egan et al., 2013). This is further supported by a recent study, which focused on surface-associated communities, that observed that the microbiomes of seagrass and seaweed species were host specific, but had broad-scale functional similarities (Roth-Schulze et al., 2016). Differences in microbial communities between sample types (leaves, roots and rhizosphere sediment) and possible functional implications We used a Bonferroni corrected Kruskall–Wallis test to test for differences in relative abundance of the orders between sample types. This showed that Saprospirales, Thiotrichales, Rhodobacterales, Desulfobacterales, Desulfuromonadales, Marinicellales, Spirochaetales, Chromatiales and Campylobacterales are significantly different between sample types (p < 0.05). Campylobacterales, Desulfobacterales, Spirochaetales and Desulfuromonadales were enriched on Z. marina roots. Thiotrichales, Rhodobacterales and Saprospirales were enriched on the leaves. Thiotrichales, Marinicellales, Chromatiales, Desulfobacterales and Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Spirochaetales were enriched in the rhizosphere. We note that many of the taxa that differ significantly between communities are closely related to ones commonly associated with various aspects of sulfur and nitrogen metabolism. This is interesting because prior studies have suggested that nitrogen and sulfur metabolism are critical functions for the seagrass associated microbiome (Lovell, 2002). For example, acquisition of nitrogen (in its many forms) is frequently a limiting factor for the health of plants, including seagrasses (Short, 1987; Elser et al., 2007) and associations with microbes are frequently critical for such acquisition (Welsh, 2000; Nielsen et al., 2001). In addition, since the reduced sulfur compounds that accumulate in aquatic sediments are known phytotoxins (Lamers et al., 2013), it is thought that sulfur metabolizing microbes could play important roles in aiding seagrass survival in such sediments (Barber & Carlson, 1993; Terrados et al., 1999; Erskine & Koch, 2000; Van der Heide et al., 2012). Sulfur and nitrogen metabolism are not necessarily independent—it has been postulated that sulfate-reducing bacteria may be responsible for most of the nitrogen fixation that occurs in seagrass sediments (Capone, 1982). Given this context, we discuss several of the specific taxa that differ between samples and their possible connection to nitrogen and/or sulfur metabolism below. For example, Campylobacterales, specifically Sulfurimonas species, from the class Epsilonproteobacteria, were enriched on Z. marina roots. Previous studies of Spartina (Thomas et al., 2014) and Z. marina (Jensen, Kühl & Priemé, 2007) also found enrichment of Epsilonproteobacteria on roots relative to the surrounding sediment. All known Sulfurimonas species are sulfur- oxidizing chemolithoautotrophs, can perform denitrification and are postulated to play significant roles in biogeochemical cycling in marine sediments (Campbell et al., 2006). Members of Campylobacterales have previously been identified as nitrogen fixers when isolated from Spartina roots (McClung & Patriquin, 1980). Additionally, Campylobacterales and Desulfobacterales, known sulfur-reducing bacteria, have been previously found to be abundant in association with plants from brackish habitats (e.g., mangroves—Gomes et al., 2010). Differences in microbial communities between sample types (leaves, roots and rhizosphere sediment) and possible functional implications Rhodobacterales which are enriched on the Z. marina leaves in our study, are purple nonsulfur bacteria, that have been identified as primary surface colonizers in marine habitats and have been shown to have the ability to fix nitrogen (Palacios & Newton, 2005; Dang et al., 2008). Desulfobacterales and Rhodobacterales species have been previously found in association with the tropical seagrass Thalassia hemprichii (Jiang et al., 2015). Thiotrichales, which are enriched in the sediment, are generally filamentous sulfur-oxidizing bacteria (Garrity, Bell & Lilburn, 2005) and are postulated to be dominant sulfur-oxiders in salt marsh sediments (Thomas et al., 2014). Variation in sediment microbial communities between locations Variation in sediment microbial communities between locations The analysis of diversity metrics reported above also showed that there are significant differences in the sediment microbial communities from different locations (inside a eelgrass patch, the edge of a patch and outside of a patch). We therefore examined in more detail the taxonomic groups that differ significantly between sediment locations and their potential functional roles (Fig. 5). Bacteroidales, Myxococcales, Thiotrichales and Chromatiales are significantly different between locations with a Bonferroni corrected Kruskall–Wallis test (p < 0.01). Thiotrichales 12/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Figure 5 Average relative abundance of taxonomic groups associated with sediment from each loca- tion (inside, edge, outside). Operational taxonomic units (OTUs) are shown grouped by taxonomic order and are colored by taxonomic class. Only orders with a mean relative abundance of at least one percent are shown. Bars represent the standard error of the mean. Figure 5 Average relative abundance of taxonomic groups associated with sediment from each loca- tion (inside, edge, outside). Operational taxonomic units (OTUs) are shown grouped by taxonomic order and are colored by taxonomic class. Only orders with a mean relative abundance of at least one percent are shown. Bars represent the standard error of the mean. and Chromatiales are enriched outside of Z. marina patches in the unvegetated sediment compared to the inside or edge of patches. In contrast, Bacteroidales and Myxococcales are enriched in the rhizosphere sediment inside and at the edge of eelgrass patches compared to the outside. The functional significance of these differences is unclear but we note a few things here. First, Thiotrichales and Chromatiales are common taxa in other marine and brackish sediments including those associated with various plants (e.g., Thomas et al., 2014). This is thought be reflective of a role in sulfur oxidation (see discussion above). Some studies have indicated that these taxa are associated with plants (e.g., seagrasses in Portugal Cúcio et al., 2016). However, other studies have indicated that these are found more in the sediment near plants but not specifically associated with plants (Thomas et al., 2014). Myxococcales, commonly found in freshwater and marsh sediments, includes microorganisms known to be involved in organic matter degradation (Bowen et al., 2012; Kou et al., 2016; Cleary et al., 2016). Variation in sediment microbial communities between locations The abundance of Myxococcales inside the eelgrass patch aligns with the expectation of higher prevalence of organic matter degradation inside the patch as opposed to surrounding unvegetated sediment. Environmental drivers of sediment communities In addition to investigating the taxonomic composition of the microbial communities of sediment collected from the inside, edge and outside of eelgrass patches, we decided to test for correlations between observed community differences and environmental factors to elucidate key factors that may be driving the microbial communities in eelgrass patches. In addition to investigating the taxonomic composition of the microbial communities of sediment collected from the inside, edge and outside of eelgrass patches, we decided to test for correlations between observed community differences and environmental factors to elucidate key factors that may be driving the microbial communities in eelgrass patches. A variety of abiotic factors were significantly different between locations including C:N ratio, TIC, dissolved oxygen, pH and sediment size fractions 710 µm and 63 µm (ANOVA, p < 0.05) (Tables S6 and S7). Unsurprisingly, eelgrass shoot density was significantly A variety of abiotic factors were significantly different between locations including C:N ratio, TIC, dissolved oxygen, pH and sediment size fractions 710 µm and 63 µm (ANOVA, p < 0.05) (Tables S6 and S7). Unsurprisingly, eelgrass shoot density was significantly 13/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Figure 6 Relationship between environmental data and microbial communities. Non-metric multi- dimensional scaling (NMDS) of Bray Curtis dissimilarities of microbial communities found in sediment samples are shown here colored by location (inside, edge, outside). Environmental factors (p < 0.055, ANOVA) were overlaid as vectors onto the NMDS using the envfit function in vegan. Figure 6 Relationship between environmental data and microbial communities. Non-metric multi- dimensional scaling (NMDS) of Bray Curtis dissimilarities of microbial communities found in sediment samples are shown here colored by location (inside, edge, outside). Environmental factors (p < 0.055, ANOVA) were overlaid as vectors onto the NMDS using the envfit function in vegan. different between locations (ANOVA, p < 0.05). To determine which pair-wise locations were driving the significant differences between location overall, we performed Tukey’s HSD tests (Tables S8 and S9). We also performed Tukey’s HSD tests on percent TOC which was marginally non significantly different (ANOVA, p = 0.0519). All pair-wise location comparisons of eelgrass density and dissolved oxygen were significantly different (Tukey’s HSD, p < 0.05). The C:N ratio and sediment fraction 63 µm were significantly different for the outside-inside comparison (Tukey’s HSD, p < 0.05). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Environmental drivers of sediment communities Percent TIC and TOC as well as sediment fraction 710 µm were significantly different for the outside-edge comparison and pH was significantly different for the inside-edge comparisons (Tukey’s HSD, p < 0.05). To test if there was a correlation between environmental measures and microbial community composition, Mantel tests were performed on Euclidean distances of environmental measures and the Bray–Curtis dissimilarities of sediment communities. A combined dataset including C:N ratio, TIC, TOC, dissolved oxygen, pH and eelgrass density was found to be significantly positively correlated with the sediment microbial community data (r = 0.1122, p = 0.0474) (Fig. 6). However, when measures were tested individually only the C:N ratio (r = 0.1701, p = 0.016) and eelgrass density (r = 0.1292, p = 0.0381) were significantly correlated with microbial community composition. The significant correlation between the sediment communities and the C:N ratio may indicate a change in ecosystem nutrient cycling at the patch edge. As Carbon (TIC and TOC) was not correlated with the sediment microbial communities, the correlation with the C:N ratio may hint at the importance of nitrogen, which was not measured in this study, to sediment community composition. Nitrogen is often a limiting terrestrial plant nutrient Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 14/25 and N-limitation has also been observed in several seagrass studies, more frequently in temperate habitats (De Boer, 2007). Terrestrial plants overcome N-limitation by having beneficial interactions with nitrogen fixing bacteria and these bacteria have previously been observed to form associations with eelgrasses (Capone & Budin, 1982; Welsh, 2000; Bagwell et al., 2002; Adhitya, Thomas & Ward, 2007; Sun et al., 2015). Nitrogen fixation can account for 5–10% of plant nitrogen requirements in temperate eelgrass patch sediments, and up to 50% in tropical patch sediments, indicating an important role for nitrogen fixation in overall patch health (Welsh, 2000). A previous study looking at forest soil microbial communities found that microbial biomass and activity were significantly lower at forest edges due to decreased litter decomposition inthe edgehabitat andthus, changesin nutrientcycling (Malmivaara-Lämsä et al., 2008). In seagrass patches, on average, vegetated sediments are significantly enriched in organic matter compared to unvegetated sediments, with carbon stocks generally higher on the inside of patches (Duarte, Holmer & Marbà, 2005; Ricart et al., 2015). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Environmental drivers of sediment communities It is possible that the observed community structure changes in the sediment between locations and the correlations with C:N ratio and eelgrass density here are indicative of a similar trend of location based nutrient cycling resulting from differing nutrient deposition and decomposition rates. Eelgrass density may have direct or indirect effects on sediment microbial communities as a result of the role eelgrass plays in its environment as a foundation species and an ecosystem engineer (Koch, 2001) Seagrasses are known to modify their surrounding habitat in a variety of ways including enhancing the input and retention of carbon and other nutrients in the sediment (Gacia et al., 2002; Duarte et al., 2005; Duarte & Cebrián, 1996), altering flow velocity and turbulence in the water column above patches (Fonseca et al., 1982; Granata et al., 2001; Folkard, 2005) which can increase sedimentation (Short & Short, 1984; Dauby et al., 1995; Gacia et al., 2002) and oxygenating the sediment using their roots (Caffrey & Kemp, 1991; Pedersen et al., 1998; Connell, Colmer & Walker, 1999). Other factors at play in the observed differences between locations as a result of eelgrass density may be the development stage of the eelgrass at the edge (if the patch is expanding or contracting) and the variable use of eelgrass as a habitat by macroorganisms. From terrestrial systems, it is known that microbial communities can vary across different stages of plant development (Chaparro, Badri & Vivanco, 2014). Seagrasses at earlier stages in development are known to have different carbon deposition rates than later stages, so if seagrass patch was in the process of expanding this may change the abiotic conditions at the patch edge, and thus might be reflected in distinct microbial communities at the edge of a patch compared to the inside (Duarte & Sand-Jensen, 1990; Cebrián et al., 2000). Additionally, seagrass patches are habitats for a large number of macroorganisms with variable abundance across seagrass patch landscapes (seagrass densities) (Tanner, 2005; Smith et al., 2008; Smith et al., 2011). Ultimately, although we see differences between locations in environmental abiotic measurements, we are unable, given the limitations of this study, to decouple these measurements from the eelgrass itself (eelgrass density), which is highly correlated with sediment community composition. 15/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 CONCLUSIONS This study provides new insights into the composition and assembly of the Z. marina microbiome. Major findings include that distinct microbial communities are associated with the leaves and roots of the plant, sediment associated communities are correlated with host plant density,and specific microbial taxa are found to have high relative abundances on particular tissues. Differences in the rhizosphere sediment community composition at the patch edge were observed and correlated with variation in environmental measurements. However, we were unable to disentangle these measures from eelgrass density, with the strongest correlated factor with community differences being presence/absence of the host plant. This is perhaps not unexpected from a field study, as eelgrass species are ecosystem engineers that actively change the sediment chemistry and landscape (Orth et al., 2006; Bos et al., 2007). Overall, we believe that the results of this study hint at a network of complex interactions between Z. marina, the microbes associated with Z. marina and biogeochemical cycling. Untangling such networks is difficult but increasingly feasible. Although Z. marina is not a model organism in the sense of Arabidopsis or poplar, we believe it can nevertheless become a model for host-microbiome-environment interaction studies. Advantages of working on this species include that there is a genome now available (Olsen et al., 2016), that there is a large network of collaborating labs focusing on this species (Zostera Experimental Network; http://zenscience.org), and that it can be used in common garden and reciprocal transplant experiments. Along these lines we have been building a library of cultured isolates associated with this species and sequencing the genomes of many of these (Lee et al., 2015a; Lee et al., 2015b; Lee et al., 2016a; Lee et al., 2016b; Alexiev et al., 2016a; Alexiev et al., 2016b). There are still areas in need of improvement (e.g., there a limited amount of full length 16S and 18S other reference data; only limited information on the in situ functions of microbes are available, there is a need for more genetic tools for the host), but given the importance of coastal marine systems and seagrasses generally, we believe continued efforts to study the host-microbiome-environment interactions in this and related species are important. ACKNOWLEDGEMENTS Illumina sequencing was performed at the DNA Technologies Core facility in the Genome Center at UC Davis, Davis, California. We thank Qingyi ‘‘John’’ Zhang for his help with the Illumina library preparation and Hannah Holland-Moritz for her help with DNA extractions. We thank Ted Grosholz and Susan Williams for hosting Sofie Voerman during her visit and for help with the sampling design. We thank Daniel Bradley for his help with sample collection. Author Contributions • Cassandra L. Ettinger analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Sofie E. Voerman conceived and designed the experiments, reviewed drafts of the paper, performed sampling. • Jenna M. Lang conceived and designed the experiments, reviewed drafts of the paper. • Jenna M. Lang conceived and designed the experiments, reviewed drafts of the paper. • John J. Stachowicz reviewed drafts of the paper, advised on experimental design, edited drafts of paper. • John J. Stachowicz reviewed drafts of the paper, advised on experimental design, edited drafts of paper. • Jonathan A. Eisen contributed reagents/materials/analysis tools, reviewed drafts of the paper, advised on data analysis, edited drafts of paper. Competing Interests Jonathan A. Eisen is an Academic Editor for PeerJ. Jenna M. Lang is an employee of Trace Genomics, Inc. DNA Deposition The following information was supplied regarding the deposition of DNA sequences: The following information was supplied regarding the deposition of DNA sequences: g g g This 16S rRNA sequencing project has been deposited at GenBank under the accession no. PRJNA350006. This 16S rRNA sequencing project has been deposited at GenBank under the accession no. PRJNA350006. Grant Disclosures Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: Gordon and Betty Moore Foundation: GBMF333. Funding g This work was supported by a grant from the Gordon and Betty Moore Foundation (GBMF333) ‘‘Investigating the co-evolutionary relationships between seagrasses and their Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 microbial symbionts.’’ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. microbial symbionts.’’ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplemental Information Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3246#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3246#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj 3246#supplemental-information peerj.3246#supplemental-information. Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: Coil, David; Eisen, Jonathan; Stachowicz, Jay; Green, Jessica; Holland-Moritz, Hannah; Lang, Jenna (2014): The Seagrass Microbiome. figshare. https://doi.org/10.6084/m9.figshare.1014334.v1. The following information was supplied regarding data availability: Coil, David; Eisen, Jonathan; Stachowicz, Jay; Green, Jessica; Holland-Moritz, Hannah; Lang, Jenna (2014): The Seagrass Microbiome. figshare. https://doi.org/10.6084/m9.figshare.1014334.v1. Coil, David; Eisen, Jonathan; Stachowicz, Jay; Green, Jessica; Holland-Moritz, Hannah; Lang, Jenna (2014): The Seagrass Microbiome. figshare. https://doi.org/10.6084/m9.figshare.1014334.v1. REFERENCES Adhitya A, Thomas FIM, Ward BB. 2007. Diversity of assimilatory nitrate reductase genes from plankton and epiphytes associated with a seagrass bed. 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Two dimensional numerical modelling and analysis of a novel electromagnetic inductive valve
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RESEARCH ARTICLE | JULY 30 2018 Two dimensional numerical modelling and analysis of a novel electromagnetic inductive valve RESEARCH ARTICLE | JULY 30 2018 Dawei Guo ; Mousen Cheng; Xiong Yang; Xiaokang Li; Moge Wang Dawei Guo ; Mousen Cheng; Xiong Yang; Xiaokang Li; Moge Wang Dawei Guo ; Mousen Cheng; Xiong Yang; Xiaokang Li; Moge Wang AIP Advances 8, 075129 (2018) https://doi.org/10.1063/1.5025415 AIP Advances 8, 075129 (2018) https://doi.org/10.1063/1.5025415 Articles You May Be Interested In Design and test of a simple fast electromagnetic inductive gas valve for planar pulsed inductive plasma thruster Rev. Sci. Instrum. (October 2017) Metal plate deformation under magnetic field pulse of complex shape High power operation of a nitrogen doped, vanadium compensated, 6H-SiC extrinsic photoconductive switch 24 October 2024 03:58:37 24 October 2024 03:58:37 AIP ADVANCES 8, 075129 (2018) Two dimensional numerical modelling and analysis of a novel electromagnetic inductive valve Dawei Guo,a Mousen Cheng, Xiong Yang, Xiaokang Li, and Moge Wang College of Aerospace Science and Engineering, National University of Defense Technology, Changsha 410073, China (Received 9 February 2018; accepted 22 July 2018; published online 30 July 2018) (Received 9 February 2018; accepted 22 July 2018; published online 30 July 2018) (Received 9 February 2018; accepted 22 July 2018; published online 30 July 2018) A novel type of electromagnetic inductive valve has been developed for the gas injector of the planar pulsed inductive plasma thruster. However, due to the com- plex operation process of the valve, the design work and the performance prediction can only be accomplished by experimentation. This paper explains a two dimensional model applied to predict the valve performance. The electromagnetic and mechan- ical aspects which dominate the valve operation process are treated as two weakly coupled problems. The equivalent circuit method is applied to analyze the electro- magnetic problem where the valve is represented by a series of sub-conductors. The Lorentz force is calculated and introduced into the mechanical problem as an ana- lytical load. Simulation and experimental validations are implemented in a prototype valve. Compared results from experiments convince that the present model can be used to predict the valve dynamic performances with satisfying accuracy. Moreover, the detailed insight with respect to the development of magnetic flux density, induced current, Lorentz force and velocity of the diaphragm is presented to better under- stand the valve operation process. The results indicate that the effective acceleration of the diaphragm occurs in the initial phase of valve opening. Only the diaphragm obtains enough kinetic energy during the first half cycle of the coil current, can the valve be fully opened. © 2018 Author(s). All article content, except where oth- erwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/1.5025415 24 October 2024 03:58:37 aElectronic mail: guodawei16@gmail.com. I. INTRODUCTION The diaphragm, mounted above the coil, can seal the working gas in the plenum under the preload exerted by the diaphragm’s deformation. The stopper is used to limit the maximum deflection of the diaphragm. When the capacitor is discharged by switch, the pulsed current in the coil induces eddy currents in the diaphragm. The diaphragm is subjected to a strong electromagnetic repulsive force (Lorentz force). Consequently, the outer part of the diaphragm is repelled away from the seal surface of the sealing plate rapidly. The high pressure working gas in the plenum expands through the annular orifice. When the deflection of the diaphragm reaches the maximum limited displacement, the diaphragm bounces off the stopper and returns to its original position, wherein the valve completes an operation cycle. vacuum at a precise time in the operating cycle.7–11 In order to support our PPIPT research, a novel pulsed electromagnetic inductive valve system has been developed. The system is fully detailed in Ref. 12. To clarify the construction and the operation process, a cross section view of the valve is shown in Fig. 2. The actuator of the valve is based on the induction dynamic mechanism and consists of a coil and a conical diaphragm. The spiral coil is embedded in the valve body and covered by a rubber sealing plate with a bulgy dam. The diaphragm, mounted above the coil, can seal the working gas in the plenum under the preload exerted by the diaphragm’s deformation. The stopper is used to limit the maximum deflection of the diaphragm. When the capacitor is discharged by switch, the pulsed current in the coil induces eddy currents in the diaphragm. The diaphragm is subjected to a strong electromagnetic repulsive force (Lorentz force). Consequently, the outer part of the diaphragm is repelled away from the seal surface of the sealing plate rapidly. The high pressure working gas in the plenum expands through the annular orifice. When the deflection of the diaphragm reaches the maximum limited displacement, the diaphragm bounces off the stopper and returns to its original position, wherein the valve completes an operation cycle. 24 October 2024 03:58:37 The valve operation process is a complex process which contains various factors can influence its performance. These include the characteristics of the electronic circuit, the magnetic field, the eddy current, the diaphragm movement/deformation, the gas flow, and the many interactions among them. I. INTRODUCTION The planar pulsed inductive plasma thruster (PPIPT) is a kind of promising spacecraft propulsion device. A typical PPIPT mainly consists of a drive coil, a gas injector, energy storage capacitors and a switch (switch array). The schematic of the typical structure and basic operation of the PPIPT is shown in Fig. 1. The electrical energy is capacitively stored and then quickly discharged through a planar coil. This forms a fast rising azimuthal current in the coil that induces a plasma current sheet within the gas propellant near the face of the coil. The interaction between the magnetic field generated by the coil current and the plasma current sheet generates a Lorentz force that expels the plasma producing a thrust that propels the spacecraft.1–3 Efficient gas propellant utilization, which is necessary for maximizing thrust efficiency and Isp (specific impulse),4 can only be achieved if the gas injection system is capable of covering the coil with a puff of propellant that has not been preceded by gas that has escaped at the time of the shot, and is not followed by any trailing gas that arrives after the shot.5 This requires that the pulsed gas valve of the gas injector can be fully opened and completely closed within a few hundred microseconds.6 Since the characters of the fast valve give a highly remarkable influence on the performances of the PPIPT, the design of a fast pulsed valve becomes very significant in the simulation and experimental research of the PPIPT. Due to the simplicity and the ultra-fast action, electromagnetic inductive valves are widely use in scientific research fields wherein a metered amount of neutral gas is required to be injected into 8, 075129-1 2158-3226/2018/8(7)/075129/14 075129-2 Guo et al. AIP Advances 8, 075129 (2018) FIG. 1. Schematic diagram of the PPIT. FIG. 1. Schematic diagram of the PPIT. vacuum at a precise time in the operating cycle.7–11 In order to support our PPIPT research, a novel pulsed electromagnetic inductive valve system has been developed. The system is fully detailed in Ref. 12. To clarify the construction and the operation process, a cross section view of the valve is shown in Fig. 2. The actuator of the valve is based on the induction dynamic mechanism and consists of a coil and a conical diaphragm. The spiral coil is embedded in the valve body and covered by a rubber sealing plate with a bulgy dam. A. Equivalent circuit method Equivalent circuit method is a simple and straightforward numerical method which can be used to solve the electromagnetic problem. In recent decades, the equivalent circuit method has been developed by many research groups and applied to model electromagnetic accelerator,13,14 high-voltage air-cored pulse transformers,15 electromagnetic launcher,16,17 ultrahigh magnetic field generators,18 magnetic resonance imaging system,19 electromagnetic forming.20 This method divides all conductive parts of the system into parallel isolated elementary volume sub-conductors, in which uniform distribution of current is assumed. The cross section of the sub-conductor must be sufficiently small for the uniform current density hypothesis to be valid. The resistance and induc- tance of the sub-conductor can be calculated, as can the mutual inductance between every possible pair of sub-conductors. An electrical circuit is associated with every sub-conductor. Accordingly, the sub-conductors form an electric circuit network. The resulting system of fist order ordinary differential equations that corresponds to the equivalent electric circuit network can be solved numer- ically. The results, in the form of time dependent currents, carry most of the information about the system. 24 October 2024 03:58:37 y A sub-conductor representation of the valve based on the equivalent circuit method is shown in Fig. 3. The necessary details to establish the equivalent circuit model are presented in the following. The influence of the spiral of the drive coil is neglected, and the coil is considered to nested concentric circular rings. Each of the rings is placed at the average position between two successive turns of the coil. All the rings are electrically connected in series. The coil current and the induced currents in the diaphragm will flow in circumferential paths. As a result, the related electromagnetic phenomena and the deformation of the diaphragm are axisymmetric. Considering the skin effect, the dimension of the sub-conductor should be much less than the equivalent skin depth for the current distribution in the cross section to be regarded as constant. In a more detail way, the diaphragm is divided into nt thin sub-layers, and each sub-layer is ulteriorly discretized into nr circular concentric sub- conductors. The simplified circular ring of the coil with round cross-section is dived into m small square elements. The main body and the stopper of the valve are subdivided to Ns and Nb non- uniform sub-conductors which near the coil and the diaphragm with a smaller cross-section. I. INTRODUCTION To analyze them thoroughly and rigorously would be much difficult for the complexity of the problem. Currently, investigation of the dynamic characteristics of electromagnetic inductive valve is mainly accomplished by experimental method. From the point view of a designer developing a simplified model which can predict the operation processes of this type of valve is essential. Early numericalstudyonelectromagneticinductivevalveemploysthelumpedmodeltoanalyzethedynamic characteristics.7 The Lorentz force acting on the movable part is calculated with neglecting Lorentz force distribution. Such approach is only applicable to the valve that the movable part subjects to FIG. 2. Cross sectional view of the valve. FIG. 2. Cross sectional view of the valve. 075129-3 Guo et al. AIP Advances 8, 075129 (2018) rigid body motion, but fail to our valve for the Lorentz force distribution significantly influences the diaphragm dynamics. This paper is undertaken to explain the numerical model that has been developed for the par- ticular type of the electromagnetic valve. An approximate but more feasible approach is to treat the operation process as a weakly coupled problem by disregarding the influence of diaphragm movement/deformation on electromagnetic phenomena, and then the Lorentz force that calculated in electromagnetic problem is applied to the mechanical problem by neglecting the influence of working gas in the gas plenum. These assumptions remain valid for that: 1) during the open- ing and closing of the valve, both the velocity and displacement of the diaphragm are small. Therefore, the effect of the movement/deformation of the diaphragm is insignificant. 2) Consid- ering the low pressure of the working gas in plenum, the force exerted on the diaphragm by the working gas is much small in comparison with the electromagnetic force and the resultant force of the diaphragm deformation, thus the force can be neglected. In the proposed model, the equivalent circuit method is employed to solve the electromagnetic problem. The coil current, the eddy currents, the magnetic field and the Lorentz force can be numerically calculated. Then, the Lorentz body force acting on the diaphragm is used in the mechanical problem of the numerical simulation. A. Equivalent circuit method The total number of the sub-conductors in the diaphragm is given by Nd=nt×nr, and the number in the coil is given by Nc=m×n, where n is the turn number of the coil. By regarding all the sub- conductors in Fig. 3 as separate coupled lumped circuits, the equivalent circuits for the valve can be treated as a primary RLC circuit coupled with several RL circuits as shown in Fig. 4. Applying Kirchhoff’s current and voltage laws, the corresponding system of first order differential equations can be expressed as: 075129-4 Guo et al. AIP Advances 8, 075129 (2018) FIG. 3. Sub-conductor representation of the valve. IcRcircuit + Lcircuit dIc dt + n X j=1 Rc (i−1)n+jic i + m X k=1 n X j=1 Mcc (i−1)n+j,(k−1)n+j dip k dt + n X j=1 Nd X k=1 Mcd (i−1)n+j,k did k dt + n X j=1 Nb X k=1 Mcb (i−1)n+j,k dib k dt + n X j=1 Ns X k=1 Mcs (i−1)n+j,k dis k dt = uc i = 1, 2, · · · , m (1) Rd i id i + Nd X j=1 Mdd i,j did j dt + m X k=1 n X j=1 Mcd i,(k−1)n+j dic k dt + Nb X j=1 Mdb i,j dib j dt + Ns X j=1 Mds i,j dis j dt = 0 i = 1, 2, · · · , Nd (2) Rb i ib i + Nb X j=1 Mbb i,j dib j dt + m X k=1 n X j=1 Mcb i,(k−1)n+j dic k dt + Nd X j=1 Mdb i,j did j dt + Ns X j=1 Mbs i,j dis j dt = 0 i = 1, 2, · · · , Nb (3) Rs iis i + Ns X j=1 Mss i,j dis j dt + m X k=1 n X j=1 Mcs i,(k−1)n+j dic k dt + Nd X j=1 Mds i,j did j dt + Nb X j=1 Mbs i,j dib j dt = 0 i = 1, 2, · · · , Ns (4) Ic = −C duc dt (5) 24 October 2024 03:58:37 FIG. 3. Sub-conductor representation of the valve. A. Equivalent circuit method Then the resistance can be given by: Ri = 2πri σiai (6) (6) where σi , ri and ai are the electrical conductivity, radiuses and the cross section area of the ith sub-conductor. where σi , ri and ai are the electrical conductivity, radiuses and the cross section area of the ith sub-conductor. A. Equivalent circuit method X j=1 X k=1 ( ) j, dt X j=1 X k=1 ( ) j, dt 1, 2, · · · , m (1) Rd i id i + Nd X j=1 Mdd i,j did j dt + m X k=1 n X j=1 Mcd i,(k−1)n+j dic k dt + Nb X j=1 Mdb i,j dib j dt + Ns X j=1 Mds i,j dis j dt = 0 i = 1, 2, · · · , Nd (2) Rb i ib i + Nb X j=1 Mbb i,j dib j dt + m X k=1 n X j=1 Mcb i,(k−1)n+j dic k dt + Nd X j=1 Mdb i,j did j dt + Ns X j=1 Mbs i,j dis j dt = 0 i = 1, 2, · · · , Nb (3) Rs iis i + Ns X j=1 Mss i,j dis j dt + m X k=1 n X j=1 Mcs i,(k−1)n+j dic k dt + Nd X j=1 Mds i,j did j dt + Nb X j=1 Mbs i,j dib j dt = 0 i = 1, 2, · · · , Ns (4) Ic = −C duc dt (5) (1) Ic = −C duc dt (5) (5) AIP Advances 8, 075129 (2018) 075129-5 Guo et al. FIG. 4. Equivalent circuit diagram for the valve. 24 October 2024 03:58:37 24 October 2024 03:58:37 FIG. 4. Equivalent circuit diagram for the valve. C and uc are the capacitance and the voltage of the capacitor. Rcircuit and Lcircuit are the distribution resistance and inductance of the circuit. Ic is the total current through the coil wire cross-section, and it is equal to the sum of current ic i . Mi,j is the mutual inductance of the ith and jth sub-conductor, the superscripts identify the inductively connected sub-conductors. For instance, Mcc is the mutual inductance between a pair of the sub-conductors belonging to the coil, and the form Mdd i,i is the self- inductance of the ith sub-conductor in the diaphragm. Ri is the resistance of the ith sub-conductor, the superscripts c, d, b and s identify the sub-conductor belongs to which part. The thermal effect is neglected for the pulsed energy storage used in this paper is not sufficient to produce a temperature rise that can significantly affect the resistance. B. Inductance calculations The mutual inductance between two circular conductors of certain cross-section can be derived by subdividing two such conductors into elements and summing elementary contributions. The general arrangement is shown in Fig. 5. Conductor 1 and conductor 2 are divided into n1 and n2 elements, respectively.21 The mutual inductance between the two conductors can be expressed as: M12 = 1 n1n2 n1 X i=1 n2 X j=1 Mi,j (7) (7) 075129-6 Guo et al. AIP Advances 8, 075129 (2018) 6 Guo et al. Guo et al. FIG. 5. Configuration of the divided conductors. FIG. 5. Configuration of the divided conductors. FIG. 5. Configuration of the divided conductors. where Mi,j is the mutual inductance between the ith element in the first conductor and the jth element in the second, and it is given by: where Mi,j is the mutual inductance between the ith element in the first conductor and the jth element in the second, and it is given by: Mi,j = µ0√rirj " 2 km −km ! K(km) −2 km E(km) # (8) k2 m = 4rirj h2 +  ri + rj 2 (9) (8) (8) 24 October 2024 03:58:37 (9) where K(·) and E(·) are complete elliptic integrals of the first and second kind; h is the axial distance between two elements ith and jth; ri and rj are the radii of the two elements, respectively. where K(·) and E(·) are complete elliptic integrals of the first and second kind; h is the axial distance between two elements ith and jth; ri and rj are the radii of the two elements, respectively. j The self-inductance of a conductor can also be calculated by dividing the conductor into squar cross-section elements, the self-inductance is given by: L = 1 n2 *. , 2 n−1 X i=1 n X j=i+1 Mi,j + n X i=1 li+/ - (10) (10) where Mi,j is the mutual inductance between the ith element and jth element in the conductor. n is the number of the elements. li is the self-inductance of the ith element and can be calculated according to Grover:22 where Mi,j is the mutual inductance between the ith element and jth element in the conductor. n is the number of the elements. li is the self-inductance of the ith element and can be calculated according to Grover:22 li = riµ0 " 0.5 1 + 1 6ci ! ln 8 ci ! B. Inductance calculations −0.84834 + 0.2041ci # (11) (11) where ci=(si 2ri)2; si and ri are the side length and the mean radius for the ith element, r where ci=(si 2ri)2; si and ri are the side length and the mean radius for the ith element, respectively. By further dividing the discretized sub-conductors into square cross-section elements, the mutual inductances between every possible pair of discretized sub-conductors can be calculated by Equation (7). The self-inductances of the discretized sub-conductors can be determined by Equation (10) and Equation (11). The optimum element number required to calculate accurately the discretized sub-conductors inductances is obtained by calculating the self-inductances for a rel- atively small number of elements, and then repeating the calculations for a progressively increased number until the differences between successive calculations are less than 0.5%. By further dividing the discretized sub-conductors into square cross-section elements, the mutual inductances between every possible pair of discretized sub-conductors can be calculated by Equation (7). The self-inductances of the discretized sub-conductors can be determined by Equation (10) and Equation (11). The optimum element number required to calculate accurately the discretized sub-conductors inductances is obtained by calculating the self-inductances for a rel- atively small number of elements, and then repeating the calculations for a progressively increased number until the differences between successive calculations are less than 0.5%. C. Magnetic field and Lorentz force density calculation where Br i and Bz i are the radial and axial components of the magnetic flux density in the sub-conductor, respectively. id i is the current and ad is cross-section area. C. Magnetic field and Lorentz force density calculation The sub-conductor currents are obtained from the solution of the system of Equations (1)–(5). Then the total magnetic flux density at a given point can be calculated by summing all the contributions of the sub-conductors present. Because of the particular shape of the sub-conductors cross section, the magnetic flux density produced by a sub-conductor has to be calculated by using the integration techniques. The magnetic flux density produced at a specific point P(rp, zp) by a particular cross 75129-7 Guo et al. AIP Advances 8, 075129 (2018) 075129-7 Guo et al. 9-7 Guo et al. Guo et al. 075129-7 section sub-conductor carrying a uniformly current density J can be expressed in the term of complete elliptic integrals of the first and the second kind:  Br(rp, zp) = µ0J 2π  A  zp −z  rp √ a + b  a a −bE(kk) −K(kk)  ds Bz(rp, zp) = µ0J 2π  A 1 √ a + b " K(kk) −a −2r2 a −b E(kk) # ds (12)  Br(rp, zp) = µ0J 2π  A  zp −z  rp √ a + b  a a −bE(kk) −K(kk)  ds Bz(rp, zp) = µ0J 2π  A 1 √ a + b " K(kk) −a −2r2 a −b E(kk) # ds (12) a = r2 p + r2 +  zp −z 2 (13) b = 2rpr (14) k2 k = 2b a + b (15) (12) a = r2 p + r2 +  zp −z 2 (13) b = 2rpr (14) k2 k = 2b a + b (15) (13) (14) (14) (15) where Br and Bz are the radial and axial components of the magnetic flux density. rp and zp are the coordinate of the point P. A is the cross section area of the sub-conductor. Inthediscretizedmodel,theLorentzforcedensityinthediaphragmcanbeobtainedbycalculating the Lorentz force density in the sub-conductors of the discretized diaphragm. The radial and axial component of Lorentz force density on the ith circular conductor of the discretized diaphragm can be given as:  f r i = id i ad Bz i (r, z) f z i = − id i ad Br i (r, z) (16) (16) 24 October 2024 03:58:37 where Br i and Bz i are the radial and axial components of the magnetic flux density in the sub-conductor, respectively. id i is the current and ad is cross-section area. A. Experimental validation To verify the numerical model, a specially designed experiment has been performed. Fig. 7 shows the schematic and the photograph of the test rig. The optic transmission technique is adopted to investigate the response characteristic of a prototype valve in normal atmospheric environment with no working gas. A laser beam passes through the gap between the diaphragm and the stopper just above the sealing surface, which is detected by a photodetector on the opposite side. The photodetector output signal drops as the gap decreases, followed by the rise during the valve close phase. The valve’s displacement (diaphragm’s deflection) from the initial sealing surface could be obtained according to the proportion between the photodetector output signal and the gap. The diameter of the laser beam is little larger compared to the maximum gap (the maximum gap is smaller than the diaphragm’s thickness), blocking the light on the photodetector when the valve is fully open. In order to focus the laser on the active area of the photodetector, a convex lens is placed between the valve and the photodetector. The coil current is monitored by a current transformer and the voltage of the capacitor is monitored by a high voltage probe. The diaphragm, the coil, the stopper and the main body are represented by Nd = 10 × 50, Nc = 20 × 6, Ns = 907, Nb = 3625 sub-conductors, respectively. The total number of differential equations to be integrated in this case is 5153. The resistances and inductances are firstly solved, and then the Runge-Kutta method is applied to solve the equivalent circuit equations based on the initial conditions. The magnetic field and the Lorentz force density are calculated at each time step. The calculation program is written using Matlab. The turnoff characteristic of the pulsed power thyristor is considered in the simulation model. When the thyristor withstands reverse bias, it continues to conduct until the reverse current reaches a maximal value. Then, the thyristor starts developing reverse blocking capability.23 In order to simplify the calculation process, the reverse process is ignored, i.e., the current immediately falls to zero after reaching the reverse maximum. For a certain discharge circuit, the maximal revers current can be predicted based on the experimental method.24 All the parameters and specifications for the experiment and the numerical simulations are presented in Table I. 24 October 2024 03:58:37 TheresponsecharacteristicobtainedbyusingthenumericalmodelispresentedinFig.8compared with the experimental results. D. Dynamical equation Once the Lorentz force density acting on the diaphragm is obtained, the calculation of the diaphragm dynamics is straight forward and the governing equation of dynamics is given by the following: ρ∂2u(r, z, t) ∂t2 = f(r, z, t)+∇σ(r, z, t) (17) (17) where u is the displacement of the diaphragm; σ is stress tensor. ρ is the density of the diaphragm. f is the Lorentz force density. The finite element software COMSOL Multiphysics® is applied to solve the dynamical equation. The model with an example mesh is presented in Fig. 6. The Lorentz force density is loaded as an analytical body load via an interior interpolation function in the COMSOL Multiphysics. Contact boundary conditions are specified on the boundaries that can come into contact to prevent the boundaries from penetrating each other. The friction between the contact boundaries is ignored. A boundary load in the axial direction is applied on the diaphragm’s outer edge to simulate the initial state of the diaphragm under the sealing force. where u is the displacement of the diaphragm; σ is stress tensor. ρ is the density of the diaphragm. f is the Lorentz force density. The finite element software COMSOL Multiphysics® is applied to solve the dynamical equation. The model with an example mesh is presented in Fig. 6. The Lorentz force density is loaded as an analytical body load via an interior interpolation function in the COMSOL Multiphysics. Contact boundary conditions are specified on the boundaries that can come into contact to prevent the boundaries from penetrating each other. The friction between the contact boundaries is ignored. A boundary load in the axial direction is applied on the diaphragm’s outer edge to simulate the initial state of the diaphragm under the sealing force. FIG. 6. Schematic of the dynamical model in COMSOL Multiphysics. FIG. 6. Schematic of the dynamical model in COMSOL Multiphysics. AIP Advances 8, 075129 (2018) AIP Advances 8, 075129 (2018) AIP Advances 8, 075129 (2018) 075129-8 Guo et al. 075129-8 A. Experimental validation The simulated currents are in good agreement with the experimental results with the negligible difference in the upslope. It can also be noted that there exists a resemblance trend between the simulated displacement curve and the photodetector signal. It is believed that the magnitude of the photodetector signal is positively related to the lift of the diaphragm for the photodetector signal is monotonic. However, due to the fact that the signal’s sensitivity to the valve- laser alignment, any quantitatively accurate calibration is determined to be outside the scope of this work to date. A spike caused by the collision of the diaphragm with the stopper can be observed in the simulated displacement curve while a similar spike is not shown in the photodetector signal. This is FIG. 7. Dynamic characteristic test rig. (a) Schematic of the test rig; (b) Photo of the test rig. FIG. 7. Dynamic characteristic test rig. (a) Schematic of the test rig; (b) Photo of the test rig. AIP Advances 8, 075129 (2018) AIP Advances 8, 075129 (2018) 075129-9 Guo et al. TABLE I. Simulation and experimental parameters for the prototype valve system. Components Parameters Unit Value Coil Pitch P mm 2 Diameter of the coil wire d mm 1.5 Conductivity S/m 5.6×107 Number of turn n - 6 Conductivity S/m 2.9×107 Circuit conditions Capacity of the capacitor C µF 350 Charge voltage u0 V 1600 Circuit inductance Lcircuit µH 1.3 Circuit resistance Rcircuit mΩ 56 Diaphragm Young’s modulus GPa 132 Poisson’s ratio - 0.36 Density kg/m3 8725 Inner radius R1 mm 38 Outer radius R2 mm 18 Thickness t mm 1.5 Height h mm 2.5 Body and Stopper Young’s modulus GPa 216 Poisson’s ratio - 0.27 Conductivity S/m 1.28×107 Inner radius of the coil groove R3 mm 24 Outer radius of the coil groove R4 mm 41 Depth of the coil groove h1 mm 7 Outer radius of the body R5 mm 50 Others h2 mm 1 h3 mm 3.5 h4 mm 0.9 TABLE I. Simulation and experimental parameters for the prototype valve system. 24 October 2024 03:58:37 mainly caused by the valve-laser misalignment, the light passing through the gap has already became zero when the diaphragm is very close to the stopper, as shown in Fig. 9. Therefore, the photodetector signal presents a top flat waveform. B. Simulation results and discussion The Lorentz force, the driving force of the valve, underlines the fact that the magnetic field and induced current in the diaphragm are essential for understanding the valve operation process and interpreting of data obtained from experiments. Accordingly, special care should be taken to account for the temporal evolution of magnetic flux density and induced current in the diaphragm. The distribution of coil current, radial component of magnetic flux density (Br) and induced current in the diaphragm, at the various times indicated in Fig. 8, are shown in Fig. 10, Fig. 11 and Fig. 12 respectively. It is found that by comparison of Fig. 11 and Fig. 10 the Br is correlated to the coil current via Ampere’s law. As can be seen by comparing Fig. 11(b) with Fig. 11(a), the increased of coil current at t = 38 µs results in an increase of Br. The current in the coil and so Br are small at t = 95 µs when the coil current Ic just reversed. It is also found in Fig. 11 that the Br is insignificant near the top surface of the diaphragm at all instances. The induced current in the diaphragm can accounted for this. At the initial phase (until t = 38 µs) of the valve opening process, the increase of the coil current leads to an increase of magnetic flux density in the diaphragm. According to the Faraday’s law of induction, the increase of the axial component of the magnetic flux density induces an electric field, which drives the eddy current proceeding along the diaphragm in reverse direction to the coil current. Initially, the coil current increases significantly but with the low magnitude because the coil current is relative small. The increase of the magnetic flux density concentrates on the bottom surface while a relative small increase occurs inside the diaphragm (see Fig. 11(a)). Consequently, the induced current occurs near the bottom surface of the diaphragm (see Fig. 11(a)) and decreases the Br inside the diaphragm. This leads to a shielding effect that blocks the further penetration of the magnetic field. When the coil current reaches its peak at t = 38 µs, the magnetic flux density becomes maximum while the growth rate is reduced (see Fig. 11(b)). Therefore, the induced current near the 24 October 2024 03:58:37 FIG. 10. Current (Ampere) distribution in the coil at the various times indicated in Fig. A. Experimental validation It can be observed in the experimental results that the signals begin to be rough and slightly oscillatory when the valve is nearly completely closed because of the dynamic behavior of the sealing plate under impact. FIG. 8. Simulated and experimental results of coil current and diaphragm dynamic response at uc = 1600 V. The displacement is the deflection of the diaphragm at r = 36 mm. FIG. 8. Simulated and experimental results of coil current and diaphragm dynamic response at uc = 1600 V. The displacement is the deflection of the diaphragm at r = 36 mm. AIP Advances 8, 075129 (2018) 075129-10 Guo et al. Guo et al. Guo et al. 075129-10 FIG. 9. The effect of valve-laser misalignment on the photodetector output signal. FIG. 9. The effect of valve-laser misalignment on the photodetector output signal. The good accordance in the simulation and experiment data confirms that the present numerical model can be successfully used to predict the particular valve dynamic performances. B. Simulation results and discussion 8. i i di d i Fi 8 FIG. 10. Current (Ampere) distribution in the coil at the various times indicated in Fig. 8. 075129-11 Guo et al. AIP Advances 8, 075129 (2018 FIG. 11. Radial magnetic flux density (Tesla) distribution in the diaphragm at the various times indicated in Fig. 8. AIP Advances 8, 075129 (2018) 75129-11 Guo et al. 075129-11 FIG. 11. Radial magnetic flux density (Tesla) distribution in the diaphragm at the various times indicated in Fig. 8. bottom surface is reduced compared with t = 15 µs. This leads to promote the further penetration of the magnetic field in the diaphragm. As a result, the induced current appears in the region far from the bottom surface (see Fig. 12(b)). With the time escaping, the magnetic flux density decreases with the attenuation of coil current (see Fig. 11(c)), then the induced current near the bottom surface begins to reduce. However, the effect of the self-induction of the diaphragm retards the induced current reduction though the magnetic flux density decreases (see Fig. 12(c)). At t = 60 µs, the direction of the induced current near the bottom surface of the diaphragm begins to reverse (see Fig. 12(d)) due to the decrease of magnetic flux density. At later instances, the reversed induced current in the diaphragm superimposes the magnetic flux density to that of the coil which leads to a further reduction of the magnetic flux near the bottom surface in the diaphragm (see Fig. 11(d)). After the coil current 24 October 2024 03:58:37 FIG. 12. Induced current (Ampere) distribution in the diaphragm at the various times indicated in Fig. 8. FIG. 12. Induced current (Ampere) distribution in the diaphragm at the various times indicated in Fig. 8. 075129-12 Guo et al. AIP Advances 8, 075129 (2018) FIG. 13. Diffusion of axial Lorentz force density (N/m3) in the diaphragm at the various times indicated in Fig. 8. AIP Advances 8, 075129 (2018) 075129-12 Guo et al. 075129-12 FIG. 13. Diffusion of axial Lorentz force density (N/m3) in the diaphragm at the various times indicated in Fig. 8. revers at t = 95 µs, similar to the above discussion, the reversed magnetic flux density generated by the coil gradually penetrates the diaphragm with increasing magnitude (see Fig. 11(e), Fig. 11(f)). B. Simulation results and discussion The reversed induced current during a short time spreads more and more inside the diaphragm with the magnitude reducing (see Fig. 12(e), Fig. 12(f)). The temporal evolution of axial Lorentz force density ( f z) in the diaphragm, deduced from Fig. 11 and Fig. 12, is plotted in Fig. 13. It is shown that f z initially distributes near the bottom surface of the diaphragm with its magnitude increasing as the coil current increasing (see Fig. 13(a) and Fig. 13(b)). The coil current begins to attenuate after t = 38 µs, then f z gradually diffuses through the thickness of the diaphragm with decreasing magnitude. It can be seen that the core of f z mainly distributes within the lower region of diaphragm in the thickness direction (see Fig. 13(a) – 13(d)). From the design perspective, this kind of f z distribution means that the diaphragm thickness matches the coil current frequency well. Further, f z near the bottom surface of the diaphragm acts in negative direction at t = 95 µs (see Fig. 13(e)). In order to more intuitively understand the effect of f z on the valve opening process, the notion of magnetic pressure is introduced.25 f z is transformed to axial magnetic pressure by integrating it along the axial direction in the diaphragm. Fig. 14 shows the radial distribution of the axial magnetic pressure acting on the bottom surface of the diaphragm. It is shown that the magnetic pressure at outer part of the diaphragm from t = 80 µs to t = 105 µs is opposite to diaphragm movement, which means that the Lorentz force tends to oppose diaphragm acceleration during this period. 24 October 2024 03:58:37 FIG. 14. Radial distribution of the axial magnetic pressure. FIG. 14. Radial distribution of the axial magnetic pressure. 075129-13 Guo et al. AIP Advances 8, 075129 (2018) FIG. 15. Velocity distribution along radial direction at various times. FIG. 15. Velocity distribution along radial direction at various times. The aforementioned f z distribution is also reflected by the velocity of the diaphragm. The axial velocity distribution of the diaphragm, indicating the valve operation process is shown in Fig. 15. At the very beginning of the process, f z is relatively small and cannot counteract the sealing force exerted by the diaphragm deformation. B. Simulation results and discussion And then, as the increasing of the coil current, the outer part of the diaphragm, just above the coil, experiences a high f z. At about t = 25 µs, the diaphragm is accelerated and the valve begins to open. At later instances, the diaphragm is further accelerated by f z until t = 80 µs when the diaphragm begins to decelerate for the reduction of f z. As f z is relatively large (see Fig. 13(a)–13(e)) and the velocity of the diaphragm has been increasing from t =25 µs to t = 80 µs, it is concluded that the opening operation is most effective in this period. Thereafter, due to the elastic deformation increasing, the reducing f z cannot counteract the increasing elastic force of the diaphragm. According to the simulated displacement result of the valve as shown in Fig. 8, the displacement at the end of the acceleration process (t = 80 µs) is 0.3 mm. This means that, the further deformation of the diaphragm is primarily dominated by the inertial force (from t = 80 µs to t = 140 µs), i.e., the converting process of kinetic energy of the diaphragm to its elastic potential energy. Therefore, in order to make the valve fully open, the diaphragm should obtain enough kinetic energy during the acceleration process. At t = 140 µs, the diaphragm collides with the stopper, the outer part of the diaphragm bounces off the stopper and moves downwards to the coil, and the valve starts to close. However, the inner part of the diaphragm remains upward unchanged for the inertia. Subsequently, the elastic potential energy stored by the deformation of the diaphragm gradually converts to kinetic energy of the diaphragm and the inner part following the outer part moves to the coil (from t = 140 µs to the end). 24 October 2024 03:58:37 IV. CONCLUSION In this paper, a simple but effective numerical model is discussed in detail for a special type electromagnetic inductive valve. Compared results from experiments convinced that the model can give reasonable predictions on the response characteristic of the valve even with adopted simplifica- tions. The model also gives a novel tool to designer for investigating the valve from the fundamental viewpoint. For the given circuit parameters and valve geometry, detailed insight with respect to the development of magnetic flux density, induced current, Lorentz force and velocity of the diaphragm is provided. The analysis indicates that a perfect match is achieved between the circuit parameter and the diaphragm of the prototype valve. The results also show that the effective acceleration of the diaphragm occurs in the initial phase of valve opening, i.e., the first half cycle of the coil current. Only the diaphragm obtains enough kinetic energy the acceleration process, can the valve be fully opened. This study would be beneficial to improve the understanding of the valve operation process. Moreover, pulsed energy, electric circuit parameter, material properties and valve geometry that can AIP Advances 8, 075129 (2018) 075129-14 Guo et al. Guo et al. 075129-14 Guo e 075129-14 affect the valve performance are considered in the calculation process, from a technological point of view, the model can facilitate the future work of optimization design with respect to a reduction of pulse energy. 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English
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Development of SNP and InDel markers by genome resequencing and transcriptome sequencing in radish (Raphanus sativus L.)
BMC genomics
2,023
cc-by
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Abstract Background  Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) are the most abundant genetic variations and widely distribute across the genomes in plant. Development of SNP and InDel markers is a val- uable tool for genetics and genomic research in radish (Raphanus sativus L.). Results  In this study, a total of 366,679 single nucleotide polymorphisms (SNPs) and 97,973 insertion-deletion (InDel) markers were identified based on genome resequencing between ‘YZH’ and ‘XHT’. In all, 53,343 SNPs and 4,257 InDels were detected in two cultivars by transcriptome sequencing. Among the InDel variations, 85 genomic and 15 transcriptomic InDels were newly developed and validated PCR. The 100 polymorphic InDels markers generated 207 alleles among 200 Chinese radish germplasm, with an average 2.07 of the number of alleles (Na) and with an average 0.33 of the polymorphism information content (PIC). Population structure and phylogenetic relationship revealed that the radish cultivars from northern China were clustered together and the southwest China cultivars were clustered together. RNA-Seq analysis revealed that 11,003 differentially expressed genes (DEGs) were identified between the two cultivars, of which 5,020 were upregulated and 5,983 were downregulated. In total, 145 flowering time-related DGEs were detected, most of which were involved in flowering time integrator, circadian clock/pho- toperiod autonomous, and vernalization pathways. In flowering time-related DGEs region, 150 transcriptomic SNPs and 9 InDels were obtained. Conclusions  The large amount of SNPs and InDels identified in this study will provide a valuable marker resource for radish genetic and genomic studies. The SNPs and InDels within flowering time-related DGEs provide fundamental insight into for dissecting molecular mechanism of bolting and flowering in radish. Keywords  Radish, InDel, Genetic diversity, Population structure, Transcriptome, Flowering BMC Genomics BMC Genomics BMC Genomics Li et al. BMC Genomics (2023) 24:445 https://doi.org/10.1186/s12864-023-09528-6 Open Access Introduction Molecular marker is a valuable tool for genetics and breeding research in plant, such as fingerprinting geno- types, genetic map construction, QTL mapping, asso- ciation analysis and marker-assisted selection (MAS) [1, 2]. In the past three decades, a large number of molec- ular markers have been successfully developed in plant, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), amplified fragment length polymorphisms (AFLPs), †Yadong Li and Xiaobo Luo contributed equally to this work. Development of SNP and InDel markers by genome resequencing and transcriptome sequencing in radish (Raphanus sativus L.) Yadong Li1,2†, Xiaobo Luo3†, Xiao Peng1,2, Yueyue Jin1,2, Huping Tan1,2, Linjun Wu1,2, Jingwei Li1,2, Yun Pei1,2, Xiuhong Xu1,2 and Wanping Zhang1,2* †Yadong Li and Xiaobo Luo contributed equally to this work. *Correspondence: Wanping Zhang 1226190368@qq.com 1 College of Agriculture, Guizhou University, Guiyang 550003, China 2 Institute of Vegetable Industry Technology Research, Guizhou University, Guiyang 550003, China 3 Guizhou Province Academy of Agricultural Sciences, Guizhou Institute of Biotechnology, Guiyang 550003, China 3 Guizhou Province Academy of Agricultural Sciences, Guizhou Institute of Biotechnology, Guiyang 550003, China © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access T In total, 47,558 InDels were identified between the two Cannabis accessions, 14 InDels were applied to perform the genetic structure analysis [7]. In mung bean, 129 InDel markers were developed and used to construct a genetic linkage map by the genome resequencing between two parents, the major effect QTL qYSC4 for young stem color on chromosome 4 was narrowed in a 347 kb interval [5]. In all, 318 InDel markers were developed across the eight chromosomes by comparing the assembled genomic sequences of two Medicago truncatula varieties, gibber- ellin 3-β-dioxygenase 1 gene for in the dwarf mutant crs were isolated [10]. response [14]. FLOWERING LOCUS T (FT) in the pho- toperiod pathway as a central floral integrator was exhib- ited to delay flowering [16]. Bolting and flowering times as two important agronomic traits determine production and quality of radish. Previous studies showed that 142 bolting and flowering time related genes were acquired by transcriptome sequencing [17]. It was found that 218 homologs of Arabidopsis flowering-time genes were obtained in radish, of which 49 genes were identified as differentially expressed in two radish accessions [18]. A total of 254 flowering genes in R. sativus were character- ized based on sequence similarities analysis [11]. Two QTLs each for bolting and flowering times were identi- fied on chromosome R06 by QTL mapping [19]. A 1627- bp insertion near the 5′ end of the first intron of RsFLC2 was associated with late-bolting trait in radish [12]. A large number of bolting and flowering genes have been reported in radish, no study on identify the genetic varia- tion in flowering genes was performed in radish. l In this study, the SNP and InDel markers were devel- oped by genome resequencing and transcriptome between early and late bolting cultivars. The InDel markers were developed and validated by PCR based markers. Population structure and clustering analysis were performed based on genotyping 200 radish culti- vars with the developed InDel markers. The differential expressed genes (DEGs) of vegetative growth were identi- fied between two cultivars. The homologs of flowering- time related gene (FTR) in radish were characterized. The transcriptomic SNP and InDel markers within flowering- time related genes were obtained. These results could provide an abundant marker for genetic and genomic studies in radish. Radish (Raphanus sativus L.) is an important root veg- etable belonging to cruciferous family. Open Access T A large number of InDel markers have been characterized and applied for genetic and genomic studies in radish [3, 11]. A total of 9,436 InDel were detected in three radish transcriptome and 40 InDel markers were used for genetic diversity analysis in 32 radish accessions [3]. In total, 99 EST-SSR and InDel markers were used to construct linkage groups and QTL mapping, QTL for late-bolting trait was located a 1.1-cM region between InDel520 and InDel535 [12]. Previous studies indicated that the R locus was identified between RsInDel4 and RsInDel11, and RsMYB90 was defined as a candidate gene underlying the taproot skin color trait [13]. However, the number of InDel markers is far from sufficient for radish genetic studies.h Open Access T Li et al. BMC Genomics (2023) 24:445 Li et al. BMC Genomics (2023) 24:445 Page 2 of 12 simple sequence repeats (SSRs), insertion/Deletions (InDels) and single-nucleotide polymorphisms (SNPs) [3, 4]. SSR and InDel are PCR-base markers with the advan- tage of bi-allelic, co-dominant, abundance and low-cost [5]. SNP and InDel are the most abundant genetic vari- ations and widely distribute across the genomes in plant [6]. With the common characteristics of SSR, InDel markers have receive more and more attention. With the development of next-generation sequencing (NGS), InDel markers has extensively developed and applied in crop breeding [7, 8]. In soybean, a total of 17,613 InDels were detected in 56 soybean accessions and a genetic map with 300 InDel markers was constructed in 20 link- age groups [9]. The promoter of Sl-ALMT9 with a 3 bp InDel was increased the expression levels of Sl-ALMT9 and fruit malate contents in cultivate tomato [8]. In total, 47,558 InDels were identified between the two Cannabis accessions, 14 InDels were applied to perform the genetic structure analysis [7]. In mung bean, 129 InDel markers were developed and used to construct a genetic linkage map by the genome resequencing between two parents, the major effect QTL qYSC4 for young stem color on chromosome 4 was narrowed in a 347 kb interval [5]. In all, 318 InDel markers were developed across the eight chromosomes by comparing the assembled genomic sequences of two Medicago truncatula varieties, gibber- ellin 3-β-dioxygenase 1 gene for in the dwarf mutant crs were isolated [10]. simple sequence repeats (SSRs), insertion/Deletions (InDels) and single-nucleotide polymorphisms (SNPs) [3, 4]. SSR and InDel are PCR-base markers with the advan- tage of bi-allelic, co-dominant, abundance and low-cost [5]. SNP and InDel are the most abundant genetic vari- ations and widely distribute across the genomes in plant [6]. With the common characteristics of SSR, InDel markers have receive more and more attention. With the development of next-generation sequencing (NGS), InDel markers has extensively developed and applied in crop breeding [7, 8]. In soybean, a total of 17,613 InDels were detected in 56 soybean accessions and a genetic map with 300 InDel markers was constructed in 20 link- age groups [9]. The promoter of Sl-ALMT9 with a 3 bp InDel was increased the expression levels of Sl-ALMT9 and fruit malate contents in cultivate tomato [8]. Genetic diversity, population structure and phylogenetic analysis Popgene 32 program was used to determine values of the expected homozygosty and expected heterozygo- sity. PowerMarker version 3.2 was employed to calcu- late the number of alleles (Na), major allele frequency (MAF), and polymorphism information content (PIC) [26]. InDel markers were divided as three types: highly informative with PIC > 0.5), moderately informa- tive with 0.25 < PIC < 0.5 and slightly informative with PIC < 0.25. Genetic structure analysis of radish acces- sions was conducted by the Structure 2.3.4 program [4]. The Bayesian model-based clustering was used to distribute individuals to groups with a predetermined number (K), which could be minimized Hardy–Wein- berg and linkage disequilibrium within each group. The number of K was set from 1 to 10 with ten independent runs and 10,000 iterations were performed for estima- tion after a 10,000 iterations burn-in period. The sub- groups and best K was estimated according to previous study [27]. Phylogenetic analysis and the dendrogram was carried out in previous studies [3]. RNA isolation and transcriptome sequencing Total RNA were extracted from the young leaves of ‘YZH’ and ‘XHT’ in vegetative growth period (after 25 days after sowing) using a Plant RNA Mini Kit (Tian- gen, Inc., China). To assess RNA integrity, the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) were used [23]. A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were constructed using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA). All libraries were sequenced on Illumina Novaseq platform. Plant materials and DNA extraction In total, 200 radish cultivars were collected from Institute of Vegetable Industry Technology Research in Guizhou University. The detail information of all cultivars are listed in Table S1. The seeds of two high-generation rad- ish inbred lines ‘YZH’ (early-flowering time, 65d) and ‘XHT’ (late-flowering time, 185d) were planted in plas- tic pots and cultured in a chamber at 22 ℃ for 14 h light and 10 h dark. After 25 days, the young leaves of ‘YZH’ and ‘XHT’ were taken for three biological replicates for genome resequencing and transcriptome sequencing. Leaves of all radish cultivars were collected and immedi- ately frozen in liquid nitrogen, and stored at -80 °C for further use. Total genomic DNA was extracted using the EasyPure Genomic DNA Kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. The DNA with a final concentration of 10 ng/ul were used to conduct PCR amplifications. fi The transition from the vegetative phase to the repro- ductive phase is the most important stages in the life cycle of flowering plants, which is control by multiple environmental signals and genetic pathway. Numerous studies have found that more than 300 genes associated with flowering time were mainly involved in six key path- ways, including photoperiod, vernalization, ambient tem- perature, age, autonomy, and gibberellin pathways [14, 15]. In Arabidopsis, FLOWERING LOCUS C (FLC) as a repressor of flower plays crucial roles in the vernalization Page 3 of 12 Li et al. BMC Genomics (2023) 24:445 Li et al. BMC Genomics (2023) 24:445 Validation of InDel markers polymorphismh The length of primer pairs of InDels were varied from 18 to 24  bp, the predicted product sizes were ranged from 100 to 200  bp by Primer Premier 5.0 program. Polymerase chain reaction (PCR) reagents and ampli- fication conditions were implemented as described previously by Luo et  al. [3]. The PCR products were separated on 8% polyacrylamide electrophoresis (PAGE) gel. All validated primers of InDel markers were shown in Table S3. Genome resequencing and identification of SNPs parameters (quality scores (QUAL) >  = 20 and quality by depth (QD) >  = 4). SnpEff (4.3q) software was imple- mented to annotate SNPs and InDels. The ‘YZH’ and ‘XHT’ in vegetative growth period was used to genome resequencing. Total genomic DNA was extracted and the quality of genomic DNA was quanti- fied prior to library construction. According to manu- facturer’s recommendation, six sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA). The DNA libraries were sequenced on Illumina platform to generate 150 bp paired-end reads. After removing low-quality reads, the clean reads were assigned to the radish reference genome using Burrows-Wheeler Aligner (BWA)-0.7.8 (parameter: mem -t 4 -k 32 -M) [20, 21]. The SNPs and InDels call- ing were performed using SAMtools-1.3.1 (mpileup -m 2 -F 0.002 -d 1000) [22]. The variants were filtered with the following criteria: (1) The depth of the variate posi- tion > 4; (2) The mapping quality > 20. The functional annotation of variants was carried out by ANNOVAR (Annotate Variation). Analysis of differential expressed genes (DEGs) and function annotation After removing adapter and low quality reads, clean reads were obtained. The clean reads were aligned to the radish reference genome (http://​radish-​genome.​org) using Hisat2 (v2.0.5) [24]. The reads numbers mapped to each gene was counted by feature Counts v1.5.0- p3. FPKM (fragments per kilobase of exon per million mapped fragments) method was applied to count the gene expression levels. The DEGs were determined with an padj <  = 0.05 and |log2(foldchange)|> = 1 using R DESeq2 package. GO pathway enrichment analysis of DEGs were performed by the cluster Profiler R pack- age (3.8.1) [25]. The P value threshold (P ≤ 0.05) was regarded as significantly enriched GO terms. Genome Analysis Toolkit (GATK, version v3.8) was used to call SNPs and InDels. SNPs and InDels were filtered with Genome resequencing g To development of molecular markers in radish, two rad- ish cultivars, ‘YZH’ and ‘XHT’ have significant differ- ences in flower time was used to genome resequencing. A total of 12.0 Gb and 12.6 Gb base pairs (bp) were gener- ated in ‘YZH’ and ‘XHT’, respectively. After filtering the adapter sequences and low-quality reads, 79,949,206 and 84,185,286 clean reads in ‘YZH’ and ‘XHT’ were obtained (Table S4), of which 92.14% and 90.61% clean reads had a coverage depth of 26 and 23.7 of the radish reference genome in ‘YZH’ and ‘XHT’, respectively [24]. In total, 366,679 SNP and 97,973 InDel were identified in two cultivars (Additional file 3 and 4). The average frequency of SNP and InDel in the radish genome were calculated to be 1 SNP/1.22  kb and 1 InDel/4.6  kb. Most 39.7% (145,691) of the SNPs were located in intergenic regions, and 23.72% (86,973) were located in exonic sequence. The length distribution of InDels were ranged from 1 to 21 bp. The InDels length with 1 to 2 bp were the two most abundant type, accounting for 66.98% (65,632) of the total InDels (Fig. S1). Nine chromosomes of rad- ish have different density, the highest number of InDels (21,734) were existed on chromosome 5, while the lowest number were displayed on chromosome 7 (6,416). Genetic diversity analysis and population structure T h i di i l i f C Genetic diversity analysis and population structure To assess the genetic diversity analysis of 200 Chinese radish accessions, 100 InDel primers were selected. The 100 polymorphic InDels markers generated 207 alleles among 200 Chinese radish germplasm. The number of alleles (Na) varied from 2 to 4, with an average of 2.07 alleles (Additional file  2). The major allele frequency (MAF) ranged from 0.48 to 0.93, with an average of 0.67. The expected homozygosity (HO) each InDel varied from 0.09 to 0.9, with an average of 0.62. The expected heterozygosity (HE) for each InDel ranged from 0.1 to 0.92, with an average of 0.38. The value of polymorphism information content (PIC) per locus ranged from 0.13 to 0.53, with an average of 0.33. Results cellular component (8 terms). A total of 53,343 SNP were identified between two cultivars (Additional file 7), with an average frequency of 1 SNP/8.43 kb. The number of InDels with 1 to 2 bp was accounted for 72.23% (3,075) of the all InDels (4,257) (Fig. S1 and Additional file 8). qRT‑PCR analysis T l RNA f qRT‑PCR analysis Total RNA of eight flowing point in times, including vegetative growth, five vernalization times (5d, 10d,15d, 20 d, 25 d, 30d) and first flowering time were extracted and the cDNA of each sample was synthesized using the SuperScript III First-Strand Synthesis System (Inv- itrogen). The qRT-PCR experiment were performed as described previously [3]. The radish Actin were used as internal controls. The double delta CT ­(2-ΔΔCt) method was carried out to calculate relative transcription levels. Three technical replicates were conducted in each sam- ple. The primer sequences were listed in Table S2. Li et al. BMC Genomics (2023) 24:445 Page 4 of 12 Screening Validation and polymorphism of developed InDel markers To validate the polymorphic InDel markers, 260 genomic and 40 transcriptomic InDels were randomly selected from genome resequencing and transcriptome sequencing of two cultivars, respectively. To facilitate the visualization of InDel markers on PAGE, the lengths of InDels greater than or equal to 3 were selected for PCR validation. The results showed that 85 genomic and 15 transcriptomic InDels were amplified successfully and exhibited polymor- phisms in two cultivars, with an 32.7% an 37.5% marker polymorphism, respectively. The polymorphism ampli- fication results of the RsInDelR4-18 primer in 200 radish cultivars were shown in Fig. 2. Consequently, the 100 poly- morphic InDel markers were used for further analysis. Transcriptome sequencing and DEG analysis To gain insights into gene expression changes and devel- opment of molecular markers in transcriptional level, a total of 6 cDNA libraries were sequenced in ‘YZH’ and ‘XHT’. In total, 29.87 Gb and 21.29 Gb clean reads were obtained (Table S7), 85.93% and 86.74% of the reads were mapped to the radish reference genome, of which 78.67% and 79.06% were uniquely mapped in ‘YZH’ and ‘XHT’, respectively. The Pearson correlation coefficient between the three replicates of each sample displayed higher val- ues (> 0.95) (Fig. 1a). The principal component analy- sis (PCA) result indicated that PC1 and PC2 explained 90.83% of the total variation (Fig. S2).l Population structure analysis was conducted based on the 200 radish accessions with 100 InDel markers using Structure 2.3.3 software. Delta K reached a maximum value at K = 2, indicating the 200 accessions could be divided into two groups (Fig. 3a and b). Apparently, six of seven accessions from Korea, one of two accessions from Japan, the majority of accessions with late flower- ing from northern China (Beijing, Shangdong, Heilongji- ang) were clustered into the same group (Fig. 3c). The majority of accessions from southwest China (Guizhou, Yunnan, Sichuan) were clustered into same group. Clus- ter analysis implied that the 200 cultivars were divided into two groups with a genetic distance of 0.58 (Fig. 4). Group I contained 122 accessions, most varieties (58.4%) were derived from the northern China, seven accessions from Korea and two Japan were clustered into the same group. Group II contained 78 accessions, most of which To explore genes associated with flowering time, genes with |log2(FoldChange)|> = 1 and padj <  = 0.05 were defined as DEGs. In total, 11,003 DEGs were identified in between the two cultivars, of which 5,020 DEGs were upregulated and 5,983 DEGs were downregulated (Fig. 1b and Additional file  5). GO annotations reveals that all DEGs were assigned into 85 significantly enriched GO terms (Fig. 1c and Additional file 6). In the molecular func- tion (43 terms), the major subcategories were iron ion bind- ing, oxidoreductase activity, catalytic activity. For biological process (34 terms), catabolic process, organic substance catabolic process, cofactor metabolic process were the dominant terms. The ‘endoplasmic reticulum’ and ‘extracel- lular region’ terms were extraordinarily remarkable in the Page 5 of 12 Li et al. BMC Genomics (20 Li et al. Transcriptome sequencing and DEG analysis c Enriched GO terms of the all DGE identified in two cultivars 1 0.965 0.97 0.561 0.564 0.551 0.965 1 0.965 0.554 0.556 0.543 0.97 0.965 1 0.566 0.571 0.555 0.561 0.554 0.566 1 0.957 0.971 0.564 0.556 0.571 0.957 1 0.954 0.551 0.543 0.555 0.971 0.954 1 XHT1 XHT2 XHT3 YZH1 YZH2 YZH3 XHT1 XHT2 XHT3 YZH1 YZH2 YZH3 0.6 0.7 0.8 0.9 1.0 R2 Pearson correlation between samples a b a 48 44 30 18 10 17 9 9 14 13 10 7 12 6 6 4 6 5 48 44 36 26 28 22 12 15 15 17 54 51 35 35 28 18 25 25 19 19 15 16 11 11 10 5 1 2 67 63 50 47 37 24 31 25 25 22 catabolic process organic substance catabolic process cofactor metabolic process protein modification by small protein conjugation or removal defense response carbohydrate catabolic process protein modification by small protein conjugation protein ubiquitination pyridine-containing compound metabolic process pyridine nucleotide metabolic process endoplasmic reticulum extracellular region organelle subcompartment apoplast proton-transporting two-sector ATPase complex preribosome nuclear chromosome part Golgi membrane iron ion binding oxidoreductase activity, acting on paired donors guanyl nucleotide binding ADP binding oxidoreductase activity, acting on CH-OH group of donors oxidoreductase activity, NAD or NADP as acceptor phosphoric ester hydrolase activity ubiquitin-protein transferase activity ubiquitin-like protein transferase activity iron-sulfur cluster binding 0 20 40 60 80 100 120 Number of genes Down Up Molecular Function Cellular component Biological process X X X Y Y Y c Fig. 1  Differentially expressed genes (DEGs) and GO enrichment analysis. a Pearson correlation coefficient with three replicates in two cultivars. b Volcano plot showed the DGEs identified in two cultivars. c Enriched GO terms of the all DGE identified in two cultivars c Biological process Molecular Function Cellular component Biological process Fig. 1  Differentially expressed genes (DEGs) and GO enrichment analysis. a Pearson correlation coefficient with three replicates in two cultivars. b Volcano plot showed the DGEs identified in two cultivars. c Enriched GO terms of the all DGE identified in two cultivars (http://​www.​phyto​syste​ms.​ulg.​ac.​be/​florid/) and aligned to radish reference genome using BLASTN. Top hits with E-values ≤ ­1E−20 and identity ≥ 80% were used to screen for the corresponding homologous genes. The 637 radish FTR genes were identified and divided into nine flowering-related pathways (Additional file 9). Transcriptome sequencing and DEG analysis BMC Genomics (2023) 24:445 48 44 30 18 10 17 9 9 14 13 10 7 12 6 6 4 6 5 48 44 36 26 28 22 12 15 15 17 54 51 35 35 28 18 25 25 19 19 15 16 11 11 10 5 1 2 67 63 50 47 37 24 31 25 25 22 catabolic process organic substance catabolic process cofactor metabolic process protein modification by small protein conjugation or removal defense response carbohydrate catabolic process protein modification by small protein conjugation protein ubiquitination pyridine-containing compound metabolic process pyridine nucleotide metabolic process endoplasmic reticulum extracellular region organelle subcompartment apoplast proton-transporting two-sector ATPase complex preribosome nuclear chromosome part Golgi membrane iron ion binding oxidoreductase activity, acting on paired donors guanyl nucleotide binding ADP binding oxidoreductase activity, acting on CH-OH group of donors oxidoreductase activity, NAD or NADP as acceptor phosphoric ester hydrolase activity ubiquitin-protein transferase activity ubiquitin-like protein transferase activity iron-sulfur cluster binding 0 20 40 60 80 100 120 Number of genes Down Up Molecular Function Cellular component Biological process 1 0.965 0.97 0.561 0.564 0.551 0.965 1 0.965 0.554 0.556 0.543 0.97 0.965 1 0.566 0.571 0.555 0.561 0.554 0.566 1 0.957 0.971 0.564 0.556 0.571 0.957 1 0.954 0.551 0.543 0.555 0.971 0.954 1 XHT1 XHT2 XHT3 YZH1 YZH2 YZH3 XHT1 XHT2 XHT3 YZH1 YZH2 YZH3 0.6 0.7 0.8 0.9 1.0 R2 Pearson correlation between samples a b c Fig. 1  Differentially expressed genes (DEGs) and GO enrichment analysis. a Pearson correlation coefficient with three replicates in two cultivars. b Volcano plot showed the DGEs identified in two cultivars. Transcriptome sequencing and DEG analysis The larg- est number of FTR genes were involved in autonomous pathway (286). The smallest number of FTR genes were involved in sugar pathway (31) (Additional file  5: Table S8). The DEGs associated with the flowering pathway between two cultivars were screened with |log2 fold (91.75%) were collected from the southwest China. The results of cluster and population structure analysis were basically consistent, but there were slight differences. These results demonstrated that the phylogenetic rela- tionships among all accessions highly correlated with their geographical origins. Identification of homologous genes for flowering timel In autonomous pathway, UBIQUITIN CARRIER PROTEIN 1 (UBC1), FLOWERING LOCUS Y (FY), AGAMOUS- LIKE 6 (AGL6), FLOWERING LOCUS VE (FVE), RELA- TIVE OF EARLY FLOWERING 6 (REF6), EMBRYONIC FLOWER 2 (EMF2) were detected. Several key genes in photoperiod pathway were also identified, such as PSEUDORESPONSE REGULATORS 3 (PRR3), PRR5, CYCLING DOF FACTOR 1 (CDF1), CDF4, CDF5, CRY2, PHYCOCHROME A (PHYA), CALCIUM-DEPEND- ENT PROTEIN KINASES 6 (CPK6), CPK33, PHYB, BBX19, CONSTANS (CO), and EARLY FLOWERING 4 (ELF4). Genes known to be involved in vernalization pathways were identified, such as VERNALIZATION INSENSITIVE 3 (VIN3), INDUCER OF CBF EXPRES- SION 1 (ICE1), FRIGIDA (FRI), AGL19. LEAFY (LFY), AGL25, AGL27, TCP18, APETALA 2 (AP2) involved in the flower development and meristem identity pathways were detected. Many vital genes in aging (SPL15), ambi- ent temperature (AGL31, PHYTOCHROME INTERACT- ING FACTOR 4 (PIF4), AGL27, hormone (GA2ox1 and GA2ox6), SUGAR SUCROSE TRANSPORTER 9 (SUC9), circadian clock (CIRCADIAN CLOCK–ASSOCIATED 1, CCA1), PRR5 and flowering time integrator genes (SUP- PRESSOR OF OVEREXPRESSION OF CONSTANS 1, SOC1) and FT were also detected. qRT‑PCR validation To assess the expression pattern of DEGs identified from RNA-Seq, ten candidate DEGs involved in the flowering pathway were selected for qRT-PCR analysis in leaves of ‘YZH’ and ‘XHT’ (Fig. 5). The expression of five DEGs ( RsAGL25, RsVIN3, RsUBC1, CO and RsPIF4) were highly expressed in ‘YZH’ during eight times. RsSOC1, RsCCA1, RsVIN3 were highly expressed in 30 d of vernalization. RsELF7, RsLFY, RsVIN3 were highly expressed in 15 d of vernalization in ‘XHT’. RsFT were highly expressed in the first flowering time. These results revealed that these DEGs might involve in the phase transition from vegeta- tive stage to bolting and flowering in radish. Identification of homologous genes for flowering timel To identify homologs of flowering-time related gene (FTR) in radish, 306 FTR genes in Arabidopsis were downloaded from the Flowering Interactive Database Li et al. BMC Genomics (2023) 24:445 Page 6 of 12 Fig. 2  Polymorphism amplification results of the RsInDelR4-18 in 200 accessions file 11). A total of 12 differentially expressed FTR genes with at least 2 SNPs were identified, such as PHYA, TEM1, VIN3, PIF5, ADG1, FVE, ELF3. A total of 3 differ- entially expressed FTR genes with at least 2 InDels were identified, including ELF3, VIL2, TPS1. These results pro- vided valuable information on the explaining significant difference in flower time between ‘YZH’ and ‘XHT’. change|> 1 and FDR < 0.05. A total of 145 flowering time genes were identified, of which 74 were upregulated and 71 were downregulated (Additional file  10). The differ- entially expressed FTR genes were mainly associated with autonomous (50), photoperiod (39), and vernaliza- tion pathway (12). Rs583930 and Rs094390, the homolog of FT plays key roles in flowering time integrator. In autonomous pathway, UBIQUITIN CARRIER PROTEIN 1 (UBC1), FLOWERING LOCUS Y (FY), AGAMOUS- LIKE 6 (AGL6), FLOWERING LOCUS VE (FVE), RELA- TIVE OF EARLY FLOWERING 6 (REF6), EMBRYONIC FLOWER 2 (EMF2) were detected. Several key genes in photoperiod pathway were also identified, such as PSEUDORESPONSE REGULATORS 3 (PRR3), PRR5, CYCLING DOF FACTOR 1 (CDF1), CDF4, CDF5, CRY2, PHYCOCHROME A (PHYA), CALCIUM-DEPEND- ENT PROTEIN KINASES 6 (CPK6), CPK33, PHYB, BBX19, CONSTANS (CO), and EARLY FLOWERING 4 (ELF4). Genes known to be involved in vernalization pathways were identified, such as VERNALIZATION INSENSITIVE 3 (VIN3), INDUCER OF CBF EXPRES- SION 1 (ICE1), FRIGIDA (FRI), AGL19. LEAFY (LFY), AGL25, AGL27, TCP18, APETALA 2 (AP2) involved in the flower development and meristem identity pathways were detected. Many vital genes in aging (SPL15), ambi- ent temperature (AGL31, PHYTOCHROME INTERACT- ING FACTOR 4 (PIF4), AGL27, hormone (GA2ox1 and GA2ox6), SUGAR SUCROSE TRANSPORTER 9 (SUC9), circadian clock (CIRCADIAN CLOCK–ASSOCIATED 1, CCA1), PRR5 and flowering time integrator genes (SUP- PRESSOR OF OVEREXPRESSION OF CONSTANS 1, SOC1) and FT were also detected. change|> 1 and FDR < 0.05. A total of 145 flowering time genes were identified, of which 74 were upregulated and 71 were downregulated (Additional file  10). The differ- entially expressed FTR genes were mainly associated with autonomous (50), photoperiod (39), and vernaliza- tion pathway (12). Rs583930 and Rs094390, the homolog of FT plays key roles in flowering time integrator. Discussioni Identification of SNP and InDel markers in radish Identification of SNP and InDel markers in radish In this study, 53,343 SNPs and 4,257 InDels were identified from transcriptomes sequencing data in two cultivars. The large discrepancy in SNPs and InDels number may be caused by different materials and SNP calling strategies. The short InDels (1–2  bp) were most prevalent types in the radish genome and transcrip- tome, which was coincided with previously studies in tea plant [4] and sesame [28]. Previous studies indicated that the InDel within the RsRf3 locus played crucial roles in involving transition to fertility in cytoplasmic male sterility radish [31]. The SNP and InDel in RsTT8 pro- moter were successfully distinguished between red and white-fleshed radish [32]. These studies demonstrated that SNP and InDel markers within functional genes were involved in important agronomic traits of rad- ish. The newly identified SNP and InDel markers could provide abundant data information for the genetics and functional genomics research in radish. with other plant species, including in Arabidopsis [29], Brassica rapa [30], tea plant [4]. The distinct filtering cri- teria and the different genetic structure among different plant species were likely to cause significant differences in SNP and InDel density [4]. In a previous study, 22,412 SNPs and 9,436 InDels were identified in three radish transcriptome [3]. In this study, 53,343 SNPs and 4,257 InDels were identified from transcriptomes sequencing data in two cultivars. The large discrepancy in SNPs and InDels number may be caused by different materials and SNP calling strategies. The short InDels (1–2  bp) were most prevalent types in the radish genome and transcrip- tome, which was coincided with previously studies in tea plant [4] and sesame [28]. Previous studies indicated that the InDel within the RsRf3 locus played crucial roles in involving transition to fertility in cytoplasmic male sterility radish [31]. The SNP and InDel in RsTT8 pro- moter were successfully distinguished between red and Identification of SNP and InDel markers in radish Identification of SNP and InDel markers in radish SNP and InDel markers were the most abundant varia- tions in the plant genomes, which had been widely uti- lized in genetic diversity analysis, gene mapping, genome wide association mapping and marker-assisted selection breeding [5, 28]. With the release of genome sequences information, providing important data reference for the resequencing of different radish varieties [21]. Although a large number of InDel markers have been extensively detected in radish, the number of InDel markers is still insufficient. In this study, 366,679 genomic SNPs and 97,973 genomic InDels were identified in ‘YZH’ and ‘XHT’, with an average frequency of 1 SNP/1.22 kb and 1 InDel/4.6 kb. The frequency of genomic SNP was signifi- cantly lower than previously described in radish (average occurrence of 3.9/kb) [21]. The frequency of genomic SNP and InDel in the radish were remarkably different Many studies reported that SNPs and InDels within or near the coding sequences (CDS) were significantly associated with important agronomic traits in crops. To identify of genetic variation in flower-related genes, SNP and InDel markers located in flower-related DGEs were detected. A total of 150 transcriptomic SNPs and 9 tran- scriptomic InDels within 30 and 6 differentially expressed FTR genes were obtained, respectively (Additional Page 7 of 12 Li et al. BMC Genomics (2023) 24:445 Li et al. BMC Genomics Fig. 3  Population structure of the 200 radish accessions analyzed by STRU​CTU​RE software. a Evaluateion of the optimal group number of △K. b L(K) (log probability of data) over ten runs for K ranging from 1 to 10. c The y-axis indicated the membership coefficient and the x-axis indicated the number of each accession Fig. 3  Population structure of the 200 radish accessions analyzed by STRU​CTU​RE software. a Evaluateion of the optimal group number of △K. b L(K) (log probability of data) over ten runs for K ranging from 1 to 10. c The y-axis indicated the membership coefficient and the x-axis indicated the number of each accession with other plant species, including in Arabidopsis [29], Brassica rapa [30], tea plant [4]. The distinct filtering cri- teria and the different genetic structure among different plant species were likely to cause significant differences in SNP and InDel density [4]. In a previous study, 22,412 SNPs and 9,436 InDels were identified in three radish transcriptome [3]. Development of InDel markers and application of germplasm resources PCR based InDels with the advantages of co-dominant, inexpensive, and highly polymorphic were favored by more and more researchers in the field of gel based geno- typing technology [33]. In this study, 85 genomic and 15 transcriptomic polymorphic InDels with an 32.7% and 37.5% marker polymorphism were validated by PCR based results, respectively, which was lower than previ- ously reported results with 70% marker polymorphism [3]. The mean PIC value of InDels was 0.33 by genotyping Li et al. BMC Genomics (2023) 24:445 Page 8 of 12 Fig. 4  Dendrogram of cluster analysis for 200 radish accessions based on the 100 newly developed InDels Fig. 4  Dendrogram of cluster analysis for 200 radish accessions based on the 100 newly developed InDels Li et al. BMC Genomics (2023) 24:445 Li et al. BMC Genomics (20 Page 9 of 12 Li et al. BMC Genomics Fig. 5  RT-qPCR validation of DEGs related to the flowering pathway in two cultivars. Each bar shows the mean ± SE of three replicate Fig. 5  RT-qPCR validation of DEGs related to the flowering pathway in two cultivars. Each bar shows the mean ± SE of three replicate RsFLC genes were no difference in vegetative phase for different cultivars. In wheat, VRN1 was upstream of FT and induced the upregulation of FT expression under LD conditions [41]. In radish, a 647-bp insertion in promoter region of RsVRN1 gene leaded the late-bolting phenotype in NAU-LB. The florigen FT played distinctive roles in regulation of the transition to reproductive development and flowering in plant [42]. The flower meristem-identity gene LFY played important roles in regulating Arabidop- sis floral development [43]. Previous studies found that a 16-bp deletion and 18-bp deletion were detected in the intron of FT and LFY gene in radish [11]. In this study, the RsFT and RsLFY gene were differentially expressed in two cultivars by transcriptome sequencing and confirmed by qRT-PCR analysis. of the 200 accessions, were lower than the PIC of the radish SSR markers, which can be explained that most InDels are single-locus, while SSRs are multi-locus [34, 35]. The alleles of single-locus markers can be positioned in the particular genomic loci, which was convenient to rapidly carry out genetic and breeding studies [36]. The genetic structure analysis of different genotypes is beneficial to develop varieties with a wide range of genetic backgrounds [7]. Development of InDel markers and application of germplasm resources In the present study, 200 radish accessions were partitioned into two groups by popula- tion structure analysis. Apparently, the radish cultivars from northern China were clustered together, the radish cultivars from southwest China were clustered together. UPGMA cluster analysis revealed the 200 accessions were also clustered into two major groups. Previous studies indicated that flowering time are increased with latitude of origin [37]. It is important to introduce high latitude late flowering genes into radish. These results indicated that the InDel markers developed in this study was an important marker for genetic diversity analysis in radish. q y In this study, SNP and InDel markers within differ- entially expressed FTR genes were identified, such as eleven SNPs and two InDels in RsELF3 gene, ten SNPs in RsTEM1 gene, five SNPs in RsVIN3 gene, two SNPs and two InDels in RsCDF5 gene, one SNPs in RsFVE gene, one SNPs in RsEMF2 gene, one SNPs in RsAGL6 gene, one SNPs in RsCPK33 gene. ELF3 gene regulated vegetative photomorphogenesis and the photoperiodic induction in Arabidopsis flower [44]. TEM1 combina- torially interacted with FT rerepressed the floral transi- tion in Arabidopsis [45]. The level of VIN3 expression was associated with the duration of cold exposure and the degree of FLC repression in Arabidopsis [46]. Previ- ous studies observed the CDF5 protein delayed flowering through directly repressing FT transcription [47]. FVE, a component of the autonomous pathway involved in a protein complex repressed FLC expression [48]. Loss of function mutations of the EMF2 genes leaded to early flower in Arabidopsis, confirming the important role of EMF2 in phase transitions by restraining reproductive development [49]. AGL6 enhanced FT expression in the flc-3 background and the expression of FLC was down- regulated in agl6-1D mutant [50]. CPK33 was an impor- tant component of the florigen complex formation by FD phosphorylation [51]. These results could provide the marker data for the research of genes involve in bolting and flowering in radish. Conclusion In this study, a large amount of genomic, transcriptomic SNPs and InDels were identified from genome and tran- scriptome sequencing between ‘YZH’ and ‘XHT’, respec- tively. A total of 100 novel InDel markers were developed and applied to genotype 200 radish accessions. Population struc- ture and phylogenetic relationship revealed that the radish cultivars from northern China were clustered together and the southwest China cultivars were clustered together. RNA- Seq analysis demonstrated that 145 flowering time-related DGEs were detected, most of which were involved in flow- ering time integrator, circadian clock/photoperiod autono- mous, and vernalization pathways. The transcriptomic SNPs and InDels within differentially expressed FTR genes were detected. These findings provide insights into the develop- ment and application of molecular markers for genetic diver- sity analysis, and provide the information of SNPs and InDels within flowering time-related DGEs in radish. SNP and InDel markers within differentially expressed FTR genes It has been widely accepted that the transition from veg- etative to reproductive growth played important roles in the life cycle of an angiosperm plant [38]. The leaf tissues of early and late flowering time cultivars at the vegetative stage was conducted to perform transcriptome sequenc- ing. A total of 145 flowering-time related DGEs were detected in radish by performing BLASTN analysis. Many studies have indicated that the InDel marker played cru- cial roles in the regulation of flowering time in radish [11, 39]. Vernalization promotes flowering in the late-flower- ing in plant. FLC encoding the MADS-box protein was a central floral repressor in Arabidopsis [40]. Previous stud- ies found that the 1627-bp insertion in the first intron of RsFLC2 gene in “Ninengo” plants, resulting in late-bolting [11]. One 9-bp deletion and two insertions (8 and 9 bp) were identified in the intron and promoter of RsFLC3 gene [39]. In this study, the RsFLC genes were expressed in two cultivars, but no differentially expressed, illustrating Li et al. BMC Genomics (2023) 24:445 Li et al. BMC Genomics (2023) 24:445 Page 10 of 12 FT FLOWERING LOCUS T Funding h k This work was supported by National Natural Science Foundation of China (31960598) and Guizhou Provincial Science and Technology Projects ([2019] 2256 and [2021] general 213). Availability of data and materials Raw sequencing reads of RNA-seq in this paper have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA874186. Authors’ contributions Xiaobo Luo and Wanping Zhang designed the work, Yadong Li, Peng Xiao, Yueyue Jin, Huping Tan and Linjun Wu conducted experiments and prepared the data, Jingwei Li, Yun Pei and Xiuhong Xu helped with the experiment. Xiaobo Luo and Wanping Zhang interpreted the results and wrote the manu- script. All authors read and approved the final manuscript. Abbreviations Abbreviations MAS Marker-assisted selection RFLPs Restriction fragment length polymorphisms RAPDs Random amplified polymorphic DNA AFLPs Amplified fragment length polymorphisms SSRs Simple sequence repeats InDels Insertion/Deletions SNPs Single-nucleotide polymorphisms QTL Quantitative trait locus NGS Next-generation sequencing FLC FLOWERING LOCUS C FT FLOWERING LOCUS T FTR Flowering-time related gene DEGs Differential expressed genes PCR Polymerase chain reaction Na Number of alleles PIC Polymorphism information content PAGE Polyacrylamide electrophoresis MAF Major allele frequency PCA Principal Component analysis bp Base pairs HE Expected heterozygosity HO Expected homozygosty UBC1 UBIQUITIN CARRIER PROTEIN 1 FY FLOWERING LOCUS Y AGL6 AGAMOUS-LIKE 6 FVE FLOWERING LOCUS VE REF6 RELATIVE OF EARLY FLOWERING 6 EMF2 EMBRYONIC FLOWER 2 PRR3 PSEUDORESPONSE REGULATORS 3 CDF1 CYCLING DOF FACTOR 1 PHYA PHYTOCHROME A CPK6 Calcium-dependent protein kinases 6 CO CONSTANS VIN3 VERNALIZATION INSENSITIVE 3 ICE1 INDUCER OF CBF EXPRESSION 1 FRI FRIGIDA LFY LEAFY AP2 APETALA 2 PIF4 PHYTOCHROME INTERACTING FACTOR4 CCA1 CIRCADIAN CLOCK–ASSOCIATED1 SOC1 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 CDS Coding sequences LD Long-day Additioanl file 7: Supplement Table S10. The information of SNPs between two radish cultivars in transcriptome data. Additional file 8: Supplement Table S11. The information of InDels between two radish cultivars in transcriptome data. Additional file 9: Supplement Table S12. Flowering time-related (FTR) genes in radish identified using Arabidopsis FTR genes as queries by BLASTN analysis. Additional file 10: Supplement Table S13. Identification of differentially expressed FTR genes in between two radish cultivars. Additional file 11: Supplement Table S14. The information of SNPs and InDels detected in differentially expressed FTR genes. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12864-​023-​09528-6. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12864-​023-​09528-6. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12864-​023-​09528-6. Additional file 1: Fig S1.The length of genomic InDel markers between two radish cultivars.Fig S2.Principal component analysis of the all samples base on the FKPM vales of all transcripts.Fig. S3.The length of transcrip- tomic InDel markers between two radish cultivars.Fig S4. The full-length gels of the RsInDelR4-18 in 200 accessions. Table S1. Radish materials used in this study.Table S2. Primer information for qRT-PCR. Table S4. Summary of genome resequencing dada in two radish cultivars. Table S7. Summary of transcriptome dada in two radish cultivars. Additional file 1: Fig S1.The length of genomic InDel markers between two radish cultivars.Fig S2.Principal component analysis of the all samples base on the FKPM vales of all transcripts.Fig. S3.The length of transcrip- tomic InDel markers between two radish cultivars.Fig S4. The full-length gels of the RsInDelR4-18 in 200 accessions. Table S1. Radish materials used in this study.Table S2. Primer information for qRT-PCR. Table S4. Summary of genome resequencing dada in two radish cultivars. Table S7. Summary of transcriptome dada in two radish cultivars. Additional file 2: Supplement Table 3. The primers of InDels used in this study and genetic diversity analysis data. Additional file 3: Supplement Table S5. The information of SNPs between two radish cultivars in resequencing data. Additional file 4: Supplement Table S6. The information of InDels between two radish cultivars in resequencing data. Additional file 5: Supplement Table S8. The information of detected DEGs between two radish cultivars. Additional file 6: Supplement Table S9. The enriched GO terms of DEGs between two radish cultivars. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12864-​023-​09528-6. Abbreviations MAS Marker-assisted selection RFLPs Restriction fragment length polymorphisms RAPDs Random amplified polymorphic DNA AFLPs Amplified fragment length polymorphisms SSRs Simple sequence repeats InDels Insertion/Deletions SNPs Single-nucleotide polymorphisms QTL Quantitative trait locus NGS Next-generation sequencing FLC FLOWERING LOCUS C FT FLOWERING LOCUS T FTR Flowering-time related gene DEGs Differential expressed genes PCR Polymerase chain reaction Na Number of alleles PIC Polymorphism information content PAGE Polyacrylamide electrophoresis MAF Major allele frequency PCA Principal Component analysis bp Base pairs HE Expected heterozygosity HO Expected homozygosty UBC1 UBIQUITIN CARRIER PROTEIN 1 FY FLOWERING LOCUS Y AGL6 AGAMOUS-LIKE 6 FVE FLOWERING LOCUS VE REF6 RELATIVE OF EARLY FLOWERING 6 EMF2 EMBRYONIC FLOWER 2 PRR3 PSEUDORESPONSE REGULATORS 3 CDF1 CYCLING DOF FACTOR 1 PHYA PHYTOCHROME A CPK6 Calcium-dependent protein kinases 6 CO CONSTANS VIN3 VERNALIZATION INSENSITIVE 3 ICE1 INDUCER OF CBF EXPRESSION 1 FRI FRIGIDA LFY LEAFY AP2 APETALA 2 PIF4 PHYTOCHROME INTERACTING FACTOR4 CCA1 CIRCADIAN CLOCK–ASSOCIATED1 SOC1 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 CDS Coding sequences LD Long-day References InDel marker development and QTL analysis of agronomic traits in mung bean [Vigna radiate (L.) 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Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 51. Kawamoto N, Sasabe M, Endo M, Machida Y, Araki T. Calcium-dependent protein kinases responsible for the phosphorylation of a bZIP tran- scription factor FD crucial for the florigen complex formation. Sci Rep. 2015;5(1):1–9. https://​doi.​org/​10.​1038/​srep0​8341. 51. 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CicLAvia and human infrastructure in Los Angeles: ethnographic experiments in equitable bike planning
Journal of transport geography
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UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ UC Irvine UC Irvine Previously Published Works Title CicLAvia and Human Infrastructure in Los Angeles: Ethnographic Experiments in Equitable Bike Planning Permalink https://escholarship.org/uc/item/3fj825jv Journal Journal of Transport Geography, 30(June) Author Lugo, Adonia E. Publication Date 2013-06-01 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title CicLAvia and Human Infrastructure in Los Angeles: Ethnographic Experiments in Equitable Bike Planning Permalink https://escholarship.org/uc/item/3fj825jv Journal Journal of Transport Geography, 30(June) Author Lugo, Adonia E. Publication Date 2013-06-01 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title CicLAvia and Human Infrastructure in Los Angeles: Ethnographic Experiments in Equitable Bike Planning Permalink https://escholarship.org/uc/item/3fj825jv Journal Journal of Transport Geography, 30(June) Author Lugo, Adonia E. Publication Date 2013-06-01 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ P i d Powered by the California Digital Library University of California eScholarship.org eScholarship.org Adonia E. Lugo University of California, Irvine Abstract Across the United States, bike movements are advocating for infrastructural changes to streets. Sustainable transport advocates and researchers expect that reshaping built environments will increase bicycle usage because people will feel safer riding with more cycling facilities in place. These strategies identify road design as the key factor in how people use streets. From an ethnographic perspective, cycling research should also consider how road users create meanings in transit. This paper looks beyond physical changes to space and explores how “human infrastructure” encourages or discourages bicycling. Tacking between observation and participation, cultural anthropology can help design experimental spaces, such as Los Angeles’ CicLAvia, that offer diverse city inhabitants an opportunity to reflect on their transport habits in situ. Experimental spaces for bicycling show that human infrastructure shapes transportation behavior, and has the potential to change it. This paper contributes to a growing ethnographic literature in mobilities research. 1. Introduction 1. Introduction On Sunday, October 10, 2010, I woke up feeling anxious after a fitful sleep and headed to the East Los Angeles neighborhood of Boyle Heights. In Hollenbeck Park, where the roar of an overhead interstate highway never fades, I joined a large group of people wearing t-shirts emblazoned with the word “CicLAvia.” I stood next to Antonio Villaraigosa, the mayor of Los Angeles, Jaime Ortíz Mariño, the founder of the ciclovía open street event in Bogotá, Colombia, and my fellow organizers as we took turns speaking to the media about what this day meant for L.A. We would be closing 7.5 miles of central Los Angeles streets to cars to encourage city residents We would be closing 7.5 miles of central Los Angeles streets to cars to encourage city residents 1 1 to walk and bike along a route that spanned from historically Chicano Boyle Heights, through Little Tokyo, into the central business district, beyond downtown into Central American MacArthur Park, and through Koreatown to the East Hollywood “bicycle district” that had grown around a bike repair collective, the Bicycle Kitchen. The organizing committee hoped that selecting a route through Los Angeles' most densely populated and diverse neighborhoods would be a clear statement that transport cycling there was possible. During the day, despite the worst fears of reluctant bureaucrats, we saw nothing but smiles on the faces of people who may have never cycled before on the streets of Los Angeles. An estimated 30,000 people came out that day and enjoyed their city streets. More events have followed, and with each iteration, CicLAvia has grown. The event now attracts over 100,000 participants and has expanded to connect more neighborhoods. In U.S. bike planning, the mantra is “if you build it, they will come,” meaning that infrastructure projects are expected to produce more cyclists (Nelson and Allen, 1997; Dill and Carr, 2003). Can open street events like CicLAvia also promote cycling? The international conversation around ciclovías has had effects in other cities, with regular ciclovías happening in Mexico City, New York City, and other metropolises in the Americas. This paper uses qualitative, participatory research on the L.A. bike movement to argue that social networks and cultural practices should be seen as “human infrastructure” supporting the rise of bicycling as a mode of transport. 1. Introduction This concept brings advocates' and planners' concerns about street design into conversation with the social scientific project to challenge divides between human and nonhuman actors. L.A. still has a very small bicycle modal share, with estimates from the American Community Survey hovering around 1% since 2008 (Alliance for Walking & Biking, 2012), but there has been an increase in cycling in the central city. At the intersection of Seventh Street and Alvarado Street, which is along the core CicLAvia route, ridership increased 165% between 2009 and 2011 (Los Angeles County Bicycle Coalition, 2012). CicLAvia has increased the visibility of cycling in Los Angeles, but its success built on existing networks of cyclists. 2 Increasingly, city governments are transitioning away from policies that frame cycling solely as a recreational activity and are instead supporting transportation cycling infrastructure as a way to promote public health and ecological sustainability (Dill, 2009; Pucher et al. 2010). In working to increase the numbers of people choosing to bike, U.S. bike advocates and researchers tend to emphasize urban form, often lobbying for northern European infrastructure models (Pucher and Buehler, 2008). In addition to “if you build it, they will come,” bike researchers are starting to talk about using “social network effects” to promote bicycling (Goetzke and Rave, 2011). There is a growing recognition that individual behavior influences other individuals' behaviors. This paper takes an ethnographic focus on the social practices that enliven cityscapes, complementing the physical infrastructure approach while working toward a qualitative understanding of the shift toward transport cycling in Los Angeles. I first outline my methods, then construct a theoretical framework for conceptualizing infrastructure, human and otherwise, based on the case study of biking in LA. If human behaviors can be infrastructure to enable certain actions, we should also consider the unintended effects physical infrastructure projects can have in social space. This paper takes an ethnographic focus on the social practices that enliven cityscapes, 2. Methods: Experimental Ethnography The field of cycling research encompasses the social world of bicyclists and the spatial designs of bike infrastructure, and academic inquiry can bring social movements to bear on spatial disciplines such as geography, planning, and urban anthropology. The research reported here attempted to bring the unstructured time of cultural anthropology's primary method, ethnography, into the more time-sensitive world of bicycle advocacy. This meant living and working alongside the cultural group under study, with the aim of developing theoretical frameworks through engaging with the everyday life of a bike 3 3 movement in a city where power plays out in the street. From September 2008 to February 2011, I was a community member at an urban ecovillage in central Los Angeles and participated in projects that aimed to support bicycling in diverse neighborhoods there. One project, City of Lights/Ciudad de Luces, was designed to engage low-income, Latino cyclists with the local bike advocacy movement. The other was CicLAvia. As part of the “mobilities turn,” there is a growing interest in urban mobility among sociologists and geographers (Sheller and Urry, 2006; Urry, 2008; Cresswell, 2010). However, despite interest by geographers in ethnographic methods, anthropologists have remained somewhat outside of this discourse. Anthropologist Farha Ghannam has argued that long-term ethnographic research can supplement the macro-scale focus of many mobility studies by emphasizing the social distinctions visible in everyday engagement, "the unexpected, fleeting, multiple, and hard-to-pin-down experiences that characterize city life” (2011:792). These things, while hard to quantify, certainly influence transport choices. Ethnography is well-suited to contribute to this area of research because it “starts with the fact of mobility," as Cresswell has argued that mobilities studies should (2010:551). The city happens through individual bodies (Sennett, 1994), so both inanimate infrastructures and living practices should be taken into account when analyzing urban mobility. Anthropology starts from the materiality of everyday life rather than taking a model social structure for granted. The growing awareness that cultural life is formed in motion has taken anthropologists into new places, and even prompted a re- evaluation of what constitutes a field site; anthropologist George Marcus argued that, "empirically following the thread of cultural process itself impels the move toward multi-sited ethnography" 4 4 (1995:97). 2. Methods: Experimental Ethnography The center is run by Marcus, who has referred to ethnography as “serious play” (2008:4). This evokes a mode of knowledge production through experimental practice rather than a fixed engagement between researcher and informant. As recently posited by anthropologist Kim Fortun, ethnography “can be designed to bring forth a future anterior that is not calculable from what we now know, a future that surprises. Ethnography thus becomes creative, producing something that didn’t exist before” (2012:450). The experimental ethnographic space creates the conditions of possibility for new articulations and forms of life to emerge. The researcher is not outside of this process, but a catalyst for it. During my fieldwork, I tacked between research and community-based advocacy, developing a theoretical framework to conceptualize cycling at the same time that I helped promote the practice in Los Angeles. I attempted to create an urban laboratory space for witnessing shifts in cultural attitudes toward mobility in Los Angeles, most notably in the case of CicLAvia. Both planning advocacy projects and watching them unfold provided ample opportunity to observe closely how people used streets in Los Angeles. 2. Methods: Experimental Ethnography Since Appadurai's call to follow things (1986) and Latour's call to follow actors (1987), anthropologists have traced phenomena such as the development of policy (Harper, 1998), the reproduction of NGOs (Riles, 2000), and the trajectory of positron emission tomography (PET) scans from labs to courtrooms (Dumit, 2004). The question of what distinguishes “multi-sited” from “mobile” ethnography merits further study. Though Cresswell characterized multi-sited ethnography as a step toward mobile ethnography (Cresswell, 2012), anthropology's enduring interest in sited milieux is not something that will fall away; it is rooted in a belief that shared cultural considerations play a significant role in what objects, individuals, and groups can travel and that these considerations can be apprehended by participating in the group's practices. This is why accounting for the situated perspective of the researcher, even as a figure in motion, is a core concern of anthropology. The discipline's defining aim has long been "to grasp the native's point of view, his relation to life, to realize his vision of his world" (Malinowski, 1966[1922]:25), but postcolonial (Chakrabarty, 2008), poststructural (Foucault, 1977), and feminist (Strathern, 1991) critiques have argued that power figures into the practice of ethnography as both fieldwork and written knowledge product. The mobile researcher does not transcend site; she becomes one, connecting ideas, people, and places that would not otherwise interact. As more anthropologists engage with mobilities frameworks, this discussion can be expanded. Studying cycling in Los Angeles allowed me to experiment with this relationship between ethnographer and field. My methods included following bicyclists around the city and following ideas about bicycling from community meetings to city policy. As I developed this project, I was influenced by conversations about ethnographic experiments happening at the University of California, Irvine's 5 5 Center for Ethnography. The center is run by Marcus, who has referred to ethnography as “serious play” (2008:4). This evokes a mode of knowledge production through experimental practice rather than a fixed engagement between researcher and informant. As recently posited by anthropologist Kim Fortun, ethnography “can be designed to bring forth a future anterior that is not calculable from what we now know, a future that surprises. Ethnography thus becomes creative, producing something that didn’t exist before” (2012:450). The experimental ethnographic space creates the conditions of possibility for new articulations and forms of life to emerge. The researcher is not outside of this process, but a catalyst for it. Center for Ethnography. 3. Bike Activism in the Streets of Los Angeles Though cycling occurs in a “remarkable plurality of lifeworlds, histories, structures and cultures" (Horton et al., 2007:1), much work on the social life of cycling has focused on users who self- identify as cyclists and who organize themselves into subcultural groups such as bike messengers (Wehr, 2009; Kidder, 2011) or activists (Carlsson, 2002; Batterbury, 2003; Horton, 2006; Mapes, 2009). Los Angeles has these networks of subcultural and politically-oriented cyclists, but on its streets and sidewalks one also encounters many “invisible riders,” immigrants from Latin America who use 6 6 6 bicycles but do not connect with these subcultural groups (Koeppel, 2005). Even if a person using a bicycle does not develop an identity around the practice, cycling carries social meanings. Zack Furness has argued that, “the bicycle, like the automobile, is an object that becomes meaningful through its relationship to an entire field of cultural practices, discourses, and social forces” (2010:9). Whitaker commented that the bicycling of aging Italian men can be seen as “meaningful rather than just health promoting” because “their motivations often stray to the social, aesthetic, and psychological realms” (2005:2). Reporting on a study of the meanings of cycling in London, Steinbach et al. (2011) noted differences in how their interviewees defined cycling according to ethnicity, calling for further work on this topic. Writing about colonial India and Vietnam, Arnold and DeWald (2011) argued that bicycles manufactured in Europe took on local meanings and values tied to colonial power relations. Nancy Hunt (1994) explored the bicycle’s association with modernity and the urban in the colony of Belgian Congo. Though the meaning of cycling is socially constructed, in certain spaces it signifies marginalization regardless of what the cyclists themselves think they are doing. A recent report on cycling identities emphasized that riding in streets dominated by motorized transport constitutes a stigmatizing act in and of itself (Aldred, 2013). In the United States, cycling has long been the practice of eccentric enthusiasts, a status sport, or a mode of transport for those too poor or too young to drive cars. The demise of L.A.'s extensive streetcar system in the mid-twentieth century created a sharp divide between those who could afford to drive and those who could not (Hutchinson, 2000). 3. Bike Activism in the Streets of Los Angeles This paper takes an inclusive view of who should count as part of the bike movement because transportation remains highly political in Los Angeles, both in terms of what gets funding at the policy level and who gets priority in everyday traffic interactions. At a May 2010 event called “Walking into the Future City,” an official at the Los Angeles County Metropolitan Transportation Authority (Metro) characterized L.A.’s implicit transport hierarchy 7 7 7 this way: this way: car > bus > bike > walking The low status of cycling in Los Angeles stirs some cyclists to political action, but even those who do not have the ability or desire to attend hearings at city hall face the same marginalization on city streets. For this reason, “bike movement” here refers not just to particular identities and politically-oriented advocacy efforts, but also to the presence of cyclists in public space. The bike social world I joined when I started fieldwork in Los Angeles dates to 1997, when a few people interested in bike commuting started a local Critical Mass ride (Lugo 2012a). Prior to this, bike advocacy in the region had been characterized by what local activist Joe Linton called “lone wolves” working singly on pet projects (interview with author, 2011). With support from the California Bicycle Coalition, Joe and Ron Milam started the first bicycle advocacy organization in Los Angeles, the Los Angeles County Bicycle Coalition (LACBC), in 1998. Still in his early 20s at that time, Ron was the founding executive director. When I arrived in Los Angeles in September 2008, they both lived at the LA Eco-Village (LAEV), an intentional community in a central L.A. neighborhood called Koreatown which has been a longtime hub of the local bike movement. Koreatown is situated on central L.A.'s urban grid that extends between Hollywood to the west and downtown to the east, which means there are many quiet side streets paralleling more congested arterials. I quickly learned that many cyclists who lived at LAEV had personalized routes that took them across the city while avoiding busy thoroughfares. One ecovillager, Randy, knew more about bike routes than anyone else. He had been a bike messenger and a participant in LA's bike social world since its start in the 1990s. Randy showed me how to ride to the Hollywood Farmer's Market from LAEV. 3. Bike Activism in the Streets of Los Angeles His route traveled west on First Street from the family-filled studio apartments of Koreatown, through the large homes of Larchmont, cutting north at Arden and Wilcox. Randy did not follow city-designated routes such as streets with bike lanes; 8 8 instead, he had amassed over many years of riding a detailed knowledge of tricks and shortcuts that avoided major streets, zig-zagging through the neighborhoods in between. Stewart argued that the bicycle can “produce speed” because its small size allows bicyclists to move through gaps in traffic (2004:154). Bicyclists can also find gaps in the built environment, cracks that one might not notice from a car or a bus. Riding a bicycle allows the individual to build a bodily spacetime that is less constrained by street design. Randy expressed a lot of satisfaction with his cycling knowledge of the city, which was for him a statement of non-conformity. Bicyclists’ perceptions of where it is appropriate to ride are influenced not just by infrastructure, but also by participation in particular social networks. Unlike Randy and other self-identified cyclists I knew, the immigrant cyclists I encountered during my fieldwork usually rode on sidewalks on arterial streets. Analyzing data from the 2001 U.S. National Household Travel Survey, Smart found that immigrants are more likely to use bicycles than the native born population and suggested that this related to the tendency for new immigrants to move into neighborhoods inhabited by co-ethnics (Smart, 2010). The people we live around influence our ideas about using urban space, and experienced cyclists become sources of knowledge for friends and family interested in trying out a bike commute. Based on this understanding, Ron started a group called BikeSage in 2008 that brought together people who rode bikes regularly to strategize about ways to get their friends on bikes. Set aside for other endeavors, the project came to an end in spring 2009. Ron described it this way when I asked him about it in early 2011: [T]he thought was there are people who ride their bikes today in Los Angeles who love it, they're completely comfortable with it, they know great places to ride. And their experience with it is completely different than people who are afraid of bicycling and terrified to bike. Like those of us who ride, we know where to go, we know how to ride, and we love it. At the same time that I was observing cyclists at LAEV, I started learning about the bike 3. Bike Activism in the Streets of Los Angeles And we deal with the existing conditions as we are. So the idea was, how can you create a network...[to] take the knowledge and experience that folks who ride in the community have, and share that with others in their social networks and inspire them to ride and sort of buddy up with them. At the same time that I was observing cyclists at LAEV, I started learning about the bike 9 9 movement through collaborating on projects as an advocate. I had visited Bogotá in August 2008, when the ciclovía stretched over 120 kilometers of streets every Sunday. Through LAEV and LACBC, I helped form a group of people interested in making something like that happen in L.A. By late 2008, we were calling ourselves the CicLAvia steering committee. The group included individuals with specialized knowledge, such as longtime environmental advocates who knew the region's policy terrain, a traffic engineer, who made maps that showed details like population density along our proposed routes, and a graphic designer, who created a consistent theme for our promotional materials. Later, a prominent event planner joined the effort. In this work, I did not maintain a divide between scholarly and advocacy activities. Our goal was to see CicLAvia become a weekly, city-run program with permanent street signage, as it was in Bogotá. We saw it as an opportunity to show elected officials that we had a bike movement and diverse cycling cultures in LA, and considered possible routes on scouting rides and in long conversations at committee meetings. On scouting rides, we took notes about potential barriers to a Sunday street closure, such as churches with parking lots that opened onto the desired street, and potential allies, such as cultural organizations. We scanned each block and thought about ways an eco- friendly dry cleaner, or a Korean radio station, or an empty park space at the base of a large corporate plaza might be transformed by crowds of bicyclists. Through our eyes, heavily congested, car-dominated stretches of Los Angeles became a series of potential sites for people on bike or on foot to see and sit. Committee members lobbied for routes that included their personal favorite symbols of Los Angeles. Each of us wondered, what would be the most transformative route that would allow people to see the same L.A. from a new perspective? 10 4. Conceptualizing the Limits of Infrastructure While we planned CicLAvia, the Los Angeles Department of Transportation (DoT) was in the process of updating their “bicycle master plan,” a document that cities must develop in order to be eligible for bike infrastructure funds such as the State of California's Bicycle Transportation Account (Los Angeles Department of City Planning, 2011). Updating the plan meant holding public meetings where bicyclists were encouraged to share ideas for routes and other infrastructural changes around the city. LACBC staff were closely involved in this process, notifying members of upcoming meetings and pressuring the city to adopt better bike policies. In October 2009, I biked with friends to a library in South Los Angeles to attend one of the bike plan update meetings. The usual crowd of bike advocates milled around, talking to DoT planners. We were encouraged to draw on maps, review plan language, and submit comment cards. Despite this implicit recognition that existing cyclists had important knowledge to share, the stated goals of the update related to “interdepartmental communication” and designing “all bicycle facilities consistently in accordance with the latest federal, state and local standards.” There was no stated intention to draw on the knowledge of existing cyclists, even though the only members of the public participating in this process were committed cyclists. At meetings like this, bike planning draws on human infrastructure without explicitly invoking it as a source of valuable insight. Even though they relied on their local networks for route advice, bike advocates I knew also seemed to overlook the importance of embodied knowledge when promoting cycling. Expert knowledge had to come from outside them, based, if possible, on some “best practice” model 11 11 circulating among bicycle and pedestrian planning professionals online. For example, in 2009 and 2011 LACBC conducted bicyclist and pedestrian counts in central Los Angeles to quantify non-motorized transport users, a task the city had failed to undertake in recent years. Advocates wanted the city to take cycling seriously, so they framed their expertise as cyclists in as technical a format as possible. Los Angeles' bike advocates had come to promote bike infrastructure as a way to ensure certain relationships between street users and urban space. 4. Conceptualizing the Limits of Infrastructure However, urban space is not a neutral zone, but rather a series of meaningful landscapes where particular groups have lived through particular Los Angeles' bike advocates had come to promote bike infrastructure as a way to ensure certain relationships between street users and urban space. However, urban space is not a neutral zone, but rather a series of meaningful landscapes where particular groups have lived through particular struggles. Individual trajectories do not happen in a vacuum; they negotiate existing built forms and the ongoing movements of others. Redrawing lines on the street does not erase legacies of segregation and disinvestment, and bike infrastructure has now been associated with gentrification (Lugo, 2012b). At that community meeting in October 2009, few people made comments on the portion of the map representing the historically Black and more recently Latino neighborhood we were standing in, presumably because most of us lived in other parts of the city. Outside the window, I saw several people of color biking down the sidewalks of busy Western Avenue. City efforts to include the public in bike planning decisions often rely on voluntary participation from cyclists, but the cyclists who have the free time to pursue political participation probably are not those who bike out of economic necessity. Bike advocates are not necessarily equipped to engage with marginalized communities and bring their concerns to city planning processes. Davey Oil, an activist who has taught bike repair for many years, has commented that he sees the bike movement as an opportunity for cyclists to develop an awareness of race, class, and gender discrimination because feeling harassed by motorists while 12 cycling may be an otherwise privileged person's first experience of discrimination (personal communication with author, 2012). However, becoming a bike advocate does not automatically prepare one to confront other forms of bias, and advocates may overlook cycling practiced by marginalized groups such as immigrants and the poor because they do not imagine themselves to belong to a shared community with these others. These empowered cyclists may become advocates and lobby for cities to install bike infrastructure. Because traveling in cars creates a divide between what Jain has called “the private space of the car and the social space of the street” (2006:66), Americans and others who habitually drive may not see roads as a shared social space. 4. Conceptualizing the Limits of Infrastructure Wayfinding signage and symbols on streets ostensibly make it possible for any individual who encounters that space to understand how to share it. It is supposed to take the social encounter out of the equation; instead of interacting with other road users, individuals interact with signals and lane markings. Bike advocates may not notice that changing street designs affect property values and long-term residents' senses of place, or they may not see this as a negative outcome. When advocates talk about bike-friendly cities, they rarely mention social equity issues such as affordable housing, as evidenced by the League of American Bicyclists' recent “bicycling means business” summit themed around using bike infrastructure as an economic development strategy. As often as bike enthusiasts note our practice's marginalization on shared streets, we usually do not mention bringing together different types of cyclists as part of the work of advocacy or planning. We assume that normalizing cycling will eliminate the gap between poverty and elite cycling, but without conceptualizing where these groups meet. Bike users who do not self-identify as cyclists and 13 who do not have the ability or desire to attend city meetings about cycling can be difficult to include in research and advocacy, but their use of street space should also be seen as part of the bike movement. who do not have the ability or desire to attend city meetings about cycling can be difficult to include in research and advocacy, but their use of street space should also be seen as part of the bike movement. We talk about infrastructure as though it can generate a population of “normal” cyclists that will fill the gap. As Spinney has remarked, in these infrastructure-oriented approaches “movement seems to have been largely ignored as a social practice generative of meaning in itself" (2007:26). Open street events like CicLAvia re-emphasize the role that individual movements play in placemaking. Rather than focusing on changes to the built environment as the key to promoting cycling, I found that witnessing the interaction of bodies with other bodies, prostheses, and environments showed their permeability, what anthropologist Thomas Csordas called “the ambiguity in the boundaries of corporeality itself” (Csordas, 1994:3). Spinney argued that, “our perceptions of our environment are informed by the goals, skills and technologies available to us" (2007:29). 4. Conceptualizing the Limits of Infrastructure To a cyclist, a street affords cycling, while to a driver, that same street might seem appropriate only for driving. The lines between social life and infrastructure can shift, as in characterizations of infrastructure that see it as relational. To Star, “infrastructure is a fundamentally relational concept, becoming real infrastructure in relation to organized practices" (1999:380). In Star’s definition infrastructure is a social phenomenon. Dourish and Bell located infrastructure in the practice of everyday life, where “the embedding of a range of infrastructures into everyday space shapes our experience of that space and provides a framework through which our encounters with space take on meaning. The experiential reading of infrastructure, then, sees infrastructure and everyday life as coextensive" (2007:417). These readings provide an opening to consider infrastructure as something more than material. Urban theorist AbdouMaliq Simone, writing about Kinshasa, Congo, noted the social power of 14 infrastructure as a medium of conveyance and articulation. It establishes a concrete framework for how residents are able to reach each other, how they are able to think about how they are positioned and located in relationship to each other. Through roads, wires, conduits, grids, and pipes, infrastructure establishes particular forms of individuation and autonomy (2009:124). In this definition, infrastructure arranges individuals, limiting who can access what. In this definition, infrastructure arranges individuals, limiting who can access what. Even in a city with inconsistent public infrastructure systems, "infrastructural fragments… enable the creation of new social spaces" (De Boeck and Plissart, 2004:230). From an ethnographic perspective, what matters about infrastructure is its impact on social life. Referring to urban infrastructures as “life support systems” for cyborgs, Matthew Gandy described them as "modes of cognition as well as processes underpinning the restructuring of urban space" (Gandy, 2005:39). The key here is that infrastructure can create spaces from which new forms of social life can “spin off in wholly unexpected directions, generating intended and unintended outcomes” (Larkin, 2008:3). Infrastructure is certainly a material interface between the wider city with social life. But there are forms of infrastructure that can only be located in social life, as Simone found. Infrastructure is certainly a material interface between the wider city with social life. But there are forms of infrastructure that can only be located in social life, as Simone found. In a city like Kinshasa, people themselves are the important infrastructure. 4. Conceptualizing the Limits of Infrastructure In other words, their selves, situations, and bodies bear the responsibility for articulating different locations, resources, and stories into viable opportunities for everyday survival" (2009:124). In a city like Kinshasa, people themselves are the important infrastructure. In other words, their selves, situations, and bodies bear the responsibility for articulating different locations, resources, and stories into viable opportunities for everyday survival" (2009:124). People can be infrastructure; they create networks in which they hold places of meaning and value. Instead of reducing movement in the street to an individual engagement with physical transport infrastructure, the concept of human infrastructure emphasizes the role of social interaction in how people move. infrastructure, the concept of human infrastructure emphasizes the role of social interaction in how people move. A commonly cited study about “safety in numbers” suggested that the more cyclists and pedestrians there are using streets, the safer they are (Jacobsen, 2003). This led Jacobsen to question 15 "whose behavior changes, the motorist's or that of the people walking and bicycling?" (2003:208). He concluded that, "motorist behavior evidently largely controls the likelihood of collisions with people walking and bicycling" (2003:209). The more people there are outside of cars, the harder it is to maintain the illusion of automobility that tells drivers they are not a part of the spaces through which they travel. People are part of the infrastructure enabling or disabling certain mobilities. What matters about this distinction between physical and social barriers to mobility is that changing social attitudes is a different project than changing built environments. In many cases, alternative social attitudes already exist, using the urban landscape in its current form, as Ron noted. Even in a car-dominated city like Los Angeles, people were already riding bikes to commute, run errands, and have fun. They co-created human infrastructure, building possibilities where physical infrastructure was lacking, and CicLAvia meant to share the mobile realities of these bike worlds with a wider group. After many months of planning and strategy, CicLAvia proposed the route described earlier to the city, and found support in Mayor Villaraigosa's office in 2010. The network activated by committee members enabled the experiment on 10-10-10, and that network grew in part from the cycling L.A. living in the practices of people like Randy. Human infrastructure works positively and negatively for cycling. That is, human infrastructure in the form of group rides, social networks of activists, and the presence of bike commuters during rush hour encourages cycling. Human infrastructure in the form of honking, yelling, and other aggressive motorist behaviors discourage cycling. When bicyclists give each other directions based not on municipal cycling maps but on their own knowledge of city streets, they are using human infrastructure. When most people do not know how they would get from point A to point B without 16 driving, and they do not know who to ask about it, they suffer from a lack of human infrastructure. Other forms of human infrastructure includes idea and events that bring people together around a particular practice, such as Bogotá's ciclovía. People can be infrastructure; they create networks in which they hold places of meaning and value. Instead of reducing movement in the street to an individual engagement with physical transport infrastructure, the concept of human infrastructure emphasizes the role of social interaction in how people move. In other words, a simple exchange of specialized knowledge or the enactment of an expectation can constitute human infrastructure. The idea that everyone, even the most stalwart New York subway rider, must rent a car upon arriving at LA International Airport is a powerful piece of infrastructure discouraging sustainable transport use in LA. The effects of CicLAvia can be seen in a New York Times article on the event, which commented that, “for years, bicyclists in Los Angeles were just another renegade subculture in a city that is teeming with all manner of subcultures. These days, they have become downright mainstream” (Nagourney, 2012). It is telling that when the city adopted the updated bike master plan in 2011, the final document included photos of CicLAvia (Los Angeles Department of City Planning, 2011). Will cosmopolitan visitors “in the know” now rent bicycles in LA? For the CicLAvia on April 21, 2013, every rental bike at local retailer Downtown LA Bicycles had been reserved days in advance. 5. Conclusion A qualitative analysis of urban transport cycling should acknowledge that the practice does not necessarily carry a stable meaning shared by all street users. Furthermore, the body and the environment do not remain discrete; on a city street, the distinction between material and immaterial infrastructures blurs as bodies become barriers. To illustrate this, I encourage the reader to visit Youtube.com and search for “ciclavia,” which will call up many videos made by participants in the events. One can witness how each user negotiates the movements of others. Bodies themselves suggest 17 ways that other bodies should move. The range of people seen on the streets during CicLAvia also challenges the idea that bicyclists are a homogenous group. The event seems to be fostering the creation of new social networks for cycling and showcasing existing sustainable transport in a city known for its love affair with the automobile. However, shifting toward cycling as transport in the U.S. is a piecemeal process, and if bike movements do not connect with other community-based networks, the infrastructure projects they promote may be perceived as serving a privileged few. As a result, cycling can come across as an act associated with gentrification, as has been reported in Brooklyn (DeSena and Shortell, 2012). There is a crucial step missing when we assume that everyone sees cycling and its infrastructure the same way. Working closely with advocates gave me considerable insight into what they believed was necessary to make change, but being a collaborator also meant that I was influencing the field under study while documenting it. While this diverges from a more traditional model that hinges on a conceptual divide between researcher and subject, the critical move I make here is acknowledging my own participation as human infrastructure. As a researcher I was not separate from the everyday intersubjectivity of shared urban space where people creatively integrate cycling into their lives. Open street events like CicLAvia invite residents to come out into the street and reimagine life there through their own bodily practice. They also provide researchers with urban laboratories in which to experiment. Acknowledgements g This paper is based on dissertation research funded by the University of California, Irvine's Community Outreach Partnership Center (2008-2009) and Eugene Cota-Robles Fellowship (2010-2011). Many thanks to my collaborators in Los Angeles for their ideas and friendship. The ideas in this paper took shape through conversations with participants at the 2012 Cycling and Society Symposium and participants at the 2012 World Cycling Research Forum. The paper also benefited from comments made by editor Rachel Aldred, Peter Wood, Ben O'Donnell, and several anonymous reviewers. 18 Csordas, T. J. (Ed), 1994. 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Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras
Journal of Generalized Lie Theory and Applications
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Introduction We aim in this paper to explore and study non-commutative ternary Nambu-Poisson algebras and their Hom-type version. The paper includes five Sections. In the first one, we summarize basic definitions of (non-commutative) ternary Nambu- Poisson algebras and discuss examples. In the second Section, we recall some basics about Hom- algebra structures and introduce the notion of (non-commutative) ternary Hom-Nambu-Poisson algebra. Section 3 is dedicated to construction of (non-commutative) ternary Hom-Nambu-Poisson algebras using direct sums and tensor products. In Section 4, we extend twisting principle to ternary Hom-Nambu- Poisson algebras. It is used to build new structures with a given ternary (Hom-) Nambu-Poisson algebra and algebra morphism. This process is used to construct ternary Hom-Nambu-Poisson algebras corresponding to the ternary algebra of polynomials where the bracket is defined by the Jacobian. We provide in the last section a classification of 3-dimensional ternary Nambu- Poisson algebras and then compute corresponding Hom-Nambu- Poisson algebras using twisting principle. Notice that a complete classification of 3-dimensional Hom-Nambu-Poisson algebras is difficult to obtain since so far the classification of 3-dimensional Hom- Nambu-Lie algebras is not known. In the 70’s, Nambu proposed a generalized Hamiltonian system based on a ternary product, the Nambu-Poisson bracket, which allows to use more than one hamiltonian [1]. More recent motivation for ternary brackets appeared in string theory and M-branes, ternary Lie type structure was closely linked to the super-symmetry and gauge symmetry transformations of the world-volume theory of multiple coincident M2-branes and was applied to the study of Bagger-Lambert theory. Moreover ternary operations appeared in the study of some quarks models. In 1996, quantizations of Nambu-Poisson brackets were investigated [2], it was presented in a novel approach of Zariski, and this quantization is based on the factorization on of polynomials of several variables. The algebraic formulation of Nambu mechanics was discussed [3] and Nambu algebras was studied [4] as a natural generalization of a Lie algebra for higher- order algebraic operations. By definition, Nambu algebra of order n over a field  of characteristic zero consists of a vector space V over together with a -multilinear skew-symmetric operation [., ,.]: Λ →  nV V , called the Nambu bracket that satisfies the following generalization of the Jacobi identity. Abstract The main purpose of this paper is to study non-commutative ternary Nambu-Poisson algebras and their Hom- type version. We provide construction results dealing with tensor product and direct sums of two (non- commutative) ternary (Hom-) Nambu-Poisson algebras. Moreover, we explore twisting principle of (non-commutative) ternary Nambu-Poisson algebras along with algebra morphism that lead to construct (non-commutative) ternary Hom- Nambu-Poisson algebras. Furthermore, we provide examples and a 3-dimensional classification of non-commutative ternary Nambu-Poisson algebras. were introduced [7]. Non-commutative Hom-Poisson algebras were discussed [8]. Likewise, n-ary algebras of Hom-type were introduced [5,9-13]. Keywords: Hom-nambu poisson algebra; Ternary nambu poisson; Non-commutative ternary; n-ary Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom- Nambu-Poisson Algebras Makhlouf A1* and Amri A2 Makhlouf A1* and Amri A2 1University of Haute Alsace, Laboratory of Mathematics, IT and Applications, 4 Rue des Freres Lumiere, France 2Badji Mokhtar University Annaba, BP 12 Department of Maths, Science Faculty, Algeria 1University of Haute Alsace, Laboratory of Mathematics, IT and Applications, 4 Rue des Freres Lumiere, France 2Badji Mokhtar University Annaba, BP 12 Department of Maths, Science Faculty, Algeria Introduction Namely, for any 1 1 ,..., n x x V −∈ define an adjoint action ad(x1, ..., xn−1 ): V → V by ad(x1 , ..., xn−1 )xn=[x1 , ..., xn−1 , xn ], xn ∈ V . Then the fundamental identity is a condition saying that the ad joint action is a derivation with respect the Nambu bracket, i.e. for all x1, ..., xn−1 , y1 , ..., yn ∈ V J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Makhlouf and Amri, J Generalized Lie Theory Appl 2015, 9:1 http://dx.doi.org/10.4172/1736-4337.1000221 Makhlouf and Amri, J Generalized Lie Theory Appl 2015, 9:1 http://dx.doi.org/10.4172/1736-4337.1000221 Makhlouf and Amri, J Generalized Lie Theory Appl 2015, 9:1 http://dx.doi.org/10.4172/1736-4337.1000221 Journal of Generalized Lie Theory and Applications Ternary (Non-Commutative) Nambu-Poisson Algebra Nambu-Poisson algebra (A, µ, {., ., .}) for which µ is commutative, then µ is commutative unless otherwise stated. A ternary algebra is given by a pair (A, m), where m is a ternary operation on A, that is a trilinear map m : A × A × A → A, which is denoted sometimes by brackets. In a (non-commutative) ternary Nambu-Poisson algebra, the ternary bracket {., ., .} is called Nambu-Poisson bracket. Definition 1.1. A ternary Nambu algebra is a ternary algebra (A, { , , }) satisfying the fundamental identity defined as Similarly, a non-commutative n-ary Nambu-Poisson algebra is a triple (A, µ, {., • • • , .}) {x1 , x2 , {x3 , x4 , x5 }}= {x1 , x2 , {x3 , x4 , x5 }}= {x1 , x2 , {x3 , x4 , x5 }}= {{x1 , x2 , x3 }, x4 , x5 } + {x3 , {x1 , x2 , x4 }, x5 } + {x3 , x4 , {x1 , x2 , x5 }} (1.1) or all x1 , x2 , x3 , x4 , x5 ∈ A. where (A, {., • • • , .}) defines an n-Lie algebra satisfying similar Leibniz rule with respect to µ. where (A, {., • • • , .}) defines an n-Lie algebra satisfying similar Leibniz rule with respect to µ. {{x1 , x2 , x3 }, x4 , x5 } + {x3 , {x1 , x2 , x4 }, x5 } + {x3 , x4 , {x1 , x2 , x5 }} (1.1) or all x1 , x2 , x3 , x4 , x5 ∈ A. A morphism of (non-commutative) ternary Nambu-Poisson algebras is a linear map that is a morphism of the underlying ternary Nambu-Lie algebras and associative algebras. This identity is sometimes called Filippov identity or Nambu identity, and it is equivalent to the identity (0.1) with n=3. A ternary Nambu-Lie algebra or 3-Lie algebra is a ternary Nambu algebra for which the bracket is skew-symmetric, that is for all σ ∈ S3 , where S3 is the permutation group, Example 1.5. Let 3 ( ) ∞ C be the algebra of ∞ C functions on 3  and x1 , x2 , x3 the coordinates on 3  . Ternary (Non-Commutative) Nambu-Poisson Algebra We define the ternary brackets as in (1.2), then ( ∞ C ( 3 ), {., ., .}) is a ternary Nambu-Lie algebra. In addition the bracket satisfies the Leibniz rule: f g , 2 3 2 3 2 3 { , , } { , , } { , , } = + fg f f f g f f f f f g where f, g, f2 , f3 ∈ 3 ( ) ∞ C and the multiplication being the point wise multiplication that is f g ( x )=f ( x ) g ( x ). Therefore, the algebra is a ternary Nambu-Poisson algebra. ( )[ ] 1 2 3 1 2 3 ( ) ( ) ( ) , , , , . σ σ σ σ    =  x x x Sgn x x x Let A and A′ be two ternary Nambu algebras (resp. Nambu-Lie algebras). A linear map f : A → ′ A is a morphism of a ternary Nambu algebras (resp. ternary Nambu-Lie algebras) if it satisfies f ({x, y, z}A )={f (x), f (y), f (z)} ′ A . This algebra was considered already in 1973 by Nambu [9] as a possibility of extending the Poisson bracket of standard hamiltonian mechanics to bracket of three functions defined by the Jacobian. Clearly, the Nambu bracket may be generalized further to a Nambu-Poisson allowing for an arbitrary number of entries. Example 1.2. is a ternary Nambu-Lie algebra. is a ternary Nambu-Lie algebra. Example 1.3. Let V= 4  be the 4-dimensional oriented Euclidian space over . The bracket of 3 vectors , ,  x y z is given by Hom-type (Non-Commutative) Ternary Nambu- Poisson Algebras Hom-type (Non-Commutative) Ternary Nambu- Poisson Algebras 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 [ , , ] , = × × =    x y z e x y z e x y z x y z x y z e x y z e In this section, we present various Hom-algebra structures. The main feature of Hom-algebra structures is that usual identities are deformed by an endomorphism and when the structure map is the identity, we recover the usual algebra structure. Where {e1,e2,e3,e4} is a basis of 4 and 3 1 = ∑   i i x x e i , 3 1 = =∑   i i y y e i and A Hom-algebra (resp. ternary Hom-algebra) is a triple (A, ν, α) consisting of a -vector space A, a bilinear map ν : A × A → A (resp. a trilinear map ν : A × A × A → A) and a linear map α : A → A. A Hom- algebra (A, µ, α) is said to be multiplicative if α o µ=µ o α⊗2 and it is called commutative if µ=µop. A ternary Hom-algebra (A, m, α) is said to be multiplicative if α o m=m o α⊗3. Classical algebras (resp. ternary algebras) are regarded as Hom-algebras (resp. ternary Hom-algebras) with identity twisting map. We will often use the abbreviation xy for µ(x, y) when there is no ambiguity. For a linear map α : A → A, denote by αn the n-fold composition of n-copies of α, with α0 ≡ I d. 1 = i i 3 1 = =∑   i i z z e i . Then (V, [.,.,.]) is a ternary Nambu – lie algebra. 3 1 = =∑   i i z z e i . Then (V, [.,.,.]) is a ternary Nambu – lie algebra. Now, we introduce the notion of (non-commutative) ternary Nambu-Poisson algebra. Definition 1.4. A non-commutative ternary Nambu-Poisson algebra is a triple (A, µ, {., ., .}) consisting of a -vector space A, a bilinear map µ : A × A → A and a trilinear map {., ., .} : A ⊗ A ⊗ A → A such that Definition 2.1. A Hom-algebra (A, µ, α) is a Hom-associative algebra if it satisfies the Hom-associativity condition, that is Definition 2.1. Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Ternary (Non-Commutative) Nambu-Poisson Algebra The polynomials of variables x1, x2, x3 with the ternary operation defined by the Jacobian function: 1 1 1 1 2 3 2 2 2 1 2 3 1 2 3 3 3 3 1 2 3 { , , } , ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ f f f x x x f f f f f f x x x f f f x x x (1.2) 1 1 1 1 2 3 2 2 2 1 2 3 1 2 3 3 3 3 1 2 3 { , , } , ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ f f f x x x f f f f f f x x x f f f x x x (1.2) In particular, the algebra of polynomials of variables x1 , x2 , x3 with the ternary operation defined by the Jacobian function in (1.2), is a ternary Nambu-Poisson algebra. (1.2) Remark 1.6. The n-dimensional ternary Nambu-Lie algebra of Example 1.3 does not carry a non-commutative Nambu-Poison algebra structure except that one given by a trivial multiplication. Ternary (Non-Commutative) Nambu-Poisson Algebra In the section we review some basic definitions and fix notations. In the sequel, A denotes a vector space over , where  is an algebraically closed field of characteristic zero. Let µ : A × A → A be a bilinear map, we denote by µop: A×2 → A the opposite map, i.e., µop=µ o τ where τ: A×2 → A×2 interchanges the two variables. 1 1 1 ,..., ,..., 1 )[ 1 ] 1 1 ( ,..., ,..., ,..., [ ( ]. ) − − = = ∑ n n n ad x x y y n k n k y ad x x y y (0.1) When n=2, the fundamental identity becomes the Jacobi identity and we get a definition of a Lie algebra. When n=2, the fundamental identity becomes the Jacobi identity and we get a definition of a Lie algebra. *Corresponding author: Makhlouf A, University of Haute Alsace, Laboratory of Mathematics, IT and Applications, 4 Rue des Freres Lumiere, 68093 Mulhouse Cedex, France, Tel: +33 3 89 33 65 00; E-mail: abdenacer.makhlouf@uha.fr Different aspects of Nambu mechanics, including quantization, deformation and various algebraic constructions for Nambu algebras have recently been studied. Moreover a twisted generalization, called Hom-Nambu algebras, was introduced [5]. This kind of algebras called Hom-algebras appeared as deformation of algebras of vector fields using σ-derivations. The first examples concerned q-deformations of Witt and Virasoro algebras. Then Hartwig, Larsson and Silvestrov introduced a general framework and studied Hom-Lie algebras [6], in which Jacobi identity is twisted by a homomorphism. The corresponding associative algebras, called Hom-associative algebras Received October 17, 2014; Accepted June 30, 2015; Published July 07, 2015 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Copyright: © 2015 Makhlouf A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Volume 9 • Issue 1 • 1000221 Page 2 of 8 Page 2 of 8 Nambu-Poisson algebra (A, µ, {., ., .}) for which µ is commutative, then µ is commutative unless otherwise stated. Now we prove the Hom-Nambu identity We recover the classical (non-commutative) ternary Nambu- Poisson algebra when α1=α2=β=I d. Tensor Product and Direct Sums 1 2 1 2 1 2 1 2 1 2 1 1 2 2 3 3 1 1 2 2 1 2 1 3 2 3 1 1 1 2 1 3 2 2 1 2 2 3 1 1 1 1 2 3 2 2 1 2 2 3 1 1 2 1 ( ( , ), ( )) ( ( , ) ( , ), ( ) ( )) ( ( , ), ( )) ( ( , ), ( )) ( ( ), ( , )) ( ( ), ( , )) ( ( ) ( µ µ α µ µ µ α α µ µ α µ µ α µ α µ µ α µ µ α α ⊕ ⊕ ⊕ ⊕ ⊕ ′ ′ ′ + + + ′ ′ ′ = + + ′ ′ ′ = + ′ ′ ′ = + ′ = + A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x 1 2 1 2 1 2 1 2 3 2 2 3 1 1 2 2 3 3 ), ( , ) ( , )) ( ( , ), ( , )) µ µ µ α µ ⊕ ⊕ ⊕ ′ ′ + ′ ′ ′ = + + A A A A A A x x x x x x x x x x 1 2 1 2 1 2 1 2 1 2 1 1 2 2 3 3 1 1 2 2 1 2 1 3 2 3 1 1 1 2 1 3 2 2 1 2 2 3 1 1 1 1 2 3 2 2 1 2 2 3 1 1 2 1 ( ( , ), ( )) ( ( , ) ( , ), ( ) ( )) ( ( , ), ( )) ( ( , ), ( )) ( ( ), ( , )) ( ( ), ( , )) ( ( ) ( µ µ α µ µ µ α α µ µ α µ µ α µ α µ µ α µ µ α α ⊕ ⊕ ⊕ ⊕ ⊕ ′ ′ ′ + + + ′ ′ ′ = + + ′ ′ ′ = + ′ ′ ′ = + ′ = + A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x 1 2 1 2 1 2 1 2 3 2 2 3 1 1 2 2 3 3 ), ( , ) ( , )) ( ( , ), ( , )) µ µ µ α µ ⊕ ⊕ ⊕ ′ ′ + ′ ′ ′ = + + A A A A A A x x x x x x x x x x The third condition is called Hom-Leibniz identity. Tensor Product and Direct Sums α α 1 1 n-1 n-1 n 2n-1 { (x ), ..., (x ), {x , ..., x }} 2 1 1 1 1 1 1 1 1 2 1 ,..., , ,..., { ( ) ( ) { } , ( , ... ) ( )} , α α α α − − − − −+ + − − = = = ∑ n n i n i n i i n i n n i n x x x x x x x α α 1 1 n-1 n-1 n 2n-1 { (x ), ..., (x ), {x , ..., x }} In this section we discuss direct sums and define tensor product of ternary (non-commutative) Hom-Nambu-Poisson algebra and a totally Hom-associative sym-metric ternary algebra. In the following, we define a direct sum of two ternary (non-commutative) Hom- Nambu-Poisson algebras. 2 1 1 1 1 1 1 1 1 2 1 ,..., , ,..., { ( ) ( ) { } , ( , ... ) ( )} , α α α α − − − − −+ + − − = = = ∑ n n i n i n i i n i n n i n x x x x x x x for all (x1 , ..., x2n−1 ) ∈ A 2n−1. for all (x1 , ..., x2n−1 ) ∈ A 2n−1. Remark 2.4. A Hom-Nambu algebra is a Hom-Nambu-Lie algebra if the bracket is skew-symmetric. Theorem 3.1. Let (A1 , µ1, {., ., .}1, α1 ) and (A2 , µ2 , {., ., .}2 , α2 ) be two ternary (non-commutative) Hom-Nambu-Poisson algebras. Let µA1 ⊕A2 be a bilinear map on A1 ⊕ A2 defined for x1 , y1 , z1 ∈ A1 and x2 , y2 , z2 ∈ A1 by µ(x1 + x2 , y1 + y2 )=µ1 (x1 , y1 ) + µ2 (x2 , y2 ), {., ., .}A1⊕A2 a trilinear map defined by 1 2 1 2 1 2 1 2 1 1 1 1 2 2 2 2 { , , } { , , } { , , } ⊕ + + + = + A A x x y y z z x y z x y z and 1 2 α ⊕ A A a linear map defined by 1 2 1 2 1 1 2 2 ( ) ( ) ( ). Now we prove the Hom-Nambu identity 1 2 1 2 1 2 1 2 1 2 1 1 2 2 3 3 4 4 5 5 1 1 2 1 1 2 2 2 3 4 5 1 3 4 5 2 1 1 1 2 3 4 5 1 1 2 1 2 2 3 4 5 2 2 1 2 { ( ), ( ),{ , , } } { ( ) ( ), ( ) ( ),{ , , } { , , } } { ( ), ( ),{ , , } } { ( ), ( ),{ , , } } {{ , , α α α α α α α α α α ⊕ ⊕ ⊕ ⊕ ⊕ ′ ′ ′ ′ ′ + + + + + ′ ′ ′ ′ ′ = + + + ′ ′ ′ ′ ′ = + = A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x 3 1 1 4 1 5 1 1 3 1 2 4 1 1 5 1 1 3 1 4 1 2 5 1 1 1 2 3 2 2 4 2 5 2 2 3 1 2 4 2 2 5 2 2 3 2 4 1 2 5 2 2 1 2 3 1 1 } , ( ), ( )} { ( ),{ , , } , ( )} { ( ), ( ),{ , , } } {{ , , } , ( ), ( )} { ( ),{ , , } , ( )} { ( ), ( ),{ , , } } {{ , , } { , α α α α α α α α α α α α + ′ ′ ′ ′ ′ + + ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ + + ′ ′ = + x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x 1 2 1 2 1 2 1 2 3 2 1 4 2 4 1 5 2 5 1 3 2 3 1 2 4 1 1 2 4 2 1 5 2 5 1 3 2 3 1 3 2 3 1 2 5 1 1 2 5 2 1 1 2 2 3 3 , } , ( ) ( ), ( ) ( )} { ( ) ( ),{ , , } { , , } , ( ) ( )} { ( ) ( ), ( ) ( ),{ , , } { , , } } {{ , , } α α α α α α α α α α α α ⊕ ⊕ ⊕ ⊕ ′ ′ ′ + + ′ ′ ′ ′ ′ + + + + ′ ′ ′ ′ ′ + + + + ′ ′ ′ = + + + A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x 2 1 2 1 2 2 1 2 1 2 1 2 2 1 2 1 2 1 2 1 2 4 4 5 5 1 3 3 1 1 2 2 4 4 5 5 1 3 3 4 4 1 1 2 2 5 5 , ( ), ( )} { ( ),{ , , } , ( )} { ( ), ( ),{ , , } } α α α α α α ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ′ ′ + + ′ ′ ′ ′ ′ + + + + + + ′ ′ ′ ′ ′ + + + + + + A A A A A A A A A A A A A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x Similarly, a non-commutative n-ary Hom-Nambu-Poisson algebra is a tuple (A, µ, β,{., … ,.}, α) where (A,{., … ,.}, α) with linear maps α= (α1, … , αn-1) that defines an n-ary Hom-Nambu-Lie algebra satisfying similar Leibniz rule with respect to (A, µ, β). We call the above condition the ternary Hom-Nambu identity. (2.4) o o . α α′ = f f Generally, the n-ary Hom-Nambu algebras are defined by an n-ary bracket and maps α1 , • • • , αn−1 , satisfying the following Hom-Nambu identity It said to be a weak morphism if hold only the two first conditions. It said to be a weak morphism if hold only the two first conditions. Tensor Product and Direct Sums When, all the linear maps are equal α=α1=α2=β, we refer to the ternary Hom-Nambu-Poisson algebra by a quadruple (A, µ, {., ., .}, α). Hom-Nambu-Poisson algebra by a quadruple (A, µ, {., ., .}, α). Remark 2.6. Notice that µ is not assumed to be commutative. When µ is a commutative multiplication, then (A, µ, β, {., ., .}, α) is said to be a ternary Hom-Nambu-Poisson algebra. J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Hom-type (Non-Commutative) Ternary Nambu- Poisson Algebras α α α α α α α α = + + x x x x x x x x x x x x x x x x x x x x (2.1) 1 1 2 2 3 4 5 1 2 3 1 4 2 5 1 3 1 2 4 2 5 1 3 2 4 1 2 5 { ( ), ( ),{ , , }} {{ , , }, ( ), ( )} { ( ),{ , , }, ( )} { ( ), ( ),{ , , }}. α α α α α α α α = + + x x x x x x x x x x x x x x x x x x x x (2.1) 1 2 3 1 2 3 ({ , , }) { ( ), ( ), ( )} ,′ = f x x x f x f x f x 1 2 3 1 2 3 ({ , , }) { ( ), ( ), ( )} ,′ = f x x x f x f x f x (2.2) 2 o o µ µ ⊗ ′ = f f (2.3) o o . α α′ = f f (2.4)i (2.3) We call the above condition the ternary Hom-Nambu identity. Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Tensor Product and Direct Sums α α α ⊕ + = + A A x x x x We introduce now the definition of non-commutative ternary Hom-Nambu-Poisson algebra in its general form, involving three linear maps. Next, we will discuss the class in which these three maps are equal. This particular case suits to provide a twisting construction. Definition 2.5. A non-commutative ternary Hom-Nambu-Poisson algebra is a tuple (A, µ, β, {., ., .},α) consisting of a vector space A, a ternary operation {., ., .} : A×A×A → A, a binary operation µ : A×A → A, a pair of linear maps α= (α1 , α2 ) Then 1 2 1 2 1 2 1 2 ( , ,{.,.,.} , ) µ α ⊕ ⊕ ⊕ ⊕ A A A A A A A A is a ternary (non- commutative) Hom-Nambu-Poisson algebra. Then 1 2 1 2 1 2 1 2 ( , ,{.,.,.} , ) µ α ⊕ ⊕ ⊕ ⊕ A A A A A A A A is a ternary (non- commutative) Hom-Nambu-Poisson algebra. Proof. The commutativity of 1 2 µ ⊕ A A is obvious since µ1 and µ2 are commutative. The skew-symmetry of the bracket follows from the skew-symmetry of {., ., .}1 and {., ., .}2. So it remains to check the Hom- associativity, the Hom-Nambu and the Hom-Leibniz identities. For Hom-associativity identity, we have where α1 , α2 : A → A, and a linear map β : A → A such that: where α1 , α2 : A → A, and a linear map β : A → A such that: (1) (A, µ, β) is a binary Hom-associative algebra, (A, µ, β) is a binary Hom-associative algebra, (1) (A,{., ., .}, α) is a ternary Hom-Nambu-Lie algebra, (A,{., ., .}, α) is a ternary Hom-Nambu-Lie algebra, (A,{., ., .}, α) is a ternary Hom-Nambu-Lie algebra, {µ(x1 , x2 ), α1 (x3 ), α2 (x4 )}=µ(β(x1 ), {x2 , x3 , x4 }) + µ({x1 , x3 , x4 }, β(x2 )). {µ(x1 , x2 ), α1 (x3 ), α2 (x4 )}=µ(β(x1 ), {x2 , x3 , x4 }) + µ({x1 , x3 , x4 }, β(x2 )). Hom-type (Non-Commutative) Ternary Nambu- Poisson Algebras A Hom-algebra (A, µ, α) is a Hom-associative algebra if it satisfies the Hom-associativity condition, that is (1) (A, µ) is a binary associative algebra, (1) (A, µ) is a binary associative algebra, (2) (A, {., ., .}) is a ternary Nambu-Lie algebra, µ(α(x), µ(y, z))=µ(µ(x, y), α(z)) for all x, y, z ∈ A. µ(α(x), µ(y, z))=µ(µ(x, y), α(z)) for all x, y, z ∈ A. (3) the following Leibniz rule (3) the following Leibniz rule Remark 2.2. When α is the identity map, we recover the classical associativity condition, then usual associative algebras. {x1 , x2 , µ(x3 , x4 )}=µ(x3 , {x1 , x2 , x4 }) + µ({x1 , x2 , x3 }, x4 ) holds for all x1 , x2 , x3 ∈ A. Definition 2.3. A ternary Hom-Nambu algebra is a triple { } , ., . . , ) ( , α A consisting of a -vector space A, a ternary map {., ., A ternary Nambu-Poisson algebra is a non-commutative ternary A ternary Nambu-Poisson algebra is a non-commutative ternary J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Volume 9 • Issue 1 • 1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Page 3 of 8 Page 3 of 8 .} : A × A × A → A and a pair of ( ) 1 2 , α α α =  where α1 , α2 : A → A, satisfying [5] (non-commutative) ternary Hom-Nambu-Poisson algebras. A linear map : ′ → f A A is a morphism of (non-commutative) ternary Hom- Nambu-Poisson algebras if it satisfies for all x1 , x2 , x3 ∈ A: .} : A × A × A → A and a pair of ( ) 1 2 , α α α =  where α1 , α2 : A → A, satisfying [5] 1 1 2 2 3 4 5 1 2 3 1 4 2 5 1 3 1 2 4 2 5 1 3 2 4 1 2 5 { ( ), ( ),{ , , }} {{ , , }, ( ), ( )} { ( ),{ , , }, ( )} { ( ), ( ),{ , , }}. Now we prove the Hom-Nambu identity In the sequel we will mainly interested in the class of non- commutative ternary Nambu-Poisson algebras where α=α1=α2=β. Definition 2.7. Let (A, µ, {., ., .}, α) be a (non-commutative) ternary Hom-Nambu-Poisson algebra. It is said to be multiplicative if Definition 2.7. Let (A, µ, {., ., .}, α) be a (non-commutative) ternary Hom-Nambu-Poisson algebra. It is said to be multiplicative if Definition 2.7. Let (A, µ, {., ., .}, α) be a (non-commutative) ternary Hom-Nambu-Poisson algebra. It is said to be multiplicative if α({x1 , x2 , x3 })={α(x1 ), α(x2 ), α(x3)}, α oµ=µ o α⊗2 . α oµ=µ o α⊗2 . If in addition α is bijective then it is called regular. If in addition α is bijective then it is called regular. Definition 2.8. Let (A, µ, {., ., .}, α) and ( , ,{.,.,.} , ) µ α ′ ′ ′ ′ A be two Definition 2.8. Let (A, µ, {., ., .}, α) and ( , ,{.,.,.} , ) µ α ′ ′ ′ ′ A be two Definition 2.8. Let (A, µ, {., ., .}, α) and ( , ,{.,.,.} , ) µ α ′ ′ ′ ′ A be two J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Volume 9 • Issue 1 • 1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. This ends the proof. Proposition 3.2. Let (A1 , µ1 , {., ., .}1 , α1 ) and (A2 , µ2 , {., ., .}2 , α2 ) be two ternary (non-commutative) Hom-Nambu-Poisson algebras. A linear map 1 2 : φ → A A is a morphism of ternary (non-commutative) Hom- Nambu-Poisson algebras if and only if 1 2 φ Γ ⊆ ⊕ A A is a Hom-Nambu- Poisson subalgebra of (3.3) 1 2 1 2 1 2 1 2 ( , ,{.,.,.} , ) µ α ⊕ ⊕ ⊕ ⊕ A A A A A A A A Where 1 1 2 {( , ( )) : } . φ φ Γ = ∈ ⊂ ⊕ x x x A A A Lemma 3.3. Let A1=(A, m, α) and 2 ( , , ) α ′ ′ ′ = A A m be two ternary Hom- algebras of given type (Hom-Nambu, totally Hom-associative). If m is symmetric and ′ m is skew–symmetric then ′ ⊗ m m is skew symmetric. Proof. Let 1 1 1 1 2 2 2 2 :( , ,{.,.,.} , ) ( , ,{.,.,.} , ) φ µ α µ α → A A be a morphism of ternary J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Now we prove the Hom-Nambu identity doi:10.4172/1736-4337.1000221 Page 4 of 8 Let A1=(A, m, α), where 1,2 ( ) α α = = i i and 2 ( , , ) α ′ ′ ′ = A A m where 1,2 ( ) α α = ′ ′ = i i be two ternary (non- commutative) Hom-algebras of a given he tensor product A1 ⊗ A2 is a ternary Hom-algebra defined by the triple ( , , ) α α ′ ′ ′ ⊗ ⊗ ⊗ A A m m where 1,2 ( ) α α α α = ′ ′ ⊗ = ⊗ i i i with 1 1 2 2 3 3 1 2 3 1 2 3 ( , , ) ( , , ) ( , , ), ′ ′ ′ ′ ′ ′ ′ ′ ⊗ ⊗ ⊗ ⊗ = ⊗ m m x x x x x x m x x x m x x x (3.1) 1 1 1 1 ( ) ( ) ( ), α α α α ′ ′ ′ ′ ⊗ ⊗ = ⊗ i i i i x x x x (3.2) Where x1, x2, x3 ∈ A and 1 2 3 2 , , ′ ′ ′ ∈ x x x A Recall that (A, m, α) is a totally Hom- associative ternary algebra if 1 1 2 2 3 4 5 1 1 2 3 4 2 5 ( ( ), ( ), ( , , )) ( ( ), ( , , ), ( )) α α α α = m x x m x x x m x m x x x x 1 2 3 1 4 2 5 ( ( , , ), ( ), ( )). α α = m m x x x x x for all x1 · · · , x5 ∈ A, and the ternary multiplication m is symmetric if (1) (2) (3) 1 2 3 ( , , ) ( , , ) σ σ σ = m x x x m x x x . (3.3) for all 3 1 2 3 , , , σ ∈ ∈ S x x x A Finally, for Hom-Leibniz identity we have Let A1=(A, m, α), where 1,2 ( ) α α = = i i and 2 ( , , ) α ′ ′ ′ = A A m where 1,2 ( ) α α = ′ ′ = i i be two ternary (non- commutative) Hom-algebras of a given he tensor product A1 ⊗ A2 is a ternary Hom-algebra defined by the triple ( , , ) α α ′ ′ ′ ⊗ ⊗ ⊗ A A m m where 1,2 ( ) α α α α = ′ ′ ⊗ = ⊗ i i i with Finally, for Hom-Leibniz identity we have 1 2 1 2 1 2 1 2 1 2 1 1 2 2 3 3 4 4 1 1 2 2 1 2 1 3 2 3 1 4 2 4 1 1 2 1 3 1 4 1 2 1 2 2 3 2 4 2 1 1 1 2 3 4 1 { ( , ), ( , ), ( , )} { ( , ) ( , ), ( ) ( ), ( ) ( )} { ( , ), ( ), ( )} { ( , ), ( ), ( )} ( ( ),{ , , } ) µ α α µ µ α α α α µ α α µ α α µ α µ ⊕ ⊕ + ⊕ ⊕ ′ ′ ′ ′ + + ′ ′ ′ ′ = + + + ′ ′ ′ ′ = + = + A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x x x 1 2 1 2 1 2 1 2 1 2 1 2 1 1 3 4 1 1 2 2 2 1 2 3 4 2 2 1 3 4 2 2 2 1 1 2 2 3 3 4 4 1 1 3 3 4 4 2 2 ({ , , } , ( )) ( ( ),{ , , } ) ({ , , } , ( )) ( ( , ),{ , , } ) ({ , , } , ( , )) α µ α µ α µ α µ α ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ′ ′ ′ ′ ′ ′ ′ ′ + + ′ ′ ′ ′ = + + + ′ ′ ′ ′ + + + + A A A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x x x y, y 1 2 1 2 1 2 1 2 1 2 1 1 2 2 3 3 4 4 1 1 2 2 1 2 1 3 2 3 1 4 2 4 1 1 2 1 3 1 4 1 2 1 2 2 3 2 4 2 1 1 1 2 3 4 1 { ( , ), ( , ), ( , )} { ( , ) ( , ), ( ) ( ), ( ) ( )} { ( , ), ( ), ( )} { ( , ), ( ), ( )} ( ( ),{ , , } ) µ α α µ µ α α α α µ α α µ α α µ α µ ⊕ ⊕ + ⊕ ⊕ ′ ′ ′ ′ + + ′ ′ ′ ′ = + + + ′ ′ ′ ′ = + = + A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x x x 1 2 1 2 1 2 1 2 1 2 1 2 1 1 3 4 1 1 2 2 2 1 2 3 4 2 2 1 3 4 2 2 2 1 1 2 2 3 3 4 4 1 1 3 3 4 4 2 2 ({ , , } , ( )) ( ( ),{ , , } ) ({ , , } , ( )) ( ( , ),{ , , } ) ({ , , } , ( , )) α µ α µ α µ α µ α ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ′ ′ ′ ′ ′ ′ ′ ′ + + ′ ′ ′ ′ = + + + ′ ′ ′ ′ + + + + A A A A A A A A A A A A x x x x x x x x x x x x x x x x x x x x x x x x x x x x a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if 1 1 2 1 1 2 ( , ) ( , ) φ µ φ µ = + ° ⊆Γ x x x x Therefore, it remains to check Hom-Nambu identity and Hom- Leibniz identity We have 1 1 1 1 2 2 2 2 3 3 4 4 5 5 { ( ), ( ),{ , , } } α α α α ⊗ ⊗ ′ ′ = ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ A B A B LHS a b a b a b a b a b 1 1 1 1 2 2 2 2 3 3 4 4 5 5 1 1 1 1 2 2 2 2 3 4 5 3 4 5 { ( ), ( ),{ , , } } { ( ) ( ), ( ) ( ),{ , , } ( , , )} α α α α α α α α τ ⊗ ⊗ ⊗ = ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ′ ′ = ⊗ ⊗ ⊗ A B A B A A B LHS a b a b a b a b a b a b a b a a a b b b 1 2 3 2 1 2 3 1 { ( ), ( ), ( )} { , , } , φ φ φ φ = x x x x x x 1 2 3 2 1 2 3 1 { ( ), ( ), ( )} { , , } , φ φ φ φ = x x x x x x and 1 1 1 1 2 2 2 2 3 4 5 3 4 5 { ( ) ( ), ( ) ( ),{ , , } ( , , )} α α α α τ ⊗ ′ ′ = ⊗ ⊗ ⊗ A A B a b a b a a a b b b 1 1 1 1 2 2 2 2 3 4 5 3 4 5 { ( ) ( ), ( ) ( ),{ , , } ( , , )} α α α α τ ⊗ ′ ′ = ⊗ ⊗ ⊗ A A B a b a b a a a b b b 1 1 2 2 3 4 5 1 1 2 2 3 4 5 { ( ), ( ),{ , , }} ( ( ), ( ), ( , , )) α α τ α α τ ′ ′ = ⊗     a a a a a b b b b b ,  1 2 1 2 )( ( )) ( ) o ( ) φ α α φ α α φ + + = + ∈Γ x x x x 1 2 1 2 )( ( )) ( ) o ( ) φ α α φ α α φ + + = + ∈Γ x x x x 1 1 ( ) o ( ) α φ α = + x x . Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if We have also 1 2 1 1 1 1 2 1 1 1 1 1 ( )( ( )) ( ) ( ) ( ) ( ), α α φ α α φ α φ α + + = + ° = + ° x x x x x x which implies that 1 2 ( ) φ φ α α + Γ ⊆Γ . (3.4) 1 2 3 4 1 2 3 4 1 3 4 2 ( ( , ), , ) ( , ( , , )) ( ( , , ), ) τ µ µ τ µ τ ′ ′ ′ = = b b b b b b b b b b b b . (3.4) 1 2 3 4 1 2 3 4 1 3 4 2 ( ( , ), , ) ( , ( , , )) ( ( , , ), ) τ µ µ τ µ τ ′ ′ ′ = = b b b b b b b b b b b b . Hom-Nambu-Poisson algebras. Hom-Nambu-Poisson algebras. Theorem 3.4. Let (A, µ, β, {., ., .}, (α1 , α2 )) be a ternary (non- commutative) Hom- Nambu-Poisson algebra, 1 2 ( , ,( , )) τ α α ′ ′ B be a totally Hom-associative symmetric ternary algebra, and ( , , ) B µ β ′ ′ be a Hom-associative algebra, then We have 1 2 1 1 2 2 3 3 1 2 3 1 1 2 3 2 { ( ), ( ), ( )} { , , } { ( ), ( ), ( )} φ φ φ φ φ φ ⊕ + + + = + A A x x x x x x x x x x x x 1 2 3 1 1 2 3 1 { , , } { , , } φ = + x x x x x x 1 2 1 1 2 2 3 3 1 2 3 1 1 2 3 2 { ( ), ( ), ( )} { , , } { ( ), ( ), ( )} φ φ φ φ φ φ ⊕ + + + = + A A x x x x x x x x x x x x 1 2 3 1 1 2 3 1 { , , } { , , } φ = + x x x x x x 1 1 2 2 ( , , ,{.,.,.} ,( , )) µ µ β β α α α α ⊗ ′ ′ ′ ′ ⊗ ⊗ ⊗ ⊗ ⊗ A B A B 1 1 2 2 ( , , ,{.,.,.} ,( , )) µ µ β β α α α α ⊗ ′ ′ ′ ′ ⊗ ⊗ ⊗ ⊗ ⊗ A B A B Then φ Γ is closed under the bracket 1 2 {.,.,.} ⊕ A A . a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if (3.4) Moreover φ Γ is closed under the multiplication indeed 1 2 1 1 2 2 1 1 2 2 1 2 ( ( ), ( )) ( , ) ( ( ), ( )) µ φ φ µ µ φ φ ⊕ + + = + A A x x x x x x x x 1 1 2 1 1 2 ( , ) ( , ) φ µ φ µ = + ° ⊆Γ x x x x Conversely, if the graph 1 2 φ Γ ⊆ ⊕ A A is a Hom-subalgebra of 1 2 1 2 1 2 1 2 ( , ,{.,.,.} , ), µ α ⊕ ⊕ ⊕ ⊕ A A A A A A A A Then we have Moreover φ Γ is closed under the multiplication indeed Moreover φ Γ is closed under the multiplication indeed 1 2 1 1 2 2 1 1 2 2 1 2 ( ( ), ( )) ( , ) ( ( ), ( )) µ φ φ µ µ φ φ ⊕ + + = + A A x x x x x x x x 1 1 2 1 1 2 ( , ) ( , ) φ µ φ µ = + ° ⊆Γ x x x x Conversely, if the graph 1 2 φ Γ ⊆ ⊕ A A is a Hom-subalgebra of 1 2 1 2 1 2 1 2 ( , ,{.,.,.} , ), µ α ⊕ ⊕ ⊕ ⊕ A A A A A A A A Then we have Proof. Since µ and µ′ are both Hom-associative multiplication whence a tensor product µ µ′ ⊗ is Hom-associative. Also the commutativity of µ µ′ ⊗ , the skew- symmetry of {., ., .} and the symmetry of τ simply the skew-symmetry of{.,.,.} ⊗ A B . a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if 1 1 2 2 3 3 1 1 4 4 2 2 5 5 {{ , , } , ( ), ( )} α α α α ⊗ ⊗ ′ ′ = ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ A B A B RHS a b a b a b a b a b 1 1 3 3 1 1 2 2 4 4 2 2 5 5 1 1 3 3 2 2 4 4 1 1 2 2 5 5 1 2 3 1 2 3 1 4 1 4 2 5 2 5 1 3 1 3 1 2 4 { ( ),{ , , } , ( )} { ( ), ( ),{ , , } } {{ , , } ( , , ), ( ) ( ), ( ) ( )} { ( ) ( ),{ , , } α α α α α α α α τ α α α α α α ⊗ ⊗ ⊗ ⊗ ⊗ ′ ′ + ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ′ ′ + ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ′ ′ = ⊗ ⊗ ⊗ ′ + ⊗ ⊗ A B A B A B A B A A B A a b a b a b a b a b a b a b a b a b a b a a a b b b a b a b a b a a a 1 2 4 2 5 2 5 1 3 1 3 2 4 2 4 1 2 5 1 2 5 1 2 3 1 4 2 5 1 2 3 1 4 2 5 1 3 1 2 4 2 5 ( , , ), ( ) ( )} { ( ) ( ), ( ) ( ),{ , , } ( , , )} {{ , , }, ( ), ( )} ( ( , , ), ( ), ( )) { ( ),{ , , }, ( τ α α α α α α τ α α τ τ α α α α ⊗ ⊗ ′ ⊗ ′ ′ + ⊗ ⊗ ⊗ ′ ′ = ⊗ +    A B A A B c d b b b a b a b a b a a a b b b a a a a a b b b b b a a a a a 1 3 1 2 4 2 5 1 3 2 4 1 2 5 1 3 2 4 1 2 5 )} ( ( ), ( , , ), ( )) { ( ), ( ),{ , , }} ( ( ), ( ), ( , , )) τ α τ α α α τ α α τ ′ ′ ⊗ ′ ′ + ⊗       e f g h b b b b b a a a a a b b b b b 1 2 1 1 2 2 1 1 2 2 1 2 ( ( ), ( )) ( , ) ( ( ), ( )) µ φ φ µ µ φ φ ⊕ + + = + A A x x x x x x x x 1 1 2 1 1 2 ( , ) o ( , ) . Indeed, we have We check the Hom-Nambu identity {βα(x1 ), βα(x2 ), {x3 , x4 , x5 }β }β=β2 {α(x1 ), α(x2 ), {x3 , x4 , x5 }} = β2 ({{x1 , x2 , x3 }, α(x4 ), α(x5 )} + {α(x3 ), {x1 , x2 , x4 }, α(x5 )} + {α(x3 ), α(x4 ), {x1 , x2 , x5 }}) = {{x1 , x2 , x3 }β , βα(x4 ), βα(x5 )}β + {βα(x3 ), {x1 , x2 , x4 }β , βα(x5 )}β + {βα(x3 ), βα(x4 ), {x1 , x2 , x5 }β }β . Then it remains to show Hom-Leibniz identity 2 1 2 3 4 1 2 3 4 { ( , ), ( ), ( )} ({ ( , ), ( ), ( )}) β β µ βα βα β µ α α = x x x x x x x x µβ (µβ (x, y), βα(z))=µβ (β(µ(x, y), βα(z)))=β2 (µ(µ(x, y), α(z))) = β2 (µ(α(x), µ(y, z)))=µβ (βα(x), µβ (y, z)). We check the Hom-Nambu identity {βα(x1 ), βα(x2 ), {x3 , x4 , x5 }β }β=β2 {α(x1 ), α(x2 ), {x3 , x4 , x5 }} = β2 ({{x1 , x2 , x3 }, α(x4 ), α(x5 )} + {α(x3 ), {x1 , x2 , x4 }, α(x5 )} + {α(x3 ), α(x4 ), {x1 , x2 , x5 }}) = {{x1 , x2 , x3 }β , βα(x4 ), βα(x5 )}β + {βα(x3 ), {x1 , x2 , x4 }β , βα(x5 )}β + {βα(x3 ), βα(x4 ), {x1 , x2 , x5 }β }β . Then it remains to show Hom-Leibniz identity 2 1 2 3 4 1 2 3 4 { ( , ), ( ), ( )} ({ ( , ), ( ), ( )}) β β µ βα βα β µ α α = x x x x x x x x µβ (µβ (x, y), βα(z))=µβ (β(µ(x, y), βα(z)))=β2 (µ(µ(x, y), α(z))) 2 2 β β ϕ µ ϕ β µ β ϕ µ β µ ϕ µ ϕ × × ′ ′ ′ ′ ′ ° = ° ° = ° ° = ° ° = ° = β2 (µ(α(x), µ(y, z)))=µβ (βα(x), µβ (y, z)). Then Aβ is multiplicative. 1 1 2 2 3 3 4 4 ( ( ),{ , , } ) µ µ β β ⊗ ′ ′ = ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ A B RHS a b a b a b a b 1 1 2 2 3 3 4 4 ( ( ),{ , , } ) µ µ β β ⊗ ′ ′ = ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ A B RHS a b a b a b a b 1 1 3 3 4 4 2 2 1 1 2 3 4 2 3 4 1 3 4 1 3 4 2 2 1 2 3 4 1 2 3 4 1 3 ({ , , } , ( )) ( ( ) ( ),{ , , } ( , , )) ({ , , } ( , , ), ( ) ( )) ( ( ),{ , , }) ( ( ), ( , , )) ({ , , µ µ β β µ µ β β τ µ µ τ β β µ β µ β τ µ ⊗ ′ ′ ′ ′ + ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ′ ′ = ⊗ ⊗ ⊗ ′ ′ + ⊗ ⊗ ⊗ ′ ′ = ⊗ +   A B c d a b a b a b a b a b a a a b b b a a a b b b a b a a a a b b b b a a 4 2 1 3 4 2 }, ( )) ( ( , , ), ( ) β µ τ β ′ ′ ′ ′ ⊗   e f a a b b b b Corollary 4.2. Let (A, µ, {., ., .}, α) be a multiplicative ternary (non- commutative) Hom-Nambu-Poisson algebra. Then ( ) ( ) ( ) 1 ( , ,{.,.,.} o o{.,.,.}, ) µ α µ α α + = = = n n n n n n A A is a multiplicative (non-commutative) ternary Hom-Nambu-Poisson algebra for each integer n ≥ 0. Proof. The multiplicativity of A implies that αn : A → A is a Nambu- Poisson algebra morphism. By Theorem 4.1 α = n n A A is a multiplicative ternary (non- commutative) Hom-Nambu-Poisson algebra. 1 1 2 2 ( , , ,{.,.,.} ,( , )) µ µ β β α α α α ⊗ ′ ′ ′ ′ ⊗ ⊗ ⊗ ⊗ ⊗ A B A B 1 1 2 2 ( , , ,{.,.,.} ,( , )) µ µ β β α α α α ⊗ ′ ′ ′ ′ ⊗ ⊗ ⊗ ⊗ ⊗ A B A B 1 1 2 2 ( , , ,{.,.,.} ,( , )) µ µ β β α α α α ⊗ ′ ′ ′ ′ ⊗ ⊗ ⊗ ⊗ ⊗ A B A B is a (non-commutative) ternary Hom-Nambu-Poisson algebra. ( , o ,{.,.,.} o{.,.,.}, ) β β β µ β µ β β = = = A A a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if We have For the Hom-Leibniz identity, we have 1 1 2 2 1 1 3 3 2 2 4 4 { ( , ), ( ), ( )} µ µ α α α α ⊗ ′ ′ ′ = ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ A B LHS a b a b a b a b 1 1 2 2 1 1 3 3 2 2 4 4 { ( , ), ( ), ( )} µ µ α α α α ⊗ ′ ′ ′ = ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ ⊗ A B LHS a b a b a b a b 2 2 2 ( )o( ) ( ) β β β βα µ βα β µ µ α β µ βα ⊗ ⊗ ⊗ = = = o o o o 1 1 2 2 1 3 1 3 2 4 2 4 { ( , ) ( , ), ( ) ( ), ( ) ( )} { ( ) ( ) ( )} ( ( ) ( ) ( )) µ µ α α α α ⊗ ′ ′ ′ = ⊗ ⊗ ⊗ ′ ′ ′ ⊗ A B a b a b a b a b b b b b 1 1 2 2 1 3 1 3 2 4 2 4 { ( , ) ( , ), ( ) ( ), ( ) ( )} µ µ α α α α ⊗ ′ ′ ′ = ⊗ ⊗ ⊗ A B a b a b a b a b 1 1 2 2 1 3 1 3 2 4 2 4 1 1 1 3 2 4 2 2 1 3 2 4 { ( , ) ( , ), ( ) ( ), ( ) ( )} { ( , ), ( ), ( )} ( ( , ), ( ), ( )) µ µ α α α α µ α α τ µ α α ⊗ ′ ′ ′ ′ ′ = ⊗ ⊗ ⊗ ′ ′ ′ = ⊗   A B A a b a b a b a b a b a b a a a b b b And And 3 o{.,.,.} o o{.,.,.} {.,.,.} o( ) β β βα βα β βα ⊗ = = J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Construction of Ternary Hom-Nambu-Poisson Algebras is a multiplicative (non-commutative) ternary Hom-Nambu-Poisson algebra. In this section, we provide constructions of ternary Hom-Nambu- Poisson algebras using twisting principle. Remark 4.4. Let (A, µ, {., ., .}, α) and ( , ,{.,.,.} , ) µ α ′ ′ ′ ′ A be two (non-commutative) ternary Nambu-Poisson algebras and : , : β β′ ′ ′ → → A A A A be ternary Nambu- Poisson algebra endomorphisms. If : ϕ ′ → A A is a ternary Nambu-Poisson algebra morphism that satisfies o o ϕ β β ϕ ′ = then Remark 4.4. Let (A, µ, {., ., .}, α) and ( , ,{.,.,.} , ) µ α ′ ′ ′ ′ A be two (non-commutative) ternary Nambu-Poisson algebras and : , : β β′ ′ ′ → → A A A A be ternary Nambu- Poisson algebra endomorphisms. If : ϕ ′ → A A is a ternary Nambu-Poisson algebra morphism that satisfies o o ϕ β β ϕ ′ = then Theorem 4.1. Let (A, µ, {., ., .}, α) be a (non-commutative) ternary Hom-Nambu- Poisson algebra and β : A → A be a weak Hom-Nambu- Poisson morphism, then Aβ=(A, {., ., .}β=β o {., ., .}, µβ=β ◦ µ, βα) is also a ternary (non-commutative)Hom-Nambu-Poisson algebra. Moreover, if A is multiplicative and β is an algebra morphism, then Aβ is a multiplicative (non-commutative) Hom-Nambu-Poisson al- gebra. :( , ,{.,.,.} , ) ( , ,{.,.,.} , ) β β β β ϕ µ βα µ β α ′ ′ ′ ′ ′ ′ ′ → A A Is a (non-commutative) ternary hom-nambu poisson algebra morphism. Proof. If µ is commutative, then clearly so is µβ . The rest of the proof applies whether µ is commutative or not. The skew-symmetry follows from the skew- symmetry of the bracket {., ., .}. It remains to prove Hom-associativity condition, Hom-Nambu-identity and Hom- Leibniz identity. Indeed And Then Aβ is multiplicative. ( , ,{.,.,.} , ) µ α ′ ′ ′ ′ A a (non-commutative) ternary Hom-Nambu-Poisson algebra if and only if φ µ φ µ = + ⊆Γ x x x x 1 2 1 1 2 2 1 1 2 2 1 2 ( ( ), ( )) ( , ) ( ( ), ( )) µ φ φ µ µ φ φ ⊕ + + = + A A x x x x x x x x 1 1 2 1 1 2 ( , ) o ( , ) . φ µ φ µ = + ⊆Γ x x x x 1 1 2 1 1 2 ( , ) o ( , ) . φ µ φ µ = + ⊆Γ x x x x 1 1 2 1 1 2 ( , ) o ( , ) . φ µ φ µ = + ⊆Γ x x x x Therefore φ is a morphism of ternary (non-commutative) Hom- Nambu-Poisson algebras. Now, we define the tensor product of two ternary Hom-algebras. Moreover, we consider a tensor product of a ternary Hom-Nambu- Poisson algebra and a totally Hom-associative symmetric ternary algebra. Volume 9 • Issue 1 • 1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Page 5 of 8 Using ternary Nambu identity of {., ., .} we have a=c + e + g, and b=d=f=h using the symmetry of τ and Hom-associativity of µ′, then the left hand side is equal to the right hand side from where the ternary Hom- Nambu identity of bracket {.,.,.}A B ⊗ is verified 2 1 2 3 4 1 3 4 2 1 2 3 4 1 3 4 2 ( ( ( ),{ , , }) ({ , , }, ( ))) ( ( ),{ , , } ) ({ , , } , ( )) β β β β β µ α µ α µ βα µ βα = + = + x x x x x x x x x x x x x x x x Therefore Aβ=(A, {., ., .}β , µβ , βα) is a ternary (non-commutative) Hom-Nambu- Poisson algebra. For the multiplicativity assertion, suppose that A is multiplicative and β is an algebra morphism. Then Aβ is multiplicative. With Hom-Leibniz identity we have ′ ′ ′ = + a c e , and using condition (3.4) we have ′ ′ ′ = = b d f , therefore the left hand side is equal to the right hand side and the Hom-Leibniz identity is proved. Then Corollary 4.3. Let (A, µ, {., ., .}) be a ternary (non-commutative) Nambu-Poisson algebra and β : A → A be a Nambu-Poisson algebra morphism. Then Indeed, we have Indeed, we have 3 3 o {.,.,.} o {.,.,.} o o{.,.,.} o{.,.,.} o {.,.,.} β β ϕ ϕ β β ϕ β ϕ ϕ × × ′ ′ ′ ′ ′ = ° = = = ° 3 3 o {.,.,.} o {.,.,.} o o{.,.,.} o{.,.,.} o {.,.,.} β β ϕ ϕ β β ϕ β ϕ ϕ × × ′ ′ ′ ′ ′ = ° = = = ° 3 3 o {.,.,.} o {.,.,.} o o{.,.,.} o{.,.,.} o {.,.,.} β β ϕ ϕ β β ϕ β ϕ ϕ × × ′ ′ ′ ′ ′ = ° = = = ° 2 2 β β ϕ µ ϕ β µ β ϕ µ β µ ϕ µ ϕ × × ′ ′ ′ ′ ′ ° = ° ° = ° ° = ° ° = ° µβ (µβ (x, y), βα(z))=µβ (β(µ(x, y), βα(z)))=β2 (µ(µ(x, y), α(z))) = β2 (µ(α(x), µ(y, z)))=µβ (βα(x), µβ (y, z)). We check the Hom-Nambu identity {βα(x1 ), βα(x2 ), {x3 , x4 , x5 }β }β=β2 {α(x1 ), α(x2 ), {x3 , x4 , x5 }} = β2 ({{x1 , x2 , x3 }, α(x4 ), α(x5 )} + {α(x3 ), {x1 , x2 , x4 }, α(x5 )} + {α(x3 ), α(x4 ), {x1 , x2 , x5 }}) = {{x1 , x2 , x3 }β , βα(x4 ), βα(x5 )}β + {βα(x3 ), {x1 , x2 , x4 }β , βα(x5 )}β + {βα(x3 ), βα(x4 ), {x1 , x2 , x5 }β }β . Then it remains to show Hom-Leibniz identity 2 1 2 3 4 1 2 3 4 { ( , ), ( ), ( )} ({ ( , ), ( ), ( )}) β β µ βα βα β µ α α = x x x x x x x x µβ (µβ (x, y), βα(z))=µβ (β(µ(x, y), βα(z)))=β2 (µ(µ(x, y), α(z))) = β2 (µ(α(x), µ(y, z)))=µβ (βα(x), µβ (y, z)). Indeed, we have − = + + = = + + + P x y z a x y a z P x y z b x P x y z c x c y c z b c a c c (9) 2 1 1 2 1 2 3 2 1 2 3 3 2 3 2 2 3 3 2 2 1 ( , , ) , ( , , ) , ( , , ) = + + + = + + = + + − a b b c P x y z x a y a z P x y z b x b y b z P x y z x c y c z b b c b c b (10) 3 2 1 2 1 2 3 2 3 3 1 2 3 2 3 2 ( , , ) , ( , , ) , ( , , ) − + = + + = = + + c a c c P x y z x a y a z P x y z b z P x y z c x c y c z b c (11) 1 1 2 2 1 2 3 1 2 1 2 2 1 1 ( , , ) , ( , , ) , ( , , ) = + + = + = + − P x y z a x a y z P x y z b x b y P x y z c x c y b c b c (12) 2 1 3 3 1 2 2 3 1 3 2 1 1 2 3 2 1 2 2 3 3 2 3 3 1 2 3 1 ( , , ) , ( , , ) , ( , , ) + − − + = + + = + + − = + + a b c a b c a b c a b c P x y z x a y a z P x y z b x b y b c b c b z P x y z c x c y c z (13) 2 1 1 3 3 2 1 2 3 3 3 1 2 2 1 3 2 1 2 2 1 ( , , ) ( ) , ( , , ) , ( , , ) = + − + = + + − = + + b P x y z a x a y a z P x y z b x b y b z P x y z b b c b c b c c x c y z b (14) The first equality shows that it is sufficient to just set α on x, y and z. Indeed, we have doi:10.4172/1736-4337.1000221 Page 6 of 8 1 1 1 2 2 2 3 3 3 ( , , ) ( , , )) ( , , ) ( , , ) ( , , ) ( , , ) 1 0, ( , , ) ( , , ) ( , , ) ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ − = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ P x y z P x y z P x y z x y z P x y z P x y z P x y z x y z P x y z P x y z P x y z x y z (4.3) Whence 1 2 3 1 2 3 1 2 3 1 0. − = a a a b b b c c c (4.4) 1 1 1 2 2 2 3 3 3 ( , , ) ( , , )) ( , , ) ( , , ) ( , , ) ( , , ) 1 0, ( , , ) ( , , ) ( , , ) ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ − = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ P x y z P x y z P x y z x y z P x y z P x y z P x y z x y z P x y z P x y z P x y z x y z (4.3) Whence 1 2 3 1 2 3 1 2 3 1 0. − = a a a b b b c c c (4.4) Theorem 4.5. Indeed, we have For the second equality, we suppose by linearity that ( , , ) , ( , , ) , ( , , ) . = = = i j k l m p q r s f x y z x y z g x y z x y z f x y z x y z ( , , ) , ( , , ) , ( , , ) . = = = i j k l m p q r s f x y z x y z g x y z x y z f x y z x y z Then we can write the second equation as follows ( ) ( ) ( ) ( ) ( ) ( ) , ( ) ( ) ( ) α α α α α α α α α α ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ f f f f f f x y z x y z g g g g g g x y z x y z h h h h h h x y z x y z which can be simplified to which can be simplified to ( ) ( ) ( ) ( ) ( ) ( ) 1 . ( ) ( ) ( ) α α α α α α α α α ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ = ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ x x x x y z y y y x y z z z z x y z (4.2) (4.2) Example 4.6. We set polynomials: Example 4.6. Indeed, we have A morphism : [ , , ] [ , , ] α →   x y z x y z which gives a structure of ternary Hom-Nambu-Poisson algebra ( [ , , ], ,{.,.,.} {.,.,.}, ) α α α α α ⋅= °⋅ = ° x y z satisfies the following equation: (4.3) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 1 0 α α α α α α α α α ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ − ∂ ∂ ∂ ∂ ∂ ∂ ∂ = ∂ x x x x y z y y y x y z z z z x y z (4.1) (4.1) 1 2 3 1 2 3 1 2 3 1 0. − = a a a b b b c c c (4.4) (4.4) Proof. Let α be a Nambu-Poisson algebra morphism, then it satisfies for all , , [ , , ] ∈ f g h x y z The polynomials P1, P2 and P3 are of one of this form ( ) ( ) ( ), { , , } { ( ), ( ), ( )}. α α α α α α α ⋅ = ⋅ = f g f g f g h f g h ( ) ( ) ( ), { , , } { ( ), ( ), ( )}. α α α α α α α ⋅ = ⋅ = f g f g f g h f g h ( ) ( ) ( ), { , , } { ( ), ( ), ( )}. α α α α α α α ⋅ = ⋅ = f g f g f g h f g h The polynomials P1, P2 and P3 are of one of this form 1 1 2 3 2 2 3 2 1 2 ( , , ) ( , , , ) , ( , , ) . Indeed, we have = + + = − = z P x y z xa ya za P x y z b y P x y z c y a c (1) 1 3 2 1 1 2 3 2 3 3 2 3 1 3 1 ( , , ) , ( , , ) , ( , , ) + = + + = + = + a b c P x y z a x a y a z P x y z y b z P x y z c y c z a c (2) 1 1 2 3 2 1 3 1 2 1 1 ( , , ) , ( , , ) , ( , , ) = + + = + = P x y z a x a y a z P x y z b x z P x y z c x a c (3) 2 3 1 1 1 2 3 2 3 3 1 3 2 3 1 ( , , ) , ( , , ) , ( , , ) −+ = + + = + = + a b c P x y z a x a y a z P x y z x b z P x y z c x c z a c (4) 2 1 3 2 1 2 3 2 1 2 3 3 3 3 ( , , ) , ( , , ) , ( , , ) + = + + = + + = a b c b P x y z a y a z P x y z b x b y b z P x y z c z c x (5) 1 2 3 2 2 3 3 3 2 3 1 ( , , ) , ( , , ) , ( , , ) = + + = + = P x y z x a y a z P x y z b y b z P x y z c z b c (6) 1 1 3 2 1 3 3 3 1 3 1 ( , , ) , ( , , ) , ( , , ) = + + = + = P x y z a x y a z P x y z b x b z P x y z c z b c (7) 1 1 2 2 1 3 1 2 1 2 1 ( , , ) , ( , , ) , ( , , ) = + + = = + P x y z a x a y z P x y z b x P x y z c x c y b c (8) 1 1 3 2 1 3 1 2 3 1 3 3 2 3 1 ( , , ) , ( , , ) , ( , , ) . J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Particular case of polynomials of degree two. We take one of the polynomials of degree two Particular case of polynomials of degree two. We take one of the polynomials of degree two Where 1 2 3 , , [ , , ] ∈ P P P x y z , and d the largest degree for each variable. We assume that a0=b0=c0=0 Where 1 2 3 , , [ , , ] ∈ P P P x y z , and d the largest degree for each variable. We assume that a0=b0=c0=0 1 1 2 3 2 1 2 3 2 3 1 2 3 4 ( , , ) ( , , ) ( , , ) = + + = + + = + + + P x y z a x a y a z P x y z b x b y b z P x y z c x c y c z c x 1 1 2 3 ( , , ) = + + P x y z a x a y a z Case of polynomials of degree one. We take Indeed, we have In the sequel, we aim to construct Hom-type version of the ternary Nambu- Poisson algebra of polynomials of three variables ( [ , , ], ,{.,.,.}) ⋅ x y z , defined in Example 1.5. The Poisson bracket of three polynomials is defined in (1.2). We check the Hom-Nambu identity The twisted version is given by a structure of ternary Hom-Nambu- Poisson algebra where ( [ , , ], o ,{.,.,.} o{.,.,.}, ) α α α α α ⋅= ⋅ = x y z where : [ , , ] [ , , ] α →   x y z x y z is an algebra morphism satisfying for all The twisted version is given by a structure of ternary Hom-Nambu- Poisson algebra where ( [ , , ], o ,{.,.,.} o{.,.,.}, ) α α α α α ⋅= ⋅ = x y z where + {α(x3 ), α(x4 ), {x1 , x2 , x5 }}) : [ , , ] [ , , ] α →   x y z x y z is an algebra morphism satisfying for all , [ , , ] ∈ f g x y z ( ) ( ) ( ) α α α ⋅ = ⋅ f g f g { , , } { ( ), ( ), ( )}. α α α α = f g h f g h { , , } { ( ), ( ), ( )}. α α α α = f g h f g h J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Volume 9 • Issue 1 • 1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. Indeed, we have We set polynomials: 1 0 , , 2 0 , , 3 0 , , ( ) ( , , ) , ( ) ( , , ) , ( ) ( , , ) , α α α ≤ ≤ ≤ ≤ ≤ ≤ = = = = = = ∑ ∑ ∑ i j k ijk i j k d i j k ijk i j k d i j k ijk i j k d x P x y z a x y z y P x y z b x y z z P x y z c x y z Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Case of polynomials of degree one. We take = − + = = + + + P x y z x a z P x y z b z P x y z c x c x c y c z b c (5) 2 1 1 3 2 1 3 4 1 3 1 3 1 ( , , ) , ( , , ) , ( , , ) = − + = = + + P x y z a x y a z P x y z b x P x y z c x c x c z b c (6) 2 3 2 1 1 3 2 1 3 4 1 2 3 1 3 3 1 ( , , ) , ( , , ) , ( , , ) − = + + + = = + + + a c P x y z a x y a z P x y z b x P x y z c x c x c y c z b c c (7) 2 1 1 2 2 1 3 4 1 2 1 2 1 ( , , ) , ( , , ) , ( , , ) = + + = = + + P x y z a x a y z P x y z b x P x y z c x c x c y b c (8) 2 3 1 1 1 2 3 2 3 3 1 3 2 3 (1 ) ( , , ) , ( , , ) , ( , , ) + = + + = + = + a b c P x y z a x a y a z P x y z x b z P x y z c x c z a c (9)i (3) (9) With Theorem 5.1. Every 3-dimensional ternary Nambu-Lie algebra is isomorphic to the ternary algebra defined with respect to basis {e1, e2, e3}, by the skew-symmetric bracket defined as (5) {e1, e2, e3}= e1 {e1, e2, e3}= e1 Moreover it defines a 3-dimensional ternary non-commutative Nambu-Poisson algebra (A, {., ., .}, µ) if and only if µ is one of the following non-commutative associative algebra defined as 5 1 1 5 2 1 2 3 5 3 1 3 ( ) , ( ) , ( ) α α α = = + + = + e ce e de e le e he e 6 6 6 6 6 6 1 2 1 2 3 1 2 3 1 3 1 3 2 1 2 3 2 2 2 1 2 3 3 3 1 2 3 ( , ) , ( , ) (1 ) ), ( ) ( , ) , ( , ) ( 1) , ( 1) ( , ) ( (1 ) ), ( , ) ( ) α α α α α α µ µ µ µ µ µ = = + + − − = = − − + − = + + − = − + − e e ace e e bde bfe a f e a b ag e e bce e e ahe b f e e b a b f e e a de fe f e e e bhe e b ag e b a (6) With 1 2 1 1 1 2 2 2 1 2 3 3 1 3 1 1 1 3 2 2 1 3 3 3 ( , ) ( , ) ( , ) ( , ) ( , ) ( , ) , µ µ µ µ µ µ = = = = = = e e ae e e ae e e ae e e be e e be e e be (1) (6) (1) where a, b are parameters. J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Case of polynomials of degree one. We take α α α = = + = + + e ce e de e e he ge e 9 9 9 1 3 1 2 3 1 3 3 3 1 2 3 ( , ) , 1 ( , ) ( ), ( , ) ( ) α α α µ µ µ = = − = + + e e ace e e a de e g e e a he ge re (9) With 1 ( ) ( ) ( ) d h 3 2 2 3 2 1 1 2 3 2 2 3 3 4 1 2 3 2 1 ( , , ) , ( , , ) , ( , , ) + = + + = + = + + + b c a b a P x y z a x y a z P x y z b y b z P x y z c x c x c y z b b (2) 2 1 2 2 3 2 2 3 4 1 2 1 2 1 ( , , ) , ( , , ) , ( , , ) . = + + = = + + + P x y z a x a y a z P x y z b y P x y z c x c x c y z a b (3) 2 2 1 1 3 2 1 3 3 4 1 2 3 3 2 3 1 ( , , ) ( ) , ( , , ) , ( , , ) = − + = + = + + + a b P x y z x a z P x y z b x b z P x y z c x c x c y c z b c b (4) 2 1 3 2 3 3 4 1 2 3 3 2 1 ( , , ) , ( , , ) , ( , , ) . Case of polynomials of degree one. We take doi:10.4172/1736-4337.1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Page 7 of 8 1 1 1 1 2 1 2 1 3 1 2 3 ( ) , ( ) , ( ) . Case of polynomials of degree one. We take α α α = = + = + + e ce e de e e he ge e 2 2 2 2 2 2 1 2 1 3 1 1 2 2 1 2 3 3 2 1 2 3 2 3 1 3 3 3 1 3 ( , ) , ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ), ( , ) ( ), α α α α α α µ µ µ µ µ µ = = = + + = + + = + = + e e ace e e bce e e a de e le e e b de e le e e a he e e e b he e (2) With 2 1 1 2 2 1 2 3 2 3 1 3 3 ( ) , ( ) , ( ) α α α = = + + = + e ce e de e le e he e e 3 3 3 3 3 3 2 1 1 3 1 1 2 2 1 2 3 3 2 1 2 3 2 2 3 1 2 3 3 3 1 2 3 ( , ) , ( , ) , ( , ) ( (1 ) ), ( , ) (1 ) , ( ) ( 1) ( , ) ( 1) ), ( , ) ( ) , α α α α α α µ µ µ µ µ µ = = = + + − = + + − − − = + − + = + + − e e ace e e bce a e e a de fe f e e e bde bfe a f e b a b ga b f e e ahe b f e e e e bhe e b ga e b a (3) With 3 1 1 3 2 1 2 3 3 3 1 2 3 ( ) ( ) , ( ) (1 ) , ( ) ( 1) α α α − = = + + − = + − + a b b ga e ce e de fe f e e he f e e b a b 4 4 4 4 4 4 1 2 1 2 3 1 2 1 3 1 3 2 1 2 3 2 2 1 2 3 3 1 2 3 ( , ) , ( , ) ( ), ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ), α α α α α α µ µ µ µ µ µ = = + = = + + = + = + + e e ace e e b de e e e bce e e a he ge e e e a de e e e b he ge e (4) With 4 1 1 4 2 1 2 4 3 1 2 3 ( ) , ( ) , ( ) . Case of polynomials of degree one. We take α α α = = + = + + e ce e de e e he ge e 5 5 5 5 5 5 1 2 1 2 3 1 2 3 1 3 1 3 2 1 3 2 2 1 2 3 3 3 1 3 ( , ) , ( , ) ( ), ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ) α α α α α α µ µ µ µ µ µ = = + + = = + = + + = + e e ace e e b de e le e e bce e e a he e e e a de e le e e b he e (5) With 5 1 1 5 2 1 2 3 5 3 1 3 ( ) , ( ) , ( ) α α α = = + + = + e ce e de e le e he e 6 6 6 6 6 6 1 2 1 2 3 1 2 3 1 3 1 3 2 1 2 3 2 2 2 1 2 3 3 3 1 2 3 ( , ) , ( , ) (1 ) ), ( ) ( , ) , ( , ) ( 1) , ( 1) ( , ) ( (1 ) ), ( , ) ( ) α α α α α α µ µ µ µ µ µ = = + + − − = = − − + − = + + − = − + − e e ace e e bde bfe a f e a b ag e e bce e e ahe b f e e b a b f e e a de fe f e e e bhe e b ag e b a (6) With 6 1 1 6 2 1 2 3 6 3 1 2 3 ( ) , ( ) (1 ) , ( ) ( 1) 8 α α α − − = = + + − = + − + a b b ag e ce e de fe f e e he f e e b a b z 7 7 7 1 3 1 2 3 1 2 3 3 3 1 2 3 ( , ) , ( , ) ( ), 1 ( , ) ( ) α α α µ µ µ = = + + + + = + + e e ace e e a de fe le g l e e a he ge e f (7) With 7 1 1 7 2 1 2 3 7 3 1 2 3 1 ( ) , ( ) , ( ) α α α + + = = + + = + + g l e ce e de fe le e he ge e f 8 8 8 1 3 1 2 3 1 2 3 3 1 2 3 ( , ) , ( , ) ( ), ( , ) ( ) α α α µ µ µ = = + = + + e e ace e e a de e e e a he ge e (8) With 8 1 1 8 2 1 2 8 3 1 2 3 ( ) , ( ) , ( ) . Case of polynomials of degree one. We take 2 1 2 3 ( , , ) = + + P x y z b x b y b z 1 1 2 3 2 1 2 3 3 1 2 3 ( , , ) , ( , , ) , ( , , ) = + + = + + = + + P x y z a x a y a z P x y z b x b y b z P x y z c x c y c z 1 1 2 3 ( , , ) , = + + P x y z a x a y a z 2 3 1 2 3 4 ( , , ) = + + + P x y z c x c y c z c x 2 1 2 3 ( , , ) , = + + P x y z b x b y b z The polynomials P1,P2 and P3 are of one of this form The polynomials P1,P2 and P3 are of one of this form 2 3 2 1 1 2 2 1 2 3 2 2 3 3 2 2 2 3 4 1 2 3 1 ( , , ) , ( , , ) , ( , , ) . = + + + = + + − = + + + a b a b P x y z x a y z P x y z b x b y b z b b c b c b P x y z c x c x c y c z (1) 2 3 2 1 1 2 2 1 2 3 2 2 3 3 2 2 2 3 4 1 2 3 1 ( , , ) , ( , , ) , ( , , ) . = + + + = + + − = + + + a b a b P x y z x a y z P x y z b x b y b z b b c b c b P x y z c x c x c y c z (1) Equation (2.5) becomes (1) Volume 9 • Issue 1 • 1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. Case of polynomials of degree one. We take = − + = = + + + P x y z x a z P x y z b z P x y z c x c x c y c z b c (5) 2 1 1 3 2 1 3 4 1 3 1 3 1 ( , , ) , ( , , ) , ( , , ) = − + = = + + P x y z a x y a z P x y z b x P x y z c x c x c z b c (6) 2 3 2 1 1 3 2 1 3 4 1 2 3 1 3 3 1 ( , , ) , ( , , ) , ( , , ) − = + + + = = + + + a c P x y z a x y a z P x y z b x P x y z c x c x c y c z b c c (7) 2 1 1 2 2 1 3 4 1 2 1 2 1 ( , , ) , ( , , ) , ( , , ) = + + = = + + P x y z a x a y z P x y z b x P x y z c x c x c y b c (8) 2 3 1 1 1 2 3 2 3 3 1 3 2 3 (1 ) ( , , ) , ( , , ) , ( , , ) + = + + = + = + a b c P x y z a x a y a z P x y z x b z P x y z c x c z a c (9)i 1 1 1 1 2 1 2 1 3 1 2 3 ( ) , ( ) , ( ) . Case of polynomials of degree one. We take α α α = = + = + + e ce e de e e he ge e 2 2 2 2 2 2 1 2 1 3 1 1 2 2 1 2 3 3 2 1 2 3 2 3 1 3 3 3 1 3 ( , ) , ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ), ( , ) ( ), α α α α α α µ µ µ µ µ µ = = = + + = + + = + = + e e ace e e bce e e a de e le e e b de e le e e a he e e e b he e (2) With 2 1 1 2 2 1 2 3 2 3 1 3 3 ( ) , ( ) , ( ) α α α = = + + = + e ce e de e le e he e e 3 3 3 3 3 3 2 1 1 3 1 1 2 2 1 2 3 3 2 1 2 3 2 2 3 1 2 3 3 3 1 2 3 ( , ) , ( , ) , ( , ) ( (1 ) ), ( , ) (1 ) , ( ) ( 1) ( , ) ( 1) ), ( , ) ( ) , α α α α α α µ µ µ µ µ µ = = = + + − = + + − − − = + − + = + + − e e ace e e bce a e e a de fe f e e e bde bfe a f e b a b ga b f e e ahe b f e e e e bhe e b ga e b a (3) With 3 1 1 3 2 1 2 3 3 3 1 2 3 ( ) ( ) , ( ) (1 ) , ( ) ( 1) α α α − = = + + − = + − + a b b ga e ce e de fe f e e he f e e b a b 4 4 4 4 4 4 1 2 1 2 3 1 2 1 3 1 3 2 1 2 3 2 2 1 2 3 3 1 2 3 ( , ) , ( , ) ( ), ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ), α α α α α α µ µ µ µ µ µ = = + = = + + = + = + + e e ace e e b de e e e bce e e a he ge e e e a de e e e b he ge e (4) With 4 1 1 4 2 1 2 4 3 1 2 3 ( ) , ( ) , ( ) . Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Case of polynomials of degree one. We take α α α = = + = + + e ce e de e e he ge e 5 5 5 5 5 5 1 2 1 2 3 1 2 3 1 3 1 3 2 1 3 2 2 1 2 3 3 3 1 3 ( , ) , ( , ) ( ), ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ) α α α α α α µ µ µ µ µ µ = = + + = = + = + + = + e e ace e e b de e le e e bce e e a he e e e a de e le e e b he e (5) With 5 1 1 5 2 1 2 3 5 3 1 3 ( ) , ( ) , ( ) α α α = = + + = + e ce e de e le e he e 6 6 6 6 1 2 1 2 3 1 2 3 1 3 1 3 2 1 2 3 ( , ) , ( , ) (1 ) ), ( ) ( , ) , ( , ) ( 1) , α α α α µ µ µ µ = = + + − − = = − − + e e ace e e bde bfe a f e a b ag e e bce e e ahe b f e e b (6) 1 1 1 1 2 1 2 1 3 1 2 3 ( ) , ( ) , ( ) . With (2) 6 1 1 6 2 1 2 3 6 3 1 2 3 ( ) , ( ) (1 ) , ( ) ( 1) 8 α α α − − = = + + − = + − + a b b ag e ce e de fe f e e he f e e b a b z The opposite algebra of (1) The multiplication which are not mentioned are equal to zero. The first statement of the Theorem is due to Filippov [4,13]. The two families are naturally not isomorphic. (7) (7) (7) Remark 5.2. The 3-dimensional ternary Nambu-Lie algebra is endowed with a commutative Nambu-Poisson algebra structure only when the multiplication is trivial. With Using the twisting principle described in Theorem 4.1, we obtain the following 3-dimensional non-commutative ternary Hom-Nambu- Poisson algebras. (8) (8) Proposition 5.3. Classification (4) In this section, we provide the classification of 3-dimensional ternary non-commutative Nambu-Poisson algebras. By straightforward calculations and using a computer algebra system we obtain the following result. Volume 9 • Issue 1 • 1000221 Case of polynomials of degree one. We take α α α = = + = + + e ce e de e e he ge e 2 2 2 2 2 2 1 2 1 3 1 1 2 2 1 2 3 3 2 1 2 3 2 3 1 3 3 3 1 3 ( , ) , ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ), ( , ) ( ), α α α α α α µ µ µ µ µ µ = = = + + = + + = + = + e e ace e e bce e e a de e le e e b de e le e e a he e e e b he e (2) With 2 1 1 2 2 1 2 3 2 3 1 3 3 ( ) , ( ) , ( ) α α α = = + + = + e ce e de e le e he e e 3 3 3 3 3 3 2 1 1 3 1 1 2 2 1 2 3 3 2 1 2 3 2 2 3 1 2 3 3 3 1 2 3 ( , ) , ( , ) , ( , ) ( (1 ) ), ( , ) (1 ) , ( ) ( 1) ( , ) ( 1) ), ( , ) ( ) , α α α α α α µ µ µ µ µ µ = = = + + − = + + − − − = + − + = + + − e e ace e e bce a e e a de fe f e e e bde bfe a f e b a b ga b f e e ahe b f e e e e bhe e b ga e b a (3) With 3 1 1 3 2 1 2 3 3 3 1 2 3 ( ) ( ) , ( ) (1 ) , ( ) ( 1) α α α − = = + + − = + − + a b b ga e ce e de fe f e e he f e e b a b 4 4 4 4 4 4 1 2 1 2 3 1 2 1 3 1 3 2 1 2 3 2 2 1 2 3 3 1 2 3 ( , ) , ( , ) ( ), ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ), α α α α α α µ µ µ µ µ µ = = + = = + + = + = + + e e ace e e b de e e e bce e e a he ge e e e a de e e e b he ge e (4) With 3 2 2 3 2 1 1 2 3 2 2 3 3 4 1 2 3 2 1 ( , , ) , ( , , ) , ( , , ) + = + + = + = + + + b c a b a P x y z a x y a z P x y z b y b z P x y z c x c x c y z b b (2) 2 1 2 2 3 2 2 3 4 1 2 1 2 1 ( , , ) , ( , , ) , ( , , ) . Case of polynomials of degree one. We take = + + = = + + + P x y z a x a y a z P x y z b y P x y z c x c x c y z a b (3) 2 2 1 1 3 2 1 3 3 4 1 2 3 3 2 3 1 ( , , ) ( ) , ( , , ) , ( , , ) = − + = + = + + + a b P x y z x a z P x y z b x b z P x y z c x c x c y c z b c b (4) 2 1 3 2 3 3 4 1 2 3 3 2 1 ( , , ) , ( , , ) , ( , , ) . Case of polynomials of degree one. We take = − + = = + + + P x y z x a z P x y z b z P x y z c x c x c y c z b c (5) 2 1 1 3 2 1 3 4 1 3 1 3 1 ( , , ) , ( , , ) , ( , , ) = − + = = + + P x y z a x y a z P x y z b x P x y z c x c x c z b c (6) 2 3 2 1 1 3 2 1 3 4 1 2 3 1 3 3 1 ( , , ) , ( , , ) , ( , , ) − = + + + = = + + + a c P x y z a x y a z P x y z b x P x y z c x c x c y c z b c c (7) 2 1 1 2 2 1 3 4 1 2 1 2 1 ( , , ) , ( , , ) , ( , , ) = + + = = + + P x y z a x a y z P x y z b x P x y z c x c x c y b c (8) 2 3 1 1 1 2 3 2 3 3 1 3 2 3 (1 ) ( , , ) , ( , , ) , ( , , ) + = + + = + = + a b c P x y z a x a y a z P x y z x b z P x y z c x c z a c (9)i 3 2 2 3 2 1 1 2 3 2 2 3 3 4 1 2 3 2 1 ( , , ) , ( , , ) , ( , , ) + = + + = + = + + + b c a b a P x y z a x y a z P x y z b y b z P x y z c x c x c y z b b (2) 2 1 2 2 3 2 2 3 4 1 2 1 2 1 ( , , ) , ( , , ) , ( , , ) . Case of polynomials of degree one. We take = + + = = + + + P x y z a x a y a z P x y z b y P x y z c x c x c y z a b (3) 2 2 1 1 3 2 1 3 3 4 1 2 3 3 2 3 1 ( , , ) ( ) , ( , , ) , ( , , ) = − + = + = + + + a b P x y z x a z P x y z b x b z P x y z c x c x c y c z b c b (4) 2 1 3 2 3 3 4 1 2 3 3 2 1 ( , , ) , ( , , ) , ( , , ) . Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal With Any 3-dimensional ternary non-commutative Hom-Nambu-Poisson algebra ( ,{.,.,.} , , ) A α α µ α obtained by a twisting defined with respect to the basis {e1, e2, e3}=e1 by the ternary bracket 1 2 3 1 { , , } e e e ce α = , where c is a parameter, is one of the following binary Hom-associative algebras defined by i α µ and a corresponding structure map With 9 1 3 1 ( , ) , α µ = e e ace 1 1 1 1 1 1 2 1 1 3 1 1 2 2 1 2 3 2 1 2 2 3 1 2 3 3 3 1 2 3 ( , ) , ( , ) , ( , ) ( ), ( , ) ( ), ( , ) ( ), ( , ) ( ), α α α α α α µ µ µ µ µ µ = = = + = + = + + = + + e e ace e e bce e e a de e e e b de e e e a he ge e e e b he ge e (1) 1 1 2 1 1 2 2 1 2 ( , ) , ( , ) ( ), α α µ µ = = + e e ace e e a de e (9) (1) With With With Volume 9 • Issue 1 • 1000221 J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal J Generalized Lie Theory Appl ISSN: 1736-4337 GLTA, an open access journal Volume 9 • Issue 1 • 1000221 Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu-Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 Page 8 of 8 7. Makhlouf A, Silvestrov SD (2008) Hom-algebra structures. Journal of Generalized Lie Theory and Applications 2: 51-64. Citation: Makhlouf A, Amri A (2015) Non-Commutative Ternary Nambu- Poisson Algebras and Ternary Hom-Nambu-Poisson Algebras. J Generalized Lie Theory Appl 9: 221. doi:10.4172/1736-4337.1000221 References 1. Nambu Y (1973) Generalized hamiltonian mechanics. Phys Rev D 7: 2405- 2412. 8. Yau D (2010) Non-commutative Hom-poisson algebras. 9. Ammar F, Makhlouf A Silvestrov S (2010) Ternary q-virasoro-witt Hom-Nambu- Lie algebras. Journal of Physics A: Mathenmetical and Theoritical. 2. Dito G, Flato M, Sternheimer D, Takhtajan L (1997) Deformation quantization and Nambu mechanics. Commun Math Phys 183: 1-22. 10. Ammar A, Mabrouk S, Makhlouf A (2011) Representations and cohomology of n-ary multiplicative Hom-Nambu-Lie algebras. J Geom Physics 61: 1898-1913. 3. Takhtajan L (1994) On foundation of the generalized Nambu mechanics. Comm Math Phys 160: 295-315. 11. Arnlind J, Makhlouf A, Silvestrov S (2010) Ternary Hom-Nambu-Lie algebras induced by Hom-Lie algebras, J Math Phys. 4. Filippov VT (1985) n-Lie algebras. Siberian Math J 26: 879-891. 5. Ataguema H, Makhlouf A, Silvestrov S (2009) Generalization of n-ary Nambu algebras and beyond. J Math Phys. 12. Yau D (2012) On n-ary Hom-Nambu and Hom-Nambu-Lie algebras. J Geom Phys 62: 506-522. 6. Hartwig JT, Silvestrov DLS (2003) Deformations of lie algebras using σ-derivations. Journal of Algebra 295: 314-361. 13. Yau D (2010) A Hom-associative analogue of n-ary Hom-Nambu algebras. References Submit your next manuscript and get advantages of OMICS Group submissions Unique features: • User friendly/feasible website-translation of your paper to 50 world’s leading languages • Audio Version of published paper • Digital articles to share and explore Special features: • 400 Open Access Journals • 30,000 editorial team • 21 days rapid review process • Quality and quick editorial, review and publication processing • Indexing at PubMed (partial), Scopus, EBSCO, Index Copernicus and Google Scholar etc • Sharing Option: Social Networking Enabled • Authors, Reviewers and Editors rewarded with online Scientific Credits • Better discount for your subsequent articles Submit your manuscript at: http://www.omicsonline.org/submission Submit your next manuscript and get advantages of OMICS Group submissions Unique features: • User friendly/feasible website-translation of your paper to 50 world’s leading languages • Audio Version of published paper • Digital articles to share and explore Special features: • 400 Open Access Journals • 30,000 editorial team • 21 days rapid review process • Quality and quick editorial, review and publication processing • Indexing at PubMed (partial), Scopus, EBSCO, Index Copernicus and Google Scholar etc • Sharing Option: Social Networking Enabled • Authors, Reviewers and Editors rewarded with online Scientific Credits • Better discount for your subsequent articles Submit your manuscript at: http://www.omicsonline.org/submission Submit your next manuscript and get advantages of OMICS Group submissions Submit your next manuscript and get advantages of OMICS Group submissions Submit your manuscript at: http://www.omicsonline.org/submission Volume 9 • Issue 1 • 1000221
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Specificity of translocator protein-targeted positron emission tomography in inflammatory joint disease
EJNMMI research
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© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. Introduction Synovial envelope of the articular joint is a critical pro- vider of synovial fluid components and articular cartilage nutrients. Synovial inflammation (synovitis) together with progressive degeneration of articular cartilage is key pathological features in a variety of inflammatory joint diseases (IJDs), as diverse as rheumatoid (RA), psoriatic, juvenile and idiopathic arthritis, lupus and gout and a sig- nificant contributor of articular cartilage degeneration in osteoarthritis (OA) [1]. Synovial inflammatory infiltrate is composed of aggressive macrophage- and fibroblast-like l Detection and treatment of subclinical or early inflam- matory arthritis is likely to prevent disease progression, permanent joint damage and associated comorbidity. However, subclinical or early disease is often difficult to detect, leading to a delay in diagnosis [4]. Furthermore, the assessment of response to treatment of inflamma- tory disease is often based on composite disease activ- ity scores, which can be highly subjective and difficult to reproduce consistently. Therefore, an unmet clinical need is to functionally evaluate the target tissue and assess changes in the infiltrate not only for quantitative assess- ment but also to evaluate targeted therapies. *Correspondence: a.saleem@hull.ac.uk 3 Hull York Medical School, Allam Medical Building, University of Hull, Cottingham Road, Hull HU6 7RX, UK Full list of author information is available at the end of the article Positron emission tomography (PET) studies with the 18 KDa translocator protein (TSPO) radioligands have Abstract Objective:  Expression of the translocator protein (TSPO) on inflammatory cells has facilitated imaging of synovitis with TSPO-targeted positron emission tomography (PET). We aimed to quantitatively assess the specificity of the second-generation TSPO PET radioligand, ­[11C]PBR28, and to generate simplified PET protocols in patients with inflammatory joint disease (IJD) in this pilot study. Methods:  Three IJD patients (two rheumatoid arthritis and one osteoarthritis) with knee involvement underwent dynamic ­[11C]PBR28-PET scans before and after administration of 90 mg of oral emapunil (XBD-173), a TSPO ligand the same day. Radial arterial blood sampling was performed throughout the scan, and total radioactivity and radioactive metabolites were obtained. A semi-automated method was used to generate regions of interest. Standardized uptake value (SUV) and SUV ratio corrected for activity in bone and blood between 50 and 70 min ­(SUVr50–70 bone, ­SUVr50–70 blood, respectively) and PET volume of distribution (VT) of the radioligand were calculated. Results:  A mean ­[11C]PBR28 radioactivity of 378 (range 362–389) MBq was administered. A significant decrease (p < 0.05) in VT, ­SUVr50–70 bone and ­SUVr50–70 blood observed after oral emapunil confirmed the TSPO specificity of ­[11C]PBR28. A decrease in SUV was not observed in the post-block scan. Conclusion:  [11C]PBR28 is TSPO-specific radioligand in IJD patients. Simplified PET protocols with static PET acquisi- tion can be used in the management and evaluation of novel therapeutics that target TSPO overexpressing cells. mesenchymal cells, macrophage-like cells, fibroblast-like synoviocytes and other inflammatory cells [2, 3].l Helo et al. EJNMMI Res (2020) 10:147 https://doi.org/10.1186/s13550-020-00736-9 Helo et al. EJNMMI Res (2020) 10:147 https://doi.org/10.1186/s13550-020-00736-9 Open Access *Correspondence: a.saleem@hull.ac.uk 3 Hull York Medical School, Allam Medical Building, University of Hull, Cottingham Road, Hull HU6 7RX, UK Full list of author information is available at the end of the article Specificity of translocator protein‑targeted positron emission tomography in inflammatory joint disease suf Helo1, Graham E. Searle1, Federica Borghese2, Sonya Abraham2 and Azeem Saleem1,3* PET scanning 11 formed b knee was severity o toid arth Table 1  Demographics, disease extent, genotype and activity of ­[11C]PB Subject number (scan) Age, gender Disease Clinical disease 1 (Baseline) 57, F RA Right (moderate 1 (Post-block) 2 (Baseline) 56, F RA Bilateral (moder 2 (Post-block) 3 (Baseline) 50, F OA Mild left 3 (Post-block) Regulatory approval and subjectsh The study was approved by the West Midlands–Black Country research ethics committee (ref. 17/WM/0082), Integrated Research Application System (ref. 216737) and Administration of Radioactive Substances Advi- sory Committee (ref. 630/3925/36195) and conducted in accordance with the Declaration of Helsinki. The study was registered with the UK National Institute of Health Research (NIHR) Clinical Research Network (No. MUSC 33816). Three subjects (two subjects with RA and satisfy- ing the American College of Rheumatology criteria with active clinical disease in the knee and one subject with knee OA) were recruited (Table 1). Participants provided written informed consent, with eligibility determined by medical history, physical examination, coagulation screen and blood genotyping for rs6971single-nucleotide polymorphism [8]. Subjects with homozygous G allele (high-affinity binders; HABs) and heterozygous G/A allele at position 439 (Ala147Thr) (medium-affinity bind- ers; MABs) were only included in the study as they have higher binding to ­[11C]PBR28 [8]. Helo et al. EJNMMI Res (2020) 10:147 Page 2 of 6 demonstrated the high expression of TSPO (formerly known as the peripheral benzodiazepine receptor; PBR) on activated macrophages at sites of inflammation, and indeed, up-regulation of TSPO has been noted in mac- rophages of inflamed synovium of animal models of inflammatory arthritis [5]. Clinical TSPO-PET studies in healthy volunteers and subjects with rheumatoid and psoriatic arthritis have reported higher TSPO radioligand uptake in IJD subjects compared to healthy volunteers [6, 7]. However, to date there have been no clinical studies to confirm that the elevated PET signal can be attrib- uted to specific binding to TSPO in IJD subjects. In this manuscript, we report on a cohort of subjects with IJD who underwent dual ­[11C]PBR28-PET scans prior to and after a heterologous oral TSPO blocking agent, emapunil (XBD-173), to assess the specificity of TSPO binding in the knee joints of the subjects scanned. PET scanning 11 g [11C]PBR28 PET scanning was performed at Invicro, London, on a Siemens PET/CT system Biograph 6 TruePoint with TrueV or a Hi-Rez Biograph 6 (Siemens Healthcare, Erlangen, Germany). Prior to the scan, all patients had a radial arterial cannulation for sampling of blood during the scan and a venous cannula for adminis- tration of radioactivity. ­[11C]PBR28 was manufactured as described previously [9]. Initially, a low-dose CT scan of the both knee joints was performed for localization and attenuation correction followed by a 90-min dynamic PET scan after intravenous administration of ­[11C]PBR28. Continuous arterial blood sampling was performed for the initial 15 min; addition- ally, discrete blood samples were performed for the full duration of the scan to obtain total blood and plasma radioactivity and the fraction of parent radiolabelled compound in the blood. A second ­[11C]PBR28 PET-CT scan was performed the same day after oral administra- tion of the TSPO ligand. Emapunil (XBD-173) was given per orally about 2 h before the second scan. manuscript, we report on a cohort of subjects with IJD who underwent dual ­[11C]PBR28-PET scans prior to and after a heterologous oral TSPO blocking agent, emapunil (XBD-173), to assess the specificity of TSPO binding in the knee joints of the subjects scanned. Methods Regulatory approval and subjects The study was approved by the West Midlands–Black Country research ethics committee (ref. 17/WM/0082), Integrated Research Application System (ref. 216737) and Administration of Radioactive Substances Advi- sory Committee (ref. 630/3925/36195) and conducted in accordance with the Declaration of Helsinki. The study was registered with the UK National Institute of Health Research (NIHR) Clinical Research Network (No. MUSC 33816). Three subjects (two subjects with RA and satisfy- ing the American College of Rheumatology criteria with active clinical disease in the knee and one subject with knee OA) were recruited (Table 1). Participants provided written informed consent, with eligibility determined by medical history, physical examination, coagulation screen and blood genotyping for rs6971single-nucleotide polymorphism [8]. Subjects with homozygous G allele (high-affinity binders; HABs) and heterozygous G/A allele at position 439 (Ala147Thr) (medium-affinity bind- ers; MABs) were only included in the study as they have higher binding to ­[11C]PBR28 [8]. administ sampling ally, disc duration radioacti compoun scan was tion of th per orally Data ana PET ima for attenu acquired ing Infor images w Synovial generated based on bone and single RO the inter (Fig. 1). Data analysis PET images were reconstructed with corrections applied for attenuation, randoms and scatter. All PET-CT images acquired were converted to the NIfTI (Neuroimag- ing Informatics Technology Initiative) format, and CT images were re-sliced to match the PET image resolution. Synovial regions of interest (ROIs) for each knee were generated using a global threshold method on CT scan, based on the Hounsfield Unit values for bone to exclude bone and adjoining tendon and muscle. For each knee, a single ROI for each knee was generated to include all of the inter-osseous area exclusive of muscle and tendons (Fig. 1). Finally, a manual review of the ROIs was per- formed by one operator and reviewed by another. Each knee was analyzed separately due to the variation in the severity of joint disease with osteoarthritis and rheuma- toid arthritis, as done previously [6]. Table 1  Demographics, disease extent, genotype and activity of ­[11C]PBR28 administered for subjects imaged Subject number (scan) Age, gender Disease Clinical disease severity and site Genotype Activity injected (MBq) 1 (Baseline) 57, F RA Right (moderate) MAB 376.7 1 (Post-block) 382.5 2 (Baseline) 56, F RA Bilateral (moderate) Left > right HAB 389.3 2 (Post-block) 361.7 3 (Baseline) 50, F OA Mild left MAB 374.8 3 (Post-block) 380.8 raphics, disease extent, genotype and activity of ­[11C]PBR28 administered for subjects imaged Table 1  Demographics, disease extent, genotype and activity of ­[11C]PBR28 administered for subje Helo et al. EJNMMI Res (2020) 10:147 Page 3 of 6 Helo et al. EJNMMI Res PET-CT images converted to NIfTI format PET-CT image resliced to match PET image resolution Global thresholding of CT to generate ROI Manual review and correction of ROI SUV50-70 for ROIs calculated Kinetic modeling. VT and SUVr derived Fig. 1  Analysis methodology flowchart illustrating the semi-automated method where a global threshold method was used to define the synovial ROI (sagittal view, pink; top right) and used to calculate uptake parameters. The bone ROI (coronal view, blue; bottom right) is also illustrated PET-CT images converted to NIfTI format Fig. 1  Analysis methodology flowchart illustrating the semi-automated method where a global threshold method was used to define the synovial ROI (sagittal view, pink; top right) and used to calculate uptake parameters. The bone ROI (coronal view, blue; bottom right) is also illustrated Fig. Data analysis 2  Time–activity plots at baseline (top panel) and post-block (lower panel) depicted as orange (right knee) and blue (left knee) dots and the model fit as continuous lines to the output data using the 2TC model for all the subjects imaged Fig. 2  Time–activity plots at baseline (top panel) and post-block (lower panel) depicted as orange (right knee) and blue (left knee) dots and the model fit as continuous lines to the output data using the 2TC model for all the subjects imaged Helo et al. EJNMMI Res (2020) 10:147 Page 4 of 6 Page 4 of 6 Tissue time–activity curves (TAC) were generated [10] as previously described for each joint synovia (Fig. 2). Radial arterial blood data obtained were used to generate plasma ­[11C]PBR28 TAC for the full duration of the scan (input function) and was modeled with the tissue data (output) (Fig. 3) to generate volume of distribution (VT) values using a two-tissue compartment model (2TC) as described previously [11, 12]. knee joints, with minimal uptake in the adjoining bone. Review of the TACs (Fig. 2) demonstrated a pla- teauing in ­[11C]PBR28 SUV between 50- and 70-min post-radioligand injection; hence, SUV and SUVr were calculated between these time points for all subjects. Variability of radioactivity uptake between the two knees post-block was minimal compared to pre-block uptake. VT was obtained in all subjects by fitting the 2TC model, apart from one subject (subject 3, Base- line), where the model did not fit the data well.h Semiquantitative uptake values (SUV) were calcu- lated in the synovium between 50 and 70 min ­(SUV50–70) and also normalized to a tissue (bone or blood) uptake between 50 and 70 min ­(SUVr50–70 blood and ­SUVr50–70 bone). i There was no change in mean (SD) ­SUV50–70 after oral XBD-173 (0.65 (0.21) versus 0.69 (0.13); p = NS). In contrast, PET parameters that account for peripheral distribution of the radioligand such as VT, ­SUVr50–70 blood and ­SUVr50–70 bone showed a significant decrease in radioligand uptake in the synovium with mean (SD) respective pre- and post-block ligand uptake for VT being (4.84 (0.54) versus 2.69 (0.80); p < 0.05), for ­SUVr50–70 blood (3.67 (1.29) versus 2.22 (0.32); p < 0.05) and for ­SUVr50–70 bone (3.12 (1.03) versus 2.10 (0.20); p < 0.05). Individual uptake values for all subjects are provided in Table  2. Figure  4 illustrates a decrease in ­[11C]PBR28 uptake in subject 2 after normalization Statistical analysis Paired t tests were used to compare VT, SUV or SUVr values within subjects. p values less than 0.05 were con- sidered statistically significant. PET analysish The composite PET image for the full duration of the scan showed radioactivity uptake in the synovia of Fig. 3  The percentage of parent ­[11C]PBR28 contributing to the total radioactivity (a) and the plasma over blood ratio (b) in arterial blood that were used to generate the plasma ­[11C]PBR28 input function for the full duration of the scan (c) for subject 1 at baseline and post-block Fig. 3  The percentage of parent ­[11C]PBR28 contributing to the total radioactivity (a) and the plasma over blood ratio (b) in arterial blood that were used to generate the plasma ­[11C]PBR28 input function for the full duration of the scan (c) for subject 1 at baseline and post-block Table 2  Uptake parameters for all the subjects imaged Subject no. (scan) SUV SUVr50–70 blood SUVr50–70 bone VT Left Right Left Right Left Right Left Right 1 (Baseline) 0.37 0.46 2.25 2.81 2.14 2.70 5.19 4.32 1 (Post-block) 0.52 0.54 1.90 1.97 1.87 1.91 3.85 2.60 2 (Baseline) 0.92 0.84 5.49 5.01 4.44 4.42 5.40 4.44 2 (Post-block) 0.82 0.80 2.66 2.59 2.36 2.31 2.14 2.18 3 (Baseline) 0.64 0.68 3.13 3.33 2.45 2.60 ND ND 3 (Post-block) 0.75 0.73 2.13 2.08 2.07 2.10 1.91 1.44 Table 2  Uptake parameters for all the subjects imaged Helo et al. EJNMMI Res (2020) 10:147 Page 5 of 6 Helo et al. EJNMMI Res Fig. 4  Trans-axial (top), coronal (middle) and sagittal (bottom) images of ­[11C]PBR28 uptake normalized for blood activity ­(SUVr50–70 blood) in subject 2 shows synovial uptake at baseline (PET1 baseline) that is reduced after administration of oral emapunil (PET2 post-block) to blood activity ­(SUVr50–70 blood) after dosing with emapunil. both blood and cancellous bone, both of which contain TSPO [15, 16] as pseudo-reference region as an estimate specific ­[11C]PBR28 tissue uptake. We found that like VT, a decrease in ­SUVr50–70 blood and ­SUVr50–70 bone was observed after TSPO blocking with XBD-173 was noted with a decrease, indicating that both ­SUVr50–70 blood and ­SUVr50–70 bone could be used to quantify ­[11C]PBR28 uptake. We also confirmed that a static ­[11C]PBR28 scan between 50 and 70 min, correcting for nonspecific bind- ing using bone SUV ­(SUVr50–70 bone) as a pseudo-ref- erence region, allows quantification of TSPO expression with ­[11C]PBR28. Discussion We conducted this study to evaluate if the increased uptake of ­[11C]PBR28 observed in the joints of patients with IJD was specific to TSPO binding and not a conse- quence of increased radioactivity in joints due to other associated pathological processes. The decrease in the ­[11C]PBR28 quantitative uptake parameter, VT, after administration of a heterologous TSPO blocking agent confirmed that the increase in ­[11C]PBR28 uptake was specific to TSPO in the tissue cellular infiltrate. How- ever, performing dynamic PET imaging with radial arte- rial sampling may not always be appropriate as the small joints of the wrist are commonly involved in RA and need to be imaged. Further, performing scans of shorter duration without arterial blood sampling would increase subject tolerability. Since the semiquantitative param- eter SUV, which does not account for changes in periph- eral metabolism of the radioligand [13], did not exhibit a decrease in uptake after blocking of TSPO, we investi- gated if we could use a pseudo-reference agent as in the brain where the cerebellum has been used [14]. We used PET analysish This is likely to help facilitate PET stud- ies that provide better subject comfort to evaluate disease and TSPO-targeting therapy and also allow assessment of disease in the small joints of the hand in subjects with IJD. Author details 1 16. Sarubin N, Baghai TC, Lima-Ojeda JM, Melchner D, Hallof-Buestrich H, Wolf L, et al. Translocator protein (TSPO) expression in platelets of depressed patients decreases during antidepressant therapy. Pharma- copsychiatry. 2016;49(5):204–9. 1 Invicro, A Konica Minolta Company, Burlington Danes Building, Ham- mersmith Hospital, Du Cane Road, London W12 0NN, UK. 2 Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. 3 Hull York Medical School, Allam Medical Building, University of Hull, Cottingham Road, Hull HU6 7RX, UK. Conclusion In conclusion, we observed that ­[11C]PBR28 behaves as a TSPO-specific radioligand in the knee joint synovia of patients with IJD. PET uptake of ­[11C]PBR28 can also be quantified using simplified acquisition of static PET scans, likely to be suitable in the evaluation of novel ther- apeutics that target TSPO overexpressing cells in IJD. Page 6 of 6 Helo et al. EJNMMI Res (2020) 10:147 2. Furuzawa-Carballeda J, Macip-Rodriguez PM, Cabral AR. Osteoarthritis and rheumatoid arthritis pannus have similar qualitative metabolic char- acteristics and pro-inflammatory cytokine response. Clin Exp Rheumatol. 2008;26(4):554–60. Availability of data and materials 11. Gunn RN, Gunn SR, Cunningham VJ. Positron emission tomography compartmental models. J Cereb Blood Flow Metab. 2001;21(6):635–52. Supporting data will be available on request. Competing interests 15. Kam WW, Meikle SR, Zhou H, Zheng Y, Blair JM, Seibel M, et al. The 18 kDa translocator protein (peripheral benzodiazepine receptor) expression in the bone of normal, osteoprotegerin or low calcium diet treated mice. PLoS ONE. 2012;7(1):e30623. GES is an employee, and AS and YH are ex-employees of Invicro, which funded and sponsored the study. Abbreviationsfi HAB: High-affinity binders; IJD: Inflammatory joint disease; OA: Osteoarthritis; MAB: Medium-affinity binders; NIfTI: Neuroimaging informatics technology ini- tiative; NIHR: National Institute of Health Research; PBR: Peripheral benzodiaz- epine receptor; PET: Positron emission tomography; RA: Rheumatoid arthritis; ROI: Region of interest; SUV: Standardized uptake value; SUVr: Standardized uptake value ratio; TAC​: Time–activity curves; TSPO: Translocator protein; VT: Volume of distribution of a radioligand; 2TC: Two-tissue compartment. 3. Allard SA, Maini RN, Muirden KD. Cells and matrix expressing cartilage components in fibroblastic tissue in rheumatoid pannus. Scand J Rheu- matol Suppl. 1988;76:125–9.i 4. Brown AK, Wakefield RJ, Conaghan PG, Karim Z, O’Connor PJ, Emery P. New approaches to imaging early inflammatory arthritis. Clin Exp Rheu- matol. 2004;22(5 Suppl 35):S18-25. Acknowledgements 5. Gent YY, Weijers K, Molthoff CF, Windhorst AD, Huisman MC, Kassiou M, et al. Promising potential of new generation translocator protein tracers providing enhanced contrast of arthritis imaging by positron emission tomography in a rat model of arthritis. Arthritis Res Ther. 2014;16(2):R70. The authors would like to express their gratitude to all patients who partici- pated in this study; Yvonne Shearley, study coordinator, Invicro; Aneta Gupta, study coordinator, Imperial College London; and Ryan Janisch, PET technolo- gist and clinical staff at Invicro and Imperial College London who facilitated the completion of the study. We would also like to thank Dr David Owen, Imperial College London, for providing emapunil (XBD-173). Infrastructure support for this study was provided by the NIHR Imperial Biomedical Research Centre and the NIHR Imperial Clinical Research Facility. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. 6. Narayan N, Owen DR, Mandhair H, Smyth E, Carlucci F, Saleem A, et al. Translocator protein as an imaging marker of macrophage and stromal activation in rheumatoid arthritis pannus. J Nucl Med. 2018;59(7):1125–32. 7. Gent YY, Ter Wee MM, Voskuyl AE, den Uyl D, Ahmadi N, Dowling C, et al. Subclinical synovitis detected by macrophage PET, but not MRI, is related to short-term flare of clinical disease activity in early RA patients: an exploratory study. Arthritis Res Ther. 2015;17:266. Authors’ contributions 8. Owen DR, Yeo AJ, Gunn RN, Song K, Wadsworth G, Lewis A, et al. An 18-kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28. J Cereb Blood Flow Metab. 2012;32(1):1–5. YH contributed to data analysis and interpretation and manuscript prepara- tion. GES performed study conception, data analysis and interpretation, and manuscript preparation. FB and SA collected and interpreted the data and reviewed the manuscript. AS helped in study conception, data collection, analysis and interpretation, and manuscript preparation. All authors read and approved the final manuscript. 9. Owen DR, Guo Q, Kalk NJ, Colasanti A, Kalogiannopoulou D, Dim- ber R, et al. Determination of [(11)C]PBR28 binding potential in vivo: a first human TSPO blocking study. J Cereb Blood Flow Metab. 2014;34(6):989–94. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Received: 3 August 2020 Accepted: 23 November 2020 Received: 3 August 2020 Accepted: 23 November 2020 Received: 3 August 2020 Accepted: 23 November 2020 Ethical approval and consent to participate 12. Innis RB, Cunningham VJ, Delforge J, Fujita M, Gjedde A, Gunn RN, et al. Consensus nomenclature for in vivo imaging of reversibly binding radioli- gands. J Cereb Blood Flow Metab. 2007;27(9):1533–9. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All participants provided writ- ten informed consent to take part in the study. 13. Woodcock EA, Schain M, Cosgrove KP, Hillmer AT. Quantification of ­[(11)C] PBR28 data after systemic lipopolysaccharide challenge. EJNMMI Res. 2020;10(1):19. 14. Lyoo CH, Ikawa M, Liow JS, Zoghbi SS, Morse CL, Pike VW, et al. Cerebel- lum can serve as a pseudo-reference region in alzheimer disease to detect neuroinflammation measured with PET radioligand binding to translocator protein. J Nucl Med. 2015;56(5):701–6. Funding 10. Saleem A, Searle GE, Kenny LM, Huiban M, Kozlowski K, Waldman AD, et al. Lapatinib access into normal brain and brain metastases in patients with Her-2 overexpressing breast cancer. EJNMMI Res. 2015;5:30. This work was supported and funded by Imanova (trading as Invicro LLC). Consent for publication All authors provide permit publication of the manuscript. 1. Bhattaram P, Chandrasekharan U. The joint synovium: a critical determi- nant of articular cartilage fate in inflammatory joint diseases. Semin Cell Dev Biol. 2017;62:86–93. References 1. Bhattaram P, Chandrasekharan U. The joint synovium: a critical determi- nant of articular cartilage fate in inflammatory joint diseases. Semin Cell Dev Biol. 2017;62:86–93.
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THE PERILS OF DOING BUSINESS ACROSS BORDERS
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BOOK REVIEWS DOI: http://dx.doi.org/10.1590/S0034-759020170610 RAE-Revista de Administração de Empresas | FGV EAESP RAE-Revista de Administração de Empresas | FGV EAESP THE PERILS OF DOING BUSINESS ACROSS BORDERS GUIDE TO COUNTRY RISK: How to identify, manage and mitigate the risks of doing business across borders. Mina Toksöz. London: Economist Books, 2014. 288 p. Mina Toksöz. London: Economist Books, 2014. 288 p. The reestablishment of the world’s economic center to Asia (China was the world’s largest economy until around 1870) has many positives. In addition to lifting hundreds of millions of people out of poverty in China and the neighboring region, growth in Asia helped mitigate the effects of the global financial crisis in 2008. Today, China and India are key drivers of the global economy with over a hundred countries listing the middle kingdom as their most important trading partner. Along with the economic revival in Asia, unprecedented global interconnectedness has also raised the level of risk exposure to virtually all businesses around the world. One example is North Korea: while an unpredictable hermit kingdom on the other side of the world would hardly have concerned Brazil’s Embraer and Vale in the early 1990s, Kim Jong Un’s current nuclear overture could have the potential to create geopolitical instability in North-east Asia, including China, an essential market to both companies. By OLIVER STUENKEL oliver.stuenkel@fgv.br Professor at Fundação Getulio Vargas, Escola de Relações Internacionais – São Paulo – SP, Brazil Country risk, which analyzes risk when conducting business across borders, also seems to be on the rise in developed countries. The rise of xenophobia and nationalist politics in Europe, Brexit, and the possibility of Donald Trump winning the U.S. presidential election all represent potential and important risks for investors in the global economy. Larry Summers recently wrote, Even the possibility of Trump becoming president is dangerous. The economy is already growing at a sub-two percent rate in substantial part because of a lack of confidence in a weak world economy. A growing sense that a protectionist demagogue could soon become President of the United States would surely introduce great uncertainty at home and abroad. The resulting increase in risk premiums might well be enough to tip a fragile US economy into recession. And a concern that the US was becoming protectionist and isolationist could easily undermine confidence in many emerging markets and set off a financial crisis. Even the possibility of Trump becoming president is dangerous. The economy is already growing at a sub-two percent rate in substantial part because of a lack of confidence in a weak world economy. THE PERILS OF DOING BUSINESS ACROSS BORDERS Indeed, many executives believe they are only rarely affected by political events that dominate the news, such as the civil wars in Syria and Iraq, Russia’s annexation of Crimea, or tensions between China and Japan. And yet, political risk covers far more than the risk of violent conflict in specific markets. In addition to geopolitical risk, the risk of terrorism, expropriation, corruption and changing regulatory policies can affect companies acting internationally. It is a useful resource for practitioners, though not necessarily for scholars, as Toksöz provides essential theoretical background and only a few references to current work on the subject. At the same time, she does not offer any overall guiding arguments as would an academic analysis. Given its manual-style format the Guide to Country Risk is written in a relatively dry and technical style and only those readers with a great interest in the topic will likely read to the end. Yet, the book presents many positives. What is perhaps most striking about Toksöz’s book is that, contrary to the current popular gloom about international political events, it lists many tangible initiatives that have reduced risk factors for international investors. For example, global transparency standards are higher today than before and anti-corruption enforcement makes prosecution more likely, as in Brazil, where post-9/11 banking laws helped the Brazilian Federal Police uncover the largest corruption scheme in the country’s history. However, the author does highlight growing concerns about the European Union (EU) Troika’s inability to handle the Greek crisis and suggests that future crises could further damage the EU. She also points out that understanding political risk analysis and the dynamics of supranational entities has become more important than ever. An interesting and detailed introduction of these themes, together with accessible analysis that explains the many facets of political risk analysis, has been written by Mina Toksöz, former head of Country Risk at Standard Bank International. It includes excellent descriptive examples of regulatory risks (including tax increases on mining corporations in Australia and local content rules for oil exploration in Brazil), convertibility risks (increasingly complex profit repatriation policy in Venezuela), and domestic political risks, such as the real possibility of Donald Trump winning the U.S. presidential election. THE PERILS OF DOING BUSINESS ACROSS BORDERS A growing sense that a protectionist demagogue could soon become President of the United States would surely introduce great uncertainty at home and abroad. The resulting increase in risk premiums might well be enough to tip a fragile US economy into recession. And a concern that the US was becoming protectionist and isolationist could easily undermine confidence in many emerging markets and set off a financial crisis. OLIVER STUENKEL oliver.stuenkel@fgv.br Professor at Fundação Getulio Vargas, Escola de Relações Internacionais – São Paulo – SP, Brazil ISSN 0034-7590 632 | © RAE | São Paulo | V. 57 | n. 6 | nov-dez 2017 | 632-633 BOOK REVIEWS | THE PERILS OF DOING BUSINESS ACROSS BORDERS BOOK REVIEWS | THE PERILS OF DOING BUSINESS ACROSS BORDERS Oliver Stuenkel Oliver Stuenkel Despite this new reality, political scientists often complain that internationally-operating companies make key investment decisions purely based on economic data alone while neglecting political risk. Executives generally believe it is too difficult to quantify political risk, and is usually left out of the equation altogether, or considered informally on a gut feeling of the situation. for populist leaders such as Julius Malema, thus increasing the risk of expropriation and a general decline in South Africa’s business environment. Unlike many available books on international affairs or risk analysis, Toksöz provides step-by-step instructions on how to assemble an actual model to assess country risk. This makes the book more of a hands-on guidebook for people working in the field than an abstract discussion of international affairs for the interested non-specialist. This book is no substitute for seeking the advice of political risk analysts to enable a company to make sense of dealing with broad political trends. However, it can be a first step for an organization that is beginning to think about risk analysis in the international business environment. An extract from Ian Bremmer and Preston Keat’s popular book on political risk, The Fat Tail, highlights this sentiment. A recent survey of executives on risk management in the financial services industry revealed that political risk was considered the least likely of all risk categories to be managed well. Geopolitical risk was also perceived as least likely to impact a corporation, and thus least likely to be included in a company’s risk management planning. 633 | © RAE | São Paulo | V. 57 | n. 6 | nov-dez 2017 | 632-633 THE PERILS OF DOING BUSINESS ACROSS BORDERS The Guide to Country Risk is a useful handbook for anyone wanting to establish a political risk analysis unit and provides remarkably detailed advice on specifics like political risk insurance and many interesting international case studies. The book also contains an extensive list of examples where many companies successfully dealt with political risk by using scenario- building exercises to develop alternative business plans. Toksöz explains clearly how macroeconomic trends affect industries in various ways and often with unexpected consequences. For example, the end of the commodity boom led to massive layoffs in South Africa’s mining sector and increased the risk of social unrest, which in turn led to stronger support 633 | © RAE | São Paulo | V. 57 | n. 6 | nov-dez 2017 | 632-633 ISSN 0034-7590
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Total versus near-total thyroidectomy in Graves’ disease and their outcome on postoperative transient hypoparathyroidism: study protocol for a randomized controlled trial?
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Open Access Open Access Total versus near-total thyroidectomy in Graves’ disease and their outcome on postoperative transient hypoparathyroidism: study protocol for a randomized controlled trial? Total versus near-total thyroidectomy in Graves’ disease and their outcome on postoperative transient hypoparathyroidism: study protocol for a randomized controlled trial? Katja Maschuw1*, Katja Schlosser1, Dirk Lubbe2, Christoph Nies3 and Detlef Klaus Bartsch3 © 2012 Maschuw et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 TRIALS Maschuw et al. Trials 2012, 13:234 * Correspondence: maschuw@med.uni-marburg.de 1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Giessen and Marburg GmbH Location Marburg, Baldingerstrasse, Marburg D-35043, Germany Full list of author information is available at the end of the article Abstract Background: Graves’ disease is an independent risk factor for transient postoperative hypoparathyroidism. Besides the disease itself, preparation techniques are influential. Transient postoperative hypoparathyroidism has severe consequences for patients’ physical and psychological state. It can be life threatening during the acute phase and may impair patients’ health, psyche and quality of life thereafter. For the surgical therapy of Graves’ disease, total thyroidectomy is recommended according to the national S2-guideline. The evidence- based on a metaanalysis- is criticized by the Cochrane diagnostic review commentary for substantial methodological deficits. Two randomized controlled trials lead to the hypothesis that a near-total resection with bilateral remnants of ≤1g on each side compared to total thyroidectomy will significantly reduce the occurrence of transient postoperative hypoparathyroidism with equal therapeutic safety. Methods/Design: Patients with Graves’ disease indicated for definite surgery are eligible for the trial. Trial-specific exclusion criteria are: conservative treatment, malignancy, previous thyroid surgery and coincident hypoparathyroidism. The trial is created for therapeutic purpose through process innovation. It is designed as a prospective randomized controlled patient and observer blinded multicentered trial in a parallel design including an active comparator and an intervention group. The intervention addresses the surgical procedure: near-total thyroidectomy leaving bilateral remnants of ≤1g on each side in the intervention group and total thyroidectomy in the control group. The occurrence of transient postoperative hypoparathyroidism is defined as primary endpoint. Secondary endpoints are: reoperations due to bleeding, recurrent laryngeal nerve palsy, permanent hypoparathyroidism, recurrent disease, changes of endocrine orbitopathy and quality of life within a one-year follow-up period. The primary efficacy analysis follows the intention-to-treat principle. A binary logistic regression model will be applied. Complications and serious adverse events will be descriptively analyzed. Discussion: The trail is expected to balance out the shortcomings of the current evidence. It will define the surgical gold standard for the surgical therapy of Graves’ disease. Patients’ safety and quality of life are assumed to be enhanced. Therapy costs are likely to be reduced and health care optimized. The conduction of the trial is feasible through the engagement and commitment of the German association of endocrine surgeons and the National Network for Surgical Trials. Background pyramidal lobes in all patients as the result of a systematic default. Two prospective randomized controlled trials by Witte et al., 2000 [2] and Barczynski et al., 2012 [8] again aimed at recurrence of Graves’ disease as the primary end- point, addressing different remaining tissue volumes and resection techniques. The trial by Witte et al. compared two interventional arms: one arm comprised a unilateral total resection and a contralateral subtotal resection with a remaining remnant of ≤4 g; the second arm comprised a bilateral subtotal resection with a remaining remnant of ≤4 g in total to the control arm total thyroidectomy. Barczynski et al. considered bilateral remnants of approxi- mately 2 g on each side. Both trials displayed a signifi- cantly lower incidence of transient hypoparathyroidism in the intervention groups. While the incidence of recurrent disease was not significant, clinically not relevant, and de- tectable in all affected patients six months after surgery, after a follow up of 18 to 58 months in the trial by Witte et al., Barczynski et al. reported significantly higher re- lapse rates within 60 months follow up. Endocrine orbito- pathy did not worsen after subtotal resection in both trials. So far, the evidence from the prospective rando- mized controlled trials seems contradictory regarding the incidence of clinically relevant recurrent disease. More- over, the S2-recommendation lacks appropriate evidence in view of comorbidities. According to our knowledge, the incidence of transient hypoparathyroidism has never been valued as primary endpoint. Current data suggest a rele- vant reduction of the incidence of transient hypoparathyr- oidism after a near-total resection if defined remnants of ≤1 g on each side are left around the posterior suspensory ligament of Berry (Ligamentum thryoihyoideum laterale). The resection will be safe compared to standard total thyr- oidectomy regarding recurrent disease, endocrine orbito- pathy and method associated complications [2,9,13]. A prospective randomized controlled trial is necessary to de- fine the gold standard resection for the surgical therapy of Graves’ disease in view of patients’ safety and quality of health care. Graves’ disease has been identified as an independent risk factor for transient postoperative hypoparathyroi- dism in retrospective trials. It was confirmed by a compre- hensive prospective study including 5,846 consecutive patients in 2003 [1]. In the only existing randomized controlled trial, transient hypoparathyroidism is reported with an incidence of 28% after total thyroidectomy for Graves’ disease [2]. Background The estimated prevalence is calcu- lated with 2,000 patients per year according to the available data [2-4]. The underlying causes are com- plex. Preparation techniques, especially the extent of resection seem to be more influential than inadvertent parathyroidectomy [1,5]. Postoperative hypoparathyr- oidism seriously affects physical health, wellbeing and quality of life. Acute hypocalcaemia can cause cram- ping, bronchial spasms, cardiac and digestive dysfunc- tion and can therefore be life threatening. Long-term effects result in cataract, conjunctivitis, pruritus, osteo- penia, calcifications of the basal ganglia (Fahr’s syn- drome), neurological and psychic impairments. Despite calcium and vitamin D substitution, wellbeing is pre- dominantly impaired by anxious and phobic psycho- logical disorders [6]. Care and treatment costs as well as the status of employee’s illness are therefore of eco- nomic relevance. At present, total thyroidectomy is favored for definite surgery in Graves’ disease instead of subtotal resection, according to the national S2-guideline issued by the Work Group of the Scientific Medical Professional Societies (AWMF) [7]. The current evidence supporting a total thyroidectomy in Graves’ disease is based on two rando- mized controlled trials [2,8], one meta-analysis [9] and four retrospective trials [10-13]. All studies evaluate the outcome after total versus subtotal thyroidectomy, in re- lation to the incidence of recurrent disease. The meta- analysis, cited in the national S2-guideline, displayed a zero-recurrence rate and an equal risk of method-asso- ciated complications [9]. However, the corresponding Co- chrane diagnostic review commentary emphasizes group inhomogeneity as a substantial methodological deficit. Four retrospective trials [10-13] demonstrated a signifi- cantly lower incidence of transient hypoparathyroidism after subtotal, compared to total resections, while the inci- dence of recurrent disease differed according to variable sizes of the remnants (mean 6.1 g, range 1 to 12 g) and follow-up periods. Among these four trials only one clearly defined the size of the remnant with a unilateral re- sidual thyroid tissue of ≤2 g when a subtotal resection was performed [13]. With this measure, the incidence of hypoparathyroidism significantly decreased, while the inci- dence of recurrent disease remained unaltered within a median follow-up of 6.7 years. The recurrence rate of 0.5% in this study was attributable to inadvertently spared Abstract Trial registration: German clinical trials register (DRKS) DRKS00004161 Keywords: Graves’ disease, Transient postoperative hypoparathyroidism, Total thyroidectomy, Near-total thyroidectomy * Correspondence: maschuw@med.uni-marburg.de 1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Giessen and Marburg GmbH Location Marburg, Baldingerstrasse, Marburg D-35043, Germany Full list of author information is available at the end of the article Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Page 2 of 5 Inclusion criteria group, the reason will be documented on the case report form. Required by the intention-to-treat principle, patients must be followed up in the group to which they are randomized. Key inclusion criteria are: indication for definite surgery according to the national guideline, no history of previ- ous thyroid and/or parathyroid surgery, normal vocal cord function, age greater than 18 years, expectancy of life greater than 12 months, and provision of informed consent. Methods/Design Eligibility Patients with Graves’ disease scheduled for definite sur- gery are eligible for the present study. Indications for definite surgery follow those of the national S2-guideline [7], in detail a high risk of recurrence (age below 40 years, male gender, thyroidea receptor anti-body (TRAK) serum levels > 10 U/l six months after conservative treatment, thyroid volume > 40 ml), persistent, recurrent or severe hyperthyroidism after primarily conservative therapy, thyroid growth, endocrine orbitopathy and refused radioactive iodine therapy. After a screening visit patients will be allocated into the trial according to the inclusion and exclusion criteria shown below. Page 3 of 5 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Secondary endpoints S d d i y p Secondary endpoints comprise the incidences of per- manent hypoparathyroidism, recurrent Graves’ disease, temporary and permanent recurrent laryngeal nerve palsy and reoperations due to bleeding, and the number of inadvertently removed parathyroid glands, as well as changes of endocrine orbitopathies and quality of life. Permanent hypoparythroidism is defined as persisting inadequate parathyroid function exceeding six months after surgery [15,16]. The incidence of recurrent Graves’ disease is assessed through laboratory thyroid function tests as thyroid stimulating hormone (TSH) < 0.34 U/l, free triiodinethyronine (fT3) > 6.5 pmol/l, thyroxine (fT4) > 21 pmol/l, and TRAK > 1.75 U/l, at 6 weeks, 6 months and 12 months after surgery, with respect to concomitant substitution or thyrostatic medications to achieve euthyrosis, determined by laboratory thyroid function tests (TSH, fT3, fT4). Cervical ultrasound fol- lows detection of elevated antibody levels to screen for recurrence. Reoperations are reported as serious adverse events. Incidences of temporary and permanent Definition of endpoints and outcome measures Primary endpoint The incidence of transient hypoparathyroidism is defined as the primary endpoint. Transient hypopara- thyroidism is defined as an inadequate parathyroid func- tion not exceeding six months after surgery [14]. Serum calcium (Ca) and parathyroid hormone (PTH) levels and substitution medication to achieve normocalcaemia are used to assess hypoparathyroidism. According to the best of our knowledge, there is no evidence- or consensus-based definition of postoperative hypopara- thyroidism based on calcium and PTH cutoff levels. In line with most experts, we define postoperative hypo- parathyroidism as calcium and PTH levels below the normal range (Ca ≤2.1 mmol/l, PTH < 11 ng/l). Post- operative hypoparathyroidism is considered to be symp- tomatic in the case of at least one of the following symptoms: carpo-pedal paresthesia, numbness or spasms, and anxiety. Application type, form and dosage of Ca and vitamin D substitution therapy to achieve nor- mocalcaemia (Ca = 2.2 to 2.7 mmol/l) are registered. Postoperative hypoparathyroidism is defined transient if calcium and vitamin-D substitution therapy to achieve normocalcaemia are required for less than six months after surgery. The parameters are recorded before sur- gery, at the day of discharge, 6 weeks, 6 months and 12 months after surgery. Exclusion criteria Exclusion criteria are: eligibility for conservative treat- ment according to the national guideline, (suspected) malignancy, coincident hyperparathyroidism, and neuro- physiological deficiencies. Intervention and control The study is designed as a prospective, randomized, con- trolled, observer- and patient-blinded, multicentered clinical trial in a parallel design, including an interven- tion group and an active comparator. The intervention addresses the surgical procedure. It is defined as near- total thyroidectomy with bilateral remnants ≤1 g on each side for inclusion in the intervention group. After ligation of the upper pole vessels, and identification and preservation of the recurrent laryngeal nerve and para- thyroid glands, the thyroid is removed from the lateral tracheal wall up to the superior suspensory ligament of Berry (ligamentum thyreohyoideum laterale). Here, the thyroid is resected leaving a piece of tissue assessed by ruler measurement accounting for 0.5 × 0.5 × 0.5 cm in size at most (≤1 g). After resection, the remnant in situ is photo documented. To ensure adequate sizes of the thyroid remnants in the intervention group, after re- moval of the specimen a representative piece of thyroid tissue (≤1 g) is excised, photo documented on a clean towel next to the ruler and weighed. This tissue frag- ment is later added to the specimen and both are sent for histopathology. Surgery in the control group com- prises a total thyroidectomy where the whole thyroid gland must be removed [7]. The surgical sites of each side will be photo documented after resection. Analysis recurrent laryngeal nerve palsies are assessed through laryngoscopy on the day of discharge and in the case of pathological findings 6 weeks, 6 months and 12 months after surgery. The number of inadvertently removed parathyroid glands will be counted from histopathology reports at the day of discharge. Changes in endocrine orbitopathy are measured using the validated clinical eyelid, exophthalmos eye muscle, optic nerve (LEMO) score [17] before and 12 months after surgery. Changes in quality of life are scored before surgery and 6 weeks thereafter using the validated multipurpose question- naire, the clinical studies 36-item medical outcomes study short-form general health survey (SF36) [18]. The primary hypothesis is whether the rate of transient hypoparathyroidism within 6 months after surgery is lower in patients after near-total compared to total thyr- oidectomy. A binary logistic regression model will be applied for the intervention comparison of the rates, adjusting for age, gender, hyperthyrosis and endocrine orbitopathy as additive covariates. The dropout rate can be assumed to be small, and therefore, few missing values are expected with respect to the primary end- point. If missing values occur their reason of omission will be explored. Depending on the properties and mag- nitude of missing values, an appropriate estimation will be specified before the analysis of the primary endpoint. Concerning other secondary endpoints, exploratory data analysis will be performed calculating appropriate sum- mary measures for the empirical distribution as well as calculation of descriptive two-sided 95% confidence intervals and P-values. Sensitivity analyses will be con- ducted for the per-protocol population as well as for appropriate subgroups. The safety analysis includes cal- culation and comparison of frequencies and rates of adverse and serious adverse events. Furthermore, statis- tical methods are used to assess the quality of data and the homogeneity of intervention groups. Randomization, allocation concealment and blinding A central randomization will be performed, stratified by the performing surgeons. Only surgeons who have per- formed at least 100 thyroidectomies working at a volume center accounting for more than 100 thyroidectomies per year will participate. A random sequence will be generated through computerized random assignment and concealed allocation. Patients and the observer are blinded to the applied surgical intervention. Intraopera- tive randomization will be performed. The surgeon will be unblinded after the skin incision, and the patient and the observer will be blinded throughout the whole study period. Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Page 4 of 5 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Analysis All assessments will be performed by personnel who are blinded to the study arms for the minimization of measurement bias. Data storage and analyses will be performed externally. Discussion Th S2 The S2-recommendation of total thyroidectomy for Graves’ disease based on the national guideline lacks appropriate evidence in view of morbidity. Total thyroi- dectomy compared to subtotal or near-total resection predisposes patients to a relevantly higher risk for transi- ent postoperative hypoparathyroidism. The consequen- ces of transient postoperative hypoparathyroidism can be severe and life threatening during the acute phase and may impair patient’s psychological state and quality of life thereafter. According to the existing evidence, near-total thyroidectomy leaving bilateral remnants of < 1g on each side is assumed to relevantly reduce the incidence of transient postoperative hypoparathyroidism with equal therapeutic safety in terms of recurrent dis- ease, deterioration of endocrine orbitopathy and method-associated complications. The trial is likely to optimize the surgical therapy of Graves’ disease. If a lower incidence of transient postoperative hypoparathyr- oidism is assumed, patient’s safety and quality of life are likely to be enhanced and postoperative care and treat- ment will be simplified. Care and treatment costs are likely to be relevantly reduced. Sample size h l The trial is designed on the basis of a critical appraisal of current evidence. According to the only existing ran- domized controlled trial by Witte et al. [1], a significant reduction in the incidence of transient hypoparathyroid- ism from 28% in the total thyroidectomy group to 12% in the near-total thyroidectomy group is suspected. Using a level of significance of 5% and a power of 80%, 97 patients per group are needed to detect the pre- specific difference when applying a chi square test (2- sided) without Yates's correction for continuity. With a dropout rate of 5% between obtaining informed consent and randomization, a total sample of 206 patients need to be recruited to the trial. Analysis of the primary end- point will be performed by an adjusted logistic regres- sion model. Therefore, it is to be expected that including covariates of prognostic importance in the logistic re- gression model will increase the power as compared to the chi square test. However, since the impact of the covariates cannot be determined a priori, a potentially liberal sample size estimation for the chi square test seems appropriate. Surgical methods l h Cervical approach, preparation, resection approach and vessel control are not limited per protocol in either group. The only binding preparation principles in either group are visualization, monitoring and protection of the recurrent laryngeal nerve, as well as the identifica- tion and protection of the parathyroid glands following the national guideline [7]. Neuromonitoring is per- formed and recorded before and after resection via the vagus nerve. If unforeseen multinodular changes, sus- pected malignancy, or frozen-section assured malig- nancy requires total thyroidectomy in the intervention group, or even additional lymphadenectomy in either Page 4 of 5 Competing interests 12. Ku CF, Lo CY, Chan WF, Kung AW, Lam KS: Total thyroidectomy replaces subtotal thyroidectomy as the preferred surgical treatment for Graves’ disease. ANZ J Surg 2005, 75:528–531. The authors hereby declare that there are no financial or non-financial competing interests within the conception or conduct of the trial. 13. Gaujoux S, Leenhardt L, Trésallet C, Rouxel A, Hoang C, Jublanc C, Chigot JP, Menegaux F: Extensive Thyroidectomy in Graves’ Disease. J Am Coll Surg 2006, 202(6):868–873. Authors’ contributions d h DKB reviewed the recommendation of the national guideline through a systematic PubMed literature search of the current evidence and generated the hypothesis. KM worked out a trial concept, created a study protocol and presented and discussed the idea within the national surgical societies (DGAV, and CAEK) and clinical trials boards (SDGC, and CHIR-Net). She applied for BMBF-funding, let the study be registered and wrote the present paper for TRIALS publication. DL carried out all statistical calculations and outlined the biometric concept. KS and CN substantially contributed to the definition and form of the trial concept and critically reviewed the trial protocol. All authors read and approved the final manuscript. 14. Shoback D: Clinical practice Hypoparathyroidism. N Engl J Med 2008, 359:391–403. 15. Kasper DL, Braunwald E, Fauci AS, Hauser S, Longo DL, Larry Jameson J: Harrisons’ principles of internal medicine. New York: Mc Graw/Hill; 2001:2629. 16. Bohrer T, Hagemeister M, Ehlert O: A clinical Chameleon: postoperative hypoparathyroidism. Langenbecks Arch Surg 2007, 392:423–426. 17. Heufelder E, Schworm D, Hofbauer C: Die endokrine Orbitopathie: Aktueller Stand zur Pathogenese, Diagnostik und Therapie. Deutsches Ärzteblatt 1996, 93:A-1336–A-1342. 18. Bullinger M, Kirchberger I, Ware J: Der deutsche SF-36 Health Survey. Übersetzung und psychometrische Testung eines krankheitsübergreifenden Instrments. München: Aktuelle Onkologie Bd. 82, W. Zuckschwerdt Verlag; 1995:35–46. Acknowledgements h d k The study group acknowledges the peer review group of the SDGC namely, Dr SC Hum Thomas Bruckner, Inga Wegener and Inga Rossion for the critical review of the trial protocol and the support given for the application for BMBF funding. We acknowledge Prof. Dr. med. Thomas Johannes Musholt (Department of General and Abdominal Surgery, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany) for support with regard to the contents. We thank the CAEK and CHIR-Net for their approval and the associated members for their declarations of commitment. doi:10.1186/1745-6215-13-234 Cite this article as: Maschuw et al.: Total versus near-total thyroidectomy in Graves’ disease and their outcome on postoperative transient hypoparathyroidism: study protocol for a randomized controlled trial?. Trials 2012 13:234. Abbreviations AWMF W k G 8. Barczynski M, Konturek A, Hubalewska-Dydejczky A, Golkowski F, Nowak W: Randomized clinical trial of bilateral subtotal versus total thyroidectomy for Graves’ disease with a 5-year follow-up. Br J Surg 2012, 99:522–523. 8. Barczynski M, Konturek A, Hubalewska-Dydejczky A, Golkowski F, Nowak W: Randomized clinical trial of bilateral subtotal versus total thyroidectomy for Graves’ disease with a 5-year follow-up. Br J Surg 2012, 99:522–523. AWMF: Work Group of the Scientific Medical Professional Societies; BMBF: German Ministry for Education and Research; Ca: Calcium; CAEK: German Society of Endocrine Surgery; CHIR-Net: National Network for Surgical Trials; DGAV: German Society of General and Visceral Surgery; fT3: Free triiodinethyronine; fT4: Thyroxine; LEMO: Eyelid exophthalmos eye muscle, optic nerve; PTH: Parathyroid hormone; SDG: Study Center of the German Surgical Society; SF36: Clinical Studies 36-Item Medical Outcomes Study Short-Form General Health Survey; TRAK: Thyroidea receptor anti-body; TSH: Thyroid stimulating hormone. y p g , 9. Pallit TK, Miller CC, Miltenburg DM: The efficacy of thyroidectomy in Graves’ disease: A meta-analysis. J Surg Res 2000, 90:161–165. 10. Miccoli P, Vitti P, Rago T, Jacconi P, Bartalena L, Bogazzi F, Fiore E, Valeriano R, Chiovato L, Rocchi R, Pinchera A: Surgical treatment for Graves’ disease: subtotal or total thyroidectomy? Surgery 1996, 120:1020–1024. discussion 1024–5. 11. Barakate MS, Agarwal G, Reeve TS, Barraclough B, Robinson B, Delbridge LW: Total thyroidectomy is now the preferred option for the surgical management of Graves’ disease. ANZ J Surg 2002, 72:321–324. References 1. Thomusch O, Machens A, Sekulla C, Ukkat J, Brauckhoff M, Dralle H: The impact of surgical technique on postoperative hypoparathyroidism in bilateral thyroid surgery: a multivariate analysis of 5846 consecutive patients. Surgery 2003, 133:180–185. 2. Witte J, Goretzki PE, Dotzenrath C, Simon D, Felis P, Neubauer M, Röher HD Surgery for Graves’ disease: total versus subtotal thyroidectomy- result of a prospective randomized trial. World J Surg 2000, 24:1303–1311. 3. Siewert JR, Rothmund M, Schumpelick V: Praxis der viszeralchirurgie: endokrine chirurgie. Springer; 2007:42. Hampel R, Külberg T, Klein K, Jerichow JU, Pichmann EG, Clausen V, S 4. Hampel R, Külberg T, Klein K, Jerichow JU, Pichmann EG, Clausen V, Schmidt I: Strumaprävalenz in Deutschland größer als bisher angenommen. Med Klin (Munich) 1995, 90:324–329. 5. Erbil Y, Barbaros U, Ozbey N, Aral F, Ozamagan S: Risk factors of incidental parathyroidectomy after thyroidectomy for benign thyroid disorders. Int J Surg 2009, 7:58–61. 6. Artl W, Fremerey C, Callies F, Reinecke M, Schneider P, Timmermann W, Allolio B: Well-being, mood and calcium homeostasis in patients with hypoparathyroidism receiving standard treatment with calcium and vitamin D. Eur J Endocrinol 2002, 146:215–222. 7. Deutsche Gesellschaft für Allgemein- und Viszeralchirurgie Autorisiert für elektronische Publikation: AWMF online HTML-Code aktualisiert: 07.09.2010; 15:50:14. http://www.awmf.org/uploads/tx_szleitlinien/003-002l_02.pdf. 7. Deutsche Gesellschaft für Allgemein- und Viszeralchirurgie Autorisiert für elektronische Publikation: AWMF online HTML-Code aktualisiert: 07.09.2010; 15:50:14. http://www.awmf.org/uploads/tx_szleitlinien/003-002l_02.pdf. Trial status The trial manuscript has been reviewed by the Study Center of the German Surgical Society (SDGC). It has been approved by the National Network for Surgical Page 5 of 5 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Maschuw et al. Trials 2012, 13:234 http://www.trialsjournal.com/content/13/1/234 Trials (CHIR-Net) of the German Society of General and Visceral Surgery (DGAV). The project was approved by the German Society of Endocrine Surgery (CAEK) at their 30th annual meeting on the 26th of November 2011 in Innsbruck, Austria. So far 20 high volume cen- ters assigned a preliminary declaration of commitment with a mean number of 23 estimated patients to be enrolled within 12 months. The local ethics committee of the Philipps-University of Marburg approved the trial giving a positive vote on the 25th of April 2012 (leading ethics committee number 35/12). The trial is registered at the German Clinical Trials Register (DRKS) (trial number DRKS 00004161). Financing is applied within the governmental funding program for clinical trials of the German Ministry for Education and Research (BMBF). The Study team is awaiting BMBF response. Received: 3 June 2012 Accepted: 19 November 2012 Published: 6 December 2012 Author details 1 1Department of Visceral, Thoracic and Vascular Surgery, University Hospital Giessen and Marburg GmbH Location Marburg, Baldingerstrasse, Marburg D-35043, Germany. 2Coordination Center for Clinical Trials-KKS, Philipps-University Marburg, Karl-von-Frisch-Strasse 4, Marburg D-35043, Germany. 3Department of General and Visceral Surgery Marien Hospital, Bischofsstrasse 1, Osnabrueck D-49074, Germany. Received: 3 June 2012 Accepted: 19 November 2012 Published: 6 December 2012
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The future of force fields in computer-aided drug design
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First draft submitted: 2 July 2019; Accepted for publication: 9 July 2019; Published online: 23 September 2019 Keywords: computational chemistry and molecular modeling • drug discovery • physical chemistry • quantum me- chanics • statistical mechanics Computational molecular simulation has the potential to answer many questions regarding the role of biological molecules in human health and disease, and make predictions about the binding affinity and selectivity of small molecules [1]. Increased access to high performance computing resources and graphical processing units, alongside improvements in sampling protocols, enables computational simulations to routinely provide dynamical informa- tion on the nanosecond to millisecond time scale. Such information can provide insight into biological function that is complementary to experimental structural biology techniques, such as x-ray crystallography and nuclear magnetic resonance. In structure-based computer-aided drug design (CADD), virtual screening aims to suggest new drug candidates by docking large libraries of small molecules into the binding site of the therapeutic target and using scoring functions to determine binding poses and estimate binding affinity [2]. While virtual screening is useful for high-throughput studies, inherent approximations limit accuracy, especially when target and/or ligand flexibility are crucial. Statistical thermodynamic methods, such as free energy perturbation (FEP), provide a more rigorous estimate of the protein–ligand binding free energy. Ultimately, the goal of FEP is to screen protein–ligand binding free energies of large numbers of potential drug candidates with sufficient accuracy that it can be used to prioritize compounds for synthesis [3]. Commentary For reprint orders, please contact: reprints@future-science.com For reprint orders, please contact: reprints@future-science.com The future of force fields in computer-aided drug design Daniel J Cole*,1 , Joshua T Horton1, Lauren Nelson1 & Vadiraj Kurdekar1 1School of Natural & Environmental Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK *Author for correspondence: daniel.cole@ncl.ac.uk “As the popularity and accessible time scales of computer simulations grow, it is becoming apparent that more accurate force fields are required to overcome limitations in predictive molecular design.” “As the popularity and accessible time scales of computer simulations grow, it is becoming apparent that more accurate force fields are required to overcome limitations in predictive molecular design.” First draft submitted: 2 July 2019; Accepted for publication: 9 July 2019; Published online: 23 September 2019 Molecular mechanics force fields Underlying many of these techniques in structure-based CADD is the molecular mechanics force field, which describes the total energy of the system as a function of the atomic coordinates. The most widely used biological force fields, AMBER, CHARMM and OPLS, share similar functional forms which have remained largely unchanged for many decades [4]. These force fields treat nonbonded interactions as a physically motivated Coulomb interaction between (usually) atom-centered point charges, supplemented by a Lennard–Jones interaction. The latter is repulsive at short interatomic distance to model overlap of electron clouds, and attractive at longer range to account for van der Waals interactions. Covalently bonded atoms are described by harmonic bond-stretching and angle-bending terms, and anharmonic torsional energies. All of these interactions require parameterization. That is, quantities such as equilibrium bond lengths, force constants and atomic charges must be assigned. Parameterization strategies vary but are characterized by extensive fitting to experimental and quantum mechanical (QM) properties of small organic molecules. As the popularity and accessible time scales of computer simulations grow, it is becoming apparent that more accurate force fields are required to overcome limitations in predictive molecular design. For small molecule validation studies, such as liquid physical properties and host-guest binding, for which sampling requirements can be readily met, the force field is often shown to limit the predictive utility of molecular modeling [5]. Turning to the calculation of protein–ligand binding free energies, it is generally assumed that FEP accuracy of 0.6–1.0 kcal/mol 10.4155/fmc-2019-0196 C⃝2019 Daniel J Cole New approaches to force field parameterization A danger associated with incremental, often labor-intensive, force field parameter updates is that accuracy will begin to stagnate. A fundamentally new approach to force field design is required, incorporating up-to-date techniques in quantum chemistry and data science. The Open Force Field Initiative (https://openforcefield.org) has recently highlighted one issue associated with legacy force field atom types, which are used to label atoms in a molecule and then assign their parameters from libraries. These atom types need to be curated by human experts and typically lead to a proliferation of redundant parameters, thus hindering automated force field development. They address this by introducing a new force field format, SMIRNOFF, which assigns parameters based on the full chemical environment of an atom (the SMIRKS pattern) through direct chemical perception [7] rather than going through the intermediate steps of atom typing. In this way, a general organic force field parameter library may be written in less than 350 lines rather than several thousand lines for typical modern force fields. While this does not immediately improve accuracy (though several inconsistencies and instances of human error in legacy force fields did surface), in combination with the open software and data infrastructures for force field design, automated protocols for parameter fitting and best practice property prediction advocated by the Open Force Field Initiative, it provides a very promising starting point for rigorous and automated parameter fitting. The force field parameter libraries described above are traditionally fit to reproduce the experimental or QM properties of a representative set of small molecules and then applied to molecules outside the training set using atom types (or SMIRKS patterns in the case of SMIRNOFF). These transferable force fields give the user the ability to rapidly parameterize diverse expanses of chemical space at low computational cost. However, there are potential disadvantages to this approach. First, experimental data may be unavailable for many systems of significant physical and chemical interest (for example, organometallic complexes or molecules in electronic excited states). Thus, the traditional approach of fitting suitably accurate force field parameters to experiment is difficult for these molecules. Second, it is well established that electron density (and hence atomic charge) polarizes in response to the atom’s environment. For example, Jorgensen et al. New approaches to force field parameterization have shown that the QM hydrogen bonding interaction between para-substituted phenols and water can vary by around 2 kcal/mol depending on the nature of the para substituent, which could have important consequences for choices of ring substituents and heterocycles during hit-to-lead optimization [8]. The observed trend could not be captured by a transferable charge set. For this reason, it is commonplace to assign ‘bespoke’ atomic charges that are computed directly for the small molecule under study. Interestingly, recent advances in quantum chemistry indicate that van der Waals interaction strengths are similarly dependent on the atomic environment. For example, Gobre and Tkatchenko studied the range, and scaling with system size, of the C6 coefficient (a measure of the van der Waals strength) in carbon nanostructures and found that it can range from around 20 atomic units (a.u.) in diamond to 150 a.u. in graphene [9]. The nature of the van der Waals interaction is less commonly studied in medicinal chemistry, but it is clear that much remains to be understood about its role in protein–ligand binding and its treatment in force fields [10]. 10.4155/fmc-2019-0196 C⃝2019 Daniel J Cole ISSN 1756-8919 Future Med. Chem. (Epub ahead of print) Commentary Cole, Horton, Nelson & Kurdekar is required to guide medicinal chemistry optimization; an accuracy that is currently only achieved in isolated best- case scenarios. Protein force fields present particular challenges, such as appropriately balancing protein–protein and protein–water interaction strengths and accounting for coupling between backbone and side chain dynamics (e.g., the optimal amino acid side chain torsion parameters may depend on whether the backbone is in an α-helix or β-sheet conformation). In long timescale simulations of proteins, recent experimental data show that state-of-the-art force fields fail to accurately model intrinsically disordered proteins and temperature-dependent folding [6]. There is evidence that employing residue-specific torsion parameters and retuning the nonbonded interaction parameters of the water model have a strong influence on these computed properties. As we shall argue, however, it may be necessary to more fully understand the underlying physical nature of these interactions rather than employing ad hoc empirical parameter adjustment, which may be masking underlying issues with the computational model. Molecule-specific force fields An alternative to the transferable force fields described above is to instead parameterize the system on a case-by-case basis. By deriving parameters specifically for the system under study, the assumptions concerning charge and van der Waals parameter transferability are reduced. To avoid experimental data curation, such force fields are necessarily derived directly from quantum mechanical (QM) calculations. The earliest QM-derived molecule-specific force fields date back several decades, but with major advances in computational resources, there is now an increasing p Future Med. Chem. (Epub ahead of print) 10.4155/fmc-2019-0196 future science group The future of force fields in computer-aided drug design Commentary Commentary range of automated derivation techniques available [11,12], which is crucial if they are to be used in high-throughput workflows in, for example, computer-aided drug design. The quantum mechanical bespoke (QUBE) force field for proteins and small molecules is one such molecule- specific force field design protocol, with a particular focus on extensibility to large, heterogeneous systems, such as protein–ligand complexes. QUBE utilizes the modified Seminario method to derive bond and angle parameters from the QM optimized structure and Hessian matrix [13]. The accuracy of the bond and angle parameters may be determined by comparing force field vibrational frequencies with QM, and QUBE is competitive with modern transferable force fields, and offers particular accuracy improvements for more complex molecules containing heterocycles. To facilitate scaling to arbitrarily large system sizes and to treat charge and Lennard–Jones parameter derivation on a consistent footing, all QUBE nonbonded parameters are derived from atoms-in-molecule electron density partitioning of a single QM calculation. QUBE parameter derivation is fully automated through the open source QUBEKit software, and the resulting force fields have been extensively benchmarked against experimental liquid properties, offering similar accuracy to transferable force fields, which have been fit to these data [14]. Following implementation in the ONETEP linear-scaling density functional theory code (www.onetep.org), QUBE nonbonded parameters may be derived for systems comprising many thousands of atoms, including entire proteins. The atomic charges reproduce the QM electrostatic potential of proteins more accurately than transferable force field charges, and they are relatively insensitive to small conformational changes; both of which are crucial requirements for flexible force field design. Torsional parameters are derived from QM dihedral scans, and a library of these parameters has been built for use in protein simulations. Molecule-specific force fields A QUBE force field has been derived for the L99A mutant of T4 lysozyme, and been shown to outperform the OPLS-AA/M force field in the prediction of absolute binding free energies of six benzene analogs to the protein (0.85 vs 1.26 kcal/mol mean unsigned errors) [15]. One disadvantage of molecule-specific force fields is of course the time cost associated with the underlying QM calculations. However, a promising way around this is to exploit advances in machine learning to train computer models to predict force field parameters at the desired QM level using only the molecular environment as input. By way of example, Riniker and coworkers have recently built a machine learning model to predict the atoms- in-molecule charges of molecules using only the 2D molecular topology as a descriptor (that is, with no QM calculation) [16]. By training on 130 k lead-like molecules, the model is able to rapidly and accurately predict the atomic charges of a test dataset of drug-like compounds (root mean square error <0.02 e). Beyond the standard functional form The ultimate goal of force field development work is to approximate as closely as possible the full solution to the Schr¨odinger equation at affordable computational expense. Of course, the definition of affordable depends strongly on the problem under investigation, and much work has been done to attempt to break down the QM interaction energy into physically meaningful and calculable components [17]. At the more accurate end of the scale (e.g., symmetry-adapted perturbation theory), the computational expense currently hinders application in medicinal chemistry. On the other hand, polarizable force fields (of which AMOEBA is a well-known example) seek to account for changes in the charge distribution of a molecule in response to the electric field while retaining the ability of the force field to simulate the dynamics of biological systems. Such considerations should be crucial when modeling, for example, the partitioning of a small molecule between a polar solvent and a hydrophobic protein binding site. However, in general, the addition of extra force field terms can also add to the labor associated with parame- terization. Visscher and Geerke address this problem by extending aspects of the quantum mechanically derived force fields described above to include both higher order van der Waals interactions (including C8 interactions) and atomic polarization [18]. This force field model is able to reproduce the liquid densities and heats of vaporization of 11 linear and branched alkanes with rms errors of 0.02 g/cm3 and 0.15 kcal/mol, respectively, and it will be interesting to see whether this level of accuracy is maintained for more polar biological building blocks. Finally, we saw above that advances in machine learning may be harnessed to accelerate parameter assignment. Though more commonly employed in the materials sciences, machine learning is now emerging as a tool, not just to extract force field parameters, but to actually learn the underlying QM potential energy surfaces of organic molecules. One such example is the ANI deep neural network potential [19], the first iteration of which was trained on a database of millions of QM calculations, spanning conformational and configurational space of more than 57 k small organic molecules containing H, C, N and O atoms. The result is, in the authors’ words, a potential game changer for molecular simulation – an interatomic potential that is chemically accurate relative to the underlying future science group 10.4155/fmc-2019-0196 Commentary Cole, Horton, Nelson & Kurdekar QM calculations, but orders of magnitude faster. Beyond the standard functional form Perhaps most importantly, the authors showed that the potential is extensible to molecules much larger than those used for training (up to 54 atoms), which already at this early stage make machine learning potentials an alternative to expensive QM methods for simulating structural properties of drug-like molecules. Open access p This article is distributed under the terms of the Creative Commons Attribution License 4.0 which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. To view a copy of the license, visit http://creativecommons.org/licenses/by/4.0// Financial & competing interests disclosure Financial & competing interests disclosure DJ Cole and V Kurdekar are grateful for financial support from EPSRC (grant number EP/R010153/1). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. No writing assistance was utilized in the production of this manuscript. Conclusion & future perspective Molecular mechanics force fields are a crucial computational technology, underpinning biological modeling and rational molecular design. Traditional force fields are undergoing continuing improvements, and gains in accuracy are apparent; yet legacy issues may limit the step changes in accuracy that are desired. Here, we have outlined some of the approaches that, in our opinion, will be vital in next generation force field design. Perhaps the most important advance will be to reduce the labor associated with force field parameterization by employing open and automated data curation, parameter fitting and force field validation approaches. As well as the expected accuracy gains, this will allow researchers to standardize parameterization protocols, so that, for example, modified amino acids, lipids and DNA/RNA are treated at the same accuracy as small molecules and proteins, and also to move away from legacy force field functional forms and rapidly investigate new physical models that more accurately capture QM interaction effects (e.g., van der Waals, polarization [20]). Allied with this, it is important to continue to investigate new methods for extracting force field parameters directly from QM. This will not only allow us to derive molecule-specific force fields, which are already showing promise in biomolecular modeling, but will also accelerate transferable force field design by reducing overfitting to experimental data. The natural and exciting progression is machine learning of the full QM potential energy surface, though it will be important to evaluate these potentials in the condensed phase where long-ranged many-body electrostatic effects are crucial and may not be sufficiently represented in small molecule training datasets. The future of force fields in computer-aided drug design Commentary The future of force fields in computer-aided drug design Commentary 11. Greff da Silveira L, Jacobs M, Prampolini G, Livotto PR, Cacelli I. Development and validation of quantum mechanically derived force-fields: thermodynamic, structural, and vibrational properties of aromatic heterocycles. J. Chem. Theory Comput. 14(9), 4884–4900 (2018). 11. Greff da Silveira L, Jacobs M, Prampolini G, Livotto PR, Cacelli I. Development and validation of quantum mechanically derived force-fields: thermodynamic, structural, and vibrational properties of aromatic heterocycles. J. Chem. Theory Comput. 14(9), 4884–4900 (2018). 12. Grimme S. A general quantum mechanically derived force field (QMDFF) for molecules and condensed phase simulations. J. Chem. Theory Comput. 10(10), 4497–4514 (2014). 12. Grimme S. A general quantum mechanically derived force field (QMDFF) for molecules and condensed phase simulations. J. Chem. Theory Comput. 10(10), 4497–4514 (2014). 13. Allen AEA, Payne MC, Cole DJ. Harmonic force constants for molecular mechanics force fields via Hessian matrix projection. J. Chem. Theory Comput. 14(1), 274–281 (2018). 13. Allen AEA, Payne MC, Cole DJ. Harmonic force constants for molecular mechanics force fields via Hessian matrix projection. J. Chem. Theory Comput. 14(1), 274–281 (2018). 13. Allen AEA, Payne MC, Cole DJ. Harmonic force constants for molecular mechanics force fields via Hessian matrix projection. J. Chem. Theory Comput. 14(1), 274–281 (2018). 14. Horton JT, Allen AEA, Dodda LS, Cole DJ. QUBEKit: automating the derivation of force field parameters from quantum mechanics. J. Chem. Inf. Model. 59(4), 1366–1381 (2019). 14. Horton JT, Allen AEA, Dodda LS, Cole DJ. QUBEKit: automating the derivation of force field parameters from quantum mechanics. J. Chem. Inf. Model. 59(4), 1366–1381 (2019). 15. Cole DJ, de Vaca IC, Jorgensen WL. Computation of protein–ligand binding free energies using quantum mechanical bespoke force fields. Med. Chem. Commun.10, 1116–1120 (2019). 15. Cole DJ, de Vaca IC, Jorgensen WL. Computation of protein–ligand binding free energies using quantum mechanical bespoke force fields. Med. Chem. Commun.10, 1116–1120 (2019). 16. Bleiziffer P, Schaller K, Riniker S. Machine learning of partial charges derived from high-quality quantum-mechanical calculations. J. Chem. Inf. Model. 58(3), 579–590 (2018). 16. Bleiziffer P, Schaller K, Riniker S. Machine learning of partial charges derived from high-quality quantum-mechanical calculations. J. Chem. Inf. Model. 58(3), 579–590 (2018). 17. Stone AJ. Intermolecular potentials. Science 321(5890), 787–789 (2008). 18. Visscher KM, Geerke DP. Deriving force-field parameters from first principles using a polarizable and higher order dispersion model. J. Chem. Theory Comput. 15(3), 1875–1883 (2019). 18. Visscher KM, Geerke DP. References 2. Gimeno A, Ojeda-Montes MJ, Tom´as-Hern´andez S et al. The light and dark sides of virtual screening: what is there to know? Int. J. Mol. Sci. 20(6), 1375 (2019). 3. Li Z, Huang Y, Wu Y et al. Absolute binding free energy calculation and design of a subnanomolar inhibitor of phosphodiesterase-10. J. Med. Chem. 62(4), 2099–2111 (2019). 4. Jorgensen WL. Foundations of biomolecular modeling. Cell 155(6), 1199–1202 (2013). 4. Jorgensen WL. Foundations of biomolecular modeling. Cell 155(6), 1199–1202 (2013). 5. Yin J, Henriksen NM, Slochower DR et al. Overview of the SAMPL5 host–guest challenge: are we doing better? J. Comput. Aided Mol. Des. 31(1), 1–19 (2017). 5. Yin J, Henriksen NM, Slochower DR et al. Overview of the SAMPL5 host–guest challenge: are we doing better? J. Comput. Aided Mol. Des. 31(1), 1–19 (2017). 6. Nerenberg PS, Head-Gordon T. New developments in force fields for biomolecular simulations. Curr. Opin. Struct. Biol. 49, 129–138 (2018). 6. Nerenberg PS, Head-Gordon T. New developments in force fields for biomolecular simulations. Curr. Opin. Struct. Biol. 49, 129–138 (2018). 7. Mobley DL, Bannan CC, Rizzi A et al. Escaping atom types in force fields using direct chemical perception. J. Chem. Theory Comput. 14(11), 6076–6092 (2018). 8. Jorgensen WL, Jensen KP, Alexandrova AN. Polarization effects for hydrogen-bonded complexes of substituted phenols chloride ion. J. Chem. Theory Comput. 3(6), 1987–1992 (2007). 9. Gobre VV, Tkatchenko A. Scaling laws for van der Waals interactions in nanostructured materials. Nat. Comm. 4, 2341 (2013). 10. Walters ET, Mohebifar M, Johnson ER, Rowley CN. Evaluating the London dispersion coefficients of protein force fields using the exchange-hole dipole moment model. J. Phys. Chem. B. 122(26), 6690–6701 (2018). p Future Med. Chem. (Epub ahead of print) 10.4155/fmc-2019-0196 future science group The future of force fields in computer-aided drug design Commentary The future of force fields in computer-aided drug design Commentary Deriving force-field parameters from first principles using a polarizable and higher order dispersion model. J. Chem. Theory Comput. 15(3), 1875–1883 (2019). 19. Smith JS, Isayev O, Roitberg AE. ANI-1: an extensible neural network potential with DFT accuracy at force field computational cost. Chem. Sci. 8(4), 3192–3203 (2017). 19. Smith JS, Isayev O, Roitberg AE. ANI-1: an extensible neural network potential with DFT accuracy at force field computational cost. Chem. Sci. 8(4), 3192–3203 (2017). 20. Manz TA, Chen T, Cole DJ, Limas NG, Fiszbein B. New scaling relationships to compute atom-in-material polarizabilities and dispersion coefficients: part 1. Theory and accuracy. RSC Adv. 9, 19297–19324 (2019). 20. Manz TA, Chen T, Cole DJ, Limas NG, Fiszbein B. New scaling relationships to compute atom-in-material polarizabilities and dispersion coefficients: part 1. Theory and accuracy. RSC Adv. 9, 19297–19324 (2019). future science group f 10.4155/fmc-2019-0196
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Splicing regulatory factors in breast cancer hallmarks and disease progression
Oncotarget
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Splicing regulatory factors in breast cancer hallmarks and disease progression Review Esmee Koedoot1, Liesanne Wolters1, Bob van de Water1 and Sylvia E. Le Dévédec1 1Division of Drug Discovery and Safety, LACDR, Leiden University, Leiden, The Netherlands Correspondence to: Sylvia E. Le Dévédec, email: s.e.ledevedec@lacdr.leidenuniv.nl Keywords: hallmarks of cancer; breast cancer; alternative splicing; splice factors; RNA sequencing Received: April 23, 2019        Accepted: August 29, 2019         Published: October 15, 2019 Copyright: Koedoot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT By regulating transcript isoform expression levels, alternative splicing provides an additional layer of protein control. Recent studies show evidence that cancer cells use different splicing events to fulfill their requirements in order to develop, progress and metastasize. However, there has been less attention for the role of the complex catalyzing the complicated multistep splicing reaction: the spliceosome. The spliceosome consists of multiple sub-complexes in total comprising 244 proteins or splice factors and 5 associated RNA molecules. Here we discuss the role of splice factors in the oncogenic processes tumors cells need to fulfill their oncogenic properties (the so-called the hallmarks of cancer). Despite the fact that splice factors have been investigated only recently, they seem to play a prominent role in already five hallmarks of cancer: angiogenesis, resisting cell death, sustaining proliferation, deregulating cellular energetics and invasion and metastasis formation by affecting major signaling pathways such as epithelial-to-mesenchymal transition, the Warburg effect, DNA damage response and hormone receptor dependent proliferation. Moreover, we could relate expression of representative genes of four other hallmarks (enabling replicative mortality, genomic instability, avoiding immune destruction and evading growth suppression) to splice factor levels in human breast cancer tumors, suggesting that also these hallmarks could be regulated by splice factors. Since many splice factors are involved in multiple hallmarks of cancer, inhibiting splice factors might provide a new layer of oncogenic control and a powerful method to combat breast cancer progression. Oncotarget, 2019, Vol. 10, (No. 57), pp: 6021-6037 Oncotarget, 2019, Vol. 10, (No. 57), pp: 6021-6037 www.oncotarget.com www.oncotarget.com Altered expression In two independent studies, the comparison of the transcriptome of human breast tumors versus matched healthy tissue revealed that 10%–50% of the protein- coding genes have altered transcript variant expression levels [24, 25]. These patient data are in line with recent in vitro findings that show a significant switch in splicing pattern during epithelial-to-mesenchymal transition (EMT) accompanied with a specific EMT splicing signature [26]. Interestingly, this shift in splicing pattern was correlated to the expression levels of specific splice factors; all three studies revealed splice factor RBFOX2 as one of the most differentially expressed between the epithelial and mesenchymal cell state [24–26]. Moreover, expression levels of MBNL1, QKI, PTBP1, ELAV1, HNRNPC, KHDRBS1, SRSF2 and TIAR were also linked to the mesenchymal state [24]. By applying a splicing motif analysis in EMT regulated alternative transcripts, Shapiro et al. concluded that the MBNL, CELF, hnRNP, or ESRP splice factors were most likely involved in the EMT splicing patterns [26]. Furthermore, depletion of the mesenchymal splice factor RBFOX2 or overexpression of the epithelial factor ESRP in mesenchymal cells induced a more epithelial morphology and reduced cell motility [26]. Altogether, these data clearly suggest that splice factors can be in control of EMT and breast cancer progression. Breast cancer is the most frequent type of cancer in women with an estimation of 268,670 new cases and 41,400 deaths in the United States in 2018 [19]. In order to develop and progress, (breast) cancer cells move through various steps to fulfill their requirements for certain oncogenic properties. These processes – the so- called ‘hallmarks of cancer’ – have been summarized by Hanahan and Weinberg in 2000 and 2011 [20, 21] and currently include ten processes essential for tumor development and progression. In this review, we will discuss the spliceosomal changes across the different hallmarks of breast cancer. Since many of the already known spliceosome target genes have already been reviewed extensively elsewhere [5–8], we will focus on the role of splice factors as potential oncogenes or tumor suppressors in breast cancer. We will highlight newly identified splice factors of which abnormal regulation is linked to the different hallmarks of breast cancer [21]. For the hallmarks that have not yet been linked to splice factors expression, we identified factors strongly related to hallmark-specific oncogenic processes using publicly available RNA sequencing data. INTRODUCTION (AS) and provides an essential layer of post-transcriptional regulation that only recently received much attention from the research community. In particular, it is becoming clear that tumor cells benefit greatly from this flexible regulatory process since many specific isoforms have been identified as promoting and supporting neoplastic transformation, tumor growth and progression. Many reports have linked AS to up-regulation of proto-oncogenes, deregulated cell division, increased survival, altered metabolism, onset of angiogenesis, increased invasion and metastasis in different cancer types including breast cancer [5–8]. During gene transcription, a pre-mature messenger RNA (pre-mRNA) molecule is generated that requires further processing to a mature in a mRNA molecule that will be translated into a protein. In this maturation step, the introns are usually removed and the exons are ligated. This process, called splicing, is one of the post-transcriptional processes essential for RNA translation into functional proteins and requires the activity of the splice factors. Besides simple intron removal and exon coupling, the activity of those splice factors enable that multiple protein isoforms can be translated out of one pre-mRNA transcript by selective incorporation of pre-mRNA parts in the mature mRNA transcript [1–4]. This is called alternative splicing Splicing is a complex multistep process catalyzed by the spliceosome, a large, dynamic, multicomponent complex consisting of five small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, U5 and U6 and many associated www.oncotarget.com Oncotarget 6021 proteins. In the human spliceosome, the 141 core factors are highly abundant and/or are specifically associated with the U1, U2, U5, U4/U6 snRNPs, or the U4/U6. U5 tri- snRNP [9, 10]. The auxiliary splice factors that are not part of the core spliceosome regulate AS and are less abundant when co-purified with the core spliceosome members [9, 10]. Splice factors are highly diverse considering both function and structure. For example, hnRNPs are characterized by a RNA Recognition Motif (RRM) domain that accommodates site-specific binding to the target RNA typically resulting in splicing inhibition by suppressing assembly of the spliceosome [11] or attraction of snRNPs [12, 13]. SR splice factors contain a domain consisting of arginine/serine repeats (RS domain) and at least one RRM domain [14–16] and facilitate recruitment of the snRNPs to the splice sites [2]. Additionally, their activity is regulated through phosphorylation by SR protein kinases (SRPKs). Post-translational modifications and chromatin structure Next to changes in expression levels, activity of certain splice factors can also be regulated by post- translational modifications (PTMs) such as acetylation, phosphorylation and ubiquitination. Strong interactions between ubiquitination and the spliceosome have been demonstrated and SR proteins are widely known to regulate the activation of other splicing factors by phosphorylation [27–29]. For example, acetylation and ubiquitination of the splicing factor SRSF5 has been shown to control tumor growth [30]. The phosphorylation status of SRSF1 and SRSF7 controls their function as only non-phosphorylated SR proteins were shown to facilitate the recruitment of mRNA to nuclear export receptors [31–34]. INTRODUCTION Currently almost 250 splice factors distributed over different classes have been identified, all playing a specific role at a specific stage of the splicing process [9, 17, 18]). suppressed intron or exon inclusion. Those deregulatory events in splicing have been shown to play a prominent role in breast cancer. Altered expression Finally, we discuss the clinical relevance of using splice factors as biomarkers and potential targets in breast cancer therapy. www.oncotarget.com SPLICE FACTOR DYSREGULATION IN BREAST CANCER Cancer-specific splicing events are established via different routes: 1) changes in expression levels, activity and localization of splice factors and/or 2) mutations in functional domains of splicing related proteins and/ or mutations in regulatory sequences, such as enhancer/ silencer sequences and branch points [22, 23]. Both processes can result in differential splice factor activity leading to differential splice site usage or increased or Moreover, the intracellular distribution can be crucial for downstream signaling events. Although most splice factors reside in the nucleus, cytoplasmic splicing has been recently shown to take place in mammalian cells implying that splice factors might have differential activities depending on their intracellular location [35]. www.oncotarget.com Oncotarget 6022 Splice factor dynamics is also highly dependent on the chromatin structure that is often disturbed in cancer cells [36]. Non-coding RNAs (ncRNAs) and in particular long ncRNAs (lncRNAs) can alter the chromatin environment preventing the recruitment of a repressive chromatin- splicing adapter complex and consequently regulate AS of the FGFR2 [37]. Moreover, histone hyper acetylation has been shown to affect the distribution of several splicing factors such as SRSF1, SRSF2 SRSF3 and U2AF65, causing accumulation in the nuclear speckles [38] and decreased spliceosomal assembly at 3′ splice sites, while calcium-mediated histone hyperacetylation regulates AS of genes important in heart development [39]. Finally, splicing can be regulated by miRNAs within the supraspliceosome that can target different RNAs via alternative base pairing, thereby regulating gene expression and quality control of AS [40]. (ESSs), intronic splicing enhancers (ISEs) and intronic splicing silencers (ISSs) [2]. Mutations can modulate activity of these elements thereby affecting AS. For instance, a point mutation in exon 18 of important tumor- suppressor gene BRCA1 disrupts an ESE resulting in exon skipping [52], while mutations in the ESEs and branchpoint recognized by SRSF2 dysregulates spliceosome assembly and result in AS in myelodysplasia [53]. Sustaining proliferation For the luminal breast cancer subtype, mutations in SF3B1 were found to be possible driver mutations [44–46]. These mutations result in a change-of-function and have been associated with hundreds of atypical splice sites at the 3′ end of the intron, thereby inducing AS of SF3B1 downstream target genes [47, 48]. Accordingly, our splice factor mutation analysis of breast cancer tumors from the COSMIC database revealed frequently mutated spliceosome genes amongst which SF3B1 (Supplementary Table 1). Interestingly, 10 splicing factors were classified as driver genes of which mutations are selected during tumor development by the Intogen database (Supplementary Table 1), 5.4% of all driver genes in breast cancer were regulating splicing, suggesting a major role for these proteins in breast cancer oncogenesis. Mutations in splice factors or regulatory sites Next to altered splice factor expression levels and activity, abnormal splicing can be caused by mutations in the genes that encode these factors. Few studies and our own analysis (Supplementary Table 1) demonstrate that the splice factors U2AF1, SRSF2 and SF3B1 are often mutated in myelodysplastic syndromes [41], but also in solid cancers amongst which breast cancer [42]. These mutations mainly caused haematopoiesis due to impaired 3′-splice site recognition followed by RNA splicing deficiencies [41]. U2AF1 mutations specifically affected AS of genes in various pathways pivotal for cancer development, such as apoptosis via CASP8, DNA damage response via ATR and FANCA and DNA methylation via DNMT3B [43]. ROLE OF SPLICE FACTORS IN THE HALLMARKS OF BREAST CANCER In order to develop and progress, (breast) cancer cells move through various oncogenic processes. These hallmarks of cancer were summarized by Hanahan and Weinberg in 2000 and 2011 and now contain ten processes essential for tumor development and progression [20, 21]. Although the splice factor research in relation to breast cancer emerged only recently, there are already five hallmarks of cancer known to be affected by splice factors: sustaining proliferation, activation of invasion and metastasis, resisting cell death, deregulating cellular energetics and angiogenesis. Here we will discuss splice factors and up –and downstream pathways important in these five hallmarks (Figure 1, Supplementary Table 2). Moreover, we could relate expression of representative genes of four other hallmarks to splice factor levels in human breast cancer tumors (Figure 2, Supplementary Table 3), suggesting that also these hallmarks might be modulated by splice factors. However, the causal relationship between splice factor levels and these hallmarks of cancer remains to be elucidated. Hormone-receptor dependent pathways Sustained proliferation is probably the most critical and studied cancer hallmark. Normal tissues precisely control cell number through many signaling pathways, amongst which the well-known MAPK and PI3K cascades [20, 54, 55]. In transformed cells, these pathways are upregulated resulting in uncontrolled growth. In breast cancer this mainly occurs via mutations resulting in overexpression of hormone receptors including the estrogen receptor (ER), androgen receptor (AR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and their corresponding ligands [20, 56, 57]. Next to mutations that could affect the functionality of splice factors, mutations in 5′- or 3′ splice site or branch point can disrupt or create splice sites [49] and thereby cause AS [50, 51]. Furthermore, specificity of AS is controlled by cis-regulatory elements that regulate the recruitment of trans-acting splicing factors to the splice site; exonic splicing enhancers (ESEs), exonic splicing silencers The ER consists of two subtypes: ERα and ERβ. Upon ligand binding ERα and/or ERβ will form homo- or heterodimers, leading to nuclear translocation and possibly transcriptional activation [58]. ERα stimulates cell proliferation and survival by regulating the transcription of hundreds of genes [59, 60]. The role of ERβ in cancer has yet to be defined as several studies report contradictory www.oncotarget.com Oncotarget 6023 results in a truncated version of HER2 named Herstatin. Herstatin binds to the extracellular domain of HER2, preventing transfer to the cell membrane and receptor dimerization and phosphorylation, thereby exhibiting anti-tumor activities [72–74]. Although HER2 and ER expression patterns are inversely correlated because of negative feedback loops, they are interconnected via downstream pathways, such as the MAPK pathway. This crosstalk between ER and HER2 seems to be important in endocrine resistance [75] and therefore treatment might be improved by identifying more pathways or proteins regulated by both ER and HER2. Since splice factors like PRPF4B fit this double activation pattern, it might be worth investigating the role of PRPF4B in hormone receptor growth resistance conditions. correlations between transcript level and patient prognosis [61–63]. Emerging evidence suggests a role for splice factors in ER cancer signaling either by direct or indirect interactions with the receptors. For instance, RNA levels of SF3B3 correlate significantly with the overall survival of patients bearing ERα-positive tumors [64], PRPF4B protein expression levels are directly regulated by ERα activation [65] and HMGA1 is directly involved in ERα splicing [66]. Nassa et al. Hormone-receptor dependent pathways discovered that the interactome of both ERα and ERβ contains multiple splice factors of which some are common for both receptor subtypes (e. g. EFTUD2, SRSF2), while others only interact with a single receptor subtype, such as SNRPD1 for ERα and SF3A1 for ERβ [67]. Introduction of ERβ expression diminished 72% of ERα induced splicing events, but also introduced distinct splicing events in 28 genes that were functionally involved in many cellular processes including DNA replication and repair, DNA transcription, cell cycle and apoptosis [68]. Whether these changes are counteracting the proliferative effects of ERα transcriptional activity is yet unknown. Hormone receptor independent pathways Next to involvement in known proliferation pathways, splice factors are also implicated in tumor growth via partly unknown mechanisms that might be independent of these hormone cascades. Examples are PTBP1 which inhibits cell growth in breast cancer cell lines [76], Tra2β, a target of transcription factor and oncogene ETS-1 that is upregulated in breast cancer and is associated with cancer cell survival [77], SRSF3 that upon inhibition decreased breast cancer cell proliferation [78], the loss of PRMT5 or WDR77 resulting in AS and loss of proliferative genes [79] and HNRNPC which is highly expressed in breast tumors and of which knockdown results in double strand breaks and reduced proliferation [80]. Furthermore, DDX3× modulates the cell cycle by In addition to being regulated by ER signaling, PRPF4B levels are also controlled by HER2 signaling. Knockdown of HER2 results in downregulation of PRPF4B, whereas HER2 upregulation increases the levels of this splice factor in breast cancer cell lines [69]. Moreover, HER2 itself can be alternatively spliced resulting in different variants exhibiting either pro- or anti-tumorigenic functions. Skipping exon 16 results in the variant HER2D16, that is linked to increased resistance to HER2-targeting therapy and associated with cancer cell dissemination [70, 71]. Inclusion of intron 8 Figure 1: The role of splice factors and their associated pathways in the five hallmarks of cancer. Core splicing factors are listed in bold. Non-core splicing factors are listed in italic. Adapted from Hanahan and Weinberg, 2011 [21]. Figure 1: The role of splice factors and their associated pathways in the five hallmarks of cancer. Core splicing factors are listed in bold. Non-core splicing factors are listed in italic. Adapted from Hanahan and Weinberg, 2011 [21]. www.oncotarget.com Oncotarget 6024 Furthermore, in line with the existing concept of breast cancer cells being addicted to oncogenes such as MYC for their proliferative and survival capacity, few recent studies have reported the dependence of breast cancer on spliceosomal components [83, 84]. In general, the overall increase in gene expression in cancer cell implies their aggravated dependence on splicing factors which open up a new strategic window for targeting affecting splicing and expression of the cell cycle repressor KLF4, resulting in G1 arrest [81], while hnRNPA2B1 knockdown affects MAPK and STAT3 signaling resulting in prolonged S-phase [82]. Activation of invasion and metastasis formation hnRNPM competes with the pro-epithelial splice factor ESRP1 for guanine-uridine rich motifs to regulate splicing of exons in genes involved in EMT-related pathways [100]. Furthermore, hnRNPM controls EMT by modulating CD44 isoform expression, which in turn increases TGFβ signaling. Elimination of hnRNPM prevents TGFβ induced breast cancer metastasis in mice by decreasing the mesenchymal-related standard CD44 isoform. hnRNPM mRNA levels were shown to correlate with aggressive breast cancer subtypes (basal and ER negative) and increased CD44 standard levels in breast cancer patients [101, 102]. Interestingly, the adhesion molecule CD44 that regulates the aggressive phenotype of breast cancer cells seem to be a common target of AS. KHDRBS1 is a factor involved in a dynamic protein complex variable in size and sensitive to EGF stimulation. EGF activation favors the smaller KHDRBS1 complex, which induces CD44 exon v5 inclusion resulting in enhanced cell migration [103]. Furthermore, SR splice factor TRA2β is overexpressed in invasive breast cancer and induces exon v4 and v5 inclusion [104], suggesting that besides the standard CD44 isoform also the v4 and v5 isoforms are related to increased invasion and metastasis formation. Because the human breasts are non-vital organs, primary tumors in breast tissue can be surgically removed without major consequences. Ultimately, it is the formation of metastatic lesions in secondary organs that causes breast cancer mortality. Metastasis formation is often described as a multi-step process, also called the invasion- metastasis cascade [87, 88]. Primary breast cancer cells start to locally invade into the surrounding tissue and often those cells undergo a phenotypic switch characterized by epithelial-to-mesenchymal transition (EMT). In this process, epithelial cells bearing strong adhesion structures can switch to a migratory mesenchymal phenotype with loss of cell polarity and cell-cell contacts [89]. Those features acquired by mesenchymal cells allow not only infiltration into adjacent tissue, but also escape into the blood or lymphatic vessels. y p Many splice factors have been linked to this EMT process. A genome-wide screen for EMT inducers identified many RNA-binding proteins, with splice factors QKI and RBFOX1 as main candidates. These factors regulated splicing of the actin binding protein FLNB, followed by release of FOXC1 leading to an intermediate mesenchymal cell state [90]. Moreover, the ratio of splice factors ESRP1/RBFOX2 was decreased during EMT and related to cancer progression and metastatic potential [91]. Hormone receptor independent pathways Altogether, we can conclude that splice factors play an important role in breast cancer sustained proliferation by either directly or indirectly activating hormone receptors and other growth associated pathways. O t t 6025 oncota get com Figure 2: Relation of splice factor expression levels to other hallmarks of cancer. (A) Steps used to link splice factor RNA expression levels to the other hallmarks of cancer. (B) Heatmap displaying the log2 fold change of genome instability markers comparing primary tumor tissue to normal tissue. NER = nucleotide excision repair, DSBR = double strand break repair, BER = base excision repair, DMMR = DNA mismatch repair. (C) Heatmap of log2 fold change of genome instability markers in ten control and hallmark patients comparing normal to primary tumor tissue. (D) Log2 fold change of splice factors in control and hallmark patients comparing normal to primary tumor tissue. Figure 2: Relation of splice factor expression levels to other hallmarks of cancer. (A) Steps used to link splice factor RNA expression levels to the other hallmarks of cancer. (B) Heatmap displaying the log2 fold change of genome instability markers comparing primary tumor tissue to normal tissue. NER = nucleotide excision repair, DSBR = double strand break repair, BER = base excision repair, DMMR = DNA mismatch repair. (C) Heatmap of log2 fold change of genome instability markers in ten control and hallmark patients comparing normal to primary tumor tissue. (D) Log2 fold change of splice factors in control and hallmark patients comparing normal to primary tumor tissue. www.oncotarget.com Oncotarget 6025 BC. Indeed, several splice factors including BUD31, SF3B1 and SRSF1 [85] are known to be gene targets of the oncoprotein MYC and inhibition or knockdown of those in MYC hyperactivated breast cancer cells impairs tumorigenesis [86]. in increased Rac1b levels and EMT [95, 99]. Moreover, breast cancer patients express high hnRNPA1 and low Rac1b levels in normal breast tissue, but low hnRNPA1 and high Rac1b levels in cancer tissue, suggesting that splicing of the Rho GTPase is also in vivo regulated by hnRNPA1 [95]. Activation of invasion and metastasis formation Next, the splice factor ESPR regulates fibroblast growth factor receptor 2 (FGFR2) splicing and thereby affects ligand binding, favoring FGFR2-IIIb which is specific to epithelial cells [92]. Genome-wide analysis of the ESRP splicing network uncovered hundreds of alternatively spliced genes that are involved in EMT related processes such as cellular adhesion and migration including ITGA6 and RALGPS2 [92]. The third hnRNP, PTBP1 is upregulated in progressively transformed human mammary epithelial cells (HMECs). Knockdown of PTBP1 impairs tumor cell growth, colony formation, in vitro invasiveness of breast cancer cell lines and transformation state of HMECs [76]. Next to the hnRNPs, the splice factor SRSF protein kinase 1 (SRPK1) was shown to be highly expressed in more aggressive basal breast cancer, correlating to less metastasis-free survival and specifically increased number of lung and brain metastases in patients. Stable knockdown of this kinase reduced metastasis to distant organs in a mouse model and inhibited focal adhesion reorganization, which were surprisingly not correlated to a downstream decrease in serine/arginine-rich (SR) splice factor phosphorylation [105]. However, an important role for these SR splice factors – in particular serine and arginine splice factor 1 (SRSF1) – cannot be excluded. SRSF1 is amplified and upregulated in breast cancer and transforms immortal cells when overexpressed [106, 107]. This transformation is mediated by SRSF1 collaboration with transcription factor MYC thereby amplifying eIF4E activation. This potential mechanism is further supported by patient data that reveal a significant co-expression of MYC and SRSF1 in human breast tumors [107]. Furthermore, SRSF1 mutants prevent tumorigenesis and soft agar colony formation by inhibiting activation of Another important class of splice factors involved in metastasis formation are the heterogeneous nuclear ribonucleoproteins (hnRNPs), which can control splice site selection by either directly antagonizing the recognition of splice sites or interfere with proteins bound to enhancers [93]. For instance, cytoplasmic localization of hnRNPA1 is associated with metastatic relapse and activates RON translation that is known to affect cell migration and dissemination [94]. Conversely, nuclear hnRNPA1 acts as a tumor suppressor and inhibits exon 3b inclusion of the small GTPase Rac1, thereby repression formation of the Rac1b isoform [95]. Rac1b has a constitutively activated GTPase domain [96, 97] and is overexpressed in breast cancer [95, 98]. Matrix metalloproteinase-3 treatment interferes with hnRNPA1-Rac1 interactions, resulting www.oncotarget.com Oncotarget 6026 favors Bcl-Xl expression [118]. Drug resistance In addition to resisting cell death due to physiological stresses, cancer cells might also gain properties resulting in resistance to cytotoxic agents. For example, overexpression of RNA-binding protein and splice factor RBM17 occurs in many cancer types and is associated with drug resistance to general chemotherapeutic agents such as doxorubicin and vincristine [120]. Next, SRPK1 inhibition increased apoptotic potential and cell killing when combined with gemcitabine and cisplatin treatments through impaired MAPK1, MAPK3 and PI3K pathways [121]. Furthermore, TRA2A overexpression results in AS of RSRC2 and decreased protein expression, contributing to paclitaxel resistance in triple-negative breast cancer patients [122]. Low levels of PRPF4B correlate to patient acquired resistance to microtubule targeting chemotherapeutics, presumably by regulating the spindle assembly checkpoint [69]. Moreover, the subunits of the SF3B complex SF3B1 and SF3B3 are upregulated in ER-α positive cells with acquired tamoxifen and fulvestrant resistance, with SF3B3 overexpression relating to a decrease in overall survival [64]. Opposite to the previous factors, spliceosome component SRSF4 induces splicing events followed by apoptosis in cancer cells when combined with the cytotoxic agent cisplatin. Knockdown of this factor reverses these splicing events and as a result significantly reduces cisplatin induced apoptosis [123]. Interestingly, this confirms that there is a dual role for different splice factors in apoptosis regulation. Resisting physiological stresses During tumorigenesis or anticancer therapy, cancer cells are exposed to numerous physiological stresses. In normal cells, these cellular stresses will cause apoptosis. However, cancer cells adapt in such environments and rewire their apoptotic program to survive. The SR related splice factor SRSF1 appears to play an important role in this process by promoting AS of crucial regulators of apoptosis BIM γ1 and γ2. Both isoforms lack the BH3 domain necessary to bind the anti-apoptotic Bcl-2 family members. Moreover, SRSF1 stimulates AS of a BIN1 isoform that is not able to bind MYC anymore, thereby losing its tumor suppressor activity leading to decreased levels of apoptosis [107]. This is in agreement with the observed upregulation of SRPK1 that contributes to the cytoplasmic accumulation of RNA-binding motif protein 4 (RBM4). This leads to the production of anti-apoptotic isoforms IR-A and MCL-1L and decreased sensitivity to apoptotic signals in breast cancer cells [112]. Furthermore, depletion of splice factor PHF5A increased apoptotic signaling by promoting expression of short truncated FAS-activated serine/threonine kinase enabling Fas- mediated apoptosis [113], while KHDRBS1 regulates exon 3 inclusion of the anti-apoptotic protein survivin that is higher expressed in advanced breast cancers [114]. The relation between splice factor levels and the ratio between the pro-apoptotic Bcl-Xs and anti-apoptotic Bcl-Xl splice variants is less evident. The activity of the splice factors PTBP1 [115], hnRNPF/H [116] and KHDRBS1 [117] increase the expression of Bcl-Xs, whereas hnRNPK Activation of invasion and metastasis formation In the end, the overall altered splicing in transformed cells is likely defined by different key factors which most probably changes during the different stages of cancer progression. the B-Raf-MEK-ERK pathway [106]. Finally, SRSF1 activates EMT and cell migration by induction of DRon, a constitutively active isoform of the Ron tyrosine kinase receptor that is causally connected to EMT [108]. Interestingly, hnRNPA1 has been shown to antagonize SRSF1-mediated EMT activation: through the inhibition of DRon production, hnRNPA1 activates the MET program at distant sites, thereby enhancing metastasis formation [108]. Transformation of normal cells into cancer cells almost invariably results in reduced genome stability. Tumor cells adapt to the load of mutations by activation of the DNA damage response (DDR) which prevents further proliferation and requires extra time to repair the lesions and might even result in apoptosis. Interestingly, splice factors TRA2α and TRA2β are clearly upregulated in breast cancer and those oncogene-like factors limit the amount of DNA damage thereby preventing cell death before entering the G2 phase. Indeed, dual knockdown of these factors results in a decreased expression of full length of CHEK1 (G2 checkpoint protein), leading to increased levels of the DNA damage marker γH2AX and decreased cell viability [77]. Moreover, upon DNA damage DDX54 interacts with pre-mRNAs containing introns with weak acceptor splice sites, leading to lower intron retention and increased survival [119]. Next to its oncogenic roles in sustaining proliferation, PRPF4B demonstrated an anti-oncogenic role in relation to EMT: loss of PRPF4B resulted in reduced EGFR degradation, increased expression of mesenchymal markers vimentin and ZEB1, detachment from the extracellular matrix and anoikis resistance [109]. Besides EMT, some splice factors have also been linked to the metastatic cascade in general, and their role in a specific step of the metastatic cascade remains to be elucidated. For example, RALY and SNW1 stimulate exon 2 inclusion in PRMT1, promoting breast cancer invasiveness [110] and CDK12 promote alternative last exon splicing of DNA damage genes ATM and DNAJB6 thereby increasing migration and invasiveness of breast cancer cells [111]. Deregulating cellular energetics Closely related to uncontrolled cell proliferation is the deregulation of cellular energetics, which is www.oncotarget.com Oncotarget 6027 angiogenic VEGFxxxb levels [134]. Treatment with SRPK1/2 inhibitors results in reduced angiogenesis, suggesting that AS regulation might provide a promising strategy to inhibit angiogenesis through depletion of pro- angiogenic components such as VEGF [135]. This is confirmed by the prediction that targeting of specific VEGF isoforms might be the best strategy to reduce free VEGF in tumors [136]. Another example is the splicing regulator Nova2 that is involved in vascular lumen formation, an essential step in angiogenesis. Nova2 targets exons implicated in the partitioning-defective (Par) complex and its regulators including Par3, Arhgef6 and Rapgap1. The Par complex interacts with tight junctions and cadherins and is important for lumen formation by endothelial cells, thereby being essential for cellular and tissue homeostasis [137–140]. Nova2 knockdown interferes with vascular lumen development in vivo and impairs endothelial cell polarity [137]. Although Nova2 has not been linked to tumorigenesis yet, it might be a potential target to inhibit angiogenesis. necessary to feed cells during growth and division. In aerobic conditions, healthy cells fuel their energy by processing glucose through glycolysis in the cytoplasm and oxidative phosphorylation in the mitochondria. Because the mitochondria consume high amounts of oxygen, energy production in anaerobic conditions relies only on glycolysis. However, cancer cells can reprogram their glucose metabolism using mainly glycolysis even in the presence of oxygen, named the Warburg effect. Splice factors that are suggested to control the Warburg effect are multiplayers PTBP1, hnRNPA1 and hnRNPA2 which not surprisingly are also involved in breast cancer growth and invasion. All three factors favor pyruvate kinase exon 10 inclusion causing higher levels of the M2 isoform (PKM2) compared to M1 (PKM1) resulting in decreased oxygen consumption contributing to the Warburg effect [76, 124]. PTBP1, hnRNPA1, hnRNPA2 levels are regulated by MYC [124]. Interestingly, hnRNPA1 also regulates MYC by regulating AS of the MYC-interacting protein Max, resulting in increased Delta Max levels in glioblastoma. Delta Max but not Max stimulates the expression of glycolytic genes and is required for tumor growth in vivo [125]. Splice factors in the other hallmarks In the last decade, splice factors levels have extensively been related to five hallmarks (Figure 1), suggesting a strong relation between splicing regulation and cancer development and progression. However, the remaining five hallmarks (genomic instability, tumor promoting inflammation, enabling replicative immortality, avoiding immune destruction and evading growth suppression) are still unaddressed. Here, we used RNA sequencing data from primary breast tumors from The Cancer Genome Atlas to investigate the potential role of splice factors in these hallmarks. For all of these hallmarks, we selected representative genes based on literature (Figure 2A, Supplementary Table 4) and calculated their log2 fold change (FC) between normal and tumor tissue. Key representative genes were selected based on their differential expression between normal and tumor tissue. For example, genome instability is characterized by a loss of repair mechanisms [21]. Comparing normal and primary tumor expression levels for genes involved in repair mechanisms, we identified four genes involved in nucleotide excision repair to be significantly downregulated in tumor tissue (Figure 2B). Next, we selected ten patients that were not affected (control patients) and ten patients that were heavily affected (hallmark patients) by the hallmark of interest (Figure 2C for genome instability). Finally, splice factor expression levels were compared between control and hallmark patients using a student’s t-test and after correction for multiple testing, splice factors significantly related to the specific hallmark could be identified (Supplementary Table 3). Interestingly, we could detect splice factors related to all remaining hallmarks, except for tumor promoting inflammation. Some of these splicing factors have already been associated with other hallmarks. PTPBP1, hnRNPA1 and hnRNPA2 are currently the only splice factors that have been related to the Warburg effect. However, since hypoxia is driving AS in breast cancer cells [126] and other splicing events of key metabolic genes such as PFKFB4 – which is responsible for retaining fructose-2,6-biphosphate, a key regulator of glycolysis – are altered in tumor tissue [127], we hypothesize that more spliceosome components are involved in cancerous cell metabolism. Angiogenesis New blood vessel formation or angiogenesis is critical for tumor progression since it i) provides the tumor with nutrients needed for growth and ii) brings the tumor cells in close proximity to blood circulation facilitating invasion and metastasis formation. Vascular endothelial growth factor (VEGF) is a key component in both physiological and pathological angiogenesis. Breast cancer patients with elevated VEGF levels have a higher risk to develop metastases or death compared to other patients [128] and therefore inhibition of this factor is a promising therapeutic strategy [129]. VEGF can be alternatively spliced by using a distal splice site selection in exon 8, resulting in the anti-angiogenic isoform VEGFxxxb bearing a different C-terminus [130–132]. Splice site selection is dependent on SRPK1/2 phosphorylation of the RNA- binding splice factor SRSF1. Furthermore, SRPK1 regulates VEGF splicing and activity in prostate cancer: SRPK1 knockdown results in up-regulation of the anti- angiogenic isoform VEGFxxxb and decreased angiogenesis in a xenograft model [133]. Accordingly, mutations in the tumor suppressor gene WT1 lead to increased SRPK1 levels and hyper phosphorylated SRSF1, reducing anti- www.oncotarget.com Oncotarget 6028 EFTUD2 expression levels are linked to markers of replicative immortality while it was previously shown to interact with ER and affect breast cancer proliferation [67]. Loss of WDR77 resulted in loss of proliferative genes and expression levels are linked to genome instability [79]. Interestingly, we also identified splice factors that have not been linked to other hallmarks in breast cancer before, such as CRNKL1, RALY and JUP. Future functional studies can use our analysis as a starting point to unravel the causal relationship between splice factor levels and these hallmarks of cancer. of breast cancer progression and for which inhibitors have been developed [105, 133, 152, 153]. Finally, pharmacological inhibition of the spliceosome would be a promising therapeutic strategy for MYC-addicted breast cancer tumors. Next to the use of small molecules, the potential of using splice-switching antisense oligonucleotides (SSOs) to modulate AS is of great therapeutic interest. SSOs are single-stranded oligonucleotides consisting of 20-30 nucleotides that bind to pre-mRNA and sterically prevent splicing factor binding, resulting in splice site switching. In contrast to normal antisense oligonucleotides (ASOs), SSOs are chemically modified to prevent breakdown of the targeted transcript to specifically target splicing without affecting total transcript levels [154]. Furthermore, SSOs are easy to synthesize and deliver, are relatively stable and can enter many different cell types [155, 156]. FUTURE PERSPECTIVES AND CONCLUSION As splice factors are frequently overexpressed in cancer compared to normal tissue, but also in highly invasive compared to less invasive tumors, splice factors might be a new promising therapeutic avenue in preventing breast cancer metastasis thereby lowering mortality in women. A possible drawback of inhibiting these factors could be the generation of adverse side effects when considering their critical function in intron removal in normal tissue. However, recent studies demonstrate that splice factor inhibition might certainly be applicable to the clinical situation. Both tumor-bearing mice and pre-diagnostic human samples of ER positive and triple negative breast cancer demonstrate autoantibody reactivity against spliceosomal proteins suggesting that at least partial inhibition for some of these factors should be possible [141, 142]. Furthermore, screens with natural products with antitumor characteristics resulted in the identification of spliceosome targeting drugs exhibiting cytostatic effects in multiple tumor cell lines by causing cell cycle arrest in G1 and G2/M phase [143–145]. Antitumor activities were confirmed in animal models and remarkably, general cytotoxicity was not observed. Moreover, these potential drugs seem to be more effective in cancer cells with some of them even targeting multidrug- resistant cells [146–148]. Recently, the natural compound resveratrol demonstrated to inhibit the oncogenic splice factor hnRNPA1 by inducing tumor suppressive miRNAs miR-424 and miR-503 via p53 thereby controlling tumor growth [149]. Also treatment with the CLK inhibitor T-025 reduced SR protein phosphorylation, which resulted in general effects on exon skipping and reduced cancer cell growth in vitro and in vivo [150]. Of note, most of the drugs described in the literature target the SF3B complex, a five-polypeptide subcomplex of the spliceosomal U2 snRNP. Small molecules affecting spliceosomal function by inhibiting different splicing complexes are known [151], but their potential role in combatting (breast) cancer has to be investigated. Other interesting targets would be the SR proteins or its upstream kinases like SRPK1 that has been demonstrated to be critical in multiple steps Since 94% of human genes are alternatively spliced [161, 162], splicing can be a very powerful new layer of oncogenic control. As discussed in detail above, splice factors demonstrated to play major roles in different hallmarks of cancer during tumorigenesis (Figure 1 and Supplementary Table 2) and some of these factors were already described as new oncogenes or tumor suppressors. Angiogenesis Although the use of SSOs was initially mainly studied in neuromuscular diseases such as Duchenne muscular dystrophy and spinal muscular atrophy [157], the therapeutic potential of SSOs as anti-cancer therapy is currently widely exploited [158]. For example, treatment with SSO stimulating exon 11 skipping caused better response to PARP inhibitors by inducing DNA double strand breaks [159]. Additionally, treatment with SSOs inducing exon 15 HER2 skipping caused downregulation of full length HER2 resulting in decreased downstream signaling, reduced cell proliferation and induction of apoptosis [160]. As a next step, clinical studies have to prove the use of these SSOs as anti-cancer therapy in breast cancer patients. FUTURE PERSPECTIVES AND CONCLUSION Since the role of many splice factors is not limited to a specific step or cancer hallmark they might provide a new approach to combat (breast) cancer progression. However, there are still many AS events that have been associated with cancer progression which cannot be attributed to specific splice factors yet. The introduction of the RNAi libraries and more recently the CRISPR Cas9 technology together with the development of high- throughput screening technologies [105, 163–165] would allow systematic evaluation of spliceosomal components in multiple aspects of breast cancer progression, such as proliferation and migration. Future studies should apply these technologies to uncover the complete signaling landscape of splice factors in breast cancer progression that then can be used to develop specific splice factor inhibitors preventing metastasis formation and patient deaths. www.oncotarget.com Oncotarget 6029 FUNDING 12. Tange TO, Damgaard CK, Guth S, Valcárcel J, Kjems J. The hnRNP A1 protein regulates HIV-1 tat splicing via a novel intron silencer element. EMBO J. 2001; 20:5748–58. https://doi.org/10.1093/emboj/20.20.5748. [PubMed] This work was supported by the ERC Advanced grant Triple-BC (grant no. 322737) and the Dutch Cancer Society project (grant nr 2011-5124). 13. House AE, Lynch KW. An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition. Nat Struct Mol Biol. 2006; 13:937–44. https:// doi.org/10.1038/nsmb1149. [PubMed] Author contributions EK performed the RNA sequencing analysis. LW performed the COSMIC mutation analysis. EK wrote the manuscript. 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Religious education for ecological sustainability: an initial reality check using the example of everyday decision-making
Journal of religious education
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* Stefan Altmeyer s.altmeyer@uni‑mainz.de Journal of Religious Education (2021) 69:57–74 https://doi.org/10.1007/s40839-020-00131-5 Journal of Religious Education (2021) 69:57–74 https://doi.org/10.1007/s40839-020-00131-5 1 Department of Religious Education, Faculty of Catholic Theology, Johannes Gutenberg- University, 55099 Mainz, Germany Religious education for ecological sustainability: an initial reality check using the example of everyday decision‑making Stefan Altmeyer1 Accepted: 8 December 2020 / Published online: 23 January 2021 © The Author(s) 2021 Accepted: 8 December 2020 / Published online: 23 January 2021 © The Author(s) 2021 For Reinhold Boschki, academic mentor and friend, on the occasion of his sixtieth birthday in May 2021. Keywords  Education for sustainable development · Religious education · Decision- making · Empirical research · Environmental education · Dilemma 1  Introduction No one can claim that the call for education to decisively contribute to the goals of sustain- able development in a consistent and interdisciplinary manner is a novel one. As early as 1992, Agenda 21 (UNCED 1992) consistently linked the issue of ecological sustainabil- ity with education, and through the World Decade of Education for Sustainable Develop- ment, the United Nations committed itself to the vision of a world “where everyone has the opportunity to benefit from education and learn the values, behaviour and lifestyles required for a sustainable future and for positive societal transformation” (UNESCO 2006, p. 24). These objectives are still valid and have again been reaffirmed in the 2030 Agenda (United Nations 2015). Nevertheless, recent developments and the increasingly impatient protests of young climate activists emphasise that the implementation of sustainability goals in educational policies and programmes has so far only begun. Unfortunately, reli- gious education appears not to be an exception to this sobering rule. In a remarkable book, the German religious education scholar Bederna (2020) recently wrote: Religious education for sustainable development is more than ethical learning with a specific subject. The contemporary diagnosis of fundamental non-sustainability is a challenge to Christian speech on God. […] Those beliefs which claim a relationship between God and the world and within it to humankind must be reformulated and translated in this light. […] The task, however, is too big. It is urgent and everything is at stake: the core problem of the future and core ideas of theology. (pp. 17–20, own translations) There is certainly no lack of good arguments that religious education—with its diverse manifestations and organisational forms worldwide1—could play an important role in transitioning towards sustainability, nor is there a shortage of normative calls to consist- ently put this contribution into practice. Although Chamberlain (2000) noted an alarming “absence of ecological insights” (p. 134) in religious education discourse 20 years ago, and Martin (2015) still criticised a veritable gap “between our appreciation of the reality of ecological devastation and our work in religious education” (p. 34) 5 years ago, a “sig- nificant uptick of activity” (Tomlinson 2019, p. 186) has recently emerged. In the context of Catholic religious education, the impulse of the encyclical Laudato Si’, in which Pope Francis (2015) clearly called for “ecological conversion” (No. 217), was probably particu- larly important for this development. 1  I am writing this paper against the background of the model of denominational religious education as a compulsory subject at state schools, as is characteristic for Germany and Austria. This context certainly influences my reflections and arguments. Nevertheless, I believe that both the topic and the results of the presented research are also relevant in numerous global contexts. Abstract There is a widely accepted consensus that religious education, together with all other school subjects, should contribute to the goals of education for sustainable development. As a result, theoretical models have been developed to profile what the specific contribu- tions of religious education might be. However, the question whether religious education can achieve the intended goals has not yet been researched in more detail. More fundamen- tally, to be able to formulate realistic goals involves the clarification of students’ predis- positions. In the light of these questions, the author focuses on an ecological sustainabil- ity dilemma, the so-called poplar dilemma, and asks how students react to this conflictive situation and what roles religious orientations play in their responses. Analysis of the responses of a sample of more than 1100, 14- to 16-year-old students at secondary schools in Germany and Austria led to the conclusion that over-reaching, action-changing environ- mental sustainability goals are probably beyond the reach of religious education as a sin- gle subject. Nevertheless, it becomes clear that ecological issues are regarded by many as religious and spiritual questions and can therefore be addressed in a focused way through religious education. The idea of religious stewardship, which implies taking responsibil- ity and addresses both religious and non-religious students, could be particularly promis- ing. Religious education could develop a characteristic profile as an area in which transla- tion between different motivations for engaging in religious stewardship occurs, and where commitments to taking responsibility are sought. Keywords  Education for sustainable development · Religious education · Decision- making · Empirical research · Environmental education · Dilemma For Reinhold Boschki, academic mentor and friend, on the occasion of his sixtieth birthday in May 2021. 1 Department of Religious Education, Faculty of Catholic Theology, Johannes Gutenberg- University, 55099 Mainz, Germany 23456789) 1 3 56789) 3 S. Altmeyer 58 1  Introduction The proposals for integrating “ecology as a constitu- ent dimension of Christian education” (Tomlinson 2019, p. 187) cover a wide range of cognitive, ethical and spiritual aspects. We can differentiate between two approaches: The first, faith-formation concept aims to teach people to be religious in a way that helps them with the “cultivation of ecologically grounded faith” (Ayres 2017, p. 64). Here, the per- sonal involvement in religious narratives, the orientation towards spiritual models and the cultivation of virtues play a central role (Tomlinson 2019; Bouma-Prediger 2016; Fleischer and DeMoor 2015; Ayres 2014; Effiong 2011; Price 2008; Dunn 1990). The second, more reflective approach, focuses on theological implications and interpretations of the sustain- ability crisis and aims to stimulate critical judgment as a basis for conscious commitment. 1 3 Religious education for ecological sustainability: an initial… 59 The focus is on the “complicated and big questions that can inspire truly critical thinking” (O’Brien 2014, p. 194), from the theology of creation and cosmology and the question of guilt and hope, to finiteness and justice (Bederna 2020; Grelle 2018; Hoven 2015; Horrell and Davis 2014; Chamberlain 2000; Bratton 1990; Hill 1990). Most aspects are grouped together in the anthropological challenge of “rethinking what it means to be human in rela- tionship to Nature” (Martin 2015, p. 25).2 However convincing the proposals may be in detail, and however fully supportable the demands made, there is a type of surplus of normativity evident in publications within both approaches, which may also hinder practical implementation. I would like to illus- trate this by taking Bederna’s above-mentioned position as an example. In her book, she argues that sustainability is not an important and ultimately contingent educational topic, but forms a universal ethical principle. By structural analogy to Kant’s categorical impera- tive, she defines the principle of sustainability as a moral obligation for all individuals to live “in such a way that what is done with ‘nature’ could always be done by anyone, any- where, everywhere” (Bederna 2020, p. 103). This “ecological imperative” (p. 104) is then no longer based solely on the acute ecological crisis and its pressure to act, but posits that the preservation of the basis of life for future generations is a universal obligation. This arises from the personal realisation of freedom, which always has to include the freedom of others, including future generations. 2  Both approaches possess advantages and disadvantages, but derive their plausibility from the educational context for which they were developed and in which they are applied. Consequently, the point at hand is not to evaluate both approaches against each other, but to present the conceptual and practical challenge with which both are equally confronted. 1  Introduction In the sense of an emancipatory understanding of edu- cation—meaning that education at its heart should empower (young) people to realise their own freedom—sustainability is thus a mandatory constituent of education for all school subjects. Religious education could contribute to this in a specific way by […] interrupting the flow of the normal and non-sustainable and by introducing and exploring a different world view – from the encounter with creation as a vision of sustainability, as common home and as a gift in which God gives himself, to the confrontation with forms of sufficiency and poverty as freedom [for example in the spirituality of mendicant orders] […] or the reflection of guilt and the enabling of conversion. (p. 272) Religious education for sustainable development, as Bederna calls it (cf. also Effiong 2011, p. 152), is therefore not required for reasons of objective norms (for example, accord- ing to the motto: ‘Christians should live in a sustainable way!’), but from a reasonable insight into the normative validity of the principle of sustainability. For Bederna, religious education for sustainable development is therefore primarily “about ethical judgement and spirituality, resilience against what is considered normal, imagination and the courage to think differently” (p. 22). f In this lucid argument, we can observe a double normativity: The demand that religious education should follow the principle of sustainability, and the thematic demonstration that it can do so through its specific content, are both presented in a normative way—the for- mer on the basis of normative ethics, the latter by means of normative theology. The more we agree with the position presented—which I would like to do explicitly—the more the following question comes to the fore: Is religious education—with its concrete personal, 1 3 S. Altmeyer 60 contextual and structural conditions—able to meet the educational expectations and the religious hopes placed upon it as religious education for sustainable development? g p p p g p Therefore, we need to perform a reality check and question what are the realistic pos- sibilities for religious education to follow the “prophetic call for sustainable development” (Effiong 2011, p. 3)? This question refers emphatically to empirical research, which has to date been extremely scant. However, research from primarily environmental sociology and psychology has shown that religious orientations play an important role in sustainability issues (for overviews cf. Tomalin et al. 2019; Jenkins et al. 2018; Basedau et al. 1  Introduction 2017; Ronan 2017; Sachdeva 2016; Haluza-DeLay 2014; Tucker 2006), although the connections are by no means clear. Whether religious orientations positively or negatively affect the formation of a sustainable environmental orientation depends on many personal and con- textual factors, and not least, on the respective religious orientation itself (Baugh 2019; Ives and Kidwell 2019; Basedau et al. 2017; Pater and Dankelman 2009; Sherkat and Elli- son 2007). Hardly any specific empirical evidence is available for this concerning young people, and despite empirical research on sustainability awareness among young people (Gifford and Nilsson 2014), the effects of religious orientations remain largely unknown. The few available studies indicate that religious orientations might positively relate to envi- ronmental awareness (Albert et al. 2019; Francis and Penny 2016; Burgert 2013). However, explicit research findings on the impact of religious education in this context are so far lacking (Kvamme 2017). In summary: the issues addressed in this introduction are as important as they are com- plex and the empirical research gap is considerable. For this reason, we need to limit the present study for it at least to make a preliminary contribution to the question of whether and in what ways religious education might contribute to education for sustainable devel- opment. On the one hand, I will limit myself to problems of environmental sustainability and to students who attend secondary school in Germany and Austria. On the other hand, I will concentrate on decision-making in everyday situations that have a dilemma structure. Some research, primarily from science education, has recommended that focus should be placed on decision-making in conflictive situations (Lee et al. 2019; Garrecht et al. 2018; Gresch et al. 2017; Jho 2015; Jho et al. 2014; Steffen and Hößle 2014; Tuncay et al. 2012; Zeidler et al. 2005). The present study addresses the factors that influence such a decision and its evaluation and the impacts of religious orientations and participation in (Christian) religious education. On this basis, I will initially discuss how religious education could realistically contribute to the emerging sustainability transitions as addressed in education for sustainable development. 2  Materials and methods To address the research questions in a focused manner, an interdisciplinary research team conducted a questionnaire study with 14- to 16-year-old students at a total of 19 secondary schools in Germany and Austria. The survey was conducted in two stages; first in Germany at the end of 2018 and then in Austria at the end of 2019. A comprehensive description of the research design and the survey instruments has already been published (Altmeyer and Dreesmann 2020a, b). Here, we restrict ourselves to describing some central characteristics of the sample and the instruments used, which were especially developed for the purpose of the study. The focal point of the survey was the so-called poplar dilemma, which was 3 1 Religious education for ecological sustainability: an initial… 61 presented to the students for evaluation as a questionnaire. The conflictive situation was this: presented to the students for evaluation as a questionnaire. The conflictive situation was this: Please imagine the following: Six months ago, a young family with two children your age bought a house in your neighbourhood and moved in. In the garden belonging to the house, is a big tree, a 50-year-old poplar. After a short time, one of the children developed a severe allergy against poplar pollen. Your new neighbours have now decided to cut down the tree. What do you think about your neighbours’ decision? Participants were invited to rate the decision to fell the poplar on a 5-point scale (from 1 = agree to 5 = disagree). A set of 32 arguments was then provided, each of which could be similarly evaluated by degrees of agreement or disagreement. Another section offered a small set of questions focused on concrete examples of nature and religious experience. The questionnaire also presented some questions that addressed different attitudes towards nature and biblical creation spirituality, most of which were taken from previous studies with adolescents in Germany (Sieg et  al. 2018; Sammet et al. 2015; Ziebertz and Riegel 2008; Brämer 2006). In the German subsample, some knowledge questions were also asked to assess the state of reflected knowledge about biblical creation theology as it could be expected according to current school curricula of Christian religious education. Additionally, we collected basic socio-biographical and contextual data (age, gender, religious affiliation, school type, participation in reli- gious education, and class level). 2  Materials and methods For the evaluations reported in this paper, I used the following variables calculated from the mentioned item sets. • Four evaluation categories as factor variables from the 32 dilemma arguments (ranging from 1 to 5, with 1 = agreement): (1) ecocentrism (α = 0.818, N = 1052), summarizing 7 items that reflect emotional, moral or rational emphasis on the value of nature; (2) anthropocentrism (α = 0.750, N = 1006), consisting of 8 items that rep- resent an orientation towards human well-being, so that the value of nature is meas- ured by its importance for human life; (3) eco-pragmatism (α = 0.700, N = 1050), representing 7 items that deal with pragmatic alternatives to conserve the ecological status quo; and (4) religious stewardship (α = 0.744, N = 1070), which summarized three items particularly characterised by the use of religious semantics (‘God’, ‘crea- tion’) to express an obligation to protect nature. Because the last category is particu- larly relevant below, I list the items that are included in the scale in “Appendix”. • Two experience scores representing experience in nature and experience with reli- gion (α = 0.769; 0.811; N = 1122) calculated from 17 items on a three-point scale; • Three attitude measurements as factor variables (ranging again from 1 to 5, with 1 = agreement): (1) eco-spirituality included nine items (α = 0.713, N = 1039), with statements that express a wide-ranging love for nature and life (cf. Sponsel 2012, 2019). This attitude included rational and, to an even greater extent, emo- tional aspects. Again, the items are listed in “Appendix”; (2) biblicism (α = 0.785, N = 1054), summarized three items that show a literal understanding of the Bible and especially the creation stories; (3) the final measurement refers to a single item that captured the personal importance of believing in a loving Creator God. The research team conducted the survey in biology and religious education classes at 18 state schools in the German federal states of Rhineland–Palatinate and Hesse, mainly around the two capitals Mainz and Wiesbaden, and one school in a mid-sized town in Lower Austria belonging to the catchment area of the Austrian capital, Vienna. Fully 3 S. 2  Materials and methods Altmeyer 62 Table 1   Admissions of religion and spirituality in the sample Religious affiliation N Experience with religion Belief in a loving creator Biblicism Eco-spirituality Frequently (%) Rarely (%) Seldom to none (%) Mean (SD) Mean (SD) Mean (SD) Christian (Catholic or Protestant) 772 22.4 25.1 52.5 3.22a (1.34) 3.64a (1.00) 2.03a (0.59) Muslim 102 58.8 25.5 15.7 2.04 (1.27) 3.26 (0.97) 2.38 (0.65) Other 67 31.3 17.9 50.7 2.85 (1.55) 3.18 (1.27) 2.17 (0.75) None 152 4.6 9.2 86.2 4.37 (1.03) 4.33 (0.92) 2.01 (0.65) Overall sample 1093 23.9 22.5 53.6 3.24 (1.42) 3.67 (1.05) 2.07 (0.63) Table 1   Admissions of religion and spirituality in the sample a Scales ranging from 1 to 5, with 1 = agreement Religious affiliation N Experience with religion Belief in a loving creator Biblicism Eco-spirituality Frequently (%) Rarely (%) Seldom to none (%) Mean (SD) Mean (SD) Mean (SD) Christian (Catholic or Protestant) 772 22.4 25.1 52.5 3.22a (1.34) 3.64a (1.00) 2.03a (0.59) Muslim 102 58.8 25.5 15.7 2.04 (1.27) 3.26 (0.97) 2.38 (0.65) Other 67 31.3 17.9 50.7 2.85 (1.55) 3.18 (1.27) 2.17 (0.75) None 152 4.6 9.2 86.2 4.37 (1.03) 4.33 (0.92) 2.01 (0.65) Overall sample 1093 23.9 22.5 53.6 3.24 (1.42) 3.67 (1.05) 2.07 (0.63) Table 1   Admissions of religion and spirituality in the sample a Scales ranging from 1 to 5, with 1 = agreement Religious affiliation N Experience with religion Belief in a loving creator Biblicism Eco-spirituality Frequently (%) Rarely (%) Seldom to none (%) Mean (SD) Mean (SD) Mean (SD) Christian (Catholic or Protestant) 772 22.4 25.1 52.5 3.22a (1.34) 3.64a (1.00) 2.03a (0.59) Muslim 102 58.8 25.5 15.7 2.04 (1.27) 3.26 (0.97) 2.38 (0.65) Other 67 31.3 17.9 50.7 2.85 (1.55) 3.18 (1.27) 2.17 (0.75) None 152 4.6 9.2 86.2 4.37 (1.03) 4.33 (0.92) 2.01 (0.65) Overall sample 1093 23.9 22.5 53.6 3.24 (1.42) 3.67 (1.05) 2.07 (0.63) 1 3 1 3 Religious education for ecological sustainability: an initial… 63 completed questionnaires were available from 1122 students, 53.2% of whom attended ninth grade and 46.8% tenth grade in secondary schools of different school types (about two-thirds were grammar schools). The participant mean age was 14.9  years (SD = 0.88), and gender was evenly distributed (47.3% female, 46.6% male, 6.1% not specified). 2  Materials and methods Regarding religious affiliation, 70.5% of respondents were Christians (Catho- lic or Protestant), 9.5% were Muslims, 6.2% belonged to other religions and 13.8% did not belong to any denomination. In total, 73.8% of students attended Christian religious education classes at school. Experience and attitude measurements provided some interesting details on religious and spiritual orientation within the sample (Table 1). A total of 261 participants (23.9%) reported having had experience with religion frequently, whereas the majority (53.6%) reported little to no religious experience. The biblicism scale indicated an overall ten- dency towards disagreement (M = 3.67, SD = 1.05). Comparably low values were found with regard to the personal significance of a belief in a Creator God. Significant differences existed, depending on religious affiliation, which were consistent with the results known from general youth studies (Albert et al. 2019). However, for the variable eco-spirituality, a high level of agreement was present, with hardly any differences in terms of religious affiliation. 3  Results On average, the students in the sample evaluated the dilemma decision positively: 54.5% thought that cutting down the tree was the right thing to do, 24.6% were undecided, and only 20.7% were critical of the decision. Analyses examined the extent to which this evalu- ation depended on religious orientations or participation in religious education and identi- fied hardly any correlations. The results of regression analyses did not provide a signifi- cant model (Table 2); therefore, we can assume that other reasons were responsible for the decision to the dilemma. The only exception was participation in religious education. This coincided with a lower agreement to the opinion that the tree should be felled in favour of the child’s health (ΔM = 0.26). More precisely, participation in religious education was Dependent variable Independent variable What do you think about your neighbours’ decision? β (stand.) Religious affiliation (Christian) Religious affiliation (Islam) Religious affiliation (other) Participation in religious education 0.099 (p = 0.003) Experience with religion Belief in loving Creator God Biblicism Model accuracy (adjusted R2) 0.006 Model significance F(7,1056) = 1.948 (p = 0.059) Table 2   Impact of religion on dilemma evaluation Table 2   Impact of religion on dilemma evaluation S. Altmeyer 64 Table 3   Explanation of dilemma decision by evaluation categories n of dilemma on categories **Significant at p < 0.001, *significant at p < 0.05 Dependent variable Independent variable What do you think about your neighbours’ deci- sion? β (stand.) Ecocentrism  − 0.248** Anthropocentrism 0.398** Eco-pragmatism  − 0.090* Religious stewardship  − 0.038 (n.s.) Model accuracy (adjusted R2) 0.381 Model significance F(4,1090) = 169.013** **Significant at p < 0.001, *significant at p < 0.05 reflected in significantly fewer votes for full approval of the pro-child decision and signifi- cantly more students chose the middle of the scale (I am undecided) (p < 0.05).f reflected in significantly fewer votes for full approval of the pro-child decision and signifi- cantly more students chose the middle of the scale (I am undecided) (p < 0.05).f However, a different picture emerged for the evaluation categories, which provided a very good model for predicting the decision in the dilemma (Table 3). Thinking in anthro- pocentric categories was clearly associated with agreement to cut down the tree, and eco- centrism was almost as clearly associated with the opposite decision. 3  Results Eco-pragmatism also led to a more critical view of the decision against the tree, although the effect was less pronounced. Particularly relevant for our question was the finding that religious steward- ship did not significantly influence the dilemma decision, which may mean that this evalu- ation category can justify a decision that is pro-child and against the tree, or vice versa. We could assume that this category is potentially oriented towards overcoming this dichotomy.i On the basis of these findings, I queried the variables on which preference for one of the four evaluation categories mentioned, depended. Additional regression analysis was used to test potential interdependencies. In addition to religious orientation and participation in religious education, the procedures also included the variables on nature orientation and gender. Table 4 summarizes the results and only lists significant coefficients. ifi For the eco-pragmatism and anthropocentrism categories, the low model accuracy indi- cates that other factors not examined here were decisive. For religious stewardship and ecocentrism, however, striking results were observed. Religious orientations showed a high impact on religious stewardship and no impact on ecocentrism. Christian students for whom the belief in a loving Creator is important and who have experience with religion were likely to align with religious stewardship. Attendance of religious education might reinforce this effect, whereas a literal understanding of the Bible had no influence. Instead, biblicism apparently promoted a more eco-pragmatic view. A more frequent experience of nature primarily resulted in an ecocentric evaluation. However, the variable eco-spirituality was particularly interesting, because it was the only variable that affected all evaluation categories and also consistently showed the highest effects. Eco-spirituality manifested very clearly in an ecocentric evaluation pattern, but might also reinforce eco-pragmatism and religious stewardship. However, anthropocentric values were not compatible with a spiritual attitude towards nature. In total, 171 students showed high approval values for both eco-spirituality and religious stewardship and did not express clear preferences in the dilemma, whereas the 119 students who scored highly for ecocentrism and eco-spirituality voted much more strongly in favour of the tree. 3  Results 1 3 Religious education for ecological sustainability: an initial… 65 1 3 Table 4   Models to explain the evaluation categories, using independent variables **Significant at p < 0.001, *significant at p < 0.05 a Positive coefficients indicate a stronger agreement with the respective category Independent variable Dependent variable Religious stewardship Ecocentrism Eco-pragmatism Anthropocentrism β (stand.)a β (stand.)a β (stand.)a β (stand.)a Religious affiliation (Christian) 0.095* Religious affiliation (Islam) 0.140**  − 0.104* Religious affiliation (other) Participation in religious education 0.073* Experience with religion 0.227** Belief in loving Creator God 0.181**  − 0.137* 0.108* Biblicism 0.228**  − 0.109* Experience in nature 0.114** Eco-spirituality 0.339** 0.464** 0.278**  − 0.161** Gender (female) 0.062* Model accuracy (adjusted R2) 0.310 0.288 0.089 0.038 Model significance F(10,1050) = 48.652** F(10,1054) = 44.058** F(10,1053) = 12.495** F(10,1053) = 5.252** 1 S. Altmeyer 66 Table 5   The experience of students with religion and degree of acceptance of religious stewardship Experience with religion Religious stewardship Total N (%) Strong agree- ment N (%) Agreement N (%) Other N (%) Frequently 116 (10.6) 98 (8.9) 51 (4.6) 265 (24.1) Seldom 65 (5.9) 110 (10.0) 72 (6.6) 247 (22.5) Little to none 92 (8.4) 209 (19.0) 286 (26.0) 587 (53.4) Total 273 (24.8) 417 (37.9) 409 (37.2) 1099 (100.0) One assumption could be that participation in religious education affects students’ religious knowledge; hence, elaborate knowledge of biblical creation theology conveyed through religious education might potentially positively affect the category of religious stewardship. Data concerning students’ knowledge of biblical creation theology were avail- able for the German subsample (N = 870), and regression analyses revealed that the vari- able ‘participation in religious education’ can be replaced by ‘religious knowledge’ and the same correlations are upheld: knowledge about biblical theology of creation influenced agreement with the category of religious stewardship in combination with the factors men- tioned above [β (stand.) = 0.092, p = 0.017]. The results allow the following conclusion to be drawn: although religious orienta- tions and participation in religious education (reflected in knowledge on the theology of creation) clearly do not influence ecocentric attitudes and only weakly impact eco- pragmatism and anthropocentrism, they clearly affect religious stewardship. However, religious stewardship is also most strongly influenced by an eco-spiritual attitude; in other words, religious stewardship is related to religion, and also to spirituality. Further analyses showed that these two areas were not identical at all. 1 3 3  Results Even a basic descriptive statistic showed that more than 60% of all students (N = 690) agreed with the concept of religious stewardship, and one-quarter of all participants strongly agreed (N = 273). However, 43.6% of these supporters (N = 301) stated that they had rarely or never had any experience with religion (Table 5). The study examined in more detail the particularly interesting group of 92 students (8.4%) who strongly agreed with the category of religious stewardship, but had had lit- tle or no contact with religion. Table 6 shows how the results of these students differed significantly from those of the others. i These students tended to be neutral in their approach to the dilemma, but at the same time were strongly in favour of the ecocentric perspective. This is a striking difference compared with the overall sample, because ecocentrism was generally associated with a decision in favour of the tree. We could imagine that for these students, the ecocen- tric perspective also includes the well-being of the child to a greater extent. In terms of religious orientations, these students were opposed to belief in the Creator, a literal understanding of the Bible, and religious anthropocentrism; they also felt that concepts of God should be reconsidered in the light of scientific knowledge. On the other hand, they revealed a special connection with nature: They acknowledged nature’s own laws, recognised a duty to protect nature, and linked these ecological questions with the ethi- cal concept of conscience. Although experience in Nature did not affect the results from these students, they scored particularly high values in eco-spirituality. From these 1 3 Religious education for ecological sustainability: an initial… 67 Table 6   Characteristics of non-religious students with a high level of agreement with religious stewardship **Significant at p < 0.001, *significant at p < 0.05 a Scales ranging from 1 to 5, with 1 = agreement. A negative deviation in the mean value corresponds to a stronger agreement on average Meana Δ Mean N Statistics Effect r What do you think about your neighbours’ decision? 4  Discussion The goals of education for sustainable development place an obligation on the whole school and thus, also on religious education. Due to its specific perspective within the school, religious education is confronted with the task of developing a very specific con- tribution to this key issue in interaction with other subjects. Ecological problems represent an important aspect of the educational and developmental goals addressed in this context. This study therefore focused on the potential contribution of religious education to eco- logical sustainability. In this still very broad area, the study focused on a sustainability dilemma from the everyday life of German and Austrian young people, and confronted students aged 14–16 years with the so-called poplar dilemma. Students were challenged to evaluate the decision of a neighbouring family that had decided to cut down a 50-year-old poplar tree that had caused a serious allergy in one of their children shortly after the fam- ily moved into the house. This everyday example raised the question concerning which factors play a role in the evaluation of this decision and whether religious orientations and participation in (Christian) religious education are significant in this context. On this basis, I would now like to ask whether religious education could contribute to educationally fos- tered sustainability transitions and to explore the nature of this contribution.il First, the study identified no direct influence of religious orientation on the decision in the dilemma. This corresponds with the finding of previous research that religion does not play a strong behavioural role for the vast majority of Central European young people in questions of everyday value orientation (overview: Riegel 2015) similar to the dilemma situation under study here. Here, other values play a much greater role—as demonstrated by the poplar dilemma instrument. The evaluation categories of ecocentrism, eco-pragmatism and anthropocentrism, which have also been discussed as being important influencing factors in other studies (Affifi 2020; Kopnina and Cocis 2017; Cocks and Simpson 2015; Kortenkamp and Moore 2001), had large and partly opposing influences on the decision. A particularly interesting fourth category was a so-called religious stewardship pattern (Graham 2020; Martin 2015; Welchman 2012). This category represented the idea that humans are obliged to assume responsibility towards nature, and this is articulated by religious semantics (concepts such as creation or God). 3  Results 3.03 0.66 1095 T(1093) =  − 5.037** 0.151 Ecocentrism 1.92  − 0.69 1104 T(113,740) = 7.979** 0.210 Anthropocentrism 3.15 0.33 1104 T(1102) = − 3.934** 0.118 Eco-pragmatism 2.21  − 0.52 1103 T(115,761) = 7.191** 0.184 Eco-spirituality 1.75  − 0.35 1097 T(122,859) = 6.612** 0.152 Belief in loving Creator God 3.68 0.48 1068 T(1066) =  − 3.069* 0.094 Biblicism 3.95 0.30 1091 T(1089) =  − 2.634* 0.080 Further single items Because he/she is in God’s image, humans are above all other living creatures 3.97 0.32 1080 T(1078) =  − 2.215* 0.067  Felling trees contradicts humans’ remit to protect nature 2.08  − 0.62 1073 T(1071) = 4.844** 0.146  I would have a guilty conscience about the tree 2.42  − 0.66 1091 T(102,798) = 4.288** 0.139  Humankind should not interfere with the laws of nature 1.93  − 0.84 1093 T(1091) = 6.677** 0.198  Scientific findings challenge us to rethink our own idea of God 2.25  − 0.47 1062 T(1060) = 3.628** 0.111 S. Altmeyer 68 results, we can conclude that these students reinterpreted the concept of religious stew- ardship individually: they agreed with religious semantics, but placed themselves out- side of the explicit (Christian) religion. For them, religious stewardship represented an expression of ecological spirituality, instead of a (traditional) religious orientation. Nevertheless, they shared with the more religious students the position that religious stewardship was a preferable response to the ecological decision-making situations in question. results, we can conclude that these students reinterpreted the concept of religious stew- ardship individually: they agreed with religious semantics, but placed themselves out- side of the explicit (Christian) religion. For them, religious stewardship represented an expression of ecological spirituality, instead of a (traditional) religious orientation. Nevertheless, they shared with the more religious students the position that religious p g p y g Nevertheless, they shared with the more religious students the position that religious stewardship was a preferable response to the ecological decision-making situations in question. 4  Discussion With regard to Christian religious education, reflected knowledge of biblical crea- tion theology seems to be important here, which additionally counteracts biblicism.i The findings extend even further, however, because the concept of religious stewardship was not limited to religious students among the young people in this study: by contrast, for a surpris- ingly high proportion of students (27.4%), religious stewardship was also compatible with a categorically non-religious attitude. Almost 9% of the students even felt that belief in a Creator God was completely unimportant to them and that conceptions of God had to be revised in the light of scientific findings, but they mainly agreed with the category of religious stewardship. Therefore, we can conclude that these students would also benefit from the religious education perspective. A precondition for this would be to offer educational opportunities for them to enter into dialogue with the more religiously oriented students, or to collectively assume ecological responsibility under the shared perspective of religious stewardship in the school community as “engaged practitioners of change” (Sandberg 2020, p. 417). A precondition for exchange and understanding might again be a reflected knowledge of Creation Theology, which also assumes and elaborates critical perspectives (Horrell and Davis 2014; O’Brien 2014), including an anthropocentric bias inherent to the stewardship metaphor (Graham 2020; Martin 2015). The tendency of individuals to orientate towards the category of religious stewardship was thus not only dependent on religious orientations, and the study identified another, more influential factor: a so-called eco-spirituality (Sponsel 2012, 2019). This consisted of a fas- cination for and love of nature, the idea of a fundamental goodness of nature, the willingness to help nature as much as possible, or the idea of a soul in nature. Eco-spirituality played a central role in the emergence of both ecocentrism and religious stewardship. In connection with religious stewardship, eco-spirituality led to a relatively open attitude towards the deci- sion in the dilemma, whereas together with ecocentrism, it led to a commitment to preserve the tree. This leads us to conclude that for religious education, a combination of the con- cepts of religious stewardship with a spiritual approach to nature (Tomlinson 2019; Mar- tin 2015; Ayres 2014; Gottlieb 2006) might “foster awareness, nurture conversation, and encourage the development of a vision of ecological development” (Effiong 2011, pp. 4–5). 4  Discussion It is particularly relevant that religious stewardship not only received the highest ratings in our sam- ple, but it also moderated in the interplay with the other categories: religious stewardship could be used to justify both the protection of the child and of the tree, so that finally, a neutral attitude towards the dilemma decision was often adopted. This finding is surprisingly congruent with more recent theological positions, which also clearly advocate that the biblical story of creation certainly does not imply a right of human beings to dominate, but instead, “calls for a caring responsibility for other creatures that reflects but does not usurp God’s own care for his crea- tion” (Bauckham 2010, p. 34; cf. Conradie 2010, pp. 305–307; even more strictly ecocentric: Habel and Wurst 2000). One conclusion from this is that through a particular focus on the idea 1 1 3 Religious education for ecological sustainability: an initial… 69 of religious stewardship, religious education could provide a good opportunity to work with students constructively (Grelle 2018). However, to expect religious education to offer far-reach- ing opportunities to change sustainability-related attitudes beyond “environmental sensitivity” (Effiong 2011, p. 124) must be regarded as rather unrealistic.ii of religious stewardship, religious education could provide a good opportunity to work with students constructively (Grelle 2018). However, to expect religious education to offer far-reach- ing opportunities to change sustainability-related attitudes beyond “environmental sensitivity” (Effiong 2011, p. 124) must be regarded as rather unrealistic.ii (fi g p ) g This thesis was substantiated and refined by further findings in this study, which showed that religious stewardship was positively influenced by religious orientations and participa- tion in religious education, although this was not the case for ecocentrism. Few meaningful correlations were observed for eco-pragmatism and anthropocentrism, which were mainly influenced by other factors. Collectively, we can conclude that religious students can be positively encouraged towards the idea of religious stewardship by participation in reli- gious education, whereas ecocentric attitudes tend to be unaffected by religious education. This finding is consistent with that of other research, which has identified an increasingly responsible, ecological but not strictly ecocentric orientation within Christianity under the label of a so-called greening of religion hypothesis (Baugh 2019; Taylor et al. 2016; Taylor 2011). 4  Discussion At the same time, even the biblical tradition, justifiably, is quite critical of idealising nature (Bauckham 2010, pp. 109–115). This resonates with current dilemmas, which consistently demonstrate that the notion of ‘innocent nature’ is an illusion, sometimes even an ideology.i Even if the above-mentioned findings only provide preliminary empirical evidence that urgently requires further research—especially through intervention studies on the actual impact of religious education, we can already make a well-founded statement: religious education offers the potential to specifically contribute to the goals of ecological education 1 3 S. Altmeyer 70 for sustainable development. For example, it can support religiously oriented students to develop an attitude of religious stewardship, but non-religious students are also amenable to this orientation. This and other studies have shown that questions of sustainable develop- ment and climate change are increasingly perceived as religious or spiritual issues—even beyond explicit religious orientations (Altmeyer and Dreesmann 2020b; Jenkins et al. 2018; Berry 2013; Sponsel 2012; Gottlieb 2006). Because of this, religious education could raise the question “how our ecological and moral imaginations might be stimulated through an encounter with these alternative [i.e. religious] perspectives on being in the world” (Grelle 2018, p. 195), and could sensitise students to take collective and personal responsibility. Theoretically, it could develop a specific translation competence with the aim of facilitating understanding of the religious/spiritual dimension in sustainability, on the basis of differing religious orientations. To explain what this ‘translation competence’ represents, I would like to take the position of the German sociologist Nassehi (2017), who has profiled the concept as fundamentally impor- tant in view of current societal challenges. For Nassehi, the central characteristic of contem- porary modern societies is their complexity: “In all areas of society, a wide variety of different authorities are now having their say at the same time. […] A complex reciprocity of non-coor- dinated voices emerges, which must be related to one another.” (p. 16, own translations) The fundamental problem of complexity is that everything always has a “multiple meaning” (p. 65) due to different logics, which are unconnected and nevertheless claim equal importance, which is why it is difficult to reach a consensus even on broadly acknowledged important goals such as sustainable development. The only solution for Nassehi is to deal with this complexity by devel- oping what he calls a translation competence. 1 3 4  Discussion When politicians, climate researchers, economists and church representatives discuss the topic of climate change on a talk show for example, it is unhelpful to assume that a common denominator could be found that would integrate the diverse standpoints and underlying world views. Communication would fail if a mandatory consensual agreement had to be reached in which the various normativities were made ultimately compat- ible. Instead, the participants in the talk show must continue to communicate and recount their different perspectives to each other. By analogy to society as a whole, what is centrally important is the “management of interruptions” (p. 199). According to Nassehi, the constantly open process of understanding must never come to a standstill. It is the ability to translate that is paramount, i.e., the ability to communicate with each other despite different normatively grounded languages through a decisive management of interruptions. The metaphor of translation thus emphasises that we must abandon the idea of being able to harmonise the heterogeneity of beliefs and world views, but still need to work onto a common basis for communicating and acting collaboratively.i g g y On this basis, we could also profile the potential contribution of religious education to sustain- able development in the context of other school subjects. The contribution of religious education would then rest less on introducing further normativity based on religion, but would rather help to translate different normativities in view of the ecological crisis, which is recognised as a reli- gious challenge. Translation competence in religious education would involve perceiving one’s own fundamental orientations with regard to everyday ecological decision-making, articulating them, and engaging in dialogue with the frequently differing orientations of others. During reli- gious education, communicative interruptions could then be addressed and a new common lan- guage sought, whereby religious and world view orientations would become explicitly thematic. A good starting point for this could be the metaphor of religious stewardship. In an ideal case, dialogue on fundamental orientations in the search for a language that bridges communicative interruptions could also help to initiate concrete commitments to personal and joint responsibil- ity. 4  Discussion To resume the example of the poplar dilemma in religious education, we would then not only 1 3 1 Religious education for ecological sustainability: an initial… 71 ask what values are competing, but how the conflict challenges us to reflect and communicate anew on what it means to be truly human and deeply religious in relation to nature. Finally, a comment on the limitations of this study. These exist and are manifold, and clearly refer to the question to what extent generalisations are possible, based on the small and locally limited sample. Alternatively, we could criticise the poplar dilemma in terms of an ethnocen- tric or middle-class bias (Nielsen et al. 2017; Henrich et al. 2010). Another important point is that the influence of religious education on ecological sustainability was investigated with- out concrete questions being asked about ecological sustainability, and without investigating what had been previously taught in religious education. Above all, solid intervention studies are needed to examine the real impact of educational programmes in religious education for sustainable development. When I became aware of all these limitations, I decided to qualify the title with the small word ‘initial’. This was to express the hope that the hypotheses and ques- tions raised by the research presented here will inspire as many people as possible to continue working on this global key question through further research and committed teaching practice. Acknowledgements  The author would like to thank all students and teachers involved in this study for their cooperation. I am grateful to Professor Dr. Daniel Dreesmann for his great cooperation on this project, to Astrid Hesse and Maren Hornberger for contributing to early versions of the questionnaire as well as to Simon Ferel for supporting initial data analyses. Finally, the excel- lent proofreading by Dr. John Chandler is greatly acknowledged. Funding  Open Access funding enabled and organized by Projekt DEAL. Conflict of interest  None. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. Funding  Open Access funding enabled and organized by Projekt DEAL. Appendix See Tables 7 and 8. See Tables 7 and 8. 1 3 Table 7   Religious stewardship Item Corrected item- scale correla- tion All life is God’s creation and has an intrinsic value that must be respected 0.655 Humans have received the task from God to deal responsibly with animals and the rest of nature and to preserve the diversity of the world 0.624 I think that humankind must actively work to preserve creation 0.450 1 3 Table 7   Religious stewardship Item Corrected item- scale correla- tion All life is God’s creation and has an intrinsic value that must be respected 0.655 Humans have received the task from God to deal responsibly with animals and the rest of nature and to preserve the diversity of the world 0.624 I think that humankind must actively work to preserve creation 0.450 Table 7   Religious stewardship 1 S. Altmeyer 72 Table 8   Eco-spirituality Item Corrected item- scale correlation I want to help nature as best I can 0.587 Why do you think nature should be protected? Out of love for nature 0.485 Insects are useful creatures 0.456 Why do you think nature should be protected? To preserve the diversity of species 0.422 If I find an insect in my room, I put it outside 0.409 I find insects fascinating 0.405 It is better for nature if I don’t travel so far away for holidays 0.329 What is natural is good 0.243 Plants have a soul 0.290 Item References Affifi, R. (2020). Anthropocentrism’s fluid binary. Environmental Education Research, 16(2), 1–18. fiil Albert, M., Hurrelmann, K., & Quenzel, G. (2019). Jugend 2019: Eine Generation meldet sich zu Wort. Shell-Jugendstudie (Vol. 18). Weinheim: Beltz.i Altmeyer, S., & Dreesmann, D. (2020a). The tree was there first – using an everyday ecological dilemma to explore the personal orientations of secondary school students in environmental decision-making. Environmental Education Research, 1–22. https​://doi.org/10.1080/13504​622.2020.18530​62 Altmeyer, S., & Dreesmann, D. (2020b). 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Gütersloh, Freiburg: Kaiser, Herder. 1 3
https://openalex.org/W2976450475
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Progesterone- primed ovarian stimulation in polycystic ovarian syndrome: An RCT
International journal of reproductive biomedicine
2,019
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Corresponding Author: Corresponding Author: Masrooreh Hoseini; Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Bouali Ave, safayeh, Yazd, Iran. Postal code: 89195999 Tel: (+98) 9171763276 Email: Abstract Corresponding Author: Masrooreh Hoseini; Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Bouali Ave, safayeh, Yazd, Iran. Postal code: 89195999 Tel: (+98) 9171763276 Email: Background: In vitro fertilization is an important therapy for women with polycystic ovarian syndrome (PCOS). The use of new ways of improving clinical results is yet required. Objective: This study was aimed to investigate the efficacy of progesterone primed ovarian stimulation (PPOS) and compare with conventional antagonist protocol in PCOS. Materials and Methods: A total of 120 PCOS women who were candidates for assisted reproductive technology treatment from August to January 2019 were enrolled in this RCT and were placed into two groups, randomly (n = 60/each). The PPOS group received 20 mg/day Dydrogesterone orally since the second day of the cycle and the control group received antagonist protocol. The pregnancy outcomes including the chemical and clinical pregnancy, the miscarriage rate, and the percent of gestational sacs/transferred embryos was compared in two groups. Results: Number of MII oocyte, maturity rate, Number of 2 pronuclei (2PN) and serum estradiol levels on trigger day were statistically lower in PPOS group (p = 0.019, p = 0.035, p = 0.032, p = 0.030), respectively. Serum LH level on trigger day in PPOS group was higher than antagonist group (p = 0.005). Although there wasn’t sever ovarian hyper simulation syndrome in any participants, mild and moderate ovarian hyper simulation syndrome was less in PPOS group (p = 0.001). Also, the chemical and clinical pregnancy rate were higher in the antagonist group, althoughit was not statistically significant (p = 0.136, p = 0.093 respectively). C l i O t d d t t th t PPOS d t i h i l d li i l Materials and Methods: A total of 120 PCOS women who were candidates for assisted reproductive technology treatment from August to January 2019 were enrolled in this RCT and were placed into two groups, randomly (n = 60/each). The PPOS group received 20 mg/day Dydrogesterone orally since the second day of the cycle and the control group received antagonist protocol. The pregnancy outcomes including the chemical and clinical pregnancy, the miscarriage rate, and the percent of gestational sacs/transferred embryos was compared in two groups. International Journal of Reproductive BioMedicine Volume 17, Issue no. 9, https://doi.org/10.18502/ijrm.v17i9.5103 Production and Hosting by Knowledge E Original Article Key words: Progesterone, Polycystic ovarian syndrome, Controlled ovarian stimulation, Frozen-thawed embryo transfer, Pregnancy rate. Registration in IRCT: IRCT20110509006420N18 Maryam Eftekhar 1,2 M.D., Masrooreh Hoseini 1 M.D., Lida Saeed 1,3 M.D. 1Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 2Abortion Research Center, Yazd Reproductive Science Institute, Shahid Sadoughi University of Medical Science, Yazd, Iran. 3Afzalipour Hospital, Kerman University of Medical Sciences, Kerman, Iran. Abstract Results: Number of MII oocyte, maturity rate, Number of 2 pronuclei (2PN) and serum estradiol levels on trigger day were statistically lower in PPOS group (p = 0.019, p = 0.035, p = 0.032, p = 0.030), respectively. Serum LH level on trigger day in PPOS group was higher than antagonist group (p = 0.005). Although there wasn’t sever ovarian hyper simulation syndrome in any participants, mild and moderate ovarian hyper simulation syndrome was less in PPOS group (p = 0.001). Also, the chemical and clinical pregnancy rate were higher in the antagonist group, althoughit was not statistically significant (p = 0.136, p = 0.093 respectively). Production and Hosting by Knowledge E cc ⃝Maryam Eftekhar et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Cheif Aflatoonian Abbas M.D. Conclusion: Our study demonstrate that PPOS does not improve chemical and clinical pregnancy rate of the infertile women with PCOS. Key words: Progesterone, Polycystic ovarian syndrome, Controlled ovarian stimulation, Frozen-thawed embryo transfer, Pregnancy rate. Registration in IRCT: IRCT20110509006420N18 How to cite this article: Eftekhar M, Hoseini M, Saeed L. “Progesterone-primed ovarian stimulation in polycystic ovarian syndrom Reprod BioMed 2019; 17: 671–676. https://doi.org/10.18502/ijrm.v17i9.5103 Page 671 International Journal of Reproductive BioMedicine Eftekhar et al. 1. Introduction Dydrogesterone (duphaston) is an artificial progesterone and is highly similar to endogenous progesterone in terms of its pharmacologic properties and molecular structure. In addition, it does not interfere with endogenous progesterone production (3). This drug is widely used to treat hormone replacement, abortion, and the luteal support in pregnancy (5). Polycystic ovarian syndrome (PCOS) is a prevalent endocrine disorder. About 6.3–21.4% of women in the reproductive age suffers from this disease. In vitro fertilization (IVF) is one of the important therapy for women with PCOS. Regardless of the higher number of retrieved oocytes in PCOS patients, low fertility rate, poor oocytes quality, and high rates of abortion are yet an important issues. Therefore, new protocols are needed to improve clinical outcomes (1). Nowadays we are observing the “freeze-all” techniques that freeze the entire number of oocytes or embryos, which we could utilize ovarian stimulation with no restriction, including adverse effects of hormones on endometrial receptivity (2). In the present study, it was hypothesized that dydrogesterone could be utilized as a substitute progestin in a PPOS protocol. We designed a randomized clinical trial (RCT) to evaluate the cycle characteristics and pregnancy outcome of individuals with PPOS and compare them with conventional antagonist. 2. Materials and Methods It has been proved that progesterone prevents pulsatile secretion of luteinizing hormone (LH) and gonadotropin releasing hormone (GnRH) (3). https://doi.org/10.18502/ijrm.v17i9.5103 2.1. Study design In order to inhibit LH increasing, progestin-primed ovarian stimulation (PPOS) was stated. In this protocol oral progesterone (P) injected from the initial day of ovarian stimulation at the follicular phase (4). Using this novel PPOS protocol, P level, are utilized as the substitutions of GnRH analogue to inhibit the early LH surge during the follicular stage (5). A total of 120 individuals with PCOS aged between 18–40 yr old and were candidate for ART treatment were enrolled in this study. The study was taken place in Yazd Research and Clinical Center for Infertility between August to January 2019. PCOS diagnosis was performed based on Rotterdam criteria (2003), including polycystic ovaries, oligo-anovulation, as well as the biochem- ical or clinical signs of hyperandrogenism (7). The effects of progestin and the freeze-all strategy suggests that progestin-primed can be used as an ideal regimen for PCOS patients who are treated with assisted reproductive technology (ART) (6). In addition, progestin administrated orally and it is more convenient. Women with the history of intrauterine abnormalities (submucosal fibroma, uterine polyp, and intrauterine adhesions), severe endometriosis, systemic diseases, and azoospermia in their husbands were excluded from the study. Grouping was done by disclosing the sealed envelopes. Freeze all embryos strategy and transfer in a subsequent cycle, can reduce the late onset ovarian hyper simulation syndrome (OHSS). All subjects received 150 subcutaneous doses of Cinnal-f (Cinnagen, Iran) from the 2nd day of the cycle. Women in progesterone primed (PPOS) group, were prescribed 20 mg oral dose of dydrogesterone (duphaston, Abbott, Netherlands) from the 2nd day of the cycle and continued until triggering day. Vaginal sonography was done for all To avoid hypothalamic pituitary ovarian hypoxia, gonadotropin releasing hormone agonist (GnRHa) with a low dose of hCG (1000IU) as the final triggering was used with low risk of moderate or severe OHSS. Selecting the appropriate progestin is essential for the PPOS protocol success (5). https://doi.org/10.18502/ijrm.v17i9.5103 Page 672 Page 672 International Journal of Reproductive BioMedicine Progesterone-primed ovarian stimulation clinical pregnancy was confirmed by detecting fetal heartbeats 2 wk following the positive β hCG. The miscarriage was defined as losing pregnancy prior to 20 wk of gestation. The implantation rate was considered as percentage of gestational sacs/transferred embryos. patients since 6th day of cycle. 2.3. Endometrium preparation Endometrial preparation process were similar in both groups. All subjects received 6 mg/day estradiol valerate (Aburaihan Co., Tehran, Iran) orally from the 2nd day of menstrual cycle. Vaginal ultrasonography was performed to measure endometrial thickness on the 13th day of menstrual cycle. When endometrial thickness reached ≥8 mm, all subjects received 400 mg of Cyclogest R ⃝vaginal peccaries (Cox Pharmaceuticals, Barnstaple, UK) twice a day until menstruation or 8 wk of gestational age in pregnant women. Embryo transfer was performed three days after progesterone administration.ering,. Estradiol and progesterone injection continued for up to 8 wk after pregnancy. 2.6. Statistical analysis The statistical analysis was performed using SPSS software (Statistical Package for the Social Sciences, version 20.0, Chicago, IL, USA). In order of identifying major differences in the two groups, both chi-square and t-test were utilized. P < 0.05 was regarded as the significance level. 2.5. Ethical consideration The Ethics Committee of Yazd Reproductive Sciences Institute approved the study protocol (code: IR.SSU.RSI.REC.1397.003). The study was registered at IRCT (Iranian Registry of Clinical Trials) under code IRCT20110509006420N18 Written informed consent was obtained from all subjects after counseling about conventional antagonist protocol and PPOS. 3. Results A total of 120 women who met the inclusion criteria enrolled the study as 2 groups (n = 60/each). Two patients failed to follow-up in the study due to familial problems group (Figure 1). The baseline characteristics were similar in both groups (Table I). There was no significant different between duration of stimulation, total dose of gonadotropin and No. of retrieved oocyte between two groups. No. of MII oocyte, maturity rate, No. of 2 pronuclei (2PN) and serum E2 levels on trigger day were lower in PPOS group (p = 0.019, p = 0.035, p = 0.032, p = 0.030 respectively). 2.1. Study design In the antagonist group, when the size of dominant follicles reached to 12–13 mm, 0.25 mg of cetrotide (Merck-Serono Germany) was injected subcutaneously daily and continued until triggering day. When dominant follicles reached 17 mm, serum LH, E2 and P were checked. Then the final triggering was performed by Subcutaneous injection of decapeptyl 0.2 mg (Ferring, Germany) and intramuscular injection of human chorionic gonadotropin (HCG) 1000 IU (Pregnyl-Germany) in both groups. Oocytes pick up was done 36 hr later. All embryos were frozen in cleavage stage and frozen embryo transfer was done 2 months later. 2.2. Embryo vitrification and warming Cryopreservation of all embryos was done by cryotop vitrification method on 2nd or 3rd day after oocyte retrieval in both groups (8). https://doi.org/10.18502/ijrm.v17i9.5103 2.4. Pregnancy outcomes Chemical pregnancy was determined by serum β hCG > 50 IU/L two wk after ET. In addition, https://doi.org/10.18502/ijrm.v17i9.5103 Page 673 Page 673 International Journal of Reproductive BioMedicine Eftekhar et al. Serum LH level on trigger day in PPOS group was higher than antagonist group (5.29 vs. 3.79; p = 0.005). Although there wasn’t sever OHSS in any patient, mild and moderate OHSS was less in PPOS group (36.5% vs. 68.3%; p = 0.001) (Table II). Chemical and clinical pregnancy were more in antagonist group which was not statistically significant (Table III). Table I. Basic characteristics of the participants in two groups Table I. Basic characteristics of the participants in two groups Table I. Basic characteristics of the participants in two groups Variable PPOS group Antagonist group P-value Female age (yr)∗ 28.47 ± 3.60 28.98 ± 3.55 0.433 Duration of infertility (yr)∗ 6.00 ± 2.84 6.88 ± 3.65 0.154 Type of infertility∗∗ Primary 48 (82.8) 46 (76.7) 0.411 Secondary 10 (17.2) 14 (23.3) AMH (IU/L)∗ 8.95 ± 3.70 9.76 ± 4.64 0.302 ∗Data presented as mean ± SD; Student’s t-test; ∗∗Data presented as n (%); Chi- square AMH: Anti mullerian hormone; PPOS: Progesterone primed ovarian stimulation; Yr: Year ∗Data presented as mean ± SD; Student’s t-test; ∗∗Data presented as n (%); Chi- square AMH: Anti mullerian hormone; PPOS: Progesterone primed ovarian stimulation; Yr: Year Variable PPOS group Antagonist group P-value Duration of stimulation (day)∗ 10.24 ± 2.39 9.53 ± 2.01 0.084 Total dose of gonadotropin (IU)∗ 1528.45 ± 413.15 1430.00 ± 354.45 0.167 No. of retrieved oocytes∗ 15.74 ± 9.88 18.65 ± 7.87 0.083 No. of MII oocytes∗ 12.50 ± 8.88 16.03 ± 6.99 0.019 Maturity rate∗∗∗ 79.90% 85.52% 0.035 No. of two pronucleus∗ 8.70 ± 7.43 11.38 ± 5.68 0.032 No. 2.4. Pregnancy outcomes of obtained embryos∗ 7.91 ± 6.63 9.48 ± 4.62 0.141 Total cycle cancelation∗∗ 4 (6.9%) 0 (0%) 0.038 E2 levels on trigger day (pg/mL)∗ 2351.55 ± 965.58 3047.68 ± 2157.04 0.030 LH levels of on trigger (IU/mL)∗ 5.29 ± 3.79 3.56 ± 2.61 0.005 P levels on trigger day (IU/mL)∗ 0.72 ± 1.25 0.81 ± 0.92 0.687 Fertilization rate∗∗∗ 63.26% 71.30% 0.073 Endometrial thickness (mm)∗ 9.20 ± 1.34 8.83 ± 1.14 0.161 OHSS (mild and moderate)∗∗∗ 36.5% 68.3% 0.001 ∗Data presented as mean ± SD; Student’s t-test; ∗∗Data presented as n (%); Chi- square; Data presented as percentages PPOS: Progesterone primed ovarian stimulation; MII: Mature oocyte II; E2: Estradiol; LH: Luteinizing hormone; P level: Progesterone ∗Data presented as mean ± SD; Student’s t-test; ∗∗Data presented as n (%); Chi- square; Data presented as percentages PPOS: Progesterone primed ovarian stimulation; MII: Mature oocyte II; E2: Estradiol; LH: Luteinizing hormone; P level: Progesterone level; OHSS: Ovarian hyper stimulation syndrome 4. Discussion development, and egg maturity. It seems that there is a level of LH capacity which the overexposure can affect the prohibition of ovulation by controlling the granulosa distribution, oocyte atresia, early luteinization and ultimately affects IVF outcome. However, the appropriate level of LH on trigger day was not determined (9). In this present study, clinical efficiency of duphaston in PPOS regimen were evaluated. Our results showed that duphaston as an FSH adjuvant to during the ovarian stimulation did not lead to similar mature oocyte retrieval. Oocyte maturity was mainly utilized to assess the oocyte quality. The maturity rate of oocytes in PPOS group was significantly lower than antagonist group. In addition, fertilization rate was lower in the PPOS group. In the present study, estradiol level on trigger day was significantly lower in PPOS group. The effect of over-physiological levels of E2 on IVF outcomes are still controversial. Some researchers have stated that the serum E2 concentrations on the day of hCG administration have a positive correlation with pregnancy outcomes. Nevertheless, some other researchers have reported adverse or no effects of high levels of E2 association between the levels of E2 serum and the IVF outcomes of (10–12). All studies that investigated the effect of estradiol on pregnancy were conducted under the fresh embryo transfer conditions (10–12). In the literature review, there was no study that made to determine the effect of estradiol serum level in trigger day on frozen embryo outcome. In the current study, the lower level of estradiol on the trigger day was correlated with the lower numbers of mature oocytes. The pregnancy results of FET in PPOS showed a lower clinical pregnancy rate, 14.6% vs. 29.9%. The implantation rate was also lower in PPOS group, although it was not statistically significant but clinically notable. Although duphaston increased the early LH rate, it did not interfere with the measurements of progesterone. However, in the present study, the mean level of LH was significantly higher in the PPOS group. Previous studies has shown that when progesterone is administered during the regular follicular phase, it decreases the the LH pulse frequency, increases the amplitude LH pulse, and decreases the mean LH levels of plasma compared with those who were not treated (3). Kuang and colleagues conducted a primary randomized study on PPOS. Variable ∗Data presented as mean ± SD; Student’s t-test; ∗∗Data presented as n (%); Chi- square; Data presented as percentages PPOS: Progesterone primed ovarian stimulation; MII: Mature oocyte II; E2: Estradiol; LH: Luteinizing hormone; P level: Progesterone level; OHSS: Ovarian hyper stimulation syndrome Variable PPOS group Antagonist group P-value# Implantation rate∗ 7.32% 15.69% 0.062 Chemical pregnancy rate/transfer∗∗ 13 (31.7) 24 (47) 0.136 Clinical pregnancy rate/transfer∗∗ 6 (14.6) 15 (29.4) 0.093 Abortion rate∗∗ 8 (61.5) 9 (37.5) 0.161 #∗Data presented as (%); ∗∗Data presented as n (%) Chi square PPOS: Progesterone primed ovarian stimulation Page 674 https://doi.org/10.18502/ijrm.v17i9.5103 https://doi.org/10.18502/ijrm.v17i9.5103 https://doi.org/10.18502/ijrm.v17i9.5103 Page 674 Page 674 International Journal of Reproductive BioMedicine Progesterone-primed ovarian stimulation Enrollment Lost to follow-up due to familial problems (give reasons) (n = 2) Follow-Up PPOS Group Allocated to intervention (n = 60) Received allocated intervention (n = 60) Antagonist Group Allocated to intervention (n = 60) Received allocated intervention (n = 60) Allocation Assessed for eligibility (n = 120) Excluded (n = 0) Lost to follow-up (give reasons) (n = 0) Randomized (n = 120) Analysed (n = 60) Analysis Analysed (n = 58) Figure 1. Consort Flow Diagram. Enrollment Figure 1. Consort Flow Diagram. References [1] Zhu X, Ye H, Fu Y. The utrogestan and hMG protocol in patients with polycystic ovarian syndrome undergoing controlled ovarian hyperstimulation during IVF/ICSI treatments. Medicine 2016; 95: e4193. [1] Zhu X, Ye H, Fu Y. The utrogestan and hMG protocol in patients with polycystic ovarian syndrome undergoing controlled ovarian hyperstimulation during IVF/ICSI treatments. Medicine 2016; 95: e4193. In another study Nanako lwami and co-workers compared the rates of ongoing and clinical pregnancies between the antagonist regimen and the PPOS protocol. They used oral dydrogesterone and HMG in the PPOS protocol. They concluded that the rates of ongoing and clinical pregnancies were similar in both groups which is in contrast with our results. However, they included normal responders in addition to hyper- responders (14). [2] Massin N. New stimulation regimens: endogenous and exogenous progesterone use to block the LH surge during ovarian stimulation for IVF. Hum Reprod Update 2017; 23: 211–220. [3] Zhu X, Ye H, Fu Y. Duphaston and human menopausal gonadotropin protocol in normally ovulatory women undergoing controlled ovarian hyperstimulation during in vitro fertilization/intracytoplasmic sperm injection treatments in combination with embryo cryopreservation. Fertil Steril 2017; 108: 505–512. [4] Huang CY, Chen GY, Shieh ML, Li HY. An extremely patient-friendly and efficient stimulation protocol for assisted reproductive technology in normal and high responders. Reprod Biol Endocrinol 2018; 16: 18. In a pilot study, a short protocol was compared with the PPOS protocol in PCOS patients. This article reported no significant differences in the number of collected oocyte and the incidence of ongoing pregnancy. However, a high dose of HMG was consumed in the PPOS group. Considering the particular risk of OHSS, two cases were reported in the short protocol group vs. none in the PPOS group (p = 0.154) (6). [5] Yu S, Long H, Chang HY, Liu Y, Gao H, Zhu J, et al. New application of dydrogesterone as a part of a progestin-primed ovarian stimulation protocol for IVF: a randomized controlled trial including 516 first IVF/ICSI cycles. Hum Reprod 2018; 33: 229–237. [6] Wang Y, Chen Q, Wang N, Chen H, Lyu Q, Kuang Y. Controlled ovarian stimulation using medroxyprogesterone acetate and hMG in patients with polycystic ovary syndrome treated for IVF: a double-blind randomized crossover clinical trial. Medicine 2016; 95: e2939. [7] Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). 5. Conclusion In conclusion, the results of the present study showed that PPOS is not appropriate for women with PCOS, however, the results of previous studies on PPOS were in contrast with this present study. It seems that further randomized clinical trials (RCTs) are required for better assessment of PPOS in PCOS. [9] Gardner DK, Weissman A, Howles CM, Shoham Z. Textbook of assisted reproductive techniques: laboratory and clinical perspectives. UK: CRC press; 2016. [10] Kosmas IP, Kolibianakis EM, Devroey P. Association of estradiol levels on the day of hCG administration and pregnancy achievement in IVF: a systematic review. Hum Reprod 2004; 19: 2446–2453. [11] Serna J, Cholquevilque JL, Cela V, Mart´ınez-Salazar J, Requena A, Garcia-Velasco JA. Estradiol supplementation during the luteal phase of IVF-ICSI patients: a randomized, controlled trial. Fertil Steril 2008; 90: 2190–2195. [12] Joo BS, Park SH, An BM, Kim KS, Moon SE, Moon HS. Serum estradiol levels during controlled ovarian hyperstimulation influence the pregnancy outcome of in vitro fertilization in a concentration-dependent manner. Fertil Steril 2010; 93: 442–446. 4. Discussion They added medroxyprogesterone acetate to gonadotropin- induced stimulation in the follicular phase, and compared this protocol with traditional short The LH level reached its highest value at the mid-cycle time and causes the meiosis I reinitiate inside pre-ovulatory follicles. The mentioned time is crucial for successful fertilization, perfect embryo https://doi.org/10.18502/ijrm.v17i9.5103 Page 675 Page 675 International Journal of Reproductive BioMedicine Eftekhar et al. protocol. They demonstrated that pregnancy, implantation, and miscarriage rates were not meaningfully different between groups (13). Acknowledgments This article was financially supported by the Research and Clinical Center for Infertility, Yazd, Iran. [13] Kuang Y, Chen Q, Fu Y, Wang Y, Hong Q, Lyu Q, et al. Medroxyprogesterone acetate is an effective oral alternative for preventing premature luteinizing hormone surges in women undergoing controlled ovarian hyperstimulation for in vitro fertilization. Fertil Steril 2015; 104: 62–70. [14] Iwami N, Kawamata M, Ozawa N, Yamamoto T, Watanabe E, Moriwaka O, et al. New trial of progestin-primed ovarian stimulation using dydrogesterone versus a typical GnRH antagonist regimen in assisted reproductive technology. Arch Gynecol Obstet 2018; 298: 663–671. References Hum Reprod 2004; 19: 41–47. [8] Eftekhar M, Rahsepar M, Rahmani E. Effect of progesterone supplementation on natural frozen-thawed embryo transfer cycles: a randomized controlled trial. Int J Fertil Steril 2013; 7: 13–20. Conflict of Interest There is no Conflict of interest. https://doi.org/10.18502/ijrm.v17i9.5103 Page 676
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The Ordinance of 1667: Ideology of Modern Codification as a Political Tool of Louis XIV
Wroclaw Review of Law, Administration & Economics
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DOI: 10.1515/wrlae-2015-0028 * The present paper has been prepared within the framework of a project financed by the National Science Centre (Narodowe Centrum Nauki) on the basis of decision no. DEC- 2013/09/B/HS5/02617. ** Department of History of Law, Faculty of Law and Administration, University of Gdańsk. 1 Slavoj Žižek, The Sublime Object of Ideology (Verso 2008) 16. 2 Rafał Mańko, ‘Koncepcja interpelacji ideologicznej a krytyczny dyskurs o prawie’ [The Notion of Ideological Interpellation and Critical Discourse on Law] (2014) 1 Archiwum Filozofii Prawa i Filozofii Społecznej 43. 3 Žižek (n 1) 138; Mańko (n 2) 43. THE ORDINANCE OF 1667: IDEOLOGY OF MODERN CODIFICATION AS A POLITICAL TOOL OF LOUIS XIV* ANNA KLIMASZEWSKA** 3 Žižek (n 1) 138; Mańko (n 2) 43. 2 Rafał Mańko, ‘Koncepcja interpelacji ideologicznej a krytyczny dyskurs o prawie’ [The Notion of Ideological Interpellation and Critical Discourse on Law] (2014) 1 Archiwum Filozofii Prawa i Filozofii Społecznej 43. 3 Ž ** Department of History of Law, Faculty of Law and Administration, University of Gdańsk. 1 Slavoj Žižek, The Sublime Object of Ideology (Verso 2008) 16. * The present paper has been prepared within the framework of a project financed by the National Science Centre (Narodowe Centrum Nauki) on the basis of decision no. DEC- 2013/09/B/HS5/02617. INTRODUCTION In his The Sublime Object of Ideology Slavoj Žižek underscores that “Ideology is not a dreamlike illusion that we build to escape insupportable reality; in its basic dimension it is a fantasy-construction which serves as a support for out ‘reality’ itself. It is an ‘illusion’ which structures our effective, real social relations and thereby masks some insupportable, real, impossible kernel […] The function of ideology is not to offer us a point of escape from our reality but to offer us the social reality itself as an escape from some traumatic, real kernel.”1 In its political dimension, ideology soothes social antagonisms, “[as it] masks the reality […], creating a fantasy-construction which allows the system to keep functioning”.2 According to Žižek, the ideological fantasy-construction fulfils the role of a “framework” which we place over the raw facts of reality in order to understand it.3 A special role in shaping these ideological frameworks is given to legal acts, which not only define accepted standards of behaviour, but also a certain vision of the world, of the political, social and economic system. The strongest expression of the created ideology is not only the final effect in the form of an enacted legal act, it is also (perhaps primarily so) the stage of preparatory works, at which the true intentions of the legislator are often manifested most evidently. g y In this article, I discuss the process of development of certain ideological frameworks serving the purpose of “masking the reality”, using the example of preparatory works leading up to the enactment of the 1667 Ordonnance sur la réformation de la justice civile, as well as the contents itself of this ordinance, enacted on the order of the French king Louis XIV, and commonly called the “Ordinance on civil procedure” (by its 128 Wroclaw Review of Law, Administration & Economics 129 [Vol 5:1 contemporaries as well as today– in textbooks, commentaries, jurisprudence, etc.). This is because a legal act, in theory passed with the purpose of regulating the civil procedure (and as such, permanently classified in the social reception), in principle consolidated the standards of judicial proceedings shaped over previous centuries. The basic innovative load that it carried was the change of entitlements and position within the system of judges, especially of those most highly placed (besides the king) within the hierarchy of the judicial system, that is of judges of the parliaments. 4 The Commercial Code of 1807 was largely based on the Ordinance on commerce of 1673 and on the Ordinance on the marine of 1681. See Anna Klimaszewska, Code de commerce – francuski Kodeks handlowy z 1807 r. [Code de Commerce: The French Commercial Code of 1807] (Arche 2011). The majority of solutions from the ordinance of 1667 were duplicated in the Code of Civil Procedure of 1806, a fact openly admitted in the materials from preparatory works of the Napoleonic codification commission who worked on the project of the code. These materials have been repeatedly published, for instance in: Code de procédure civil, motifs et rapports, edition stereotype faite au moyen de matrices mobiles en cuivre, procédé d’Herhan (Garnery, Librairie Stéréotype Nicolle 1806). Even the materials from works on the reform of substantive civil law, conducted at the consent of Louis XIV, were later used to a large extent in drafting of the Code civil. According to J.P. Royer, the principle of copying regulations from the ordinances of Louis XIV by Napoleonic codification commissions also applied to penal procedure; Jean-Pierre Royer, Histoire de la justice en France (PUF 2001) 38. INTRODUCTION Its promulgation marked an important step towards the building of an absolute monarchy in France. The catchy propaganda phrases used by Louis XIV and the people employed by him for the task basically oscillated between steps taken up in social interest, for the good of the subjects (elimination of the abuses of the justice system, abolishment of arbitrary decisions made by judges and reduction of the costs of proceedings), as well as around the lofty work of codification of the law, which is marked with an atmosphere of taking the law a notch higher, of modernization and modernity. This is because codification, even when employed in order to solidify the ruler’s authority, is inherently linked with the paradigm of progress, positively viewed by the society regardless of the legislator’s original intentions. This article comprises three major parts: first I explain the significance of the 1667 Ordinance, I then move onto the political circumstances in which it was enacted, before finally considering the steps taken in the battle for power of Louis XIV and his acolytes, in the course of which one of the tools was the legislative works on civil procedure. I. SIGNIFICANCE OF THE 1667 ORDINANCE In April of 1667, Louis XIV issued the Ordonnance sur la réformation de la justice civile, which regulated civil procedure. This was an unprecedented occurrence for many reasons. It was the first act in the series of so-called great ordinances, which constituted the first complex attempt at codification of French law, leading it onto a path of unification aimed at the level of national authority. Some of the Napoleonic codes went on to copy the contents of ordinances drafted under the reign of Louis XIV to various extents.4 The objective of this monarch, long before Bonaparte, was to “work THE ORDINANCE OF 1667: IDEOLOGY OF MODERN CODIFICATION AS A POLITICAL TOOL OF LOUIS XIV 2015] 130 on the system of justice in his kingdom” (de travailler à la justice de son royame) and to develop a “great collection of French laws” (grand corps de droit français). The longevity of the Ordinance on civil procedure was also remarkable. From a formal perspective, it remained in force until 1806, when the Code de procedure civile was enacted, however this code largely repeated the norms contained in the ordinance. Therefore, it would be difficult to speak of a new code; de facto it was rather an amendment of the ordinance of Louis XIV. As a result, it constituted an absolutely ground-breaking act in terms of the regulation of the law of civil procedure, laying the foundations for the French model in this legal field. g The preamble to the Ordinance emphasized the significance that the ruler attached to the principles of the work of the justice system, denominating this sector of the state authority as “the most solid foundation ensuring the continuation of the State, peace of the families and happiness of the nation.”5 However, it was simultaneously admitted that in this regard the state was not functioning properly. The very same preamble condemned the arbitrariness of courts, the excessive procedures and costs of proceedings, due to which the judiciary was ineffective in satisfying the needs of the subjects.6 The ordinance was viewed as the necessary remedy to this situation, especially since it was to have the semblance of the effect of a consensus reached by the ruler and the jurists, who to some extent represented the society. 5 Preamble to the ordinance, version used here: Ordonnance de Louis XIV. Roy de France et de Navarre. Donnée à Saint Germain en Laye au mois d’Avril 1667 (Associez choisis par ordre de Sa Majesté pour l’impression de ses nouvelles ordonnances 1667) 1. 6 ibid 2. 7 Legists – scholars of the Roman law, as opposed to specialists in canon law (canonists or decretalists); in France the legists were king’s counsellors (usually officials originating from the small gentry or from the bourgeoisie), who strove to extend the king’s power and who justified the need for a strong royal authority based on Roman law texts. They aided in the consolidation of the principles of succession to the throne, of the integrity of the king’s domain and of those defining the king’s general position within the monarchy. They are referred to as “the first creators of absolutism in France”. I. SIGNIFICANCE OF THE 1667 ORDINANCE However, the propagandist and catchy phrases of the preamble, focusing on the social interest did not mean that the political interests of the monarch, who aimed to strengthen the state and his own absolute power, did not underlie the codification of the civil procedure. Both these objectives did not have to be mutually exclusive, after all. An ordinance on civil procedure could very well both further the interests of the state and constitute an important modernising tool. The codification may certainly be perceived in the categories of progress, a phenomenon that fostered the interests of the general society. II. POLITICAL BACKGROUND The principles drafted by legists7 as early as the 13th century clearly underscored the sovereign and absolute nature of the king's power (“the king is emperor in his realm”), also in the domain of justice (“the king is the fountain of all justice”). As a result of the expansion of royal judiciary, a Wroclaw Review of Law, Administration & Economics [Vol 5:1 131 separate judicial body emerged in the 13th century from the Royal Council, called the Parliament (Parlement) from the 14th century onwards.8 It had become impossible for the Council and the king to resolve the growing influx of cases. At the same time, it was inadvisable to bestow these competences onto unprofessional vassals. As a result, a group of legists was formed. They were educated jurists with a permanent delegation to adjudicate in the name of the king and in his absence. Since the early 14th century, Parliament functioned regularly as the supreme royal court with its permanent seat in the Cité, with professional judges (irremovable as of the 15th century). With the Parliament of Paris increasingly overburdened, kings begun to create new parliaments in individual provinces.9 Formally, the position of all parliaments as supreme courts was equal. In practice, the one in Paris, with jurisdiction over more than half of the kingdom, played a central role.10 Besides judicial competences, parliaments eventually acquired very serious legislative powers, connected to the procedure of registration of legal acts issued by the king. Following this registration, each of these acts was then published and it came into effect within the jurisdictions of separate tribunals. In principle, this took place through the reading of an act aloud in open sessions, followed by its copying into the register and sending a printed version to subordinate courts. However, as time passed, parliaments began to desire a more significant role, claiming that the process of registration far exceeded the simple procedural steps, as it consisted in legalizing each separate legal act by its acceptance and publication. In order to justify the claims of parliaments, an entire ideological foundation was required.11 The 16th-century school of constitutionalists played a key role in this respect. For the larger part, they shared the views of the theorists on absolutism. They accepted, for example, the absolute power of the king, who wielded it in accordance with God-given and natural law. 8 The subject of the genesis, structure, composition and competences of the Parliament of Paris has been the focus of numerous publications, e.g. Ernest Désiré Glasson, Le Parlement de Paris. Son rôle politique depuis le règne de Charles VII jusqu'à la Révolution, vol 1-2 (Librairie Hachette 1901); Philippe Payen, Les arrêts de règlement du Parlement de Paris au XVIIIe siécle. Dimension et doctrine. Les grandes thèses du droit français (PUF 1997); Jean- Baptiste Dubédat, Histoire du Parlement de Toulouse, vol 1-2 (A Rousseau 1885); Gustave Saulnier de la Pinelais, Le gens du roi au Parlement de Bretagne 1553-1790 (J Plihon et L Hommay, A Picard 1902); also in English: John Hurt, Louis XIV and the parlements (Manchester University Press 2002); Joseph Hugh Shennan, The Parlement of Paris (Cornell University Press 1968); Bailey Stone, The Parlement of Paris, 1774-1789 (University of North Carolina Press 1981). 9 ) 9 Among others: in Toulouse (1443) for Languedoc and neighbouring territories;in Grenoble (1453) for Dauphiny; in Bordeaux (1462) for Guyenne and Western Gascony; in Dijon (1476) for Burgundy; in Rouen (1499) for Normandy; in Aix (1501) for Provence; in Rennes (1554) for Britanny; in Pau (1620) for Béarn and Navarre and in Metz (1633) for the dioceses of Metz, Toul and Verdun. 10 11 Cf Étienne Pasquier, Les recherches de la France (Laurens Sonnius 1621); Bernard de la Roche-Flavin, Treize livres des Parlements de France (M Berjon 1621); Claude de Seyssel, La grand’ monarchie de France (E Groulleau 1558); William Farr Church, Constitutional Thought in the Sixteenth-Century France (Harvard University Press 1941). 10 It was also composed of a higher number of judges – over two hundred, while the provincial parliaments usually had between fifty and one hundred. 11 8 The subject of the genesis, structure, composition and competences of the Parliament of Paris has been the focus of numerous publications, e.g. Ernest Désiré Glasson, Le Parlement de Paris. Son rôle politique depuis le règne de Charles VII jusqu'à la Révolution, vol 1-2 (Librairie Hachette 1901); Philippe Payen, Les arrêts de règlement du Parlement de Paris au XVIIIe siécle. Dimension et doctrine. Les grandes thèses du droit français (PUF 1997); Jean- Baptiste Dubédat, Histoire du Parlement de Toulouse, vol 1-2 (A Rousseau 1885); Gustave Saulnier de la Pinelais, Le gens du roi au Parlement de Bretagne 1553-1790 (J Plihon et L Hommay, A Picard 1902); also in English: John Hurt, Louis XIV and the parlements (Manchester University Press 2002); Joseph Hugh Shennan, The Parlement of Paris (Cornell University Press 1968); Bailey Stone, The Parlement of Paris, 1774-1789 (University of North Carolina Press 1981). 9 Among others: in Toulouse (1443) for Languedoc and neighbouring territories;in Grenoble (1453) for Dauphiny; in Bordeaux (1462) for Guyenne and Western Gascony; in Dijon (1476) for Burgundy; in Rouen (1499) for Normandy; in Aix (1501) for Provence; in Rennes (1554) for Britanny; in Pau (1620) for Béarn and Navarre and in Metz (1633) for the dioceses of Metz, Toul and Verdun. 10 It was also composed of a higher number of judges – over two hundred, while the provincial parliaments usually had between fifty and one hundred. 11 Cf Étienne Pasquier, Les recherches de la France (Laurens Sonnius 1621); Bernard de la Roche-Flavin, Treize livres des Parlements de France (M Berjon 1621); Claude de Seyssel, La grand’ monarchie de France (E Groulleau 1558); William Farr Church, Constitutional Thought in the Sixteenth-Century France (Harvard University Press 1941). y ) North Carolina Press 1981). 9 12 This examination took place at plenary sessions of the combined chambers, at which a given act was read aloud in its entirety, analyzed and scrutinized through discussion. Subsequently, the members of the chambers would express their approval or lack thereof in a free vote (liberté de suffrages). Additionally, according to the constitutionalists, if a new law did not stand the procedure of verification, the parliament was entitled to strike the articles it did not accept. Parliaments could also, in their opinion, modify the law by adding articles drafted on the spot in order to solve any disputed issues. Both these procedures were referred to as modification. Moreover, the constitutionalists deemed it natural that parliaments that modified laws should not bother the king with information about it. Another manoeuvre employed by the parliaments in order to obstruct regulations they did not approve of was to drag out the procedure of registration for as long as possible. If this was not detected for long enough, the new regulations would often fall into oblivion. Additionally, a serious delay could also influence the king’s willingness to accept certain modifications in return for effective registration. The right of remonstrance (droit de remontrances) was another measure of blocking new legal acts. It consisted in lodging oral or written objection to the registration of a given act. Simply passing this idea through voting would freeze the registration process, while the subsequent steps, that is drafting, verification, accepting and sending the remonstrance to the king were usually a very lengthy process. The king could order the registration of a law by way of a special writ (lettre de jussion). The parliaments, however, usually tried to wrestle the king by sending more remonstrances, which could only be overridden with the king’s personal appearance at the parliament’s “bed of justice” (lit de justice). Therefore, the parliaments were able to recourse to a sort of suspending veto. 13 Sovereign courts were understood to be all those tribunals which delivered sovereign II. POLITICAL BACKGROUND Unlike the absolutists, however, the constitutionalists raised the argument that the king’s power was additionally limited by French law and customs. In their opinion, the essential nature of this power was not breached by this limitation, THE ORDINANCE OF 1667: IDEOLOGY OF MODERN CODIFICATION AS A POLITICAL TOOL OF LOUIS XIV 2015] 132 as it was accepted voluntarily. Therefore, they perceived the king's power to be both absolute and limited. According to their outlook, the king’s power could only be executed following its verification (vérification) by the parliaments. This process, they claimed, required the detailed examination of legal acts, aimed to detect possible contradictions in abstracto with the broadly understood notions of reason and justice, in concreto with the king’s ordinances and jurisprudence of precedential nature.12 Such a far-reaching interpretation of the role of parliaments could not be meet with the approval of an absolute ruler. He had no resort but to accept it, however, as the stance of the parliaments, in clear opposition to the monarch, gained widespread acclaim in society. Especially since the Estates- General were not summoned, the parliaments assumed the role of guards of fundamental rights. Following the death of Louis XIII, the scales seemed to definitely tip in favour of the parliaments and other central bodies of authority. They attempted to use the tumultuous political situation which ensued in connection with the minority of the new ruler, Louis XIV. They began to act much more forcefully than before. In 1648 Chief Minister of France, Cardinal Mazarin, who co-ruled the country in the period of minority of Louis XIV, struggled against the Great Council (Grand Conseil). The Chamber of Accounts (Chambres de comptes), the Board of Excise (Cour des aides) and the Parliament of Paris demonstrated their solidarity by establishing an assembly of the delegates of sovereign courts of Paris (l’assemblée de la chambre Saint Louis), on the basis of Arrêt d’Union passed in May of the same year13. Of course, such a wilful act of defiance went against the already solidified absolutist systemic principles, and it was designed to permanently tip the balance of power in favour of the parliaments: the king was to maintain the authority to propose, and the sovereign courts – to decide. The monarch’s 13 Sovereign courts were understood to be all those tribunals which delivered sovereign rulings in the last instance, in cases entrusted to them by the king. II. POLITICAL BACKGROUND Their judgments could be repealed only by the king or his Council. Sovereign courts included the Great Council, the Chamber of Accounts, the Excise Board, the Cour de Monnaies and the parliaments. Wroclaw Review of Law, Administration & Economics 133 [Vol 5:1 situation was not improved by the views disseminated without any obstacles by Omer Talon, avocat général of the Parliament of Paris14, who claimed that sovereign courts should, pursuant to the constitution of the kingdom, contribute to limiting royal power, as designated for the exercise of the “second power” (puissance seconde), which they did through refusal to register and through exercising their right of remonstrance15. 14 Avocats généraux, (usually two of them per each tribunal), similarly to procureur général, were considered the so-called “king’s men” (gens du roi), appointed by him personally. They usually functioned collectively as the public prosecution, responsible for enforcement of king’s law and for his interests. Unofficially, they were headed by procureur général, who drafted the written documentation, while avocat général spoke before the tribunal. 15 Dominique Bernard Rives (ed) Œuvres d’Omer et de Denis Talon, avocats-généraux au Parlement de Paris, publiées sur les manuscrits autographes, vol 1 (A Égron 1821) 217. 16 Charles Louis Dreyss, Mémoires de Louis XIV pour l’instruction du Dauphin, vol 2 (Didier 1860) 384. 17 Louis XIV, ‘Mémoires pour l’instruction du Dauphin (Mémoires pour l’année 1661)’ in Pierre Goubert, Mémoires pour l’instruction du Dauphin (Imprimerie nationale 1992) 66. 18 ibid 400. 19 ibid. 20 Pierre Clément, Lettres, instructions et mémoires de Colbert, vol 4 (Imprimerie impériale 1867) 27-43. 14 Avocats généraux, (usually two of them per each tribunal), similarly to procureur général, were considered the so-called “king’s men” (gens du roi), appointed by him personally. They usually functioned collectively as the public prosecution, responsible for enforcement of king’s law and for his interests. Unofficially, they were headed by procureur général, who drafted the written documentation, while avocat général spoke before the tribunal. 15 20 Pierre Clément, Lettres, instructions et mémoires de Colbert, vol 4 (Imprimerie impériale 1867) 27-43. 17 Louis XIV, ‘Mémoires pour l’instruction du Dauphin (Mémoires pour l’année 1661)’ in Pierre Goubert, Mémoires pour l’instruction du Dauphin (Imprimerie nationale 1992) 66. 18 ibid 400. 19 15 Dominique Bernard Rives (ed) Œuvres d’Omer et de Denis Talon, avocats-généraux au Parlement de Paris, publiées sur les manuscrits autographes, vol 1 (A Égron 1821) 217. p g p , ( g ) 16 Charles Louis Dreyss, Mémoires de Louis XIV pour l’instruction du Dauphin, vol 2 (Didier 1860) 384. 17 III. REACTION OF LOUIS XIV Following the death of Cardinal Mazarin in 1661, Louis XIV proclaimed his personal rule. Over the subsequent months, the ruler took a series of steps to strengthen his position by reclaiming the lost instruments of governance (instruments de gouvernement). Among the tasks that he had set for himself, reforming the judicial system was of pivotal importance.16 “Excessive elevation of the parliaments was dangerous for the entire kingdom in the period of my childhood. They must be humiliated, not only in the context of the harm they have already done, but particularly in the context of harm they may still cause in the future.”17 The first king's move was to dismiss and exile some of the “haughtiest” (plus hardi) and most defiant judges of the Excise Board (Cour des aides).18 The fundamental idea behind this move was to set a harsh example for the entire system of justice, with particular emphasis on the parliaments.19 In September 1663 Jean-Baptiste Colbert, finance controller general closely connected to the king, went even further, by issuing Instruction pour les maîtres des requêtes, commissaires départis dans les provinces,20 which included guidelines for the intendants, commissaires and maîtres des requêtes regarding the statistical data they were to collect. Besides gathering maps and general information on individual regions, their task was to secure documentation and draft reports on ecclesiastical, military and financial matters, as well as on issues related to the functioning of the judicial authorities, with particular stress on instances of corruption and abuses of power by the judiciary, as well as the jurisprudence approach adopted by judges and possible complaints in connection with these. This document was a harbinger of change, foretelling the move away from the “judicial monarchy” and toward the “administrative monarchy”. It indicated that the 20 Pierre Clément, Lettres, instructions et mémoires de Colbert, vol 4 (Imprimerie impériale 1867) 27-43. THE ORDINANCE OF 1667: IDEOLOGY OF MODERN CODIFICATION AS A POLITICAL TOOL OF LOUIS XIV 2015] 134 centre of gravity in the field of power was about to shift from judges21 to intendants (commissaires) who were fully dependent on the king. Colbert had high expectations for the intendants and for their influence on the local judiciary. He also hoped that the Instruction would standardize, unify and order the views and practical methods of action. Colbert wished to fit the general course of action into this one text. III. REACTION OF LOUIS XIV Intendants, as civil servants who enjoyed a broad scope of competences, were to represent an extension of the sovereign’s will22. They were his agents in contact with the common people; their role was to find out about the needs and capabilities of the local communities and to evaluate the atmosphere within them. Moreover, as part of the division of retained justice23 (justice retenue), an intendant, based on the right of evocation, could preside over chosen cases at his discretion. At the same time, his competences overlapped24 with the competences of the first presidents of local parliaments, which often turned into embers of conflict. This was clearly visible both in the old, previously integrated provinces, and in the territories newly conquered by Paris. Especially in the latter, in places such as Alsace, Roussillon, Artois and Franche-Comté, the reform of state structures was facing numerous challenges, particularly in what concerned the system of justice. A serious crisis broke out, among others, in 1628 in Bordeaux, where the members of parliament opposed the claims of intendant Servien to judiciary functions and refused to register his lettres de commission.25 By nominating Colbert a royal minister, the king also secured his institutional influence on the parliaments. His original intention was to reduce their role in French public life. In particular, he aimed to create a situation wherein the role of parliaments regarding the registration of legal acts would be purely passive, coming down to their automatic acceptance and entering in the registers, without any freedom to modify their contents or to delay these actions. Colbert's plan was also to exert a moderate, long-term pressure, in order to influence the parliaments gradually yet consistently. 21 All of the judiciary (with the exception of patrimonial, church and municipal courts) was king’s; the king was the supreme judge and on this account, he had the right to judge personally. The existing courts exercised their power only in his name and only on the basis of a royal delegation. The king, however, reserved the right to suspend such delegation and to adjudicate in cases chosen by him. As a result, there was a distinction between delegated justice (justice déléguée), exercised by royal courts (prevotal, bailliage, presidial and the parliaments) and retained justice (justice retenue), reserved for the king personally or for the King’s Council. The King could also freely decide on the competence of a court for chosen cases. 24 Intendants were to monitor the activities of courts outside of parliaments, which often resulted in conflicts. They also exercised the right of evocation, consisting in the withdrawal of any case, regardless of the advancement of proceedings, and remanding it to another court “for the good of the justice system”. Intendants were also allowed to adjudicate, with the assistance of royal magistrates. 25 Charles Bon François Boscherondês Portes, Histoire du Parlement de Bordeaux, vol 1 (Charles Lefebvre 1877) 446 ff. 22 Intendants were nominated by the king in the Council; they were not subject to any local control and fell directly under the King’s Council. Their decisions could be appealed against only to the Council. 23 23 Compare with information above. 24 21 All of the judiciary (with the exception of patrimonial, church and municipal courts) was king’s; the king was the supreme judge and on this account, he had the right to judge personally. The existing courts exercised their power only in his name and only on the basis of a royal delegation. The king, however, reserved the right to suspend such delegation and to adjudicate in cases chosen by him. As a result, there was a distinction between delegated justice (justice déléguée), exercised by royal courts (prevotal, bailliage, presidial and the parliaments) and retained justice (justice retenue), reserved for the king personally or for the King’s Council. The King could also freely decide on the competence of a court for chosen cases. Through the appointment of commissary court, he could indicate special judges to examine a given case. 22 Intendants were nominated by the king in the Council; they were not subject to any local control and fell directly under the King’s Council. Their decisions could be appealed against only to the Council. 23 Compare with information above. 24 Intendants were to monitor the activities of courts outside of parliaments, which often resulted in conflicts. They also exercised the right of evocation, consisting in the withdrawal of any case, regardless of the advancement of proceedings, and remanding it to another court “for the good of the justice system”. Intendants were also allowed to adjudicate, with the assistance of royal magistrates. 25 Charles Bon François Boscherondês Portes, Histoire du Parlement de Bordeaux, vol 1 (Charles Lefebvre 1877) 446 ff. III. REACTION OF LOUIS XIV I expect assistance and cooperation from you, for this task to be successful as I expect it to be, especially since I have decided to handle it with zeal and since the Council assembled today is not to stay for a year or a few years. I hope to use its services and to summon it to me until the end of my life.”26 y g g p “For a long time now [...] it has been my desire to meet in this circle in order to bring justice to my kingdom. I know that my country needs it desperately for reason of chaos caused by the war and the years of my minority. I have long searched for the resources to remedy this chaos. This has never seemed an impossible feat to me, although I do appreciate the numerous challenges, which oblige me even more to engage in solving this matter. A ruler of ambition must put all his efforts into difficult affairs. I expect assistance and cooperation from you, for this task to be successful as I expect it to be, especially since I have decided to handle it with zeal and since the Council assembled today is not to stay for a year or a few years. I hope to use its services and to summon it to me until the end of my life.”26 As a consequence, the Council of Justice (Conseil de justice) was made permanent by Louis XIV. Colbert’s uncle and member of the State Council, Henri Pussort,27 presided over its sessions. The fate of the ordinance on civil procedure, and especially of its provisions contained in title I, which were of key importance for the king's permanent advantage over the parliaments, hinged in particular on the ruler’s ability to neutralize the opposition on the side of the Parliament of Paris. Initially, Louis XIV attempted to win the favour of the members of this court, and in particular of its first president. As previous practice had shown, the presidents usually had a strong influence over the remaining judges, although in reality their clout depended on the individual predispositions and skills. It was a commissary post, due to which the Parliament's president was perceived as a representative of the king, and accountable to him only. 26 Francis Monnier, Guillaume de Lamoignon et Colbert. Essai sur la législation française au XVIIe siècle (Didier 1862) 46-47. 27 Colbert himself, even if he attended some sessions, categorically refused to preside over them; ibid 70-71. 28 Vie de M. Premier Président de Lamoignon, écrit d’après le mémoires du temps et les papiers de la famille (Nyon 1781) xxxiv-xxxv. III. REACTION OF LOUIS XIV Through the appointment of commissary court, he could indicate special judges to examine a given case. 22 22 Intendants were nominated by the king in the Council; they were not subject to any local control and fell directly under the King’s Council. Their decisions could be appealed against only to the Council. 23 24 Intendants were to monitor the activities of courts outside of parliaments, which often resulted in conflicts. They also exercised the right of evocation, consisting in the withdrawal of any case, regardless of the advancement of proceedings, and remanding it to another court “for the good of the justice system”. Intendants were also allowed to adjudicate, with the assistance of royal magistrates. 25 Charles Bon François Boscherondês Portes, Histoire du Parlement de Bordeaux, vol (Charles Lefebvre 1877) 446 ff. 135 [Vol 5:1 Wroclaw Review of Law, Administration & Economics The turning point in this regard was the issuance of the ordinance on civil procedure, under the telling title of Ordonnance sur la réformation de la justice civile. Even the organization of the legislative work was planned so as to strengthen the position of Louis XIV in the field of power at the expense of the parliaments. The project was elaborated by a committee of counsellors of state, with the participation of outstanding and renowned lawyers, such as Barthélemi Auzanet. The fundamental ideological core of the project had been highlighted numerous times before, starting with the speech delivered b th ki hi lf d i i i L 25 S t b 1665 by the king himself during a session in Louvre on 25 September 1665: “For a long time now [...] it has been my desire to meet in this circle in order to bring justice to my kingdom. I know that my country needs it desperately for reason of chaos caused by the war and the years of my minority. I have long searched for the resources to remedy this chaos. This has never seemed an impossible feat to me, although I do appreciate the numerous challenges, which oblige me even more to engage in solving this matter. A ruler of ambition must put all his efforts into difficult affairs. III. REACTION OF LOUIS XIV On the other hand, however, the Parliament's president needed the support of other judges, for which reason he could not manifest his dependency on the king too ostentatiously while working for the good of his office, by definition marked to a certain extent by the independence of the king's power. The first president of the Parliament of Paris, Guillaume de Lamoignon, appointed in 1658, subjected himself to the king formally, declaring his full devotion and loyalty. His actions, however, were marked by a high degree of independence. He refused bribes,28 fiercely defended the parliament's prerogatives and the judges' freedom of adjudication; he also THE ORDINANCE OF 1667: IDEOLOGY OF MODERN CODIFICATION AS A POLITICAL TOOL OF LOUIS XIV 2015] 136 criticized the king's grandiose military ambitions that put a devastating financial burden on the state.29 It was therefore no wonder that Louis XIV, fearing attacks not only from the first president, but also from the other judges, came to the conclusion that their active participation in works on the ordinance on civil procedure was inadvisable. They were initially kept a secret, but finally they came to Lamoignon’s attention. Nevertheless, all of his attempts to actively join the drafting of the ordinance were met with the king’s unfaltering resistance.30 But the participation of judges, even if only symbolic, was necessary to present the ordinance to society as an effect of consensus and broad consultations with various legal circles. Therefore, the text drafted by Pussort's commission was simply read out loud by the chancellor, article by article, to the delegates of the Parliament, in the presence of the members of the State Council, to enable the judges a limited opportunity to participate in introducing changes and in the process of unification while maintaining all the disciplinary rules of the session.31 The remarks made by judges were sometimes accepted32 in the scope directly regarding to the civil procedure, but any of their objections as to the provisions of political nature were always nipped in the bud with the same statement uttered by Pussort: “Such is the king’s will.”33 Characteristically, discussion over Title I of the ordinance, which contained limitations of the parliaments’ prerogatives, did not take place until the very last session.34 p y However, Title I of the ordinance had already been drafted by the Council of Justice in 1665. Four articles from this title were subjected to registration procedure. 34 In the fifteenth and last session of 17 March 1667: Procès-verbal des conférences tenues par ordre du Roi pour l’examen des articles de l’Ordonnance civile du mois d’Avril 1667 et de l’Ordonnance criminelle du mois d’Avril 1670 (Associez choisis par ordre de Sa Majesté pour l’impression de ses nouvelles ordonnances 1757) 472 ff. 32 Although Lamoignon advocated that the principle of written form be observed, Pussort managed to render the proceedings more oral. For example, the presentation of disputed items in an articulated form (appointment) was reduced only to the most complex cases. Otherwise, evidence was presented during an oral hearing (Title XIV, art. 6). 33 33 Monnier (n 26) 89. 29 Cf Discours de Guillaume de Lamoignon, the manuscripts at the National Library in Paris (Bibliothèque Nationale – BN), sign.: vol 1 – BN Fr 14051, vol 2 – BN Fr 14052. 30 The king’s answer was always the same: “Mr. Colbert currently employs Mr. Pussort for this job. See mister Colbert and follow his instructions” ibid. 31 The king, as he confessed in his Mémoires, was keen to avoid any “annoying obstacles” (quelques chicane qui me fachât); Jean de Gain-Montagnac, Mémoires de Louis XIV, écrits par lui-même, composés pour le Grand Dauphin, son fils et adressés a ce prince, vol 2 (Garnery, Librairie Stéréotype Nicolle 1806) 6. 32 Although Lamoignon advocated that the principle of written form be observed, Pussort managed to render the proceedings more oral. For example, the presentation of disputed items in an articulated form (appointment) was reduced only to the most complex cases. Otherwise, evidence was presented during an oral hearing (Title XIV, art. 6). 33 Monnier (n 26) 89. 34 In the fifteenth and last session of 17 March 1667: Procès-verbal des conférences tenues par ordre du Roi pour l’examen des articles de l’Ordonnance civile du mois d’Avril 1667 et de l’Ordonnance criminelle du mois d’Avril 1670 (Associez choisis par ordre de Sa Majesté pour l’impression de ses nouvelles ordonnances 1757) 472 ff. 29 Cf Discours de Guillaume de Lamoignon, the manuscripts at the National Library in Paris (Bibliothèque Nationale – BN), sign.: vol 1 – BN Fr 14051, vol 2 – BN Fr 14052. j 31 The king, as he confessed in his Mémoires, was keen to avoid any “annoying obstacles” (quelques chicane qui me fachât); Jean de Gain-Montagnac, Mémoires de Louis XIV, écrits par lui-même, composés pour le Grand Dauphin, son fils et adressés a ce prince, vol 2 (Garnery, Librairie Stéréotype Nicolle 1806) 6. 32 ( q ), g , 30 The king’s answer was always the same: “Mr. Colbert currently employs Mr. Pussort for this job. See mister Colbert and follow his instructions” ibid. 29 Cf Discours de Guillaume de Lamoignon, the manuscripts at the National Library in Paris (Bibliothèque Nationale – BN), sign.: vol 1 – BN Fr 14051, vol 2 – BN Fr 14052. 30 The king’s answer was always the same: “Mr. Colbert currently employs Mr. Pussort for this job. See mister Colbert and follow his instructions” ibid. 31 The king, as he confessed in his Mémoires, was keen to avoid any “annoying obstacles” (quelques chicane qui me fachât); Jean de Gain-Montagnac, Mémoires de Louis XIV, écrits par lui-même, composés pour le Grand Dauphin, son fils et adressés a ce prince, vol 2 (Garnery, Librairie Stéréotype Nicolle 1806) 6. 32 l h h i d d h h i i l f i f b b d III. REACTION OF LOUIS XIV Of them, the most important was article IV, which regarded the registration duty. It enabled the king personally, but also through others acting as his proxies, to expedite the registration procedure. The so- called “bed of justice in the king’s absence” (lit de justice roi non présent) was established. Thus, article IV allowed the king to introduce laws without any reservations or limitations foreseen by the practice of parliaments pertaining to the registration procedure, supported by the theoretical grounds provided by constitutionalists. In this way, Louis XIV forced through a reform that none of his predecessors managed to. 137 [Vol 5:1 Wroclaw Review of Law, Administration & Economics One of the most strongly affirmed objectives of the legislative works was the replacement of old regulations with uniform norms regarding procedure, and thus the elimination of all provisions that stood in the way of generally shortening the length of civil proceedings. This was not about some fundamental restructuring of the heretofore functioning procedure, but rather about setting out its rules in an explicit and concrete manner. In this sense, the ordinance was the crown jewel in the evolution of the French model of civil procedure which had started some time before. Avoidance of abrupt changes in the ages-old matter, as well as the emphasis put on the pro-civic direction of the legislative works served, at the same time, as the ideological justification for actions which, incidentally, also limited the role of the parliaments in the whole process, thus debilitating their position. The king’s moves were in fact part of his consistent and long-term drive for power, played out under the ruse of a lofty reform of the justice system. In the 1667 ordinance, the term “sovereign courts” was replaced by “supreme courts”. This was because, in an absolutist state, there was no space for the existence of any sovereign entities other than the monarch himself. Therefore, this small modification was in fact a momentous one in its ideological aspect. On a practical level, the provisions laying down disciplinary measures against judges who disregarded the norms set out in the royal legislation35 had a similar weight. 35 Earlier, the parliaments had at their disposal the right to apply disciplinary measures against their members. 36 Claude Joseph de Ferrière, Dictionnaire de droit et de pratique, contenant l’explication des termes de droit, d’ordonnances, de coutumes et de pratique: avec les juridictions de France, vol 2 (J Dupleix 1779) 408. More Serge Dauchy, Les voies de recours extraordinaires : proposition d’erreur et requête civile (de l’ordonnance de Saint Louis jusqu’à l’ordonnance de 1667 (PUF 1989). III. REACTION OF LOUIS XIV This was clearly expressed in Article 6 of Title I: “Our will is for all of our ordinances, edicts, declarations and lettres patentes to be abided both in judgments and in other decisions, without any opposition; we do not wish for the courts or judges who represent us to feel exempt from them, nor to feel competent to modify their disposition, regardless of the case and matter, even under the pretence of rightfulness, public good or expedition of justice.” Nevertheless, the reform regarding the extraordinary appeal measures was much more important from the perspective of the judiciary. It abolished the tremendously strict proposition d’erreur,36 introducing instead the much more far-sighted cassation (cassation), a measure that made such a great carrer all over the world and became the hallmark of the French procedure. The very term “cassation” was not expressly employed until the decree of 1684, but its practical application was already suggested in Article 7 of Title I of the ordinance: “If, in the process of adjudication in a case pending before our parliaments or other courts, any doubts or difficulties that arise in connection with the execution of any of our ordinances, edicts, declarations, lettres patentes, we forbid their interpretation; in such cases it is our will for the [courts] to refer to us to be acquainted with our intention”. The legal enquiries – designed as a tool for ongoing control of the judges’ jurisprudential work – were lodged with the Council of Parties, which formed part of the King's Council. This institution bore fruit in the form of cassation as a competence to repeal (delete) final and binding judgements. Initially, this THE ORDINANCE OF 1667: IDEOLOGY OF MODERN CODIFICATION AS A POLITICAL TOOL OF LOUIS XIV 2015] 138 was a method to withdraw a judgement delivered on the basis of a fact-related error, not a legal one, as the idea that supreme courts could apply the existing law incorrectly was inadmissible.37 y Louis XIV concluded his game in the field of power by solving the problem of the parliaments dragging out the registration procedure, which he accomplished by way of articles 2 and 5 of Title I of the ordinance. From then on, the procedure had to be initiated immediately following the receipt of the legal act by courts, which were to “abandon all other matters”. III. REACTION OF LOUIS XIV The right of remonstrance was limited to eight days for the Parliament of Paris and to six weeks for the others. Due to the “varied interpretations” of these regulations, a royal declaration was issued in 1673 (24 February), unequivocally upholding the abolishment of remonstrance prior to registration of legal acts.38 Thus, the only effective way for the parliaments to obstruct the king’s will was disarmed. Always the “master of pragmatism”,39 Louis XIV did not in fact deprive them altogether of this right, for centuries deeply rooted in tradition; he rather shifted the center of gravity of how this right was exercised. Legal acts regarding the “proper movement” (propre mouvement) of the king had to be registered by obligation and without delay. Only later could the courts, if such need arose, draft a remonstrance. Although, according to Michel Antoine, the parliaments still maintained their security shield against the monarchy, and the introduced limitations failed to silence them altogether,40 it is clear that the right of remonstrance lost much of its force. 40 Cf Michel Antoine, ‘Les remontrances des Cours supérieures sous le règne de Louis XIV (1673-1715)’ (1993) 151 Bibliothèque de l’École des chartes 87-122. 37 See Marguerite Boutel-Sautel, ‘La cassation sous l’Ancien Régime’ in Le tribunal et la Cour de cassation (1790-1990) (Association des magistrats et anciens magistrats de la Cour de cassation 1990) 1-24; idem, Vivre au Royaume de France (PUF 2010) 453-476. 38 François-André Isambert, Decrusy, Alphonse Honoré Taillandier, Recueil général des anciennes lois françaises, depuis l’an 420, jusqu'à la Révolution de 1789, vol 19 (Librairie de Plon Frères 1829) 70-73. 39 Royer (n 4) 34 37 See Marguerite Boutel-Sautel, ‘La cassation sous l’Ancien Régime’ in Le tribunal et la Cour de cassation (1790-1990) (Association des magistrats et anciens magistrats de la Cour de cassation 1990) 1-24; idem, Vivre au Royaume de France (PUF 2010) 453-476. 38 François-André Isambert, Decrusy, Alphonse Honoré Taillandier, Recueil général des anciennes lois françaises, depuis l’an 420, jusqu'à la Révolution de 1789, vol 19 (Librairie de Plon Frères 1829) 70-73. 39 Royer (n 4) 34. 40 Cf Michel Antoine, ‘Les remontrances des Cours supérieures sous le règne de Louis XIV (1673-1715)’ (1993) 151 Bibliothèque de l’École des chartes 87-122. 39 Royer (n 4) 34. CONCLUSIONS The enactment of the 1667 Ordinance marked a very significant reform of the law, which led France into a new era of codification in the domain of civil procedure. It was from that point on that France joined the avant-garde of states exemplary in terms of the direction of evolution of the law in the entire world. It was also a fundamental blow in the political battle between the king and the judicial authority, which should not be forgotten within the context of this legal act. The employment of clever social propaganda in this process constituted an element of a well-planned strategy which was to help curtail, without any social upheaval, the competences of parliaments, which were widely considered a bastion of opposition against the royal power; a strategy of which the 1667 Ordinance was one of the tools, as the reform of civil procedure set the official framework for actions taken 139 Wroclaw Review of Law, Administration & Economics [Vol 5:1 by Louis XIV, it enabled the proper “interpretation of the raw facts of reality”.41 Unable to explicitly declare his intention to deprive the parliaments of their competences, Louis XIV “claded” his objectives in the framework of progressive codification. Without excessively reforming the procedure itself, he revolutionized the position of judges within the legal system, as an addition to the great legislative work designed to protect citizens from the abuses of the judicial system. 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Hurt JJ, Louis XIV and the parlements (Manchester University Press 2002) 41 Žižek (n 1 ) 138; Mańko (n 2) 43. THE ORDINANCE OF 1667: IDEOLOGY OF MODERN CODIFICATION AS A POLITICAL TOOL OF LOUIS XIV 2015] 140 Isambert FA, Decrusy, Taillandier AH, Recueil général des anciennes lois françaises, depuis l’an 420, jusqu'à la Révolution de 1789, vol 19 (Librairie de Plon Frères 1829). Klimaszewska A, Code de commerce – francuski Kodeks handlowy z 1807 r. [Code de Commerce: The French Commercial Code of 1807] (Arche 2011). Mańko R, ‘Koncepcja interpelacji ideologicznej a krytyczny dyskurs o prawie’ [The Notion of Ideological Interpellation and Critical Discourse on Law] (2014) 1 Archiwum Filozofii Prawa i Filozofii Społecznej. Monnier F, Guillaume de Lamoignon et Colbert. Essai sur la législation française au XVIIe siècle (Didier 1862). Ordonnance de Louis XIV. Roy de France et de Navarre. Donnée à Saint Germain en Laye au mois d’Avril 1667 (Associez choisis par ordre de Sa Majesté pour l’impression de ses nouvelles ordonnances 1667). É Pasquier É, Les recherches de la France (Laurens Sonnius 1621). Payen P, Les arrêts de règlement du Parlement de Paris au XVIIIe siécle. Dimension et doctrine. Les grandes thèses du droit français (PUF 1997). References Procès-verbal des conférences tenues par ordre du Roi pour l’examen des articles de l’Ordonnance civile du mois d’Avril 1667 et de l’Ordonnance criminelle du mois d’Avril 1670 (Associez choisis par ordre de Sa Majesté pour l’impression de ses nouvelles ordonnances 1757). Rives DB (ed), Œuvres d’Omer et de Denis Talon, avocats-généraux au Parlement de Paris, publiées sur les manuscrits autographes, vol 1 (A Égron, 1821). Roche-Flavin B de la, Treize livres des Parlements de France (M Berjon 1621). Royer JP, Histoire de la justice en France (PUF 2001). Saulnier de la Pinelais G, Le gens du roi au Parlement de Bretagne 1553- 1790 (J Plihon et L Hommay, A Picard 1902). Seyssel C de, La grand’ monarchie de France (E Groulleau 1558). Shennan JH, The Parlement of Paris (Cornell University Press 1968). Stone B, The Parlement of Paris, 1774-1789 (University of North Carolina Press 1981). Vie de M. Premier Président de Lamoignon, écrit d’après le mémoires du temps et les papiers de la famille (Nyon 1781). Žižek S, The Sublime Object of Ideology (Verso 2008).
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English
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Observation of the decay Λ b 0 → pK − μ + μ − and a search for CP violation
˜The œJournal of high energy physics/˜The œjournal of high energy physics
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cc-by
18,690
, Aaij, R., Adinolfi, M., Buchanan, E., Cook, A., Dalseno, J., Harnew, S., Kariuki, J., Naik, P., Petridis, K., Pomery, G., Price, E., Prouve, C., Quagliani, R., Rademacker, J., & Skidmore, N., & Velthuis, J. (2017). Observation of the decay Λb0 → pK−μ+μ− and a search for CP violation. Journal of High Energy Physics, 108. Advance online publication. https://doi.org/10.1007/JHEP06(2017)108 , Aaij, R., Adinolfi, M., Buchanan, E., Cook, A., Dalseno, J., Harnew, S., Kariuki, J., Naik, P., Petridis, K., Pomery, G., Price, E., Prouve, C., Quagliani, R., Rademacker, J., & Skidmore, N., & Velthuis, J. (2017). Observation of the decay Λb0 → pK−μ+μ− and a search for CP violation. Journal of High Energy Physics, 108. Advance online publication. https://doi.org/10.1007/JHEP06(2017)108 Keywords: Hadron-Hadron scattering (experiments), Particle and resonance production, proton-proton scattering, QCD, Quarkonium Open Access, Copyright CERN, University of Bristol – Bristol Research Portal General rights University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Published for SISSA by Springer Received: December 23, 2016 Accepted: May 25, 2017 Published: June 9, 2017 Published for SISSA by Springer Received: December 23, 2016 Accepted: May 25, 2017 Published: June 9, 2017 Received: December 23, 2016 Accepted: May 25, 2017 Published: June 9, 2017 Received: December 23, 2016 Accepted: May 25, 2017 Published: June 9, 2017 for the benefit of the LHCb Collaboration. Measurement of the J/ψ pair production cross-section in pp collisions at √s = 13 TeV JHEP06(2017)047 The LHCb collaboration Abstract: The production cross-section of J/ψ pairs is measured using a data sample of pp collisions collected by the LHCb experiment at a centre-of-mass energy of √s = 13 TeV, corresponding to an integrated luminosity of 279±11 pb−1. The measurement is performed for J/ψ mesons with a transverse momentum of less than 10 GeV/c in the rapidity range 2.0 < y < 4.5. The production cross-section is measured to be 15.2 ± 1.0 ± 0.9 nb. The first uncertainty is statistical, and the second is systematic. The differential cross-sections as functions of several kinematic variables of the J/ψ pair are measured and compared to theoretical predictions. Keywords: Hadron-Hadron scattering (experiments), Particle and resonance production, proton-proton scattering, QCD, Quarkonium ArXiv ePrint: 1612.07451 doi:10.1007/JHEP06(2017)047 doi:10.1007/JHEP06(2017)047 Contents 1 Introduction 1 2 Detector and data set 3 3 Cross-section determination 4 4 Systematic uncertainties 5 5 Results and comparison to theory 6 6 Summary 17 A Fits to the differential cross-sections with SPS and DPS components 18 The LHCb collaboration 33 Contents 1 Introduction 1 2 Detector and data set 3 3 Cross-section determination 4 4 Systematic uncertainties 5 5 Results and comparison to theory 6 6 Summary 17 A Fits to the differential cross-sections with SPS and DPS components 18 The LHCb collaboration 33 JHEP06(2017)047 33 1 Introduction After having been first observed by the NA3 collaboration in pion-nuclear and proton-nuclear interac- tions [55, 56], J/ψ pair production has been measured in pp collisions by the LHCb [57] and CMS [58] experiments at √s = 7 TeV and by the ATLAS experiment [59] at √s = 8 TeV. The D0 experiment [60] measured it using pp collision data at √s = 1.96 TeV. JHEP06(2017)047 Within the DPS mechanism, two quarkonia are produced independently in different partonic interactions. Neglecting the parton correlations in the proton, the contribution of this mechanism is estimated according to the formula [61–63] σDPS (J/ψJ/ψ) = 1 2 σ (J/ψ)2 σeff , (1.1) (1.1) where σ(J/ψ) is the inclusive prompt J/ψ production cross-section, the factor 1/2 accounts for two identical particles in the final state, and σeffis an effective cross-section, which provides a proper normalisation of the DPS cross-section estimate. The effective cross- section is related to the transverse overlap function between partons in the proton, and is thought to be universal for all processes and energy scales. Most of the measured values of σefflie in the range 12 −20 mb [43, 54, 64], which supports the expectation that σeffis universal for a large range of processes with different kinematics and scales, and for a wide spectrum of centre-of-mass energies in pp and pp collisions. The LHCb measurement of σ (J/ψJ/ψ) = 5.1 ± 1.0 ± 1.1 nb at √s = 7 TeV is not pre- cise enough to distinguish between the SPS and DPS contributions [65, 66]. The SPS contribution is calculated to be 4.0 ± 1.2 nb [67, 68] and 4.6 ± 1.1 nb [39] in the lead- ing-order NRQCD CS approach, and 5.4+2.7 −1.1 nb [39] using complete next-to-leading or- der NRQCD CS approach. The DPS contribution is estimated to be 3.8 ± 1.3 nb with eq. (1.1) using σ (J/ψ) from ref. [69] and σeff= 14.5 ± 1.7+1.7 −2.3 mb from ref. [44]. The large number of reconstructed J/ψ pair events in the CMS data [58] allowed for study of J/ψ correlations [70]. The observation of events with a large separation in rapidity of two J/ψ mesons indicates a significant DPS contribution, leading to σeff= 8.2 ± 2.2 mb [70], somewhat lower than the majority of other σeffmeasurements. A similarly small value, σeff= 6.3 ± 1.9 mb, is obtained by the ATLAS collaboration using a data-driven mod- el-independent approach [59]. 1 Introduction The production mechanism of heavy quarkonia is a long-standing and intriguing prob- lem in quantum chromodynamics (QCD), which is not fully understood even after over forty years of study. The colour-singlet model (CSM) [1–10] assumes the intermediate QQ state to be colourless and to have the same JPC quantum numbers as the final quarko- nium state. Leading-order calculations in the CSM underestimate the J/ψ and ψ(2S) production cross-sections at high transverse momentum, pT, by more than one order of magnitude [11]. The gap between CSM predictions and experimental measurements is reduced when including next-to-leading-order corrections, but the agreement is still not satisfactory [12–14]. The non-relativistic QCD (NRQCD) model takes into account both colour-singlet (CS) and colour-octet (CO) states of the QQ pair [15–17]. It either describes the production cross-sections and polarisations at large pT or it describes the produc- tion cross-section at all pT values, but then fails to predict the polarisation [18–33]. This puzzle can be probed via the production of pairs of quarkonia [34–39], where the inter- pretation of the measured cross-section could be simpler. In quarkonium-pair production, the selection rules in the CS process of leading-order (LO) NRQCD forbid the feed-down from cascade decays of excited C-even states. This feed-down from C-even states, e.g. χc →J/ψγ or χb →Υγ, plays an important role in single quarkonium production. It significantly complicates the precise comparison between data and model predictions, and makes the interpretation of polarisation measurements difficult. Besides the single parton scattering (SPS) process, the process of double parton scatter- ing (DPS) can also contribute to quarkonium pair production. The DPS process is of great – 1 – importance since it can provide information on the transverse momenta of the partons and their correlations inside the proton, and can help in understanding various backgrounds, e.g. Z + bb, W + + W −, multi-jets etc., in searches for new physics. The DPS processes have been studied in several final states, e.g. 4-jets by the AFS [40], UA2 [41], CDF [42], and ATLAS [43] collaborations, γ + 3-jets by the CDF [44] and D0 [45, 46] collaborations, 2γ +2-jets by the D0 [47] collaboration, W +2-jets [48] and Υ +Υ [49] by the CMS collabo- ration, J/ψ +W [50] and J/ψ +Z [51] by the ATLAS collaboration, and double charm [52], Z + open charm [53] and Υ + open charm [54] by the LHCb collaboration. 2 Detector and data set The LHCb detector [72, 73] is a single-arm forward spectrometer covering the pseudorapidity range 2 < η < 5, designed for the study of particles containing b or c quarks. The detector includes a high-precision tracking system consisting of a silicon- strip vertex detector surrounding the pp interaction region, a large-area silicon-strip de- tector (TT) located upstream of a dipole magnet with a bending power of about 4 Tm, and three stations of silicon-strip detectors and straw drift tubes placed downstream of the magnet. The tracking system provides a measurement of momentum, p, of charged particles with a relative uncertainty that varies from 0.5% at low momentum to 1.0% at 200 GeV/c. The minimum distance of a track to a primary vertex (PV), the impact parame- ter, is measured with a resolution of (15+29/pT) µm, where pT is in GeV/c. Different types of charged hadrons are distinguished using information from two ring-imaging Cherenkov detectors. Photons, electrons and hadrons are identified by a calorimeter system consisting of scintillating-pad and preshower detectors, an electromagnetic calorimeter and a hadronic calorimeter. Muons are identified by a system composed of alternating layers of iron and multiwire proportional chambers. The online event selection is performed by a trigger [74], which consists of a hardware stage (L0), based on information from the calorimeter and muon systems, followed by a software stage, which applies a full event reconstruction. The L0 trigger requires two muons with pT(µ1) × pT(µ2) > (1.3 GeV/c)2. In the first stage of the software trigger (HLT1), two muons with pT > 330 MeV/c and p > 6 GeV/c are required to form a J/ψ candidate with invariant mass M(µ+µ−) > 2.7 GeV/c2; alternatively, the event can also be accepted when it has a good quality muon with pT > 4.34 GeV/c and p > 6 GeV/c. In the second stage of the software trigger (HLT2), the two J/ψ mesons are reconstructed from µ+µ−pairs with good vertex-fit quality and invariant masses within ±120 MeV/c2 of the known J/ψ mass [75], using algorithms identical to the offline reconstruction. In the offline selection, all four muons in the final state are required to have pT > 650 MeV/c, 6 < p < 200 GeV/c and 2 < η < 5. Each track must have a good-quality track fit and be identified as a muon. The four muon tracks are required to originate from the same PV. 1 Introduction A small value of σeff= 4.8 ± 2.5 mb is also obtained by the D0 collaboration [60] using the separation of the two J/ψ mesons in pseudorapid- ity to distinguish SPS and DPS contributions. Together with an even smaller value of σeff= 2.2 ± 1.1 mb, determined by the D0 collaboration from the measurement of the si- multaneous production of J/ψ and Υ mesons [71], and the estimate of σeff= 2.2−6.6 mb by – 2 – the CMS collaboration from the production of Υ pairs [49], these results question the uni- versality of σeff. In this paper, the J/ψ pair production cross-section is measured using pp collision data collected by the LHCb experiment in 2015 at √s = 13 TeV with both J/ψ mesons in the rapidity range 2.0 < y < 4.5, and with a transverse momentum pT < 10 GeV/c. The polarisation of the J/ψ mesons is assumed to be zero since there is as yet no knowledge of the polarisation of J/ψ pairs, and all the LHC analyses indicate a small polarisation for the quarkonia [29–33]. The J/ψ mesons are reconstructed via the µ+µ−final state. In the following, the labels J/ψ1 and J/ψ2 are randomly assigned to the two J/ψ candidates. JHEP06(2017)047 2 Detector and data set This reduces to a negligible level the number of pile-up candidates, i.e. J/ψ pairs from two independent pp interactions. The reconstructed J/ψ mesons are required to have a good-quality vertex and an invariant mass in the range 3000 < M(µ+µ−) < 3200 MeV/c2. Only events explicitly – 3 – triggered by one of the J/ψ candidates at the L0 and the HLT1 stages are retained. For events with multiple candidates, in particular where the four muons can be combined in two different ways to form a J/ψ pair, which account for 1.4% of the total candidates, one randomly chosen candidate pair is retained. Simulated J/ψ samples are generated to study the behaviour of the signal. In the simulation, pp collisions are generated using Pythia8 [76, 77] with a specific LHCb con- figuration [78]. Decays of hadronic particles are described by EvtGen [79], in which final-state radiation is generated using Photos [80]. The interaction of the generated par- ticles with the detector, and its response, are implemented using the Geant4 toolkit [81] as described in ref. [82]. JHEP06(2017)047 3 Cross-section determination The inclusive J/ψ pair production cross-section is measured as σ(J/ψJ/ψ) = Ncor L × B(J/ψ →µ+µ−)2 , (3.1) (3.1) where Ncor is the number of signal candidates after the efficiency correction, B(J/ψ → µ+µ−) = (5.961 ± 0.033)% is the branching fraction of the J/ψ →µ+µ−decay [75], and L = 279 ± 11 pb−1 is the integrated luminosity, determined using the beam-gas imaging and van der Meer scan methods [83]. The total detection efficiency of the J/ψ pair is estimated as εtot = εacc × εrec&sel × εPID × εtrig, (3.2) (3.2) where εacc is the geometrical acceptance, εrec&sel is the reconstruction and selection effi- ciency for candidates with all final-state muons inside the geometrical acceptance, εPID is the muon particle identification (PID) efficiency for selected candidates, and εtrig is the trigger efficiency for selected candidates satisfying the PID requirement. The first three efficiencies of the J/ψ pair, εacc, εrec&sel and εPID, are factorized as ε (J/ψJ/ψ) = ε (J/ψ1 ) × ε (J/ψ2 ) . (3.3) (3.3) Since the HLT2 trigger selection is performed using the same reconstruction algorithm as the offline selection and the selection criteria of the HLT2 trigger are a subset of those used in the final selection, the corresponding trigger efficiency for the reconstructed and selected events is 100%. Since at least one of the two J/ψ meson candidates is required to have passed the L0 and HLT1 trigger, the efficiency εtrig of the J/ψ pair can be expressed as Since the HLT2 trigger selection is performed using the same reconstruction algorithm as the offline selection and the selection criteria of the HLT2 trigger are a subset of those used in the final selection, the corresponding trigger efficiency for the reconstructed and selected events is 100%. Since at least one of the two J/ψ meson candidates is required to have passed the L0 and HLT1 trigger, the efficiency εtrig of the J/ψ pair can be expressed as (3.4) εtrig (J/ψJ/ψ) = 1 −(1 −εtrig (J/ψ1 )) × (1 −εtrig (J/ψ2 )) . (3.4) All terms in the single J/ψ efficiency are estimated in bins of pT and y of the J/ψ mesons using the simulation. The track reconstruction and muon PID efficiency are corrected using data-driven techniques, as described in section 4, and the trigger efficiency measurement is validated on data. 3 Cross-section determination The (black and magenta) dashed lines represent the background components due to the combination of a real J/ψ with a combinatorial candidate. The (green) shaded area shows the purely combinatorial background. The signal yield is determined by performing an extended unbinned maximum likeli- hood fit to the efficiency-corrected two-dimensional (M(µ+ 1 µ− 1 ), M(µ+ 2 µ− 2 )) mass distribu- tion. The total detection efficiency is applied individually on an event-by-event basis. The signal is modelled by the sum of a double-sided Crystal Ball (DSCB) function [84] and a Gaussian function, which share the same mean value. The power law tail parameters of the DSCB, the relative fraction and the difference between the widths of the DSCB and the Gaussian function are fixed to the values obtained from simulation, leaving the peak value and the core width of the DSCB as free parameters. The combinatorial background is described by an exponential function. Since the labels J/ψ1 and J/ψ2 are assigned ran- domly, the fit function is symmetric under the exchange of the J/ψ1 and J/ψ2 masses. The fit projections on M(µ+ 1 µ− 1 ) and M(µ+ 2 µ− 2 ) are shown in figure 1. The corrected yield1 of J/ψ pairs is determined to be Ncor = (15.8 ± 1.1) × 103. After the fit, the residual contamination, where either one or both J/ψ mesons come from b-hadron decays, must be corrected for. The fraction of background is evaluated with the help of simulation validated with data and normalized using the measured prompt J/ψ and inclusive bb →J/ψ production cross-sections within the LHCb acceptance at √s = 13 TeV [85]. The fraction of candidates with J/ψ mesons from b-hadron decays is determined to be 4.5%. 1The corresponding fit of the efficiency-uncorrected sample gives (1.05 ± 0.05) × 103 signal events. 3 Cross-section determination – 4 – 3000 3050 3100 3150 3200 1000 2000 3000 4000 5000 N cor/(5 MeV/c2) M(µ+ 1 µ− 1 )  MeV/c2 LHCb 13 TeV Data Total fit J/ψ1 + J/ψ2 bkg + J/ψ2 J/ψ1 + bkg bkg + bkg 3000 3050 3100 3150 3200 1000 2000 3000 4000 5000 N cor/(5 MeV/c2) M(µ+ 2 µ− 2 )  MeV/c2 LHCb 13 TeV Data Total fit J/ψ1 + J/ψ2 bkg + J/ψ2 J/ψ1 + bkg bkg + bkg Figure 1. Projections of the fit to the efficiency-corrected distribution of the reconstructed J/ψ mass for (left) M(µ+ 1 µ− 1 ) and (right) M(µ+ 2 µ− 2 ). The (black) points with error bars represent the data. The (blue) solid line is the total fit function. The (red) cross-hatched area shows the signal distribution. The (black and magenta) dashed lines represent the background components due to the combination of a real J/ψ with a combinatorial candidate. The (green) shaded area shows the purely combinatorial background. 3000 3050 3100 3150 3200 1000 2000 3000 4000 5000 N cor/(5 MeV/c2) M(µ+ 1 µ− 1 )  MeV/c2 LHCb 13 TeV Data Total fit J/ψ1 + J/ψ2 bkg + J/ψ2 J/ψ1 + bkg bkg + bkg 3000 3050 3100 3150 3200 1000 2000 3000 4000 5000 N cor/(5 MeV/c2) M(µ+ 2 µ− 2 )  MeV/c2 LHCb 13 TeV Data Total fit J/ψ1 + J/ψ2 bkg + J/ψ2 J/ψ1 + bkg bkg + bkg JHEP06(2017)047 Figure 1. Projections of the fit to the efficiency-corrected distribution of the reconstructed J/ψ mass for (left) M(µ+ 1 µ− 1 ) and (right) M(µ+ 2 µ− 2 ). The (black) points with error bars represent the data. The (blue) solid line is the total fit function. The (red) cross-hatched area shows the signal distribution. The (black and magenta) dashed lines represent the background components due to the combination of a real J/ψ with a combinatorial candidate. The (green) shaded area shows the purely combinatorial background. Figure 1. Projections of the fit to the efficiency-corrected distribution of the reconstructed J/ Figure 1. Projections of the fit to the efficiency-corrected distribution of the reconstructed J/ψ mass for (left) M(µ+ 1 µ− 1 ) and (right) M(µ+ 2 µ− 2 ). The (black) points with error bars represent the data. The (blue) solid line is the total fit function. The (red) cross-hatched area shows the signal distribution. 4 Systematic uncertainties Several sources of systematic uncertainties on the J/ψ pair production cross-section are studied and summarized in table 1. The uncertainty due to the signal shape description is estimated by replacing the nominal model with two alternative models, the Hypatia function [86] and a kernel estimate for the underlying probability distribution function of – 5 – the simulated sample convolved with a Gaussian function [87]. The relative difference of 1.6% with respect to the nominal result is taken as a systematic uncertainty. A difference between simulation and data, in particular in the fit quality of the can- didates when constraining the muons to the PV, can lead to a bias in the efficiency de- termination. This is estimated by comparing the vertex-fit quality of the reconstructed J/ψ candidates between the simulated and the data samples, where the background is sub- tracted using the sPlot technique [88]. Data and simulation agree within 1.0%, which is taken as a systematic uncertainty. The track reconstruction efficiency is studied in data using a tag-and-probe tech- nique [89]. In this method, one of the muons from the J/ψ is fully reconstructed as the tag track, and the other muon track, the probe track, is reconstructed using only informa- tion from the TT detector and the muon stations. The tracking efficiency is taken as the fraction of J/ψ candidates whose probe tracks match fully reconstructed tracks. The sim- ulated sample is corrected to match the track multiplicity of events in the data. The ratio of tracking efficiencies between data and simulation is taken as the correction factor. A systematic uncertainty of 0.8% per track is assigned for the difference in event multiplicity between data and simulation. JHEP06(2017)047 The muon PID efficiency is also determined using a tag-and-probe method [90], where only one track of the J/ψ is identified as a muon, i.e. the tag track. The single muon PID efficiency, defined as the fraction of J/ψ candidates with the other track (probe track) identified as a muon, is determined in bins of p and η of the probe track. Systematic effects arising from the choice of the binning scheme and for the difference in event multiplicity between data and simulation are studied. In total, the muon PID efficiency uncertainty is determined to be 2.3%. 4 Systematic uncertainties The trigger efficiency εtrig (J/ψ) measured with simulation is compared with the result obtained in data for inclusive J/ψ events using a tag-and-probe method [74]. A difference of 1.0% between the two results is observed and is taken as the systematic uncertainty. An uncertainty of 1.0% is assigned to the determination of the fraction of candidates from b-hadron decays, which accounts for the uncertainty of the prompt J/ψ and bb produc- tion cross-sections. The uncertainty introduced by the limited statistics of the simulated samples used to determine the efficiencies is estimated to be negligible. The 1.1% uncer- tainty on B(J/ψ →µ+µ−) is propagated to the cross-section. The systematic uncertainty due to the luminosity calibration is 3.9%. The total systematic uncertainty is 6.1%. 5 Results and comparison to theory The J/ψ pair production cross-section where both J/ψ mesons are in the region 2.0 < y < 4.5 and pT < 10 GeV/c is measured to be σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, assuming negligible polarisation of the J/ψ mesons. The detection efficiency of J/ψ mesons can be affected by the polarisation, especially by the polarisation parameter λθ in the helicity frame [32, 85]. If a value of λθ = ±20% is assumed for both of the J/ψ mesons, assuming negligible polarisation of the J/ψ mesons. The detection efficiency of J/ψ mesons can be affected by the polarisation, especially by the polarisation parameter λθ in the helicity frame [32, 85]. If a value of λθ = ±20% is assumed for both of the J/ψ mesons, – 6 – Source Uncertainty[%] Signal shape 1.6 Data/simulation difference 1.0 Tracking efficiency 0.8 × 4 Muon PID efficiency 2.3 Trigger efficiency 1.0 Fraction of J/ψ from b-hadron candidates 1.0 B(J/ψ →µ+µ−) 1.1 Luminosity 3.9 Total 6.1 JHEP06(2017)047 Table 1. Summary of the systematic uncertainties on the measurement of the J/ψ pair production cross-section. the J/ψ pair production cross-section changes by ±7%. The ratio of the production cross- section of the J/ψ pair to that of the inclusive prompt J/ψ is calculated to be the J/ψ pair production cross-section changes by ±7%. The ratio of the production cross- section of the J/ψ pair to that of the inclusive prompt J/ψ is calculated to be σ(J/ψJ/ψ) σ(J/ψ) = (10.2 ± 0.7 (stat) ± 0.9 (syst)) × 10−4, (5.1) (5.1) where the production cross-section of prompt J/ψ mesons in the range 2.0 < y < 4.5 and pT < 10 GeV/c is σ(J/ψ) = 14.94 ± 0.02 (stat) ± 0.91 (syst) µb [85], and the systematic un- certainties of σ(J/ψJ/ψ) and σ(J/ψ) are treated as uncorrelated. According to eq. (1.1), the ratio where the production cross-section of prompt J/ψ mesons in the range 2.0 < y < 4.5 and pT < 10 GeV/c is σ(J/ψ) = 14.94 ± 0.02 (stat) ± 0.91 (syst) µb [85], and the systematic un- certainties of σ(J/ψJ/ψ) and σ(J/ψ) are treated as uncorrelated. According to eq. (1.1), the ratio 1 2 σ(J/ψ)2 σ(J/ψJ/ψ) = 7.3 ± 0.5 (stat) ± 1.0 (syst) mb. (5.2) (5.2) can be interpreted as σeffif all J/ψ pairs are produced through the DPS process. The results on J/ψ pair production are compared with a data-driven prediction for the DPS mechanism and several calculations performed within the SPS mechanism. The DPS prediction is calculated via eq. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, σ(J/ψJ/ψ) [nb] no pT cut pT > 1 GeV/c pT > 3 GeV/c LO CS [92] 1.3 ± 0.1+3.2 −0.1 — — LO CO [95, 96] 0.45 ± 0.09+1.42+0.25 −0.36−0.34 — — LO kT [102] 6.3+3.8+3.8 −1.6−2.6 5.7+3.4+3.2 −1.5−2.1 2.7+1.6+1.6 −0.7−1.0 NLO∗CS′ [92] — 4.3 ± 0.1+9.9 −0.9 1.6 ± 0.1+3.3 −0.3 NLO∗CS′′ [70, 93–96] 15.4 ± 2.2+51 −12 14.8 ± 1.7+53 −12 6.8 ± 0.6+22 −5 NLO CS [39] 11.9+4.6 −3.2 — — DPS [44, 85, 91] 8.1 ± 0.9+1.6 −1.3 7.5 ± 0.8+1.5 −1.2 4.9 ± 0.5+1.0 −0.8 Data 15.2 ± 1.0 ± 0.9 13.5 ± 0.9 ± 0.9 8.3 ± 0.6 ± 0.5 JHEP06(2017)047 Table 2. Summary of the theoretical predictions and the measurement of σ(J/ψJ/ψ) for different regions of transverse momentum of the J/ψ pair. For SPS predictions, the first uncertainty accounts for the variation of PDFs and gluon densities, while the second one corresponds to the variation of the factorisation and renormalisation scales. For the LO CO predictions the third uncertainty corresponds to the choice of LDMEs from refs. [25, 113–119]. For NLO CS predictions [39] the un- certainty corresponds to the variation of the factorisation and renormalization scales. For the DPS prediction the first uncertainty is due to the measured prompt J/ψ production cross-section [85] and the second is due to the uncertainty in σeff[44, 91]. Gluon densities from refs. [104–108] are used for the LO kT approach, while CT 14 parton distribution functions (PDF) [109] are used for LO CS and NLO∗CS′ calculations, NNPDF 3.0 NLO PDFs with αs(MZ) = 0.118 [110] are used for LO CO and NLO∗CS′′ pre- dictions, and CTEQ6L1 and CTEQ6M PDFs [111, 112] are used for NLO CS computa- tions. For LO CO predictions the long-distance matrix elements (LDMEs) are taken from refs. [25, 113–119] and a smearing of transverse momenta of initial gluons, similar to that used in NLO∗CS′′, is applied. The production cross-section of J/ψ pairs is sensitive to the choice of parameters; for example, it varies by a factor between 0.8 and 3 when vary- ing the factorisation and renormalisation scales by a factor of two, or increases if the CTEQ 6L PDF set [120] is used instead of the nominal PDFs. The contribution of LO CO is very sensitive to the choice of the LDME; the absolute cross-section varies from the min- imum of 0.11 nb, based on LDME set from ref. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, (1.1) using the measured J/ψ production cross-section at √s = 13 TeV [85] and the effective cross-section σeff= 14.5 ± 1.7+1.7 −2.3 mb from refs. [44, 91]. Theoretical predictions of the production cross-section of J/ψ pairs are summarized in table 2. The contribution from the SPS mechanism is calculated using several approaches: the state-of-art complete NLO colour-singlet (NLO CS) computations [39]; the incom- plete (no-loops) next-to-leading-order colour-singlet (NLO∗CS) calculations [70, 92–96]; leading-order colour-singlet (LO CS) [92] and colour-octet (LO CO) [95, 96] calculations and the approach based on the kT-factorisation method [97–101], with the leading-order colour-singlet matrix element (LO kT) [102, 103]. Even with the leading-order matrix el- ement, the LO kT approach includes a large fraction of higher-order contributions via the evolution of parton densities [102]. Since NLO∗CS calculations are divergent at small transverse momentum of the J/ψ pair, two approaches are used: a simple cut-off for pT(J/ψJ/ψ) [92] (denoted as NLO∗CS′), and a cut on the mass of any light parton pair (NLO∗CS′′) [70, 93–96]. – 7 – σ(J/ψJ/ψ) [nb] no pT cut pT > 1 GeV/c pT > 3 GeV/c LO CS [92] 1.3 ± 0.1+3.2 −0.1 — — LO CO [95, 96] 0.45 ± 0.09+1.42+0.25 −0.36−0.34 — — LO kT [102] 6.3+3.8+3.8 −1.6−2.6 5.7+3.4+3.2 −1.5−2.1 2.7+1.6+1.6 −0.7−1.0 NLO∗CS′ [92] — 4.3 ± 0.1+9.9 −0.9 1.6 ± 0.1+3.3 −0.3 NLO∗CS′′ [70, 93–96] 15.4 ± 2.2+51 −12 14.8 ± 1.7+53 −12 6.8 ± 0.6+22 −5 NLO CS [39] 11.9+4.6 −3.2 — — DPS [44, 85, 91] 8.1 ± 0.9+1.6 −1.3 7.5 ± 0.8+1.5 −1.2 4.9 ± 0.5+1.0 −0.8 Data 15.2 ± 1.0 ± 0.9 13.5 ± 0.9 ± 0.9 8.3 ± 0.6 ± 0.5 Table 2. Summary of the theoretical predictions and the measurement of σ(J/ψJ/ψ) for different regions of transverse momentum of the J/ψ pair. For SPS predictions, the first uncertainty accounts for the variation of PDFs and gluon densities, while the second one corresponds to the variation of the factorisation and renormalisation scales. For the LO CO predictions the third uncertainty corresponds to the choice of LDMEs from refs. [25, 113–119]. For NLO CS predictions [39] the un- certainty corresponds to the variation of the factorisation and renormalization scales. For the DPS prediction the first uncertainty is due to the measured prompt J/ψ production cross-section [85] and the second is due to the uncertainty in σeff[44, 91]. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, [113] to the maximum of 0.70 nb, calculated using LDME set from ref. [116], while most of the predictions cluster around 0.5 nb. The feed-down from ψ(2S) →J/ψX decays is included in the LO kT, LO CO and NLO∗CS′′ calculations and not in the LO CS and NLO∗CS′ calculations. Likewise, a tiny contri- bution from J/ψχc production with subsequent decay χc →J/ψγ [92] is included in the NLO∗CS′ and LO CO results but neglected in the NLO∗CS′′ calculations. While the predictions for the production cross-section of J/ψ pairs are significantly affected by the theory uncertainties, the shapes of the differential cross-sections are very stable and practically invariant with respect to the choice of PDFs, scales and LDMEs. – 8 – 0 5 10 0 1 2 3 4 5 6 7 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c SPS: LO CO⟨kT⟩=0.5 GeV/c SPS: LO CO⟨kT⟩=2 GeV/c ×××××SPS: NLO CS Figure 2. Comparisons between measurements and theoretical predictions for the differential cross-sections as a function of pT(J/ψJ/ψ). The (black) points with error bars represent the mea- surements. 0 5 10 0 1 2 3 4 5 6 7 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c SPS: LO CO⟨kT⟩=0.5 GeV/c SPS: LO CO⟨kT⟩=2 GeV/c ×××××SPS: NLO CS JHEP06(2017)047 Figure 2. Comparisons between measurements and theoretical predictions for the differential cross-sections as a function of pT(J/ψJ/ψ). The (black) points with error bars represent the mea- surements. In contrast, the smearing of gluon transverse momenta for NLO∗CS′′ and LO CO models does not affect the production cross-section, but significantly affects some differential dis- tributions. The measured differential production cross-sections of J/ψ pairs as a function of several kinematic variables are compared to the theoretical predictions. For each variable v, the differential production cross-section of J/ψ pairs is calculated as dσ(J/ψJ/ψ) dv = 1 L × B(J/ψ →µ+µ−)2 × ∆Ncor i ∆vi , where ∆Ncor i is the number of efficiency-corrected signal candidates in bin i, and ∆vi is the corresponding bin width. The luminosity uncertainty and the uncertainty introduced by B(J/ψ →µ+µ−) are common to all bins and are fully correlated. The tracking efficiency and muon PID efficiency uncertainties are strongly correlated. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, In figures 2–8 of the differ- ential cross-sections, only the statistical uncertainties are shown as the systematic ones are negligibly small and almost 100% correlated. The comparison between measurements and theoretical predictions is performed for the following kinematical variables: transverse momentum and rapidity of the J/ψ pair, transverse momentum and rapidity of each J/ψ meson, differences in the azimuthal angle and rapidity between the two J/ψ mesons (|∆φ| and |∆y|), the mass of the J/ψ pair and the transverse momentum asymmetry, defined as AT ≡ pT(J/ψ1 ) −pT(J/ψ2 ) pT(J/ψ1 ) + pT(J/ψ2 ) . The distributions for the whole pT(J/ψJ/ψ) range are presented in figures 2, 3 and 4, for pT(J/ψJ/ψ) > 1 GeV/c in figures 5 and 6, and for pT(J/ψJ/ψ) > 3 GeV/c in figures 7 and 8. The DPS predictions are obtained using a large number of pseudoexperiments, where two uncorrelated J/ψ mesons are produced according to the measured differential distribu- tions d2σ (J/ψ) /dpTdy [85] for single prompt J/ψ production, uniformly distributed over – 9 – 0 2 4 6 8 10 0 1 2 3 4 5 6 7 8 1 2 dσ(J/ψJ/ψ) dpT(J/ψ)  nb GeV/c  pT(J/ψ) [GeV/c] LHCb 13 TeV DPS SPS: LO kT SPS: LO CS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c SPS: LO CO⟨kT⟩=0.5 GeV/c SPS: LO CO⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 14 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 1 2 dσ(J/ψJ/ψ) dy(J/ψ) [nb] y(J/ψ) LHCb 13 TeV Figure 3. Comparisons between measurements and theoretical predictions for the differential cross-sections as functions of (top left) pT(J/ψ), (top right) y(J/ψJ/ψ) and (bottom) y(J/ψ). The (black) points with error bars represent the measurements. 0 2 4 6 8 10 0 1 2 3 4 5 6 7 8 1 2 dσ(J/ψJ/ψ) dpT(J/ψ)  nb GeV/c  pT(J/ψ) [GeV/c] LHCb 13 TeV DPS SPS: LO kT SPS: LO CS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c SPS: LO CO⟨kT⟩=0.5 GeV/c SPS: LO CO⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 14 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV JHEP06(2017)047 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 1 2 dσ(J/ψJ/ψ) dy(J/ψ) [nb] y(J/ψ) LHCb 13 TeV Figure 3. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, Comparisons between measurements and theoretical predictions for the differential cross-sections as functions of (top left) pT(J/ψ), (top right) y(J/ψJ/ψ) and (bottom) y(J/ψ). The (black) points with error bars represent the measurements. the azimuthal angle φ. For LO CO and NLO∗CS′′ models two values of Gaussian smearing of the initial transverse momentum of gluon kT are used, namely ⟨kT⟩= 0.5 and 2 GeV/c. The pT(J/ψJ/ψ) distribution, shown in figure 2, demonstrates the large dependence of the shape on the choice of the ⟨kT⟩parameter. For the NLO∗CS′′ approach [70, 93–96], relatively large smearing of the initial gluon transverse momenta ⟨kT⟩= 2 GeV/c is re- quired to eliminate peaking structures in the distribution. The distributions of the variables pT(J/ψJ/ψ), |∆φ| and AT, predicted by the LO CS model, are trivial, pT(J/ψJ/ψ) ∼0, |∆φ| ∼π and AT ∼0, and omitted from the plots. A similar trivial pattern is expected for the LO CO model, but due to the kT-smearing, the actual shape of the distributions strongly depends on the choice of the ⟨kT⟩parameter. The NLO∗CS′′ model also demon- strates a large dependence on the ⟨kT⟩parameter for |∆φ| /π distribution. Neither the DPS model with the given value of the σeffparameter, nor any of the SPS models can describe simultaneously the measured cross-section and the differential shapes. However, the sum of the DPS and SPS contributions can adequately describe both the measured production cross-sections and the differential distributions. To discriminate between the SPS and DPS contributions, the differential distribution for each variable v is – 10 – 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO kT SPS: LO CS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c SPS: LO CO⟨kT⟩=0.5 GeV/c SPS: LO CO⟨kT⟩=2 GeV/c ×××××SPS: NLO CS 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 40 45 50 πdσ(J/ψJ/ψ) d |∆φ| [nb] |∆φ| /π LHCb 13 TeV 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 40 45 50 dσ(J/ψJ/ψ) dAT [nb] AT LHCb 13 TeV 6 8 10 12 14 0 2 4 6 8 10 12 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV Figure 4. Comparisons between measurements and theoretical predictions for the differential cross-sections as functions of (top left) |∆y|, (top right) |∆φ|, (bottom left) AT and (bottom right) m(J/ψJ/ψ). σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, The (black) points with error bars represent the measurements. 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 40 45 50 πdσ(J/ψJ/ψ) d |∆φ| [nb] |∆φ| /π LHCb 13 TeV 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO kT SPS: LO CS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c SPS: LO CO⟨kT⟩=0.5 GeV/c SPS: LO CO⟨kT⟩=2 GeV/c ×××××SPS: NLO CS JHEP06(2017)047 6 8 10 12 14 0 2 4 6 8 10 12 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 40 45 50 dσ(J/ψJ/ψ) dAT [nb] AT LHCb 13 TeV Figure 4. Comparisons between measurements and theoretical predictions for the differential cross-sections as functions of (top left) |∆y|, (top right) |∆φ|, (bottom left) AT and (bottom right) m(J/ψJ/ψ). The (black) points with error bars represent the measurements. fitted with the simple two-component model dσ dv = σDPSFDPS(v) + σSPSFSPS(v), (5.3) (5.3) where FDPS and FSPS are templates for the DPS and SPS models and σDPS and σSPS are floating fit parameters representing the DPS and SPS contributions. The theory normali- sation is not used in the fits. The DPS fraction fDPS is defined as fDPS ≡ σDPS σSPS + σDPS . (5.4) (5.4) Some distributions give little discrimination between SPS and DPS. The percentages of the DPS component obtained from the fits for the most discriminating variables are presented in table 3. The fit results are presented in the appendix. All the fits indicate a large DPS contribution to the J/ψ pair production process. The inclusion of the CO component in the fit does not have a large effect on the determination of the DPS fraction fDPS, and the fraction of the CO component determined in such a fit procedure is significantly smaller than the CS contribution. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, The value of σSPS, calculated as (1−fDPS)×σ(J/ψJ/ψ), – 11 – 0 2 4 6 8 10 0 1 2 3 4 5 6 1 2 dσ(J/ψJ/ψ) dpT(J/ψ)  nb GeV/c  pT(J/ψ) [GeV/c] LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c 2 2.5 3 3.5 4 4.5 0 1 2 3 4 5 6 7 8 9 10 1 2 dσ(J/ψJ/ψ) dy(J/ψ) [nb] y(J/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c Figure 5. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 1 GeV/c for the differential cross-sections as functions of (top left) pT(J/ψ), (top right) y(J/ψJ/ψ) and (bottom) y(J/ψ). The (black) points with error bars represent the measurements. 0 2 4 6 8 10 0 1 2 3 4 5 6 1 2 dσ(J/ψJ/ψ) dpT(J/ψ)  nb GeV/c  pT(J/ψ) [GeV/c] LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 2 4 6 8 10 12 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c JHEP06(2017)047 2 2.5 3 3.5 4 4.5 0 1 2 3 4 5 6 7 8 9 10 1 2 dσ(J/ψJ/ψ) dy(J/ψ) [nb] y(J/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c Figure 5. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 1 GeV/c for the differential cross-sections as functions of (top left) pT(J/ψ), (top right) y(J/ψJ/ψ) and (bottom) y(J/ψ). The (black) points with error bars represent the measurements. is smaller than expectations from the NLO∗CS′′ [70, 93–96] and NLO CS [39] approaches and roughly agrees with the NLO∗CS′ [92] and LO kT [102] predictions. is smaller than expectations from the NLO∗CS′′ [70, 93–96] and NLO CS [39] approaches and roughly agrees with the NLO∗CS′ [92] and LO kT [102] predictions. The value σDPS determined with eq. (5.3) is converted to σeff, σeff= 1 2 σ (J/ψ)2 σDPS , (5.5) (5.5) where σ(J/ψ) is the production cross-section of prompt J/ψ mesons from ref. [85]. The values obtained for σeffare summarized in table 4. Values between 10.0 and 12.5 mb are found for the models considered in this analysis. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, These values are slightly larger than those measured from central J/ψ pair production at LHC, σeff= 8.2 ± 2.2 mb [70] and σeff= 6.3 ± 1.9 mb [59], and significantly exceed the values obtained by the D0 collabo- ration from analysis of J/ψ pair production, σeff= 4.8 ± 2.5 mb [60], and ΥJ/ψ produc- tion, σeff= 2.2 ± 1.1 mb [71]. On the other hand, they are smaller than the values of σeff measured by the LHCb collaboration in the processes of multiple associated heavy quark production [52, 54], in particular σeff∼15 mb measured for various J/ψ + cc produc- tion processes [52] and σeff= 18.0 ± 1.8 mb measured for the Υ(1S) + D0,+ production processes [54]. – 12 – Variable LO CS LO kT NLO∗CS′ NLO∗CS′′ NLO CS ⟨kT⟩= 2 GeV/c ⟨kT⟩= 0.5 GeV/c no pT(J/ψJ/ψ) cut pT(J/ψJ/ψ) — 78 ± 3 — 88 ± 56 81 ± 7 — y(J/ψJ/ψ) 83 ± 39 — — 75 ± 37 68 ± 34 — m(J/ψJ/ψ) 76 ± 7 74 ± 7 — 78 ± 7 77 ± 7 |∆y| 59 ± 21 61 ± 18 — 63 ± 18 61 ± 18 69 ± 16 pT(J/ψJ/ψ) > 1 GeV/c y(J/ψJ/ψ) — — 75 ± 24 71 ± 38 68 ± 34 — m(J/ψJ/ψ) — 73 ± 8 76 ± 7 88 ± 1 — |∆y| — 57 ± 20 59 ± 19 60 ± 18 60 ± 19 — pT(J/ψJ/ψ) > 3 GeV/c y(J/ψJ/ψ) — — 77 ± 18 64 ± 38 64 ± 35 — m(J/ψJ/ψ) — 76 ± 10 84 ± 7 87 ± 2 — |∆y| — 42 ± 25 53 ± 21 53 ± 21 53 ± 21 — Table 3. Percentages of the DPS component, fDPS, determined with the simple two-component fit to different distributions for different SPS models. JHEP06(2017)047 Table 3. Percentages of the DPS component, fDPS, determined with the simple two-component fit to different distributions for different SPS models. Table 3. Percentages of the DPS component, fDPS, determined with the simple two-component fit to different distributions for different SPS models. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, Variable LO kT NLO∗CS′′ NLO CS ⟨kT⟩= 2 GeV/c ⟨kT⟩= 0.5 GeV/c pT(J/ψJ/ψ) 11.3 ± 0.6 10.1 ± 6.5 10.9 ± 1.2 — y(J/ψJ/ψ) — 11.9 ± 7.5 10.0 ± 5.0 — m(J/ψJ/ψ) 10.6 ± 1.1 10.2 ± 1.0 10.4 ± 1.0 |∆y| 12.5 ± 4.1 12.2 ± 3.7 12.4 ± 3.9 11.2 ± 2.9 Table 4. Summary of the σeffvalues (in mb) from DPS fits for different SPS models. The uncertainty is statistical only, originating from the statistical uncertainty in σDPS (and dσ (J/ψJ/ψ)/dv). The common systematic uncertainty of 12%, accounting for the systematic un- certainty of σ (J/ψJ/ψ) and the total uncertainty for σ(J/ψ), is not shown. Table 4. Summary of the σeffvalues (in mb) from DPS fits for different SPS models. The uncertainty is statistical only, originating from the statistical uncertainty in σDPS (and dσ (J/ψJ/ψ)/dv). The common systematic uncertainty of 12%, accounting for the systematic un- certainty of σ (J/ψJ/ψ) and the total uncertainty for σ(J/ψ), is not shown. – 13 – 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 πdσ(J/ψJ/ψ) d |∆φ| [nb] |∆φ| /π LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 dσ(J/ψJ/ψ) dAT [nb] AT LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c 6 8 10 12 14 0 1 2 3 4 5 6 7 8 9 10 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c Figure 6. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 1 GeV/c for the differential cross-sections as functions of (top left) |∆y|, (top right) |∆φ|, (bot- tom left) AT and (bottom right) m(J/ψJ/ψ). The (black) points with error bars represent the measurements. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c d (J/ψJ/ψ) 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 πdσ(J/ψJ/ψ) d |∆φ| [nb] |∆φ| /π LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c JHEP06(2017)047 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c 6 8 10 12 14 0 1 2 3 4 5 6 7 8 9 10 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 dσ(J/ψJ/ψ) dAT [nb] AT LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c Figure 6. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 1 GeV/c for the differential cross-sections as functions of (top left) |∆y|, (top right) |∆φ|, (bot- tom left) AT and (bottom right) m(J/ψJ/ψ). The (black) points with error bars represent the measurements. – 14 – 0 2 4 6 8 10 0 0.5 1 1.5 2 2.5 3 3.5 4 1 2 dσ dpT(J/ψ)  nb GeV/c  pT(J/ψ) [GeV/c] LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 1 2 3 4 5 6 7 8 9 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c 2 2.5 3 3.5 4 4.5 0 1 2 3 4 5 6 7 1 2 dσ(J/ψJ/ψ) dy(J/ψ) [nb] y(J/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c Figure 7. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 3 GeV/c for the differential cross-sections as functions of (top left) pT(J/ψ), (top right) y(J/ψJ/ψ) and (bottom) y(J/ψ). The (black) points with error bars represent the measurements. σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, 2 2.5 3 3.5 4 4.5 0 1 2 3 4 5 6 7 8 9 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c 0 2 4 6 8 10 0 0.5 1 1.5 2 2.5 3 3.5 4 1 2 dσ dpT(J/ψ)  nb GeV/c  pT(J/ψ) [GeV/c] LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c JHEP06(2017)047 2 2.5 3 3.5 4 4.5 0 1 2 3 4 5 6 7 1 2 dσ(J/ψJ/ψ) dy(J/ψ) [nb] y(J/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c Figure 7. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 3 GeV/c for the differential cross-sections as functions of (top left) pT(J/ψ), (top right) y(J/ψJ/ψ) and (bottom) y(J/ψ). The (black) points with error bars represent the measurements. – 15 – 0 0.5 1 1.5 2 2.5 0 2 4 6 8 10 12 14 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12 14 16 18 20 πdσ(J/ψJ/ψ) d |∆φ| [nb] |∆φ| /π LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12 14 16 18 20 dσ(J/ψJ/ψ) dAT [nb] AT LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c Figure 8. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 3 GeV/c for the differential cross-sections as functions of (top left) |∆y|, (top right) |∆φ|, (bot- tom left) AT and (bottom right) m(J/ψJ/ψ). The (black) points with error bars represent the measurements. 6 Summary The J/ψ pair production cross-section with both J/ψ mesons in the region 2.0 < y < 4.5 and pT < 10 GeV/c is measured to be 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, using pp collision data collected by LHCb at √s = 13 TeV, corresponding to an integrated luminosity of 279 pb−1. The differential production cross-sections as functions of pT(J/ψJ/ψ), pT(J/ψ), m(J/ψJ/ψ), y(J/ψJ/ψ), y(J/ψ), |∆φ|, |∆y| and AT are compared to theoretical predic- tions. A fit to the differential cross-sections using simple DPS plus SPS models indicates a significant DPS contribution. The data can be reasonably well described with a sum of DPS and SPS colour-singlet contributions, with no evidence for a large SPS colour-octet contribution. The obtained SPS contribution is overestimated in the NLO∗CS′′ [70, 93–96] and NLO CS [39] approaches and roughly agrees with the NLO∗CS′ [92] and LO kT [102] predictions. Good agreement with the data for the differential cross-sections calculated within the LO kT [102] and NLO∗CS′ [92] approaches indicates that a significant part of high-order contributions can be properly accounted via the evolution of parton densi- ties [102]. Relatively large smearing of initial gluon transverse momenta ⟨kT⟩= 2 GeV/c is preferred over ⟨kT⟩= 0.5 GeV/c for the NLO∗CS′′ approach [70, 93–96]. An improve- ment in the precision for SPS predictions is needed for a better discrimination between the different theory approaches. A large DPS contribution results in values of σeffthat are smaller than the values of σeffmeasured previously by the LHCb collaboration in the pro- cesses of multiple associated heavy quark production [52, 54], and slightly larger than those measured from central J/ψ pair production at the CMS [58] and ATLAS [59] experiments. JHEP06(2017)047 σ(J/ψJ/ψ) = 15.2 ± 1.0 (stat) ± 0.9 (syst) nb, 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12 14 16 18 20 πdσ(J/ψJ/ψ) d |∆φ| [nb] |∆φ| /π LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c 0 0.5 1 1.5 2 2.5 0 2 4 6 8 10 12 14 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT SPS: NLO∗CS′ SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c JHEP06(2017)047 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12 14 16 18 20 dσ(J/ψJ/ψ) dAT [nb] AT LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c Figure 8. Comparisons between measurements and theoretical predictions with pT(J/ψJ/ψ) > 3 GeV/c for the differential cross-sections as functions of (top left) |∆y|, (top right) |∆φ|, (bot- tom left) AT and (bottom right) m(J/ψJ/ψ). The (black) points with error bars represent the measurements. – 16 – Acknowledgments We would like to thank K.-T. Chao, J.-P. Lansberg, A.K. Likhoded and A.V. Luchinsky for interesting discussions on quarkonia and quarkonium-pair production, and S.P. Baranov, S.V. Poslavsky, H.-S. Shao and L.-P. Sun for providing the SPS calculations. We express our gratitude to our colleagues in the CERN accelerator departments for the excellent performance of the LHC. We thank the technical and administrative staffat the LHCb institutes. We acknowledge support from CERN and from the national agencies: CAPES, CNPq, FAPERJ and FINEP (Brazil); NSFC (China); CNRS/IN2P3 (France); BMBF, DFG and MPG (Germany); INFN (Italy); FOM and NWO (The Netherlands); MNiSW and NCN (Poland); MEN/IFA (Romania); MinES and FASO (Russia); MinECo (Spain); SNSF and SER (Switzerland); NASU (Ukraine); STFC (United Kingdom); NSF (U.S.A.). We acknowledge the computing resources that are provided by CERN, IN2P3 (France), KIT and DESY (Germany), INFN (Italy), SURF (The Netherlands), PIC (Spain), GridPP (United Kingdom), RRCKI and Yandex LLC (Russia), CSCS (Switzerland), IFIN-HH (Romania), CBPF (Brazil), PL-GRID (Poland) and OSC (U.S.A.). We are indebted to the communities behind the multiple open source software packages on which we depend. Individual groups or members have received support from AvH Foundation (Germany), EPLANET, Marie Sk lodowska-Curie Actions and ERC (European Union), Conseil G´en´eral de Haute-Savoie, Labex ENIGMASS and OCEVU, R´egion Auvergne (France), RFBR and – 17 – Yandex LLC (Russia), GVA, XuntaGal and GENCAT (Spain), Herchel Smith Fund, The Royal Society, Royal Commission for the Exhibition of 1851 and the Leverhulme Trust (United Kingdom). Yandex LLC (Russia), GVA, XuntaGal and GENCAT (Spain), Herchel Smith Fund, The Royal Society, Royal Commission for the Exhibition of 1851 and the Leverhulme Trust (United Kingdom). A Fits to the differential cross-sections with SPS and DPS component The results of fits used for the determination of σeffare shown in figures 9, 10 and 11. The fits used only for determination of fDPS in pT(J/ψJ/ψ) > 1 GeV/c and pT(J/ψJ/ψ) > 3 GeV/c regions are shown in figures 12, 13, 14 and 15. JHEP06(2017)047 – 18 – 0 5 10 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: LO kT 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV DPS SPS: LO CS 0 5 10 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 0 5 10 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c Figure 9. Result of templated DPS fit for dσ(J/ψ J/ψ ) dpT(J/ψ J/ψ ) and dσ(J/ψ J/ψ ) dy(J/ψ J/ψ ). The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. A Fits to the differential cross-sections with SPS and DPS component 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV DPS SPS: LO CS 0 5 10 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: LO kT JHEP06(2017)047 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c / ] V/c 0 5 10 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 0 5 10 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) dpT(J/ψJ/ψ)  nb GeV/c  pT(J/ψJ/ψ) [GeV/c] LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c Figure 9. Result of templated DPS fit for dσ(J/ψ J/ψ ) dpT(J/ψ J/ψ ) and dσ(J/ψ J/ψ ) dy(J/ψ J/ψ ). The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. – 19 – 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS SPS: LO CS 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS SPS: LO kT 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS ×××××SPS: NLO CS Figure 10. Result of templated DPS fit for dσ(J/ψ J/ψ ) dm(J/ψ J/ψ ). The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. A Fits to the differential cross-sections with SPS and DPS component 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS SPS: LO CS 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS SPS: LO kT JHEP06(2017)047 14  6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS ×××××SPS: NLO CS 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c Figure 10. Result of templated DPS fit for dσ(J/ψ J/ψ ) dm(J/ψ J/ψ ). The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. – 20 – JHEP06(2017)047 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO CS 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO kT 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS ×××××SPS: NLO CS Figure 11. Result of templated DPS fit for dσ(J/ψ J/ψ ) d|∆y| . The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. A Fits to the differential cross-sections with SPS and DPS component 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO CS 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO kT 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO kT 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: LO CS JHEP06(2017)047 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c d (J/ψJ/ψ) dσ(J/ψJ/ψ) [nb] | y| | y| 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS ×××××SPS: NLO CS 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV DPS ×××××SPS: NLO CS Figure 11. Result of templated DPS fit for dσ(J/ψ J/ψ ) d|∆y| . The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. A Fits to the differential cross-sections with SPS and DPS component – 21 – 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′ 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′ 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c Figure 12. Result of templated DPS fit for dσ(J/ψ J/ψ ) dy(J/ψ J/ψ ) and dσ(J/ψ ) dm(J/ψ J/ψ ) for the pT(J/ψJ/ψ) > 1 GeV/c region. The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. A Fits to the differential cross-sections with SPS and DPS component 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′ JHEP06(2017)047 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′ 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 6 8 10 12 14 0 1 2 3 4 5 6 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c Figure 12. Result of templated DPS fit for dσ(J/ψ J/ψ ) dy(J/ψ J/ψ ) and dσ(J/ψ ) dm(J/ψ J/ψ ) for the pT(J/ψJ/ψ) > 1 GeV/c region. The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. – 22 – 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′ 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c Figure 13. A Fits to the differential cross-sections with SPS and DPS component Result of templated DPS fit for dσ(J/ψ J/ψ ) d|∆y| for the pT(J/ψJ/ψ) > 1 GeV/c region. The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′ 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: LO kT JHEP06(2017)047 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 1 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c d (J/ψJ/ψ) Figure 13. Result of templated DPS fit for dσ(J/ψ J/ψ ) d|∆y| for the pT(J/ψJ/ψ) > 1 GeV/c region. The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. A Fits to the differential cross-sections with SPS and DPS component – 23 – 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′ 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′ 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c Figure 14. Result of templated DPS fit for dσ(J/ψ J/ψ ) dy(J/ψ J/ψ ) and dσ(J/ψ J/ψ ) dm(J/ψ J/ψ ) for the pT(J/ψJ/ψ) > 3 GeV/c region. The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. A Fits to the differential cross-sections with SPS and DPS component 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′ JHEP06(2017)047 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′ 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 6 8 10 12 14 0 0.5 1 1.5 2 2.5 3 dσ(J/ψJ/ψ) dm(J/ψJ/ψ)  nb GeV/c2  m(J/ψJ/ψ)  GeV/c2 LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 2 2.5 3 3.5 4 4.5 0 0.5 1 1.5 2 2.5 3 3.5 dσ(J/ψJ/ψ) dy(J/ψJ/ψ) [nb] y(J/ψJ/ψ) LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c Figure 14. Result of templated DPS fit for dσ(J/ψ J/ψ ) dy(J/ψ J/ψ ) and dσ(J/ψ J/ψ ) dm(J/ψ J/ψ ) for the pT(J/ψJ/ψ) > 3 GeV/c region. The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. – 24 – 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′ 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c Figure 15. Result of templated DPS fit for dσ(J/ψ J/ψ ) d|∆y| for the pT(J/ψJ/ψ) > 3 GeV/c region. References [1] C.E. Carlson and R. 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The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: LO kT 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′ JHEP06(2017)047 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=0.5 GeV/c 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 3.5 4 dσ(J/ψJ/ψ) d |∆y| [nb] |∆y| LHCb 13 TeV pT(J/ψJ/ψ) > 3 GeV/c DPS SPS: NLO∗CS′′ ⟨kT⟩=2 GeV/c dσ(J/ψJ/ψ) Figure 15. Result of templated DPS fit for dσ(J/ψ J/ψ ) d|∆y| for the pT(J/ψJ/ψ) > 3 GeV/c region. The (black) points with error bars represent the data. The total fit result is shown with the thick (red) solid line and the DPS component is shown with the thin (orange) solid line. – 25 – Open Access. 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Martinez Santos39, F. Martinez Vidal69, D. Martins Tostes2, L.M. Massacrier7, A. Massafferri1, R. Matev40, A. Mathad50, Z. Mathe40, D. Martins Tostes2, L.M. Massacrier7, A. Massafferri1, R. Matev40, A. Mathad50, Z. Mathe C. Matteuzzi21, A. Mauri42, E. Maurice7,b, B. Maurin41, A. Mazurov47, M. McCann55,40, A. McNab56, R. McNulty13, B. Meadows59, F. Meier10, M. Meissner12, D. Melnychuk29, A. McNab56, R. McNulty13, B. Meadows59, F. Meier10, M. Meissner12, D. Melnychuk29 A. Mogini8, J. Molina Rodriguez1, I.A. Monroy66, S. Monteil5, M. Morandin23, P A. Mogini8, J. Molina Rodriguez1, I.A. Monroy66, S. Monteil5, M. Morandin23, P. Morawski28, A. Mord`a6, M.J. Morello24,t, O. Morgunova68, J. Moron28, A.B. Morris52, R. Mountain61, F. Muheim52, M. Mulder43, M. Mussini15, D. M¨uller56, J. M¨uller10, K. M¨uller42, V. M¨uller10, S. Neubert12, N. Neufeld40, M. Neuner12, T.D. Nguyen41, C. Nguyen-Mau41,n, S. Nieswand9, R. Niet10, N. Nikitin33, T. Nikodem12, A. Nogay68, A. Novoselov37, D.P. O’Hanlon50, A. Oblakowska-Mucha28, V. Obraztsov37, S. Ogilvy19, R. Oldeman16,f, C.J.G. Onderwater70 J.M. Otalora Goicochea2, A. Otto40, P. Owen42, A. Oyanguren69, P.R. Pais41, A. Palano14,d, M. Palutan19, A. Papanestis51, M. Pappagallo14,d, L.L. Pappalardo17,g, W. Parker60, C. Pa G. Passaleva18, A. Pastore14,d, G.D. Patel54, M. Patel55, C. Patrignani15,e, A. Pearce40, A. Pellegrino43, G. Penso26, M. Pepe Altarelli40, S. Perazzini40, P. Perret5, L. Pescatore47, K. Petridis48, A. Petrolini20,h, A. Petrov68, M. Petruzzo22,q, E. Picatoste Olloqui38, B. Pietrzyk M. Pikies27, D. Pinci26, A. Pistone20, A. Piucci12, V. Placinta30, S. Playfer52, M. Plo Casasus39 T. Poikela40, F. Polci8, A. Poluektov50,36, I. Polyakov61, E. Polycarpo2, G.J. Pomery4 p , p , p , , , , , J. Prisciandaro39,40, A. Pritchard54, C. Prouve48, V. Pugatch46, A. Puig Navarro42, G. Punzi24,p M.S. Rangel2, I. Raniuk45, F. Ratnikov35, G. Raven44, F. Redi55, S. Reichert10, A.C. dos Reis1, C. Remon Alepuz69, V. Renaudin7, S. Ricciardi51, S. Richards48, M. Rihl40, K. Rinnert54, V. Rives Molina38, P. Robbe7,40, A.B. Rodrigues1, E. Rodrigues59, J.A. Rodriguez Lopez66, P. Rodriguez Perez56,†, A. Rogozhnikov35, S. Roiser40, A. Rollings57, V. Romanovskiy37, A. Romero Vidal39, J.W. Ronayne13, M. Rotondo19, M.S. Rudolph61, T. Ruf40, P. Ruiz Valls69, – 34 – J.J. Saborido Silva39, E. Sadykhov32, N. Sagidova31, B. Saitta16,f, V. Salustino Guimaraes1, C. Sanchez Mayordomo69, B. Sanmartin Sedes39, R. Santacesaria26, C. Santamarina Rios39, Sanchez Mayordomo69, B. Sanmartin Sedes39, R. Santacesaria26, C. Santamarina Rios39, y , , , , Santimaria19, E. Santovetti25,j, A. Sarti19,k, C. Satriano26,s, A. Satta25, D.M. Saunders48, y , , , , M. Santimaria19, E. Santovetti25,j, A. The LHCb collaboration Sarti19,k, C. Satriano26,s, A. Satta25, D.M. Saunders48 Savrina32,33, S. Schael9, M. Schellenberg10, M. Schiller53, H. Schindler40, M. Schlupp10, D. Savrina32,33, S. Schael9, M. Schellenberg10, M. Schiller53, H. Schindler40, M. Schlupp10, M. Schmelling11, T. Schmelzer10, B. Schmidt40, O. Schneider41, A. Schopper40, K. Schubert10, M. Schubiger41, M.-H. Schune7, R. Schwemmer40, B. Sciascia19, A. Sciubba26,k, A. Semennikov M. Schubiger41, M.-H. Schune7, R. Schwemmer40, B. Sciascia19, A. Sciubba26,k, A. Semennikov32 A. Sergi47, N. Serra42, J. Serrano6, L. Sestini23, P. Seyfert21, M. Shapkin37, I. Shapoval45, Y. Shcheglov31, T. Shears54, L. Shekhtman36,w, V. Shevchenko68, B.G. Siddi17,40, Y. Shcheglov31, T. Shears54, L. Shekhtman36,w, V. Shevchenko68, B.G. Siddi17,40, T. Skwarnicki61, E. Smith55, I.T. Smith52, J. Smith49, M. Smith55, H. Snoek43, l. Soares Lavra1, T. Skwarnicki61, E. Smith55, I.T. Smith52, J. Smith49, M. Smith55, H. Snoek43, l. Soares Lavra1, M.D. Sokoloff59, F.J.P. Soler53, B. Souza De Paula2, B. Spaan10, P. Spradlin53, S. Sridharan40, JHEP06(2017)047 F. Stagni40, M. Stahl12, S. Stahl40, P. Stefko41, S. Stefkova55, O. Steinkamp42, S. Stemmle12, F. Stagni40, M. Stahl12, S. Stahl40, P. Stefko41, S. Stefkova55, O. Steinkamp42, S. Stemmle12, O. Stenyakin37, H. Stevens10, S. Stevenson57, S. Stoica30, S. Stone61, B. Storaci42, S. Stracka24,p M. Straticiuc30, U. Straumann42, L. Sun64, W. Sutcliffe55, K. Swientek28, V. Syropoulos44, M. Szczekowski29, T. Szumlak28, S. T’Jampens4, A. Tayduganov6, T. Tekampe10, G. Tellarin F. Teubert40, E. Thomas40, J. van Tilburg43, M.J. Tilley55, V. Tisserand4, M. Tobin41, S. To L. Tomassetti17,g, D. Tonelli40, S. Topp-Joergensen57, F. Toriello61, E. Tournefier4, S. Tourne K. Trabelsi41, M. Traill53, M.T. Tran41, M. Tresch42, A. Trisovic40, A. Tsaregorodtsev6, Trabelsi41, M. Traill53, M.T. Tran41, M. Tresch42, A. Trisovic40, A. Tsaregorodtsev6, P. Tsopelas43, A. Tully49, N. Tuning43, A. Ukleja29, A. Ustyuzhanin35, U. Uwer12, C. Vac Tsopelas43, A. Tully49, N. Tuning43, A. Ukleja29, A. Ustyuzhanin35, U. Uwer12, C. Vacca16,f, V. Vagnoni15,40, A. Valassi40, S. Valat40, G. Valenti15, R. Vazquez Gomez19, V. Vagnoni15,40, A. Valassi40, S. Valat40, G. Valenti15, R. Vazquez Gomez19, P. Vazquez Regueiro39, S. Vecchi17, M. van Veghel43, J.J. Velthuis48, M. Veltri18,r, G. Veneziano5 A. Venkateswaran61, M. Vernet5, M. Vesterinen12, J.V. Viana Barbosa40, B. Viaud7, D. Vieira6 M. Vieites Diaz39, H. Viemann67, X. Vilasis-Cardona38,m, M. Vitti49, V. Volkov33, A. Vollhardt42 B. Voneki40, A. Vorobyev31, V. Vorobyev36,w, C. Voß9, J.A. de Vries43, C. V´azquez Sierra39, 67 50 13 24 61 49 54 R. Waldi67, C. Wallace50, R. Wallace13, J. Walsh24, J. Wang61, D.R. Ward49, H.M. Wark54, M. Wilkinson61, M. Williams40, M.P. Williams47, M. Williams58, T. Williams47, F.F. Wils J. Wimberley60, J. The LHCb collaboration Wishahi10, W. Wislicki29, M. Witek27, G. Wormser7, S.A. Wotton49, K. Wraight53, K. Wyllie40, Y. Xie65, Z. Xing61, Z. Xu4, Z. Yang3, Y. Yao61, H. Yin65, J X. Yuan36,w, O. Yushchenko37, K.A. Zarebski47, M. Zavertyaev11,c, L. Zhang3, Y. Zhang7 Y. Zhang63, A. Zhelezov12, Y. Zheng63, X. Zhu3, V. Zhukov33, S. Zucchelli15. 1 Centro Brasileiro de Pesquisas F´ısicas (CBPF), Rio de Janeiro, Brazil 2 Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 2 Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3 Center for High Energy Physics, Tsinghua University, Beijing, China 3 Center for High Energy Physics, Tsinghua University, Beijing, China 4 LAPP, Universit´e Savoie Mont-Blanc, CNRS/IN2P3, Annecy-Le-Vieux, France 6 CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 6 CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 7 LAL, Universit´e Paris-Sud, CNRS/IN2P3, Orsay, France 7 LAL, Universit´e Paris-Sud, CNRS/IN2P3, Orsay, France 8 LPNHE, Universit´e Pierre et Marie Curie, Universit´e Paris Diderot, CNRS/IN2P3, Paris, France 9 I. The LHCb collaboration Physikalisches Institut, RWTH Aachen University, Aachen, Germany 10 Fakult¨at Physik, Technische Universit¨at Dortmund, Dortmund, Germany 11 Max-Planck-Institut f¨ur Kernphysik (MPIK), Heidelberg, Germany Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 13 School of Physics, University College Dublin, Dublin, Ireland 16 Sezione INFN di Cagliari, Cagliari, Italy 17 Sezione INFN di Ferrara, Ferrara, Italy 18 Sezione INFN di Firenze, Firenze, Italy – 35 – 19 Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy 20 Sezione INFN di Genova, Genova, Italy 21 Sezione INFN di Milano Bicocca, Milano, Italy 22 Sezione INFN di Milano, Milano, Italy 23 Sezione INFN di Padova, Padova, Italy 24 Sezione INFN di Pisa, Pisa, Italy 25 Sezione INFN di Roma Tor Vergata, Roma, Italy 26 Sezione INFN di Roma La Sapienza, Roma, Italy 27 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Krak´ow, Polan 28 27 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academ 28 AGH - University of Science and Technology, Faculty of Physics and Applied Computer Science, Krak´ow, Poland 29 National Center for Nuclear Research (NCBJ), Warsaw, Poland 30 National Center for Nuclear Research (NCBJ), Wa JHEP06(2017)047 JHEP06(2017)047 30 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania 31 Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 32 Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia itute of Theoretical and Experimental Physics (ITEP 33 Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia 34 Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow, Russia 34 Institute for Nuclear Research of the Russian Academy of Sciences (INR RA Yandex School of Data Analysis, Moscow, Russia 36 Budker Institute of Nuclear Physics (SB RAS), Novosibirsk, Russia 37 Institute for High Energy Physics (IHEP), Protvino, Russia 38 ICCUB, Universitat de Barcelona, Barcelona, Spain 39 Universidad de Santiago de Compostela, Santiago de Compostela, Spain Universidad de Santiago de Compostela, Santiago d 40 European Organization for Nuclear Research (CERN), Geneva, Switzerland 41 Institute of Physics, Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland hysik-Institut, Universit¨at Z¨urich, Z¨urich, Switzerlan 43 Nikhef National Institute for Subatomic Physics, Amsterdam, The Netherlands ikhef National Institute for Subatomic Physics, Amst 43 Nikhef National Institute for Subatomic Physics, Amsterdam, The Netherlands 44 Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam, Th Netherlands 45 NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukraine 46 Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine 47 University of Birmingham, Birmingham, United Kingdom 49 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 49 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom avendish Laboratory, University of Cambridge, Camb 50 Department of Physics, University of Warwick, Coventry, United Kingdom 50 Department of Physics, University of Warwick, Coventry, United Kingdom 51 STFC Rutherford Appleton Laboratory, Didcot, United Kingdom School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom Physics and Astronomy, University of Edinburgh, E 52 School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 53 School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 54 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom Imperial College London, London, United Kingdom 56 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 56 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom School of Physics and Astronomy, University of Ma niversity of Manchester, Manchester, United Kingdo 57 Department of Physics, University of Oxford, Oxford, United Kingdom 57 Department of Physics, University of Oxford, Oxford, United Kingdom 58 Massachusetts Institute of Technology, Cambridge, MA, United States 58 Massachusetts Institute of Technology, Cambridge, MA, United States 59 University of Cincinnati, Cincinnati, OH, United States 59 University of Cincinnati, Cincinnati, OH, United States 60 University of Maryland, College Park, MD, United States 60 University of Maryland, College Park, MD, United States 61 Syracuse University, Syracuse, NY, United States 62 Pontif´ıcia Universidade Cat´olica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, associated to 2 62 Pontif´ıcia Universidade Cat´olica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, associated to 2 63 University of Chinese Academy of Sciences, Beijing, China, associated to 3 63 University of Chinese Academy of Sciences, Beijing, China, associated to 3 64 School of Physics and Technology, Wuhan University, Wuhan, China, associated to 3 65 Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China, associated to 3 – 36 – 66 Departamento de Fisica , Universidad Nacional de Colombia, Bogota, Colombia, associated to 8 67 Institut f¨ur Physik, Universit¨at Rostock, Rostock, Germany, associated to 12 68 National Research Centre Kurchatov Institute, Moscow, Russia, associated to 32 69 Instituto de Fisica Corpuscular (IFIC), Universitat de Valencia-CSIC, Valencia, Spain, associated to 38 70 Van Swinderen Institute, University of Groningen, Groningen, The Netherlands, associated to 43 a Universidade Federal do Triˆangulo Mineiro (UFTM), Uberaba-MG, Brazil b Laboratoire Leprince-Ringuet, Palaiseau, France c P.N. Telecommunications, Krak´ow, Poland m LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain m LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain n Hanoi University of Science, Hanoi, Viet Nam n Hanoi University of Science, Hanoi, Viet Nam o Universit`a di Padova, Padova, Italy o Universit`a di Padova, Padova, Italy p Universit`a di Pisa, Pisa, Italy p Universit`a di Pisa, Pisa, Italy q Universit`a degli Studi di Milano, Milano, Italy q Universit`a degli Studi di Milano, Milano, Italy r Universit`a di Urbino, Urbino, Italy s Universit`a della Basilicata, Potenza, Italy t Scuola Normale Superiore, Pisa, Italy t Scuola Normale Superiore, Pisa, Italy u Universit`a di Modena e Reggio Emilia, Modena, Italy u Universit`a di Modena e Reggio Emilia, Modena, Italy v Iligan Institute of Technology (IIT), Iligan, Philippines v Iligan Institute of Technology (IIT), Iligan, Philippines w Novosibirsk State University, Novosibirsk, Russia w Novosibirsk State University, Novosibirsk, Russia † Deceased The LHCb collaboration Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia d c P.N. Lebedev Physical Institute, Russian Academ c P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia d Universit`a di Bari, Bari, Italy d Universit`a di Bari, Bari, Italy d Universit`a di Bari, Bari, Italy e Universit`a di Bologna, Bologna, Italy JHEP06(2017)047 f Universit`a di Cagliari, Cagliari, Italy f Universit`a di Cagliari, Cagliari, Italy g Universit`a di Ferrara, Ferrara, Italy h Universit`a di Genova, Genova, Italy i Universit`a di Milano Bicocca, Milano, Italy j Universit`a di Roma Tor Vergata, Roma, Italy k Universit`a di Roma La Sapienza, Roma, Italy k Universit`a di Roma La Sapienza, Roma, Italy l AGH - University of Science and Technology, Faculty of Computer Science, Electronics and l AGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Krak´ow, Poland l AGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Krak´ow, Poland † Deceased – 37 –
https://openalex.org/W2339528563
https://europepmc.org/articles/pmc4833865?pdf=render
English
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The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules
Nature communications
2,016
cc-by
10,971
ARTICLE Received 22 Oct 2015 | Accepted 8 Mar 2016 | Published 13 Apr 2016 1 Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Davidson Building Room 324, Glasgow G12 8QQ, UK. 2 Section for Cell & Neurobiology, Department of Biology, University of Copenhagen, Universitetsparken 15, Copenhagen DK-2100, Denmark. 3 MRC—University of Glasgow Centre for Virus Research, Henry Wellcome Building, 464 Bearsden Road, Glasgow G61 1QH, UK. 4 Cancer Research UK | Beatson Institute, Garscube Estate, Switchback road, Glasgow G61 1BD, UK. 5 Section of Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, Copenhagen DK-2100, Denmark. 6 Institut fu¨r Neuro- und Verhaltensbiologie, Universita¨t Mu¨nster, Badestrasse 9, 48149 Mu¨nster, Germany. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to K.A.H. (email: kahalberg@bio.ku.dk) or to J.A.T.D. (email: Julian.Dow@glasgow.ac.uk). NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 They are composed of two morphologically and physiologically distinct cell types, the principal ‘type 1’ cell and the stellate ‘type 2’ cell; the larger principal cells are the sites of active cation transport, energized by an apical plasma membrane V-ATPase driving an K þ/H þ exchanger, whereas the smaller stellate cells are in control of chloride and water fluxes13–15. Like all transporting epithelia, the MTs possess a prominent brush border16, which serves to increase the membrane surface area available to the transport machinery that drives transepithelial transport17. Indeed, the insect MT moves fluid faster on a per-cell basis than any other epithelium18. However, fundamental questions on how the MT brush border is maintained, how microvillar length is controlled and what regulates brush border organization remain unanswered. Here we report that Fas2 plays a critical role in regulating brush border length and organization in renal tubules of Drosophila. We show that Fas2 extracellular interactions are necessary and sufficient to mediate these effects, and that the intracellular domain is dispensable in this regard. Furthermore, we demonstrate that the transport capacity of the epithelium is correlated with Fas2-induced changes to microvillar length. Our data are thus consistent with a model in which Fas2-dependent adhesion complexes promote microvillar growth and organization, and propose that Fas2-homotypic intermicrovillar links act to stabilize the brush border against shear stress associated with the uniquely high secretion rates of the tissue. Fas2 localizes extracellularly between microvilli. As Fas2 has been shown to be a key mediator of axon fasciculation through homophilic adhesion5,7,21, we rationalized that Fas2 might be involved in the structural stabilization of microvilli, by forming homotypic intermicrovillar links that help to maintain their ‘fasciculation’. To test this hypothesis, we performed super- resolution confocal microscopy. This revealed that although Fas2 colocalizes with F-actin throughout the proximal-distal axis of the brush border concentrating distally (Fig. 3a,b), the Fas2 signal appears to predominantly interpolate between discrete F-actin signals (Fig. 3c). This is consistent with F-actin microfilaments marking the intracellular region of the microvilli, and the GFP778 tag labelling the extracellular domain of Fas2 (Fig. 2a,b), resulting in a limited overlap between signals. These findings indicate that Fas2 localizes extracellularly, and is properly positioned to contribute to intermicrovillar links. Fas2 expression affects microvilli length and organization. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 Yet, whether this localization pattern remains static throughout tubule development is unknown. We therefore investigated the spatio-temporal expression pattern of Fas2 during tubule morphogenesis, by utilizing a series of Fas2 exon trap insertion lines that recognize different Fas2 isoforms (Fig. 2a,b). Each fusion protein—known to behave as native protein in the CNS20,21—reported the same distribution pattern (Fig. 2c): Fas2 localizes to the lateral cell junctions in MTs of late stage embryos (stage 16o), but abruptly switches to the apical brush border (area between arrows; Fig. 2c) of the principal cells during early larval development, where it remains until pupation. In the transport incompetent pupal tubules—evident by the involuted brush border and reduced tubule lumen22—Fas2 completely vanishes, then reappears as the adult microvilli form and remains throughout adulthood (Fig. 2c). The observed changes in brush border appearance during tubule development (see Fig. 6a) is consistent with that previously described at an ultrastructural level22. The localization of Fas2 to the apical brush border was confirmed by immunocytochemistry using two separate anti-Fas2 monoclonal antibodies (Supplementary Fig. 1). These data thus show a highly dynamic expression pattern of Fas2 during tubule development, and indicate that MT transport competence partly depends on Fas2 localization to the brush border. C omprehensive tissue expression atlases allow gene function to be reassessed across the whole organism1–4, and many genes show unexpected expression patterns outside the tissues in which they have been traditionally studied. One such example is Fasciclin 2 (Fas2), which has been extensively studied for its role in axon guidance and neuronal development during embryogenesis5–8. Fas2 is a cell adhesion molecule that directs axon fasciculation through homophilic cell–cell recognition, which helps to establish a regular neuronal scaffold, on which the developing nervous system is organized5,9–11. Surprisingly, the online resource flyatlas.org2,4 reports high postembryonic levels of expression in both larvae and adults, suggesting a lifelong function of Fas2, in addition to its well-characterized role in early development. Intriguingly, expression levels have been reported to be highest in both larval and adult renal (Malpighian) tubules2, yet the role Fas2 serves in this non-neuronal tissue is remarkably unexplored. The insect Malpighian tubules (MTs) constitute the functional analogue of the vertebrate kidney, and offer a unique opportunity for the study of epithelial function and control12. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 To gain further insight into the functional role of Fas2 in renal tubules, we restrictively modulated expression levels of Fas2 by driving either RNAi or overexpressor constructs in the principal cells with the GAL4/UAS system23,24. Confocal microscopy of tubules stained with phalloidin (F-actin) confirms that wild type (WT) tubules have a strict organized, dense population of microvilli at the brush border of the principal cells (Canton S, Fig. 3d). On significant reduction (one-way analysis of variance (ANOVA), Po0.05) of Fas2 transcript and protein levels (Supplementary Fig. 2), the brush border becomes sparse, shorter and less organized (Fas2-RNAi and Fas2EB112, Fig. 3d). Conversely, increased levels of Fas2 result in the opposite phenotype, where the brush border appears denser and longer (Fas2-EP, Fig. 3d). Scanning electron microscopy on tubules from the same genetic backgrounds (Fig. 3e) confirmed these results, and additionally allowed quantification of microvilli length, which demonstrated that Fas2 depletion (UroGAL44Fas2-RNAi and Fas2EB112) results in a significant decrease in microvilli length compared with both parental lines (Fas2-RNAi/ þ , UroGal4/ þ and Fas2-EP/ þ ) and WT (Canton S), respectively (Fig. 3e–g). Conversely, increased levels of Fas2 cause a significant increase in microvilli length compared with The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules Kenneth A. Halberg1,2,*, Stephanie M. Rainey1,3,*, Iben R. Veland4,5, Helen Neuert6, A Christian Kla¨mbt6, Shireen-Anne Davies1 & Julian A.T. Dow1 Kenneth A. Halberg1,2,*, Stephanie M. Rainey1,3,*, Iben R. Veland4,5, Helen Neuert6, Anthony J. Dornan1, Christian Kla¨mbt6, Shireen-Anne Davies1 & Julian A.T. Dow1 Multicellular organisms rely on cell adhesion molecules to coordinate cell–cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border. 1 Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Davidson Building Room 324, Glasgow G12 8QQ, UK. 2 Section for Cell & Neurobiology, Department of Biology, University of Copenhagen, Universitetsparken 15, Copenhagen DK-2100, Denmark. 3 MRC—University of Glasgow Centre for Virus Research, Henry Wellcome Building, 464 Bearsden Road, Glasgow G61 1QH, UK. 4 Cancer Research UK | Beatson Institute, Garscube Estate, Switchback road, Glasgow G61 1BD, UK. 5 Section of Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, Copenhagen DK-2100, Denmark. 6 Institut fu¨r Neuro- und Verhaltensbiologie, Universita¨t Mu¨nster, Badestrasse 9, 48149 Mu¨nster, Germany. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to K.A.H. (email: kahalberg@bio.ku.dk) or to J.A.T.D. (email: Julian.Dow@glasgow.ac.uk). 1 NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications URE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www ARTICLE NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 Fas2-RA Fas2-RB Fas2-RC d Sequence localization 4,030 kb 4,045 kb 4,100 kb Tubule 452 0 226 Ovary 86 0 43 Testis 0 86 43 Head 0 200 100 Whole fly 100 0 50 c Expression ratio compared with whole fly 0 2 4 6 8 * * Whole fly Tubule Head Fas2-RA 0 5 10 15 Whole fly Tubule Head Fas2-RC 0 50 100 150 200 * * Whole fly Tubule Head Fas2-RB 0 20 40 60 80 Whole fly Tubule Head * * All Fas2 Expression ratio compared with whole fly a Brain Head Eye TA ganglion Salivary gland Crop Midgut Tubule Hindgut Heart Fat body Ovary Testis Male accessory glands Virgin spermatheca Mated spermatheca Adult carcass Larval CNS Larval salivary gland Larval midgut Larval tubule Larval hindgut Larval fat body Larval trachea Larval carcass Whole fly 0 200 400 600 800 1,000 1,200 1,400 1,600 1,800 Fas 2 spatial expression Mean affymetrix signal Low High Fas2 relative expression CNS Tubule b Signal strength (FPKM) Figure 1 | Transcriptomic profiling of Fas2 expression. (a) Mean normalized Affymetrix signal±s.e.m. (N ¼ 4 tissue samples) showing the Fas2 spa expression pattern across major tissues from both larval and adult Drosophila (flyatlas.org). (b) Overview of Drosophila anatomy with superimposed h maps of the spatial expression pattern of Fas2. (c) Microarray and qPCR analyses of the dominant Fas2 isoforms across different tissues. The Affyme whole fly signal (dark grey bars) was set to an a.u. of 1, with head and tubule signals being expressed as a ratio to whole fly. The mean±s.d. qPCR expression ratios (light grey bars) largely reiterate the microarray data, albeit show much higher Fas2-RB transcript enrichment in MTs compared with Affymetrix signal. *Significantly different (one-way ANOVA, Po0.05) compared with whole fly. (d) RNA-Seq data comparing Fas2 transcript levels ac different tissues. The combined transcriptomic meta-analysis reveal that Fas2-RA, RB and RC are the main isoforms expressed in Drosophila, with isof Fas2-RB being the dominant splice variant expressed in the MTs. Results T Transcriptomic analysis of Fas2 expression. To verify the spatial expression profile reported by FlyAtlas.org (Fig. 1a,b), and to identify the Fas2 splice variant expressed in Drosophila MTs, we used both quantitative reverse transcription (RT)–PCR and whole transcriptome sequencing (RNA-Seq). Both approaches independently confirmed the data reported by FlyAtlas, and further showed that although all major Fas2 isoforms are expressed, Fas2-RB is the dominant splice variant expressed in Drosophila renal tubules (Fig. 1c,d). Dynamic expression of Fas2 during tubule development. To gain insight into what role Fas2 serves in MTs of Drosophila, we initially identified the subcellular localization of Fas2 protein using genetic and immunocytochemical approaches. During embryogenesis, Fas2 is known to localize to the lateral junctions of the developing MTs19, which is consistent with its role in cell–cell recognition in the central nervous system (CNS)5. NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 (N ¼ 4 tissue samples) showing the Fas2 spatial expression pattern across major tissues from both larval and adult Drosophila (flyatlas.org). (b) Overview of Drosophila anatomy with superimposed heat maps of the spatial expression pattern of Fas2. (c) Microarray and qPCR analyses of the dominant Fas2 isoforms across different tissues. The Affymetrix whole fly signal (dark grey bars) was set to an a.u. of 1, with head and tubule signals being expressed as a ratio to whole fly. The mean±s.d. qPCR expression ratios (light grey bars) largely reiterate the microarray data, albeit show much higher Fas2-RB transcript enrichment in MTs compared with the Affymetrix signal. *Significantly different (one-way ANOVA, Po0.05) compared with whole fly. (d) RNA-Seq data comparing Fas2 transcript levels across different tissues. The combined transcriptomic meta-analysis reveal that Fas2-RA, RB and RC are the main isoforms expressed in Drosophila, with isoform Fas2-RB being the dominant splice variant expressed in the MTs. Genetic dissection of Fas2 function. All Fas2 isoforms contain an extracellular domain with multiple Ig-binding domains; yet the isoforms differ in their membrane-anchoring or intracellular regions (Fig. 2a,b). In the developing CNS, Fas2-RA is capable of signalling intracellularly through its cytoplasmic domain5. In contrast, the Fas2-RB isoform is predicted to lack a transmembrane region25, and instead link to the membrane via a GPI anchor26. Because the MTs express all three major splice variants (Fig. 1c,d), the microvillar stabilization observed could result from either intracellular signalling to the cytoskeleton, direct homophilic binding between microvilli or both. As the tubule shows highest expression of the Fas2-RB transcript (Fig. 1c,d), we rationalized that homotypic binding of ectodomains acts to stabilize microvillar length by forming extracellular links of Fas2, analogous to the intercellular homophilic interactions demonstrated in the developing CNS and neuromuscular junctions5,7. To test this model, we selectively overexpressed either the extracellular or intracellular domains in tubule principal cells (Fig. 4c–e). When comparing tubules with controls (Fig. 3e,f); these findings were reiterated using two separate principal cell-specific GAL4 drivers (Supplementary Fig. 3). Further analysis of the microvillar protrusions indicated that the organization (in addition to the length) of the microvilli also was disrupted on manipulation of Fas2 levels (Fig. 4a,b; Supplementary Fig. 4). Control tubules (Fas2-RNAi/ þ and Fas2- EP/ þ ) have a highly organized structure of microvilli with a tendency for small, well-distributed clusters. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 a Brain Head Eye TA ganglion Salivary gland Crop Midgut Tubule Hindgut Heart Fat body Ovary Testis Male accessory glands Virgin spermatheca Mated spermatheca Adult carcass Larval CNS Larval salivary gland Larval midgut Larval tubule Larval hindgut Larval fat body Larval trachea Larval carcass Whole fly 0 200 400 600 800 1,000 1,200 1,400 1,600 1,800 Fas 2 spatial expression Mean affymetrix signal Low High Fas2 relative expression CNS Tubule b 00 1,800 Low High Fas2 relative expression CNS Tubule b b a Fas2 relative expression Mean affymetrix signal Fas2-RA Fas2-RB Fas2-RC d Sequence localization 4,030 kb 4,045 kb 4,100 kb Tubule 452 0 226 Ovary 86 0 43 Testis 0 86 43 Head 0 200 100 Whole fly 100 0 50 Signal strength (FPKM) c Expression ratio compared with whole fly 0 2 4 6 8 * * Whole fly Tubule Head Fas2-RA 0 50 100 150 200 * * Whole fly Tubule Head Fas2-RB d c c Sequence localization 4,045 kb Signal strength (FPKM) W 0 5 10 15 Whole fly Tubule Head Fas2-RC W 0 20 40 60 80 Whole fly Tubule Head * * All Fas2 Expression ratio compared with whole fly Figure 1 | Transcriptomic profiling of Fas2 expression. (a) Mean normalized Affymetrix signal±s.e.m. (N ¼ 4 tissue samples) showing the Fas2 spatial expression pattern across major tissues from both larval and adult Drosophila (flyatlas.org). (b) Overview of Drosophila anatomy with superimposed heat maps of the spatial expression pattern of Fas2. (c) Microarray and qPCR analyses of the dominant Fas2 isoforms across different tissues. The Affymetrix whole fly signal (dark grey bars) was set to an a.u. of 1, with head and tubule signals being expressed as a ratio to whole fly. The mean±s.d. qPCR expression ratios (light grey bars) largely reiterate the microarray data, albeit show much higher Fas2-RB transcript enrichment in MTs compared with the Affymetrix signal. *Significantly different (one-way ANOVA, Po0.05) compared with whole fly. (d) RNA-Seq data comparing Fas2 transcript levels across different tissues. The combined transcriptomic meta-analysis reveal that Fas2-RA, RB and RC are the main isoforms expressed in Drosophila, with isoform Fas2-RB being the dominant splice variant expressed in the MTs. Figure 1 | Transcriptomic profiling of Fas2 expression. (a) Mean normalized Affymetrix signal±s.e.m. (N ¼ 4 tissue Figure 1 | Transcriptomic profiling of Fas2 expression. (a) Mean normalized Affymetrix signal±s.e.m. NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 Arrows indicate splice variants recognized by each exon trap insertion line. The differences between tran Fas2-RA, -RB and -RC are highlighted in a,b using the same colour code. PEST, PESTdomain; SP, signal peptide. (c) In stage 16 embryos, Fas2 localiz junctions of the developing MTs; however, Fas2 switches abruptly to the apical brush border prior to eclosion of the first instar larva, where it throughout larval development. As the third instar larva pupates, the MTs become transport incompetent, the microvilli shorten, tubule lumen i 1 kb Fas2 RB RC GFP397 GFPCB03613 GFP778 RA a b GFP397 GFP778 GFPCB03613 Fas2-RA Fas2-RB Fas2-RC SP Ig FNIII PEST Intracellular Extracellular TM TM GPI b a c Dynamic expression of Fas2 during MT developme Figure 2 | Fas2 is dynamically expressed during tubule development. (a) Fas2 genomic region illustrating the positions of the GFPCB03613, GFP397 and GFP778 exon trap insertions. (b) Fas2 is expressed in multiple isoforms that all comprise five immunoglobulin (Ig) and two fibronectin type III (FNIII) domains, yet have distinct C-termini. Arrows indicate splice variants recognized by each exon trap insertion line. The differences between transcripts Fas2-RA, -RB and -RC are highlighted in a,b using the same colour code. PEST, PESTdomain; SP, signal peptide. (c) In stage 16 embryos, Fas2 localizes to cell junctions of the developing MTs; however, Fas2 switches abruptly to the apical brush border prior to eclosion of the first instar larva, where it remains throughout larval development. As the third instar larva pupates, the MTs become transport incompetent, the microvilli shorten, tubule lumen is reduced and Fas2 is no longer expressed. As the brush border reform in the adult tubule, Fas2 is again abundantly expressed in the MT, where it localizes to the apical brush border (area between arrows). This expression pattern is consistently reported by each fusion protein, and is commensurate with the transcription profile of each Fas2 isoform. Inserts: single optical section of the selected region shown as separate (small circle) and merged (large circle) signals. Scale bars, 25 mm. Fas2-RA splice variant, is not involved in regulating microvilli length or organization (Fig. 4d). Together, these observations support the hypothesis that Fas2 extracellular domains are necessary to stabilize the brush border. endogenous levels of Fas2 (UroGAL4), overexpression of only the extracellular domain (UroGAL44Fas2-Extra) resulted in longer microvilli, comparable to the overexpression phenotype of the full-length transcript (Figs. 3f, 4d). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 However, decreased levels of Fas2 (UroGAL44Fas2-RNAi) resulted in less dense and more disorganized microvilli (Fig. 4a; Supplementary Fig. 4), whereas overexpression of Fas2 (UroGAL44Fas2-EP) frequently gave rise to both larger and longer bundles of microvilli (Fig. 4a; Supplementary Fig. 4). Moreover, there is a significant increase in the distance between individual microvilli when Fas2 is decreased, and a slight, but significant, decrease in intermicrovillar distance when Fas2 levels are increased (Fig. 4b). Taken together these results indicate that endogenous levels of Fas2 are required for the development of microvillar protrusions with stereotypic length and organization. controls (Fig. 3e,f); these findings were reiterated using two separate principal cell-specific GAL4 drivers (Supplementary Fig. 3). Further analysis of the microvillar protrusions indicated that the organization (in addition to the length) of the microvilli also was disrupted on manipulation of Fas2 levels (Fig. 4a,b; Supplementary Fig. 4). Control tubules (Fas2-RNAi/ þ and Fas2- EP/ þ ) have a highly organized structure of microvilli with a tendency for small, well-distributed clusters. However, decreased levels of Fas2 (UroGAL44Fas2-RNAi) resulted in less dense and more disorganized microvilli (Fig. 4a; Supplementary Fig. 4), whereas overexpression of Fas2 (UroGAL44Fas2-EP) frequently gave rise to both larger and longer bundles of microvilli (Fig. 4a; Supplementary Fig. 4). Moreover, there is a significant increase in the distance between individual microvilli when Fas2 is decreased, and a slight, but significant, decrease in intermicrovillar distance when Fas2 levels are increased (Fig. 4b). Taken together these results indicate that endogenous levels of Fas2 are required for the development of microvillar protrusions with stereotypic length and organization. 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 DAPI F-actin DAPI F-actin GFP397 GFPCB03613 Dynamic expression of Fas2 during MT development DAPI F-actin GFP778 Fas2-RA Fas2-RB Fas2-RC 1 kb Fas2 RB RC GFP397 GFPCB03613 GFP778 RA a b GFP397 GFP778 GFPCB03613 c Fas2-RA Fas2-RB Fas2-RC SP Ig FNIII PEST Intracellular Extracellular TM TM GPI re 2 | Fas2 is dynamically expressed during tubule development. (a) Fas2 genomic region illustrating the positions of the GFPCB03613, GFP397 an 78 exon trap insertions. (b) Fas2 is expressed in multiple isoforms that all comprise five immunoglobulin (Ig) and two fibronectin type III (FNIII) ains, yet have distinct C-termini. Arrows indicate splice variants recognized by each exon trap insertion line. The differences between transcripts RA, -RB and -RC are highlighted in a,b using the same colour code. PEST, PESTdomain; SP, signal peptide. (c) In stage 16 embryos, Fas2 localizes to ce ions of the developing MTs; however, Fas2 switches abruptly to the apical brush border prior to eclosion of the first instar larva, where it remain ghout larval development. As the third instar larva pupates, the MTs become transport incompetent, the microvilli shorten, tubule lumen is reduce Fas2 is no longer expressed. As the brush border reform in the adult tubule, Fas2 is again abundantly expressed in the MT, where it localizes to th l brush border (area between arrows). This expression pattern is consistently reported by each fusion protein, and is commensurate with the cription profile of each Fas2 isoform. Inserts: single optical section of the selected region shown as separate (small circle) and merged (large circl ls Scale bars 25 mm DAPI F-actin DAPI F-actin GFP397 GFPCB03613 ent DAPI F-actin GFP778 Fas2-RA Fas2-RB Fas2-RC 1 kb Fas2 RB RC GFP397 GFPCB03613 GFP778 RA a b GFP397 GFP778 GFPCB03613 c Fas2-RA Fas2-RB Fas2-RC SP Ig FNIII PEST Intracellular Extracellular TM TM GPI DAPI F-actin DAPI F-actin GFP397 GFPCB03613 Dynamic expression of Fas2 during MT development DAPI F-actin GFP778 Fas2-RA Fas2-RB Fas2-RC 1 kb Fas2 RB RC GFP397 GFPCB03613 GFP778 RA a b GFP397 GFP778 GFPCB03613 c Fas2-RA Fas2-RB Fas2-RC SP Ig FNIII PEST Intracellular Extracellular TM TM GPI Figure 2 | Fas2 is dynamically expressed during tubule development. (a) Fas2 genomic region illustrating the positions of the GFPCB03613, GF GFP778 exon trap insertions. (b) Fas2 is expressed in multiple isoforms that all comprise five immunoglobulin (Ig) and two fibronectin type III domains, yet have distinct C-termini. NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 (a) Super-resolution confocal microscopy (Airyscan) on MT from Fas2–GFP778 stained with anti-GFP, suggests that Fas2 localizes to the brush border, where it appears concentrated distally. Zoom: single optical section of the indicated region shown as separate and merged signals. Arrows indicate base (solid) and tip (line) orientations of the brush border. PC, principal cell; SC, stellate cell. Scale bar, 25 mm. (b) Mean normalized fluorescent intensity profiles for F-actin and Fas2 signals from N ¼ 12 brush border regions confirm that Fas2 is concentrated at the distal tip of the F-actin-based protrusions. (c) Magnification of the white square in ‘a zoom’, and a perpendicular view (‘90’) on the brush border, suggests that Fas2 does not strictly colocalize with intracellular F-actin (phalloidin), but is positioned extracellularly between individual microvilli. Scale bar, 0.5 mm. (d) Inverse coloured optical sections of Alexa-488-phalloidin (black) stained MTs from Fas2 knockdown flies (Fas2-RNAi and Fas2EB112) showed a marked decrease in length and density of the microvillar brush border compared with WT (Canton S) tubules. Conversely, overexpression of Fas2 (Fas2-EP) produced notably longer and denser microvilli. Scale bars, 15 mm. (e) Scanning electron microscopy (SEM) analysis of MT cross-sections (brush border pseudo-coloured in cyan) from adult Drosophila using the principal cell-specific UroGAL4 driver to drive both RNAi and overexpressor constructs confirmed these results. Individual microvilli were measured (see inserts) from (N ¼ 10–13) cross-sections with (N ¼ 400–520) microvilli measured in total for each Fas2 genetic background. Scale bars, 20 mm. (f,g) Tukey box and whisker plots of microvilli length from the different Fas2 genetic backgrounds. Genetic manipulations of Fas2 expression levels significantly reduced or increased (*, one-way ANOVA, Po0.05) microvilli length compared with parentals or WT. Solid squares indicate mean values; open circles symbolize data outliers. Fas2 levels allows direct testing of this model; if the longer microvilli in Fas2 overexpressors are physiologically functional, these tubules should be able to secrete fluid at a higher rate. This can be quantitatively tested in Drosophila, by measuring fluid secretion rates12 both at rest and under stimulation. Tubules actively transport cyclic nucleotides, and so can be stimulated by addition of extracellular cyclic AMP (cAMP)12,31. Accordingly, secretion rates in control tubules are clearly elevated on cAMP addition (Fig. 5a–d). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 However, overexpression of the extracellular domain (UroGAL44Fas2-Extra) also resulted in areas of the brush border, demonstrating aberrant microvilli organization compared with parental controls (UroGAL4; Fig. 4c), which may suggest that the GPI anchor of the full-length protein is important for controlling how the brush border is organized. In contrast, when only the intracellular domain is overexpressed (UroGAL44Fas2-Intra), the microvilli are indistinguishable from those from control tubules (Fig. 4c), indicating that the intracellular domain, of the less abundant Microvilli length correlates with MT transport capacity. Microvilli are classically observed on the apical surface of transporting epithelia, where they are thought to increase the membrane area available for transmembrane transport27,28. Consistent with this, when microvilli are compromised, for example, in coeliac disease or in Usher syndrome, malabsorption and/or renal tubular dysfunction results29,30. Manipulation of NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 Fas2EB112 UroGal4>Fas2-RNAi UroGal4>Fas2EP Canton S 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 F-actin DAPI Fas2778 SC PC Zoom F-actin Fas2778 Merge Zoom Zoom F-actin 1.0 0.8 0.6 0.4 0.2 0.0 0.0 2.5 1.0 Fas2778 Merge Fas2778 F-actin Zoom Zoom 90° 90° 90° Zoom 2.0 1.5 0.5 f d e 1.53 µm 2.17 µm g UroGal4>Fas2-RNAi UroGal4>Fas2EP Fas2EP/+ Fas2-RNAi/+ UroGal4/+ Microvilli length (µm) * * N =520 Fas2EB112 Canton S N =400 a F-actin b c x y x z Base Tip Normalized distance Normalized intensity 1.13 µm 1.15 µm * Figure 3 | Genetic manipulation of Fas2 impacts microvilli length. (a) Super-resolution confocal microscopy (Airyscan) on MT from Fas2–GFP778 stained with anti-GFP, suggests that Fas2 localizes to the brush border, where it appears concentrated distally. Zoom: single optical section of the indicated region shown as separate and merged signals. Arrows indicate base (solid) and tip (line) orientations of the brush border. PC, principal cell; SC, stellate cell. Scale bar, 25 mm. (b) Mean normalized fluorescent intensity profiles for F-actin and Fas2 signals from N ¼ 12 brush border regions confirm that Fas2 is concentrated at the distal tip of the F-actin-based protrusions. (c) Magnification of the white square in ‘a zoom’, and a perpendicular view (‘90’) on the brush border, suggests that Fas2 does not strictly colocalize with intracellular F-actin (phalloidin), but is positioned extracellularly between individual microvilli. Scale bar, 0.5 mm. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 (d) Inverse coloured optical sections of Alexa-488-phalloidin (black) stained MTs from Fas2 knockdown flies (Fas2-RNAi and Fas2EB112) showed a marked decrease in length and density of the microvillar brush border compared with WT (Canton S) tubules. Conversely, overexpression of Fas2 (Fas2-EP) produced notably longer and denser microvilli. Scale bars, 15 mm. (e) Scanning electron microscopy (SEM) analysis of MT cross-sections (brush border pseudo-coloured in cyan) from adult Drosophila using the principal cell-specific UroGAL4 driver to drive both RNAi and overexpressor constructs confirmed these results. Individual microvilli were measured (see inserts) from (N ¼ 10–13) cross-sections with (N ¼ 400–520) microvilli measured in total for each Fas2 genetic background. Scale bars, 20 mm. (f,g) Tukey box and whisker plots of microvilli length from the different Fas2 genetic backgrounds. Genetic manipulations of Fas2 expression levels significantly reduced or increased (*, one-way ANOVA, Po0.05) microvilli length compared with parentals or WT. Solid squares indicate mean values; open circles symbolize data outliers. | Fas2EB112 UroGal4>Fas2-RNAi UroGal4>Fas2EP Canton S 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 F-actin DAPI Fas2778 SC PC Zoom F-actin Fas2778 Merge Zoom Zoom F-actin 1.0 0.8 0.6 0.4 0.2 0.0 0.0 2.5 1.0 Fas2778 Merge Fas2778 F-actin Zoom Zoom 90° 90° 90° Zoom 2.0 1.5 0.5 f d e 1.53 µm 2.17 µm g UroGal4>Fas2-RNAi UroGal4>Fas2EP Fas2EP/+ Fas2-RNAi/+ UroGal4/+ Microvilli length (µm) * * N =520 Fas2EB112 Canton S N =400 a F-actin b c x y x z Base Tip Normalized distance Normalized intensity 1.13 µm 1.15 µm * 778 d F-actin DAPI Fas2778 SC PC Zoom F-actin Fas2778 Merge Zoom Zoom a a Fas2EB112 UroGal4>Fas2-RNAi UroGal4>Fas2EP Canton S d e 1.53 µm 2.17 µm F-actin 1.13 µm 1.15 µm F-actin e e F-actin 1.0 0.8 0.6 0.4 0.2 0.0 0.0 2.5 1.0 Fas2778 2.0 1.5 0.5 b Base Tip Normalized distance Normalized intensity b 0.0 0.5 1.0 1.5 2.0 2.5 3.0 f UroGal4>Fas2-RNAi UroGal4>Fas2EP Fas2EP/+ Fas2-RNAi/+ UroGal4/+ Microvilli length (µm) * * N =520 0.0 0.5 1.0 1.5 2.0 2.5 3.0 g Fas2EB112 Canton S N =400 * f g Microvilli length (µm) Normalized distance Merge Fas2778 F-actin Zoom Zoom 90° 90° 90° Zoom c x y x z c Figure 3 | Genetic manipulation of Fas2 impacts microvilli length. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 Although basal secretion rates are unaffected by manipulation of Fas2 expression—the utility of the increased membrane area is not exploited unless the tissue is stimulated— knockdown of Fas2 (red) resulted in a significantly reduced (*, one-way ANOVA, Po0.05) stimulated rate of secretion (Fig. 5a). Conversely, overexpression of either the full-length (green) or extracellular domain (blue) of Fas2 significantly increased (*, one-way ANOVA, Po0.05) the stimulated response (Fig. 5b–d); the apparent discrepancy in brush border organization between overexpressing the full-length and the extracellular domain, respectively (Fig. 4a,c), likely explains the observed difference in the maximal performances of the tubules from the two genotypes (Fig. 5b–d). By contrast, overexpression of just the intracellular domain (magenta) had no effect (Fig. 5c). Together, these results indicate that the transport capacity of the tissue is proportional to Fas2-induced changes to brush border size, and that the increased membrane area is functionally significant. NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications Discussion Intermicrovill distance was measured (see inserts) from (N ¼ 6–10) cross-sections with (N ¼ 90–150) distances measured in total for each Fas2 genetic backgro Solid squares indicate mean values; open circles symbolize data outliers. (c) Independent overexpression of the Fas2 extracellular domain (Fas2-Extra results in a phenotype comparable to overexpressing the full-length transcript (although areas of the brush border showed an excess of adhesion-a compared with parental controls), whereas overexpression of the Fas2 intracellular domain (Fas2-Intra–YFP) results in microvilli being indistinguis from controls. Top tier images represent face-on views, whereas bottom tier images are cross-sectional views of the brush border from the various ge backgrounds. SC, stellate cell (yellow). Scale bars, 5 mm and 1 mm, respectively. (d) Tukey box and whisker plot of microvilli length from the select overexpression of the Fas2 extracellular and intracellular domains, respectively. Overexpression of the extracellular domain significantly changes (*, one-way ANOVA, Po0.05) the average microvillar length compared with overexpression of the intracellular domain and parental controls. Indi microvilli were measured from (N ¼ 5–10) cross-sections with (N ¼ 90–520) microvilli measured in total for each Fas2 genetic background. Solid sq indicate mean values; open circles symbolize data outliers (e) Schematic diagram of native full length Fas2 RA (PEST ) and the YFP tagged Fas2 Intr c UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4 SC b Intermicrovillar distance (µm) UroGal4>Fas2-RNAi Fas2-RNAi/+ Fas2-EP/+ UroGal4>Fas2EP 0.0 0.1 0.2 0.3 0.4 0.5 0.6 * N = 90–150 * 0.24 µm 0.38 µm 0.23 µm 0.16 µm d 1.0 1.5 2.0 2.5 Microvilli length (µm) 3.0 * 3.5 UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4/+ 0.0 0.5 N = 90–520 e Fas2-Intra Fas2-Extra Intracellular Extracellular SP Ig FNIII YFP Fas2-RA (PEST-) b Intermicrovillar distance (µm) 0.1 0.2 0.3 0.4 0.5 0.6 * N = 90–150 * b c UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4 SC c d d 1.0 1.5 2.0 2.5 Microvilli length (µm) 3.0 * 3.5 UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4/+ 0.0 0.5 N = 90–520 Fas2-Intra Fas2-Extra Intracellular Extracellular SP Ig FNIII YFP Fas2-RA (PEST-) e Extracellular Microvilli length (µm) Figure 4 | Genetic manipulation of Fas2 impacts microvilli organization. (a) Scanning electron microscopy (SEM) analysis of microvillar organization. Microvilli (pseudo-coloured light brown) from parentals (Fas2-RNAi/ þ and Fas2-EP/ þ ) show distinct well-organized bundles of microvilli, whereas in Fas2 knockdown flies (UroGAL44Fas2-RNAi) the microvilli tend to appear as single distinct protrusions. Overexpressing Fas2 makes the bundles more obvious, and the microvilli often appearing more densely packed (UroGAL44Fas2-EP). Discussion How microvillar protrusions are assembled and maintained in different epithelial tissues is still largely unknown. In this study, we show that the cell adhesion molecule Fas2 is dynamically 5 ARTICLE c UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4 SC a UroGal4>Fas2-RNAi Fas2-RNAi/+ UroGal4>Fas2EP Fas2-EP/+ b Intermicrovillar distance (µm) UroGal4>Fas2-RNAi Fas2-RNAi/+ Fas2-EP/+ UroGal4>Fas2EP 0.0 0.1 0.2 0.3 0.4 0.5 0.6 * N = 90–150 * 0.24 µm 0.38 µm 0.23 µm 0.16 µm d 1.0 1.5 2.0 2.5 Microvilli length (µm) 3.0 * 3.5 UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4/+ 0.0 0.5 N = 90–520 e Fas2-Intra Fas2-Extra Intracellular Extracellular SP Ig FNIII YFP Fas2-RA (PEST-) Figure 4 | Genetic manipulation of Fas2 impacts microvilli organization. (a) Scanning electron microscopy (SEM) analysis of microvillar organ Microvilli (pseudo-coloured light brown) from parentals (Fas2-RNAi/ þ and Fas2-EP/ þ ) show distinct well-organized bundles of microvilli, whe F 2 k kd fli (U GAL4 F 2 RNAi) h i illi d i l di i i O i F 2 k h b dl ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomm NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 a UroGal4>Fas2-RNAi Fas2-RNAi/+ UroGal4>Fas2EP Fas2-EP/+ 0.24 µm 0.38 µm 0.23 µm 0.16 µm a c UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4 SC a UroGal4>Fas2-RNAi Fas2-RNAi/+ UroGal4>Fas2EP Fas2-EP/+ b Intermicrovillar distance (µm) UroGal4>Fas2-RNAi Fas2-RNAi/+ Fas2-EP/+ UroGal4>Fas2EP 0.0 0.1 0.2 0.3 0.4 0.5 0.6 * N = 90–150 * 0.24 µm 0.38 µm 0.23 µm 0.16 µm d 1.0 1.5 2.0 2.5 Microvilli length (µm) 3.0 * 3.5 UroGal4>Fas2-Intra UroGal4>Fas2-Extra UroGal4/+ 0.0 0.5 N = 90–520 e Fas2-Intra Fas2-Extra Intracellular Extracellular SP Ig FNIII YFP Fas2-RA (PEST-) Figure 4 | Genetic manipulation of Fas2 impacts microvilli organization. (a) Scanning electron microscopy (SEM) analysis of microvillar organiz Microvilli (pseudo-coloured light brown) from parentals (Fas2-RNAi/ þ and Fas2-EP/ þ ) show distinct well-organized bundles of microvilli, where Fas2 knockdown flies (UroGAL44Fas2-RNAi) the microvilli tend to appear as single distinct protrusions. Overexpressing Fas2 makes the bundles obvious, and the microvilli often appearing more densely packed (UroGAL44Fas2-EP). Inter-microvilli distances were measured (see inserts) from (N ¼ 6–10) cross-sections with (N ¼ 90–150) microvilli measured in total for each Fas2 genetic background. Scale bars, 1 mm. (b) Tukey box and w plot of intermicrovillar distances from the different fas2 genetic backgrounds. Genetic manipulations of Fas2 expression levels significantly change (*, one-way ANOVA, Po0.05) the average distance between the individual microvillar protrusions compared with parental controls. NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 (c) Overexpression of only the Fas2 intracellular domain (magenta) has no significant effect on the cAMP-induced secretion rates (NS, one-way ANOVA, Po0.05), while (d) overexpression of the extracellular domain alone (blue) significantly increased (*, one-way ANOVA, Po0.05) stimulated fluid secretion rates compared with parental lines. Arrows indicate point of cAMP (10  4 M) addition. Data are expressed as mean±s.e.m. of N ¼ 6–7. universal role in regulating the assembly and maintenance of actin-based cellular protrusions. Furthermore, the apparent diversity of cell adhesion molecules recruited to perform seemingly identical tasks in different epithelia (for example, Cad99C in Drosophila follicle cells34,35 and Fas2 in Drosophila tubule cells) indicates that different adhesion molecules likely help define structural properties specific to individual tissues. extends. As such, stabilization of the plasma membrane envelope by intermicrovillar links might help alleviate some of this tension, and thus promote actin-core filament growth and microvilli extension34. Alternatively, the length of microvilli protrusion in insect MTs could be physically constrained by the shear stress caused by high luminal flux rates, in which case a scaffold composed of Fas2-homotypic links might help drive actin-core assembly/disassembly dynamics towards microvilli elongation. Both models—which are not mutually exclusive—could explain the correlation between microvilli length and organization and Fas2 expression levels. Whether additional factors are involved in regulating this mechanism, for example, targeting Fas2 to the brush border, anchoring Fas2 to the actin core, or regulating Fas2 adhesive activity—as demonstrated for other ‘scaffolding’ proteins30–36—remains unknown. In Drosophila follicle cells34,35, as well as in the vertebrate inner ear and intestinal epithelia29,32, members of the cadherin superfamily of cell adhesion molecules have been shown to contribute to heterophilic adhesion complexes between adjacent F-actin-based protrusions. In contrast, Fas2 has been demonstrated to be capable of forming homophilic complexes in both in vitro5 and in vivo7 studies (Fig. 6b). As our data suggest Fas2 is positioned extracellularly between individual F-actin bundles (Fig. 3a–c), it seems highly likely that Fas2 is involved in forming homotypic intermicrovillar links in the Drosophila MT (Fig. 6c). Through genetic dissection of Fas2 function, we further found that membrane-anchored Fas2 ectodomains are sufficient to mediate the observed changes in brush border length and integrity, and that the cytoplasmic domain of the less abundant Fas2-RA isoform is dispensable (Fig. 4). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 ARTICLE UroGAL4>Fas2EP UroGAL4>Fas2-Extra-YFP UroGAL4>Fas2-RNAi UroGAL4>Fas2-Intra-YFP UroGAL4/+ UroGAL4/+ UroGAL4/+ UroGAL4/+ a Secretion rate (nl min–1) % change in secretion 100 200 300 400 500 * 0 Secretion rate (nl min–1) 1 2 3 4 5 Fas2EP/+ 0 0 20 40 60 0.0 0.5 1.0 1.5 0 20 40 60 Fas2-RNAi/+ % change in secretion 50 100 150 200 * 0 Secretion rate (nl min–1) Secretion rate (nl min–1) 0.0 0.5 1.0 1.5 0 20 40 60 Fas2-Extra-YFP/+ % change in secretion * 50 100 150 200 0 250 Time (Min) Time (Min) c 20 40 60 0 0.0 0.5 1.0 1.5 Fas2-Intra-YFP/+ % change in secretion 50 100 150 0 b d NS Figure 5 | Genetic manipulation of Fas2 impacts renal function. (a) Knock down (red) of Fas2 results in a significant reduction, while (b) overexpression (green) of Fas2 causes a significant increase, in cAMP-stimulated rates of fluid secretion compared with parental controls (*, one-way ANOVA, Po0.05). (c) Overexpression of only the Fas2 intracellular domain (magenta) has no significant effect on the cAMP-induced secretion rates (NS, one-way ANOVA, Po0.05), while (d) overexpression of the extracellular domain alone (blue) significantly increased (*, one-way ANOVA, Po0.05) stimulated fluid secretion rates compared with parental lines. Arrows indicate point of cAMP (10  4 M) addition. Data are expressed as mean±s.e.m. of N ¼ 6–7. UroGAL4>Fas2-RNAi UroGAL4/+ a Secretion rate (nl min–1) 0.0 0.5 1.0 1.5 0 20 40 60 Fas2-RNAi/+ % change in secretion 50 100 150 200 * 0 UroGAL4>Fas2EP UroGAL4/+ % change in secretion 100 200 300 400 500 * 0 Secretion rate (nl min–1) 1 2 3 4 5 Fas2EP/+ 0 0 20 40 60 b b a UroGAL4>Fas2-Intra-YFP UroGAL4/+ Secretion rate (nl min–1) Time (Min) c 20 40 60 0 0.0 0.5 1.0 1.5 Fas2-Intra-YFP/+ % change in secretion 50 100 150 0 NS UroGAL4>Fas2-Extra-YFP UroGAL4/+ Secretion rate (nl min–1) 0.0 0.5 1.0 1.5 0 20 40 60 Fas2-Extra-YFP/+ % change in secretion * 50 100 150 200 0 250 Time (Min) d d Time (Min) Figure 5 | Genetic manipulation of Fas2 impacts renal function. (a) Knock down (red) of Fas2 results in a significant reduction, while (b) overexpression (green) of Fas2 causes a significant increase, in cAMP-stimulated rates of fluid secretion compared with parental controls (*, one-way ANOVA, Po0.05). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 These observations are strongly mirrored by the molecular function of Cad99C in Drosophila follicle cells34,35; a protein that is also known to interact with the unconventional motor protein Myosin VIIA (Myo7A), to further control the length and organization of microvilli36. Interestingly, both Cad99C and Myo7A, according to flyatlas.org2, are most highly enriched in larval and adult MTs, respectively, indicating that these proteins might similarly play a role in brush border assembly and/or maintenance in Drosophila renal tubules. Yet, irrespective of the binding partners involved, how might adhesion complexes control microvilli length? The lengthening or shortening of microvilli actin-core filaments depends on the assembly versus disassembly rates of actin polymerization, through a process called treadmilling37. According to this model, the force created by net-positive treadmilling rates has to overcome the opposite force generated by tension of the plasma membrane as the actin-based protrusion p Based on our collective findings, we propose a model for Fas2 function, in which Fas2 homophilic binding between adjacent microvilli forms intermicrovillar links, which help stabilize the brush border against shear stress (Fig. 6c). As argued, this model is both a logical extension of what is known about the Fas2 cell adhesion properties5,7 and insect renal tubule physiology18, and is furthermore consistent with the concentration-dependent effect observed between Fas2 activity on microvilli length and organization (Figs 3 and 4). Our study thus provides a radically new view on the function, outside the CNS, of one of the best- known neural development molecules, and moreover suggests that while actin involvement is known to be critical for microvillar formation, the molecular machinery responsible for brush border assembly and maintenance in different epithelial systems is far from understood. Discussion Inter-microvilli distances were measured (see inserts) from (N ¼ 6–10) cross-sections with (N ¼ 90–150) microvilli measured in total for each Fas2 genetic background. Scale bars, 1 mm. (b) Tukey box and whisker plot of intermicrovillar distances from the different fas2 genetic backgrounds. Genetic manipulations of Fas2 expression levels significantly change (*, one-way ANOVA, Po0.05) the average distance between the individual microvillar protrusions compared with parental controls. Intermicrovillar distance was measured (see inserts) from (N ¼ 6–10) cross-sections with (N ¼ 90–150) distances measured in total for each Fas2 genetic background. Solid squares indicate mean values; open circles symbolize data outliers. (c) Independent overexpression of the Fas2 extracellular domain (Fas2-Extra–YFP) results in a phenotype comparable to overexpressing the full-length transcript (although areas of the brush border showed an excess of adhesion-activity compared with parental controls), whereas overexpression of the Fas2 intracellular domain (Fas2-Intra–YFP) results in microvilli being indistinguishable from controls. Top tier images represent face-on views, whereas bottom tier images are cross-sectional views of the brush border from the various genetic backgrounds. SC, stellate cell (yellow). Scale bars, 5 mm and 1 mm, respectively. (d) Tukey box and whisker plot of microvilli length from the selective overexpression of the Fas2 extracellular and intracellular domains, respectively. Overexpression of the extracellular domain significantly changes (*, one-way ANOVA, Po0.05) the average microvillar length compared with overexpression of the intracellular domain and parental controls. Individual microvilli were measured from (N ¼ 5–10) cross-sections with (N ¼ 90–520) microvilli measured in total for each Fas2 genetic background. Solid squares indicate mean values; open circles symbolize data outliers. (e) Schematic diagram of native full-length Fas2-RA (PEST-) and the YFP-tagged Fas2-Intra and Fas2-Extra constructs (adapted from ref. 7). expressed during MT development—localizing to the brush border of transport competent renal tubules (Fig. 6a)—and demonstrate that tissue-specific genetic manipulation of Fas2 expression levels elicits concentration-dependent effects on microvillus length and brush border organization (Figs 3 and 4). Similar observations have been made on other cell adhesion molecules in both vertebrate32,33 and invertebrate34,35 epithelial systems, which suggests that cell adhesion molecules likely play a 6 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 Fly strains. All fly lines were cultured on standard medium over a 12:12 h photoperiod at 45–55% humidity at 22 C. Ectopic gene expression using the GAL4/UAS system was carried out at 26 C. The Fas2 exon trap insertions Fas2GFP397 and Fas2GFP778 (this study) were isolated in exon trap screens21, while Fas2GFPCB03613 was obtained from the Spradling lab38. The UASFas2-Intra–YFP and UASFas2-Extra–YFP lines were a kind gift from the Nose lab7, whereas the Fas2EB112 hypomorph was obtained from the Goodman lab5. The Fas2 overexpressor Fas2EP39 was obtained from the Bloomington Stock Center, and the NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications Methods Fl t i The lower aqueous phase was then gently removed, and 5 ml of a fresh solution of 9:1 MeOH (100%) and 50 mM EGTA (pH 8) was added, and the sample gently shaken for 3–5 min. Devitellinized embryos, which appeared in the MeOH/EGTA phase, were collected and placed in PBS before being mounted and imaged. In contrast, acutely dissected MTs from both larvae and adults, were fixed in 4% paraformaldehyde in PBS for 12 min, and incubated in PBS with 10% NGS and 0.1% Triton X-100 (PBT) and rabbit-anti- green fluorescent protein (GFP) conjugated to Alexa Fluor 488 (1:200; Molecular Probes, OR, USA, cat# A21311) where necessary. Alternatively, fixed tissues were incubated overnight in PBT with either mouse anti-Fas2 1D4 (1:80; DSHB, deposited by Goodman) or 34B3 (1:40; DSHB). Following several washes in PBT, the tissues were then incubated with Alexa Fluor 488-conjugated goat-anti-mouse secondary antibody (1:200; Thermo Fisher Scientific, MA, USA, cat# A-10667), DAPI (40,6-diamidino-2-phenylindole, 1 mg ml  1; Thermo Fisher Scientific, cat# D1306) and Rhodamine-coupled phalloidin (1:100; Thermo Fisher Scientific, cat# R415) in PBT for a minimum of 2 h. Next, samples were washed repeatedly in PBS before being mounted on poly-L-Lysine (0.1% w/v in H2O, Sigma-Aldrich, MO, USA) covered 35 mm glass bottom dishes (MatTek Corporation, MA, USA) in Vectashield (Vector Laboratories Inc, CA, USA) and image acquisition was performed on an inverted Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany). Quantitative RT–PCR. Fas2 expression levels were verified using qPCR. A two-step qPCR was carried out using the fluorescent double-stranded DNA dye DyNAmo SYBR Green (Finnzymes, Finland). Prior to carrying out these experiments, complementary DNA was synthesized from the tissue of interest using SuperScript IV (Invitrogen, CA, USA). For each experiment, four biological replicates were generated, and three technical replicates were loaded for each. Primers were designed to produce products o500 bp, and where possible, to span exon/intron boundaries of the gene of interest. Primers were also designed against the housekeeping gene alpha-tubulin, to normalize samples (forward: 50-AGG GTATGGAGGAGGGAGAGTTC-30; reverse: 50-TGCGATTGGAAGCGTAAA CAC-30). Template amplicons were generated for each primer pair, and standards ranging from 10  1 to 10  7 ng were created by serial dilution, allowing for absolute quantification of gene expression. Cycling was performed in Opticon 3 thermal cycler (BioRad, UK). Following amplification, Opticon 3 software was used to generate a standard curve. Methods Fl t i Growth cone Axon b – – – + + + + Fas2-RA interaction 0 0 18 90 16 24 4 6 8 10 12 14 2 Minutes Hours Days a 0.0 0.5 1.0 MT transport capacity Pupa Adult Fas2 localization Larva Embryo Brush border Junctions b a a MT c Lumen MV length Fas2 expression Transport capacity Fas2-RB interaction c MV length Fas2 expression Transport capacity Figure 6 | Hypothesis for Fas2 function in the Drosophila MT. (a) Schematic representation of the spatio-temporal expression pattern of Fas2 (green) during tubule development. Fas2 does not localize to the microvilli when the tubules are transport incompetent, but are abruptly recruited to the microvillar brush border when the tissue develops transport competence (blue). (b) Schematic representation of how Fas2 is proposed to provide attractive forces through homophilic adhesion on the axon relative to the environment that promotes axon fasciculation and growth cone guidance (based on ref. 11). (c) Proposed model for Fas2 function in the adult Drosophila MT. By contrast to the Drosophila nervous system and neuromuscular junction, where Fas2 mediates intercellular interactions, we hypothesize that Fas2 acts as homotypic bridging protein within the same cell of the renal tubule, where it stabilizes the microvillar brush border against shear stress caused by uniquely high flux rates. Knockdown of Fas2 results in shorter and disorganized microvilli with the opposite being the case when Fas2 is overexpressed. Fas2RNAi line was acquired from the Vienna Drosophila RNAi Centre (VDRC). The UroGAL4 (ref. 40) and CapaRGAL4 (ref. 41) drivers, both specific to principal cells of MTs, were developed in-house and used to drive all constructs. Fas2RNAi line was acquired from the Vienna Drosophila RNAi Centre (VDRC). The UroGAL4 (ref. 40) and CapaRGAL4 (ref. 41) drivers, both specific to principal cells of MTs, were developed in-house and used to drive all constructs. Immunocytochemistry. For embryo collection, adult females were allowed to lay eggs on grape juice agar plates for B16–24 h at 26 C. Embryos were next collected and dechorionated in a fresh solution of 50:50 bleach (10%) and ddH2O for exactly 3 min, and then washed thoroughly with ddH2O. Using a fine brush, the embryos were subsequently transferred to 5 ml of heptane (embryos should sink), and 5 ml of 4% PFA in PBS was added, before shaking the liquid to saturate the mixture. Samples were left for 30 min on a shaker to fix. Methods Fl t i Fly strains. All fly lines were cultured on standard medium over a 12:12 h photoperiod at 45–55% humidity at 22 C. Ectopic gene expression using the GAL4/UAS system was carried out at 26 C. The Fas2 exon trap insertions Fas2GFP397 and Fas2GFP778 (this study) were isolated in exon trap screens21, while Fas2GFPCB03613 was obtained from the Spradling lab38. The UASFas2-Intra–YFP and UASFas2-Extra–YFP lines were a kind gift from the Nose lab7, whereas the Fas2EB112 hypomorph was obtained from the Goodman lab5. The Fas2 overexpressor Fas2EP39 was obtained from the Bloomington Stock Center, and the URE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 MT c Lumen 0 0 18 90 16 24 4 6 8 10 12 14 2 Minutes Hours Days a MV length Fas2 expression Transport capacity Fas2-RB interaction Growth cone Axon b – – – + + + + Fas2-RA interaction 0.0 0.5 1.0 MT transport capacity Pupa Adult Fas2 localization Larva Embryo Brush border Junctions Figure 6 | Hypothesis for Fas2 function in the Drosophila MT. (a) Schematic representation of the spatio-temporal expression pattern of Fas2 (green) during tubule development. Fas2 does not localize to the microvilli when the tubules are transport incompetent, but are abruptly recruited to the microvillar brush border when the tissue develops transport competence (blue). (b) Schematic representation of how Fas2 is proposed to provide attractive forces through homophilic adhesion on the axon relative to the environment that promotes axon fasciculation and growth cone guidance (based on ref. 11). (c) Proposed model for Fas2 function in the adult Drosophila MT. By contrast to the Drosophila nervous system and neuromuscular junction, where Fas2 mediates intercellular interactions, we hypothesize that Fas2 acts as homotypic bridging protein within the same cell of the renal tubule, where t stabilizes the microvillar brush border against shear stress caused by uniquely high flux rates. Knockdown of Fas2 results in shorter and disorganized microvilli with the opposite being the case when Fas2 is overexpressed. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11266 in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 90 min15,44. The tissue was then rinsed repeatedly in ddH2O before being dehydrated through a graded ethanol series, and desiccated using an Autosamdri-815 critical point dryer (Tousimis Research Corporation, Maryland, USA). The tubules were then transferred to aluminium stubs, fractioned and coated with platinum (70 s B12 nm thickness) in a JEOL JFC-2300HR high-resolution fine coater (Jeol, Tokyo, Japan) and examined with a JEOL JSM-6335-F scanning electron microscope (Jeol). Quantification of microvilli length (mm) and intermicrovillar distance (mm) was done post image acquisition for each relevant genotype. Length was measured from base to tip of 40 microvilli from each cross-section, with 10–13 cross-sections providing the mean microvilli length for each genotype. The distance between individual microvilli was measured from the centre of each adjacent microvillus with minimum 15 intermicrovillar distances measured per cross-section and a total of 6–10 cross-sections from each genotype. All measurements were performed exclusively on microvilli in which these metrics were clearly discernable (see Fig. 3e, inserts; Fig. 4a, inserts) and only on principal ‘type I’ cells of the main segment22. 8. Mao, Y. & Freeman, M. Fasciclin 2, the Drosophila orthologue of neural cell-adhesion molecule, inhibits EGF receptor signalling. Development 136, 473–481 (2009). 9. Harrelson, A. L. & Goodman, C. S. Growth cone guidance in insects: fasciclin II is a member of the immunoglobulin superfamily. Science 242, 700–708 (1988). 10. Garcia-Alonso, L., VanBerkum, M. F., Grenningloh, G., Schuster, C. & Goodman, C. S. Fasciclin II controls proneural gene expression in Drosophil Proc. Natl Acad. Sci. USA 92, 10501–10505 (1995). 11. Araujo, S. J. & Tear, G. Axon guidance mechanisms and molecules: lessons from invertebrates. Nat. Rev. Neurosci. 4, 910–922 (2003). , ( ) 12. Dow, J. A. T. et al. The Malpighian tubules of Drosophila melanogaster: a novel 12. Dow, J. A. T. et al. The Malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control. J. Exp. Biol. 197, 421–428 (1994). 13. Terhzaz, S. et al. Isolation and characterization of a leucokinin-like peptide of Drosophila melanogaster. J. Exp. Biol. 202, 3667–3676 (1999). 14. Cabrero, P. et al. Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Proc. Natl Acad. Sci. USA 111, 14301–14306 (2014). Ramsay fluid secretion assay. ARTICLE Intact MTs were dissected from native tissue and set up as in vitro preparations by isolating them in drops of a freshly prepared mixture of Schneider’s medium and insect saline (1:1, v/v) under water-saturated liquid paraffin, with one end being wrapped around a metal pin and the other end submerged in the saline drop12,15. Secreted fluid would then accumulate as a discrete droplet from the common ureter. A drop of secreted fluid was subsequently collected at appropriate intervals, and the diameter measured using an eyepiece graticule. The volume of each droplet was calculated as (4/3)pr3, where r is the radius of the droplet, and secretion rates plotted against time. Secretion was measured under basal conditions to establish a steady rate of secretion, prior to stimulation with 10  4 M cAMP. Per cent change from basal rates of fluid secretion immediately prior to cAMP application, compared with 30 min after stimulation, was statistically tested between the parentals and the different Fas2 genetic backgrounds. 15. Halberg, K. A. et al. Tracing the evolutionary origins of insect renal function. Nat. Commun. 6, 6800 doi: 10.1038/ncomms7800 (2015). 16. Du, J. et al. The SzA mutations of the B subunit of the Drosophila vacuolar H þ ATPase identify conserved residues essential for function in fly and yeast. J. Cell Sci. 119, 2542–2551 (2006). 17. Overend, G. et al. A comprehensive transcriptomic view of renal function in the malaria vector, Anopheles gambiae. Insect Biochem. Mol. Biol. 67, 47–58 (2015). 18. Maddrell, S. H. P. The fastest fluid-secreting cell known: the upper Malpighian tubule cell of Rhodnius. Bioessays 13, 357–362 (1991). 19. Campbell, K., Knust, E. & Skaer, H. Crumbs stabilises epithelial polarity during tissue remodelling. J. Cell Sci. 122, 2604–2612 (2009). 20. Morin, X., Daneman, R., Zavortink, M. & Chia, W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl Acad. Sci. USA 98, 15050–15055 (2001). 21. Silies, M. & Kla¨mbt, C. APC/C(Fzr/Cdh1)-dependent regulation of cell adhesion controls glial migration in the Drosophila PNS. Nat. Neurosci. 13, 1357–1364 (2010). Western blotting. For each genetic background, MTs from 470 flies were dissected under Schneider’s medium and subsequently transferred to 100 ml RIPA buffer (150 mM NaCl, 10 mM TrisHCL ph 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) with HALT protease inhibitor (Thermo Fisher Scientific). Samples were homogenized using a Microson XL2000 sonicator (Misonix Inc. ARTICLE NY, USA), and ultra centrifuged (17,000g) at 4 C for 5 min, before transferring the supernatant to fresh tubes. Protein contents were measured using the Bradford assay (Precision Red, Cytoskeleton). Approximately 15 mg protein from each sample was electro- phoresed on a NuPage 4–12% Bis-Tris gel (Invitrogen), and blotted onto nitro- cellulose using the Novex system (Invitrogen). Blots were probed with monoclonal anti-Fas2 34B3 (1:500) and anti-GAPDH (1:1,000, Abcam, UK, cat# ab125247) antibodies, and developed with DyLight800-conjugated goat anti-mouse IgG (1:10,000, Thermo Fisher Scientific, cat# SA5-35521) and Alexa Fluor 680-con- jugated goat anti-rabbit IgG (1:10,000, Thermo Fisher Scientific, cat# A-21109) secondary antibodies, using a Li-Cor CLx Odyssey. Fas2 expression was quantified relative to GAPDH levels using Image Studio Lite software (Li-Cor Biosciences), and the data represented as mean±s.e.m. based on three independent experi- mental repeats. The full scan is provided in Supplementary Fig. 5. 22. Wessing, A. & Eichelberg, D. in The Genetics and Biology of Drosophila (eds Ashburner, A & Wright, T. R. F.) (Academic Press, 1978). 23. Brand, A. H. & Perrimon, N. Targetted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993). 24. Duffy, J. B. GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34, 1–15 (2002). 25. Letunic, I., Doerks, T. & Bork, P. SMART: recent updates, new developments and status in 2015. Nucleic Acids Res. 43, D257–D260 (2015). 26. Eisenhaber, B., Bork, P. & Eisenhaber, F. Sequence properties of GPI-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase. Protein Eng. 11, 1155–1161 (1998). 27. Lange, K. Regulation of cell volume via microvillar ion channels. J. Cell. Physiol. 185, 21–35 (2000). 28. Lange, K. Microvillar ion channels: cytoskeletal modulation of ion fluxes. J. Theor. Biol. 206, 561–584 (2000). Statistics. Data were tested using one-way ANOVA followed by Tukey’s multiple comparisons of means with a significance level of Po0.05 (*significant; not significant (NS)). The statistical tests were performed using the data analysis programs OriginPro 7.5 (OriginLab, MA, USA) or Prism 6.0. 29. Green, P. H. et al. 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The Fas2 gene is predicted to be differentially spliced into multiple isoforms (Fas2-RA to Fas2-RH; Metazoa.Ensembl.org) of which only Fas2-RA ( þ /  PEST), -RB and -RC have been experimentally confirmed21. To identify which isoforms of Fas2 are expressed in MTs of Drosophila, we therefore performed RNA-Seq. In brief, MTs were dissected on ice, and total RNA was extracted using a Qiagen RNAeasy kit. RNA library preparation was performed using directional RNA (polyA selection) Illumina library preparation kit according to manufacturer’s protocol, and samples were subsequently run on a Solexa Genome Analyzer II RNA-Seq system with 10M paired-end reads (75 bp). The resultant FASTQ files were processed using the open-source software tools TopHat and Cufflinks42, using the latest version of the Drosophila reference genome sequence43. Super-resolution microscopy. To improve visualization of the subcellular localization of Fas2, we performed super-resolution microscopy using a Zeiss LSM 880 inverted confocal microscope equipped with Airyscan (kindly lent by Zeiss), providing an optical resolution of B140 nm. Tissue samples were prepared as described above. Line scans and normalized fluorescent intensity profiles of the F-actin and Fas2 signals across the MT brush border were generated using the ZEN blue software (Zeiss). Scanning electron microscopy. MTs were dissected from 7-day-old flies (males and females) under Schneider’s insect medium (Invitrogen), and subsequently fixed NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. 43. Hoskins, R. A. et al. The Release 6 reference sequence of the Drosophila melanogaster genome. Genome Res. 25, 445–458 (2015). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 44. Halberg, K. A., Jørgensen, A. & Møbjerg, N. Desiccation tolerance in the tardigrade Richtersius coronifer relies on muscle mediated structural reorganization. PLoS ONE 8, e85091 (2013). How to cite this article: Halberg, K. A. et al. 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Genetics 175, 1505–1531 (2007). 9 Additional information Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 42. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012). Competing financial interests: The authors declare no competing financial interests. NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications Acknowledgements This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work was supported by funding from the BBSRC (Grant no. BB/L002647/1). K.A.H. and I.R.V. were funded by individual postdoctoral fellowships from the Danish Council for Independent Research Natural Sciences (Grant no. 0602-02523B and 4002-00278B). K.A.H. was additionally funded by The Carlsberg Foundation (Grant no. CF14-0204). 10 NATURE COMMUNICATIONS | 7:11266 | DOI: 10.1038/ncomms11266 | www.nature.com/naturecommunications
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Guaranteeing Correctness of Bulk Operations in Outsourced Databases
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To cite this version: Luca Ferretti, Michele Colajanni, Mirco Marchetti. Guaranteeing Correctness of Bulk Operations in Outsourced Databases. 30th IFIP Annual Conference on Data and Applications Security and Privacy (DBSec), Jul 2016, Trento, Italy. pp.37-51, ￿10.1007/978-3-319-41483-6_3￿. ￿hal-01633666￿ Distributed under a Creative Commons Attribution 4.0 International License {luca.ferretti,michele.colajanni,mirco.marchetti}@unimore.it Abstract. The adoption of public cloud services, as well as other data outsourcing solutions, raises concerns about confidentiality and integrity of information managed by a third party. By focusing on data integrity, we propose a novel protocol that allows cloud customers to verify the correctness of results produced by key-value databases. The protocol is designed for supporting efficient insertion and retrieval of large sets of data through bulk operations in read and append-only workloads. In these contexts, the proposed protocol improves state-of-the-art by re- ducing network overheads thanks to an original combination of aggregate bilinear map signatures and extractable collision resistant hash functions. Keywords: database · outsourcing · cloud · integrity · authenticity · complete- ness · aggregate · signature · accumulator · bilinear map · ECRH · BLS Department of Engineering “Enzo Ferrari” University of Modena and Reggio Emilia Department of Engineering “Enzo Ferrari” University of Modena and Reggio Emilia Department of Engineering “Enzo Ferrari” University of Modena and Reggio Emilia HAL Id: hal-01633666 https://inria.hal.science/hal-01633666v1 Submitted on 13 Nov 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Guaranteeing Correctness of Bulk Operations in Outsourced Databases Luca Ferretti, Michele Colajanni, and Mirco Marchetti 1 Introduction The adoption of cloud services and other data outsourcing solutions is often hindered by data confidentiality needs and by limited trust about the correct- ness of operations performed by the service provider. Data confidentiality issues are addressed by several proposals based on encryption schemes (e.g., [10, 12, 21]). The correctness may be guaranteed through standard authenticated data structures [15, 24] based on message authentication codes [1] and digital signa- tures [19] that are affected by large network overheads and by limited database operations. Recent proposals, such as [13, 16, 17, 20], improve standard protocols but they cannot be adopted to guarantee results correctness in outsourced key- value databases because they incur either in network overheads [13, 16, 20] or in high computational costs [16, 17, 9]. For these reasons, we propose Bulkopt, a novel protocol that allows us to detect unauthorized modifications on outsourced data, as well as the correctness of all results produced by a cloud database ser- vice. Bulkopt guarantees authenticity, completeness and freshness of results pro- duced by outsourced databases including cloud related services. It is specifically designed to work efficiently in read and append-only workloads possibly charac- terized by bulk operations, where large amounts of records may be inserted in the key-value database through one write operation. Moreover, Bulkopt supports efficient fine-grained data retrievals by reducing network overhead related to the verification of bulk read operations in which multiple, possibly dispersed, keys are retrieved at once. Closer cryptographic protocols [8, 14] proposed for memory checking data model [5] efficiently support operations on large numbers of records, but they do not support standard database queries and they cannot be immediately ex- tended to database outsourcing scenarios. Bulkopt supports standard insert and read operations on key-value databases and limits communication overhead and verification costs of bulk operations. It recasts the problem of verifying the cor- rectness of results produced by an untrusted database in terms of set operations by leveraging an original combination of bilinear map aggregate signatures [7] and extractable collision resistant (ECR) hash functions [4, 8]. The remainder of the paper is structured as following. Section 2 outlines the system and threat models assumed by the Bulkopt protocol. Section 3 describes the main ideas behind the Bulkopt protocol and outlines the high-level design of the solution. Section 4 proposes the implementation based on aggregate signa- tures and ECR hash functions. 1 Introduction Section 5 outlines the Bulkopt main contributions and compares it with related work. Finally, Section 6 concludes the paper and outlines future work. 3 Protocol overview We describe the formal model used by Bulkopt to represent data and operations (Section 3.1) and to express authenticity and completeness guarantees as set operations (Sections 3.2 and 3.3). We note that since in this version of the protocol we do not consider delete and updates, the server can only violate freshness if he returns results that are both authentic and complete, but that refer to an old version of the database. As a result, clients can detect freshness violations by always using updated cryptographic digest to compute authenticity and completeness proofs. For details about verification operations please refer to the candidate implementation of the protocol described in Section 4. 2 System and threat models We adopt popular terminology for database outsourcing [23]. We identify a data owner that stores data on a database server managed by an untrusted service provider, and many authorized users that retrieve data from the server. The server offers a query interface that can be accessed by the data owner and the authorized users to retrieve values by providing a set of keys. We consider a publicly verifiable setting [23] and assume that only the data owner knows his private key, that is required to insert data into the database, and that authorized users know the public key of the owner that is required to verify results produced by the server. We note that in this first version of the protocol, we do not consider delete and update operations and focus on efficient insert and read database operations. Our threat model assumes that the owner and all users are honest, while the server is untrusted. In particular we assume that the server (or any other unauthorized party, that does not have legitimate access to the private key) may try to insert, modify and delete data on behalf of the owner. The Bulkopt protocol allows all users and the owner to verify the correctness of all results produced by the server. We distinguish three types of results violations: – authenticity: results that contain records that have never been previously inserted by the data owner or that have been modified after insertion; – completeness: results that do not include all keys requested by the client but that have been previously inserted by the data owner; – freshness: results that are based on an old version of the database. In the considered operation workload the server can only violate freshness if he returns results that are both authentic and complete, but refer to an old version of the database. 3.1 Data model We model the key-value database as a set of tuples D = {(k, v)}, where k is the key and v is the value associated to k. The owner populates the key-value database by executing one or more insert operations. For each insert operation the owner sends a set of tuples Bi = {(k, v)}, where i is an incremental counter that uniquely identifies an insert operation. The set Bi contains at least one tuple, and may contain several tuples in case of bulk insertions. Without loss of generality, in the following we refer to each set of tuples Bi as a bulk. We define as Ki the set of keys included in Bi, and Dn = ∪n i=1Bi the set of records stored in the database after n bulk insertions. We assume that the server has access to a lookup function that given a set of keys {k} allows him to retrieve the set of insert operation identifiers {i} in which these keys were sent by the owner. Such function can be obtained by deploying any standard indexing data structure of preference (e.g., a B-tree). ( ) Any client (including the owner) can issue a read operation requesting an arbitrary set of keys X = {k}. If the server behaves correctly he must return the subset of the database A, defined as: A = {(k, v) ∈Dn | k ∈X} (1) (1) We define R as the set of keys included in A, that is: R = {k ∈X | (k, v) ∈A} (2) (2) While executing read operations issued by clients, the server distinguishes two different sets of keys: T and ¯T. T is the union of all sets Ki that contain at least one key among those requested by a client: T = [ Ki | Ki ∩X ̸= ∅ (3) (3) Within each Ki we identify two subsets of keys: Ri = Ki ∩X and Qi = Ki\Ri. We define Q as the union of all sets Qi, and we note that the union of all sets Ri is equal to set R (see Equation (2)). Thus, set Q is the complement of R in T. Within each Ki we identify two subsets of keys: Ri = Ki ∩X and Qi = Ki\Ri. Within each Ki we identify two subsets of keys: Ri = Ki ∩X and Qi = Ki\Ri. 3.1 Data model We define Q as the union of all sets Qi, and we note that the union of all sets Ri is equal to set R (see Equation (2)). Thus, set Q is the complement of R in ¯T is the union of all sets Ki that do not contain any key among those re- quested by a client: ¯T = [ Ki | Ki ∩X = ∅ (4) (4) To better explain how these sets are built and the relationships among them, we refer to a simple example shown in Figure 1. In this example we have a key- Fig. 1. Example of sets computed over a key-value database. Fig. 1. Example of sets computed over a key-value database. value database on which the owner already executed five bulk insert operations, each involving a different amount of tuples. The keys included in the database are represented by sets K1 to K5. We assume that a legitimate client executes a read operation, asking to retrieve six keys belonging to three different bulks. The set of keys requested is represented by X. Since X includes keys belonging to bulks K1, K3 and K4, all keys of these bulks belong to T, while ¯T includes all keys belonging in the remaining bulks (K2 and K5). Sets R1, R3 and R5 include only the keys requested by the client and belonging to K1, K3 and K5, respectively. Set R includes all the keys belonging to the union of R1, R3 and R5. Sets Q1, Q3 and Q5 include only the keys that were not requested by the client and that belong to K1, K3 and K5, respectively. Finally, set Q includes all the keys belonging to the union of Q1, Q3 and Q5. Sets Q and ¯T are the main building blocks that Bulkopt leverages to identify a violation of the security properties or to prove the correctness of results produced by the server. 3.2 Authenticity Bulkopt builds proofs of authenticity by demonstrating that: (5) R ∪Q ∪¯T = KD (5) where KD represents the set of keys included in Dn. We recall from Section 2 that authenticity is violated if the server produces a result containing a key that has not been inserted by the owner. Let us assume that R includes a fake key kf that has been created by the server but does not belong to KD. Then it is obvious that Equation (5) does not hold, since R is not a subset of KD. An obvious solution to demonstrate that R is a subset of KD would be for the client to have the complete set KD. Of course this is not applicable, since it would require all clients to maintain a local copy of the whole key-value database. To overcome this issue, Bulkopt requires the owner to maintain a crypto- graphic accumulator σ(KD) that represents the state of the keys stored in the database Dn. This accumulator is updated after each insert operation and has to be available to all users. Moreover, the server builds two witness data struc- tures WQ and W ¯T that represent the sets Q and ¯T, and sends them to the client together with its response A. We remark that cryptographic accumulators and witnesses are small and fixed-size data structures, that can be transmitted with minimal network overhead [3, 6]. To verify Equation (5) a client can extract the set of keys R from A, and use two accumulators verification functions. In particular, it checks whether the wit- ness data structures received by the database validates the results with respect to the requested data and the current state of the database that is maintained locally. Intuitively, the client verification process can be represented as following: verify (verify (σ(R), WQ) , W ¯T ) ?= σ(KD) (6) (6) (6) where verify denotes accumulators verification functions. where verify denotes accumulators verification functions. If Equation (6) is verified, then the user knows that the two witnesses pro- duced by the server are correct and that Equation (5) is also verified. Hence R is a subset of KD and authenticity holds. On the other hand, if Equation (6) is not verified, either the witnesses produced by the server are not correct or R is not a subset of KD. 3.3 Completeness Bulkopt builds proofs of completeness by demonstrating that: X ∩(KD\R) = ∅ (7) (7) that is, the set of keys requested by the client X and the set of keys not returned by the server KD\R share no common keys. We recall that KD\R is equal to Q ∪¯T, hence Equation (7) can be expressed as the following equation: X ∩(Q ∪¯T) = ∅ (8) (8) Bulkopt proves such conditions by leveraging properties of ECR hash functions. In particular, as shown by [8], ECR hash functions can be used to efficiently express set intersections by using polynomial representations of sets. That is, an empty intersection between sets correspond to polynomials having great common divisor (gcd) equal to 1 (that is, informally we say that since the sets do not share any common elements, the corresponding polynomials do not have common roots). ) Let us denote as CM(s) a polynomial representation of a generic set M w.r.t. variable s [8, 11], and a set P = Q ∪¯T. To prove that the gcd of the polynomials is 1, the server must generate two polynomials ˙p, ˙x such that: CP · ˙p + CX · ˙x = 1, (9) (9) The server sends witnesses WP , W ˙p and W ˙x in addition to WQ and W ¯T that were already sent to prove authenticity. A user can now exploit verification functions of the considered cryptographic signature to verify Equation (9). If Equation (9) is verified, then the client knows that the witnesses produced by the server are correct and that Equation (7) is also verified. Hence R includes all keys X requested by the client that are available in the server database, and completeness holds. On the other hand, if Equation (9) is not verified, either the witnesses produced by the server are not correct or X shares common elements with sets of keys Q or ¯T that were not sent by the server, thus violating completeness. In both cases, the client is able to efficiently detect a misbehavior of the server. 3.2 Authenticity In both cases, the client is able to efficiently detect a misbehavior of the server. 4.1 Setup and key generation Setup. Let g be a generator of the cyclic multiplicative group G of prime order p, GT a cyclic multiplicative group of the same order and ˆe : G×G →GT be the pairing function that satisfies the following properties: bilinearity: ˆe(ma, nb) = ˆe(m, n)ab ∀m, n ∈G, a, b ∈Z∗ p; non-degeneracy: ˆe(g, g) ̸= 1; computability: there exists an efficient one-way algorithm to compute ˆe(m, n), ∀m, n ∈G. Let h be a cryptographic hash function and hz(·), hg(·) be two full domain hash functions (FDH) secure in the random oracle model [2, 7] defined as follow- ing: hz : {0, 1}∗→Z∗ p (10) hg : {0, 1}∗→G (11) (10) (11) (11) Let us denote as CM(s) the characteristic polynomial that uniquely repre- sents the set M, generated by using as roots of the polynomial the sum opposite of the elements of the set and as variable the secret key s [22]. Polynomial CM(s) can be computed as following: CM(s) = Y m∈M (m + s) (12) (12) Let FM = (f(M), f ′(M)) be the output of an extractable collision resis- tant (ECR) hash function [4] with secret key (s, α) ∈Z∗ p × Z∗ p and public key [g, gs, . . . , gsq, gα, gαs, . . . , gαsq], where M denotes a set of values m ∈Z∗ p. The output of the function can be computed through two different algorithms de- pending on the knowledge of the secret key s. For this reason, we denote as fsk(M), fsk ′ (M)  the computation of (f(M), f ′(M)) with knowledge of the se- cret key and fpk(M), fpk ′ (M)  the computation of (f(M), f ′(M)) with only knowledge of the public key. We will use notation FM, f(M) and f ′(M) to identify the black-box outputs of the functions when it is indifferent if they were computed with or without knowledge of the secret key. Functions fsk(M) and fsk(M) can be computed by using straightforwardly the polynomial CM(s) shown in Equation (12) as following: fsk(M) = gCM(s) = g Q|M| i=1(mi+s), (13) fsk ′ (M) = gαCM(s) = gα Q|M| i=1(mi+s), (14) (13) (14) (13) (14) Functions fpk(M) and fpk ′ (M) can be computed by using the coefficients of the polynomial CM(s). 4 Protocol Implementation In this section we describe the Bulkopt protocol by referring to its main three phases: setup and key generation (Section 4.1), insert operations (Section 4.2) and read operations (Section 4.3). 4.1 Setup and key generation That is, if we consider the set of the coefficients {ai}i=[1,...,|M|] of the polynomial CM(s) such that CM(s) = P|M| i=1 ai · si, fpk(M) and fpk ′ (M) can be computed as following: can be computed as following: fpk(M) = |M| Y i=1  gsiai (15) fpk ′ (M) = |M| Y i=1  gαsiai (16) (15) (16) Although functions (fsk(·), fsk ′ (·)) and (fpk(·), fpk ′ (·)) have the same behavior, computing of (fsk(·), fsk ′ (·)) is more efficient due to the computation of only one exponentiation in the group G. Without knowledge of the secret key, ECR hash functions can be verified as following: ˆe(f(M), gα) ?= ˆe(f ′(M), g) (17) (17) Otherwise, the secret key allows a more efficient verification: Otherwise, the secret key allows a more efficient verification: f(M)α ?= f ′(M) (18) (18) Although knowledge of the secret key improves the algorithm efficiency, it allows one to cheat in the computation of the hash function. Hence, it cannot be given to parties that have advantages in breaking the security of the ECR hash function. K G i W d h ’ d bli k k d one to cheat in the computation of the hash function. Hence, it cannot be given to parties that have advantages in breaking the security of the ECR hash function. Key Generation. We denote the owner’s secret and public keys as sk and pk and generate them as follows: Key Generation. We denote the owner’s secret and public keys as sk and pk and generate them as follows: sk = (u, s, α), (u, s, α) R←−Z∗ p × Z∗ p × Z∗ p (19) pk = (U, [gs, . . . , gsq, gα, gαs, . . . , gαsq]), U = gu (20) (19) (20) (19) (20) (20) where q ∈N must be greater than or equal to the maximum number of records involved for each insert or read operation, and u, s and α be different from each other. 4.2 Insert operations The owner issues an insert operation by sending the tuple (Bi, σi, Γi), where: – i ∈N is the operation identifier, that is the incremental counter maintained locally by the owner and by the server that identifies the insert operation (see Section 3); – Bi = {(k, v)} is the set of keys and records inserted in the database at operation i. We also denote as Ki the set of the keys {k} inserted in this operation; p ; – σi is the bulk signature of the set of keys Ki inserted at operation i. It is computed by the tenant as: – σi is the bulk signature of the set of keys Ki inserted at operation i. It is computed by the tenant as: σi(Ki) = [hg(i) · fsk(Ki)]u,  hg(i) · fsk ′ (Ki) u = = h hg(i) · g Q k∈Ki(k+s)iu , h hg(i) · gα Q k∈Ki(k+s)iu (21) (21) (21) – Γi is the set of the record signatures of the records Bi, computed by using a BLS aggregate signature scheme [7]: Γi(Bi) = {γi(k, v)}(k,v)∈Bi (22) γ(k, v) = hg(k ∥v)u (23) (22) (23) (23) where ∥denotes the concatenation operator. We assume that the concatena- tion of the values k and v does not compromise the security of hg(·). If the security of the candidate implementation of hg(·) in this context, one should apply a collision resistant hash function or a message authentication code algorithm on the value v previous to the concatenation operation [1]. We note that the bulk signature σi (Equation (21)) is similar to the compu- tation of a bilinear map accumulator [18]. The original scheme would compute the signature of fsk(Ki) as fsk(Ki)u. Our scheme differs for the factor hg(i)u, that could be seen as a BLS signature of the operation identifier i. This variant allows us to bind the bulk signature σi(Ki) to the operation identifier i in which the insert operation is executed. As we describe in Section 4.3, this design choice also allows us to verify correctness of the server answers by using security proofs that were originally proposed for the memory checking setting [8]. Both the owner and the server keep track of the operation identifier i locally, without exchanging it in each insert operation. 4.2 Insert operations As a result, the value of FDlast after the last insert operation is the following: FDlast = (g Qlast i=1 CKi(S), gα Qlast i=1 CKi(S)) (25) (25) 4.2 Insert operations After each insert operation, the server stores all records Bi, the bulk signatures σi and the record signatures Γi in the database associated to the operation identifier i. The owner does not store any bulk signature σi or record Γi, but he main- tains a cryptographic structure of constant size to keep track of the state of the database. We call it the database signature D = (σ⋆last, FDlast), where last is the value of the operation identifier i for the last insert operation executed on the server, and σ⋆ last and FDlast are the bulk signature and ECR hash function of all the keys inserted in the database. The owner computes the bulk signature σ⋆ last as following: – after the first insertion (i = 1) he sets the initial value of the database signature as σ⋆ 1 = σ1; – after any other insert operation (i > 1), the owner computes the database signature σ⋆ i by computing the product of the current version of the database signature σ⋆ i−1 and the bulk signature σi of the last executed insert operation as σ⋆ i = σ⋆ i−1 · σi−1. As a result, the value of the database signature σ⋆ last is equal to the product of all the bulk signatures σi ever sent by the owner to the server: σ⋆ last = i=last Y i=1 σi (24) (24) The owner computes the database ECR hash function FDlast as following: The owner computes the database ECR hash function FDlast as following: – after the first operation (i = 1), the database accumulator is equal to the ECR hash function of the keys included in the first bulk of data, that is FD1 = (fsk(K1), fsk ′ (K1)); – after the first operation (i = 1), the database accumulator is equal to the ECR hash function of the keys included in the first bulk of data, that is FD1 = (fsk(K1), fsk ′ (K1)); 1 (f ( ), f ( )); – after any other operation (i > 1), the database accumulator is computed as FDi = F CKi(s) Di−1 . 1 ( ( ) ( )) – after any other operation (i > 1), the database accumulator is computed as FDi = F CKi(s) Di−1 . FDi = F CKi(s) Di−1 . 4.3 Read operations The server generates each bulk witness FQi by computing the ECR hash function fpk (see Equation (16)) on the set complement Qi of Ri with respect to Ki, as following: FQi = fpk(Qi), fpk ′ (Qi)  = fpk(Ki\Ri), fpk ′ (Ki\Ri)  = =  gCKi\Ri(s), gα·CKi\Ri(s) , ∀i ∈I (28) Moreover, the server computes the aggregate bilinear signature W ¯T as the wit- ness for bulks that do not include any keys requested by the client by aggregating the owner signatures as following: W ¯T = Y i∈I σi(Ki) = "Y i∈I hg(i)gCKi #u (29) (29) The client verifies authenticity of the keys {Ri} returned by the server by using values included in the authentication proof πauth and the database sig- nature σ⋆ last stored locally (see Equation (24)). The client verifies correctness of the ECR hash function FT by using Equation (17). Then, the client verifies that the ECR hash function FT is built correctly with respect to the aggregate bilinear signature W ¯T by using the locally maintained database signature σ⋆ last, as following: ˆe (FT , U) ?= ˆe σ⋆ last W ¯T , g  (30) (30) Finally, the client uses FT to verify authenticity of the returned records {Ri}i∈I by using the bulk witnesses {FQi}i∈I, as following: ˆe Y i∈I hg(i), g ! Y i∈I ˆe (fpk(Ri), FQi) ?= ˆe (FT , g) (31) (31) After this verification process the client is sure about the following guarantees: – FT is a valid witness for the bilinear aggregate signature W ¯T , as the proba- bility of generating or extracting any other owner signature would break the non-extractability guarantees of aggregate bilinear signatures [7]; – all the returned keys {Ri}i∈I are authentic, because the server proved exis- tence of the witnesses Qi with respect to bulks aggregate hash function FT and generating false witnesses would break extractable collision resistance (ECR) guarantees of the ECR hash function (f(·), f ′(·)) [8]; – all the operation identifiers i ∈I sent by the client are authentic, as gen- erating identifiers that satisfy Equation (31) would break either the FDH function hg(·) or the collision resistance guarantees of aggregate bilinear sig- natures [7]. Keys completeness. As described in Section 3.3, to prove completeness of the response the server must produce witnesses that prove disjunction the requested keys X with respect to the complement sets Q and ¯T. 4.3 Read operations To execute a read operation a client must send a set of keys X = {k} to the server. The server returns the following tuple: response (X) := (I, A, πauth, πcomp, πrec) (26) (26) where I = {i} is the set of the operation identifiers associated to the bulks that include at least one of the keys X requested by the client; A = {Ai}i∈I is the set of the key-value records that compose the actual response to the client, grouped by the corresponding operation identifier i from which the server retrieved it; πauth, πcomp and πrec are the keys authenticity proof, the keys completeness proof and the records authenticity proof used to prove keys authenticity, completeness for the returned keys and authenticity of the values associated to the keys, respec- tively. Although from a security perspective keys authenticity and completeness proofs depend on each other, we distinguish them for the sake of clarity. We also observe that guaranteeing records correctness does not require any completeness proof because we are considering a key-value database where projection queries are not allowed. We recall from Section 3 that the elements of each set of the response Ai is a key-value tuple (k, v), and we denote as Ri the set of the keys included in the set Ai. In the following we describe separately the generation and the verification processes for keys authenticity proofs, keys completeness proofs and records authenticity proofs. Keys authenticity. The keys authenticity proof is a tuple that includes the fol- lowing values: πauth = ({FQi}i∈I, FT , W ¯T ), (27) (27) where {FQi}i∈I is the set of the bulk witnesses, FT is the aggregate ECR hash function of bulks that include at least one of the keys requested by the client, W ¯T is the aggregate bilinear signature of the bulks that do not include any of the keys requested by the client. 4.3 Read operations The completeness proof is a tuple that includes such witnesses, and additional values that allow the client to verify that the server generated them correctly: πcomp = (FP , F ˙p, F ˙x), (32) (32) where FP is the ECR hash function of the set union including the complement sets Q and ¯T, (F ˙q and F ˙x) the witnesses that prove disjunction of the set of the requested keys X with respect to sets ¯T and Q. First, the server computes the ECR hash function of Q ∪¯T as: FP = fpk(Q ∪¯T), fpk ′ (Q ∪¯T)  =  gCQ∪¯ T (s) , gα·CQ∪¯ T (s) (33) (33) The two witnesses F ˙p and F ˙x of polynomials ˙x and ˙p are generated by the server to show that the gcd between the characteristic polynomials CX and CQ∪¯T of sets X and Q ∪¯T is 1, that is equivalent to prove disjunction of sets X, Q and ¯T, as shown in [8]: ˙x, ˙p : CX(s) · ˙x + CP (s) · ˙p = 1 (34) F ˙p = fpk( ˙p), fpk ′ ( ˙p)  (35) F ˙x = fpk( ˙x), fpk ′ ( ˙x)  (36) (34) (35) (36) ( ) (36) (36) The client verifies correctness of the ECR hash functions FP , F ˙q and F ˙x sent by the server by using Equation (17). Then, he verifies whether FP represents the set complement of R with respect to D by checking the value of FP against the database accumulator FDlast (see Equation (25)) publicly distributed by the owner: ˆe (fpk(R), FP ) ?= ˆe (FDlast, g) (37) (37) Now that the client verified the correct generation of the witnesses FP , he can verify disjunction of X, Q and ¯T by testing Equations (34) as following: Now that the client verified the correct generation of the witnesses FP , he can verify disjunction of X, Q and ¯T by testing Equations (34) as following: ˆe(fpk(X), F ˙x) · ˆe(FP , F ˙p) ?= ˆe(g, g) (38) (38) Records authenticity. 5 Related Work Most literature related to security of data outsourcing and cloud services aims to protect data confidentiality of tenant data against malicious insiders of cloud providers. These works typically assume the honest-but-curious threat model where an insider within the cloud provider may access and copy tenant data without corrupting or deleting them. To solve this issue several works already proposed in the literature leverage architectures based on partially homomorphic and property preserving encryptions that allow cloud computations and efficient retrieval on encrypted data (e.g., [10, 12, 21]). Unlike these works, in this paper we do not trust the cloud provider to behave correctly, but we assume a threat model where the cloud provider can violate authenticity and completeness of tenant data, either due to hardware/software failures or deliberate attacks. The main problem in this context is to combine authenticity and completeness guar- antees without affecting the database performance and functionalities. As an example, standard message authentication codes or digital signatures can guar- antee authenticity of outsourced data. However, they cannot guarantee results completeness without incurring in great network overhead. A well-known solution to guarantee results correctness is to adopt Merkle hash trees [9], that allow to build efficient proofs for range queries by authenti- cating the sorted leafs of the tree with respect to an index defined at design time. However, they do not support efficient queries on arbitrary values and efficient proofs on dispersed key values. Other solutions allow the tenant to verify authen- ticity and completeness of outsourced data by means of RSA accumulators [16, 17, 13]. Although RSA accumulators provide constant asymptotic complexity for read and update operations, their high constant computational overhead often prevent their practical application in most scenarios [9]. A different approach is proposed in [25], that relies on the insertion of a number of fake records in the database. These records are then retrieved to verify their presence, and possibly identify completeness violations. However, since no cryptographic verification is executed on the real database, such a solution provides lower security guarantees based on probabilistic completeness verification. The protocols proposed in [8] guarantees authenticity of operations in a memory-checking model by maintain- ing an N-ary tree of constant height. Since only the values of the nodes change (but not the number of cells), these protocols can produce proofs of constant size with respect to the cardinality of the sets stored in each memory cell. 4.3 Read operations The server computes the proof of authenticity πrec by aggregating all the record signatures γk,v = γ(k, v) previously received by the owner for all the records returned to the client, as following: πrec = Y (k,v)∈Ai,∀Ai∈A γk,v (39) (39) The client verifies authenticity of the response A given the server integrity proof πint and the owner public key U by verifying the following condition: The client verifies authenticity of the response A given the server integrity proof πint and the owner public key U by verifying the following condition: ˆe   Y (k,v)∈Ai,∀Ai∈A hg(k ∥v), U  ?= ˆe(πrec, g) (40) (40) This concludes the description of the protocol: any client that is enabled to query the database and that knows the owner’s public key pk and the state of the database D can verify correctness of the results by using the described verification operations. We recall that if a client knows the secret key sk, such as in symmetric settings, he can verify results correctness more efficiently by using the secret exponents u and α. 5 Related Work How- ever, their proposal cannot be easily adopted in the data outsourcing scenario because the amount of sets is not constant and the tree structure would require expensive re-balancing operations. 6 Conclusion This paper proposes Bulkopt, a novel protocol that provides authenticity and completeness guarantees for key-value databases. Bulkopt is specifically designed for providing data security guarantees in the context of cloud-based services sub- ject to read/write workloads, and efficiently support bulk insert operations, as well as read requests that involve the retrieval of multiple and not contiguous keys at once. Efficient verification of bulk operations is achieved by modeling data security constraints in terms of set operations, and by leveraging crypto- graphic proofs based for set operations. In particular, Bulkopt is the first protocol that combines extractable collision resistant hash functions and aggregate bilin- ear map signatures to achieve novel cryptographic constructions that allow the verification of authenticity and completeness over large sets of data by relying on small cryptographic proofs. More work is needed to tune the protocol per- formance by using data structures to cache partial proofs at the server side, as well as further developments to also support update operations. Acknowledgments. The authors acknowledge the support of HORIZON 2020 FCT-1-2015 project ASGARD “Analysis System for Gathered Raw Data”. References 1. M. Bellare, R. Canetti, and H. Krawczyk. Keying hash functions for message authentication. In Proc. 1996 IACR Int’l Conf. Advances in Cryptology. Springer. 2. M. Bellare and P. Rogaway. The exact security of digital signatures-how to sign with rsa and rabin. In Proc. 1996 Int’l Conf. Advances in Cryptology. Springer. 3. J. Benaloh and M. De Mare. One-way accumulators: A decentralized alternative to digital signatures. In Proc. 1993 IACR Int’l Conf. Advances in Cryptology. Springer. 4. N. Bitansky, R. Canetti, A. Chiesa, and E. Tromer. From extractable collision resistance to succinct non-interactive arguments of knowledge, and back again. In Proc. 2012 ACM Third Int’l Conf. Innovations in Theoretical Computer Science. 5. M. Blum, W. Evans, P. Gemmell, S. Kannan, and M. Naor. Checking the correct- ness of memories. Algorithmica, 12(2-3), 1994. 6. D. Boneh, C. Gentry, B. Lynn, and H. Shacham. Aggregate and verifiably en- crypted signatures from bilinear maps. In Advances in cryptology. Springer, 2003. 7. D. Boneh, B. Lynn, and H. Shacham. Short signatures from the weil pairing. In Proc. 2001 IACR Int’l Conf. Advances in Cryptology. Springer. 8. R. Canetti, O. Paneth, D. Papadopoulos, and N. Triandopoulos. Verifiable set operations over outsourced databases. In Proc. 2014 IACR Int’l Conf. Public-Key Cryptography. Springer. 9. S. A. Crosby and D. S. Wallach. Authenticated dictionaries: Real-world costs and trade-offs. ACM Trans. Information and System Security, 14(2):17, 2011. 10. L. Ferretti, M. Colajanni, and M. Marchetti. Distributed, concurrent, and inde- pendent access to encrypted cloud databases. IEEE Transactions on Parallel and Distributed Systems, 25(2), 2014. ( ) 11. M. J. Freedman, K. Nissim, and B. Pinkas. Efficient private matching and set intersection. In Proc. 2004 IACR Int’l Conf. Advances in Cryptology. Springer. 12. H. Hacig¨um¨u¸s, B. Iyer, C. Li, and S. Mehrotra. Executing sql over encrypted data in the database-service-provider model. In Proc. 2002 ACM SIGMOD Int. Conf. Management of Data, 2002. 13. J. Hong, T. Wen, Q. Gu, and G. Sheng. Query integrity verification based-on mac chain in cloud storage. In Proc. 13th IEEE/ACIS Int. Conf. Computer and Information Science, 2014. 14. A. E. Kosba, D. Papadopoulos, C. Papamanthou, M. F. Sayed, E. Shi, and N. Triandopoulos. Trueset: Faster verifiable set computations. In Proc. 23rd USENIX Int’l Conf. Security Symposium, 2014. 15. A. Miller, M. Hicks, J. Katz, and E. Shi. Authenticated data structures, generically. ACM SIGPLAN Notices, 49(1), 2014. 16. E. Mykletun, M. Narasimha, and G. Tsudik. References Authentication and integrity in outsourced databases. ACM Trans. Storage, 2(2), May 2006. 17. M. Narasimha and G. Tsudik. Dsac: An approach to ensure integrity of outsourced databases using signature aggregation and chaining. Cryptology ePrint Archive, Report 2005/297, 2005. / 18. L. Nguyen. Accumulators from bilinear pairings and applications. In Proc. 2005 Int’l Conf. Topics in Cryptology. Springer, 2005. 19. Nist. Digital signature standard. Technical report, Jul. 2013. 20. R. A. Popa, J. R. Lorch, D. Molnar, H. J. Wang, and L. Zhuang. Enabling security in cloud storage slas with cloudproof. In Proc. USENIX Annual Technical Conf., 2011. 21. R. A. Popa, C. M. S. Redfield, N. Zeldovich, and H. Balakrishnan. CryptDB: protecting confidentiality with encrypted query processing. In Proc. 23rd ACM Symp. Operating Systems Principles, Oct. 2011. 22. F. P. Preparata and D. V. Sarwate. Computational complexity of fourier transforms over finite fields. Mathematics of Computation, 31(139), 1977. 23. P. Samarati and S. D. C. di Vimercati. Data protection in outsourcing scenar- ios: Issues and directions. In Proc. 5th ACM Symp. Information, Computer and Communications Security, 2010. 24. R. Tamassia. Authenticated data structures. In Algorithms-ESA. Springer, 2003. 25. M. Xie, H. Wang, J. Yin, and X. Meng. Integrity auditing of outsourced data. In Proc. 33rd Int. Conf. Very Large Data Bases. VLDB Endowment, 2007.
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An Algorithm for Making Informed Decisions about Patient Care from Interactions between Genetic and Environmental Factors
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An Algorithm for Making Informed Decisions about Patient Care from Interactions between Genetic and Environmental Factors Sybil Prince Nelson∗, Viswanathan Ramakrishnan, Paul Nietert, Diane Kamen, Paula Ramos, Bethany Wolf Washington and Lee University, Sybil Prince Nelson∗, Viswanathan Ramakrishnan, Paul Nietert, Diane Kamen, Paula Ramos, Bethany Wolf Washington and Lee University, Sybil Prince Nelson∗, Viswanathan Ramakrishnan, Paul Nietert, Diane Kamen, Paula Ramos, Bethany Wolf Washington and Lee University, Abstract The use of statistics in medicine is important for making informed decisions for patient care and discovering possible new treatments or cures. Thus, it is often useful to find what factors lead to increased risk of disease. In statistics, the association of one factor with out- come, may differ in the presence of another factor. This interaction, if not addressed, may cause the association with the outcome to go undetected. Interactions are often difficult to distinguish with tradi- tional methods because of the need to state the possible interactions a priori. If there are a large number of factors, the possible inter- actions quickly become infeasible to enumerate. Decision trees such as CART eliminate the need to state the interactions a priori and is often used to identify interactions associated with outcome. CART, however, tends to be biased toward the inclusion of continuous fac- tors over binary ones such as those found in genetic data. This paper proposes a method called C.Logic that eliminates the need to state interactions a priori and can effectively identify which interactions are associated with outcome without being biased toward the inclusion of continuous factors into the model. 1 1 1 Introduction Researchers are often interested in investigating genetic variants that are as- sociated with disease outcome. With the completion of the Human Genome Project and the International HapMap, along with many technological ad- vancements in gene mapping, researchers are able to perform genome-wide association studies (GWAS). GWAS involves analyzing whole samples of genomes to look for genetic variations associated with disease. In a GWA study, it is common practice to gather DNA samples from both cases and controls and scan for genetic variations, or Single Nucleotide Polymorphisms (SNPs). SNPs that are more frequently found in cases than controls are studied further. Though GWA studies have found many common variants associated with diseases such as in Celiac’s Disease and Crohn’s Disease [1] among others, these variants typically explain only a fraction of the inherited contribution or heritability of disease risk. [2] It is possible, however, that certain SNPs are associated with disease outcome only in the presence of other factors. These possible interactions will go undiscovered if variables are eliminated based solely main effect or if genetic factors are not considered in connection with environmental factors. These interactions could explain at least a portion of the remaining variance in disease outcome. Neglect of possible gene-by-environment interactions in GWA studies has led to the emergence of specifically designed gene-by-environment experi- ments in order to find missing heritability [3]. In these randomized controlled experiments, the environment is manipulated in standard ways. For exam- ple, in studying dopamine receptor D4 (DRD4) gene, researchers noticed that toddlers who carried this allele showed reduced destructive behavior after a parenting class was administered versus toddlers who did not carry this allele [4, 3]. Another way to explain missing heritability or variance could be to identify these interactions with statistical methods. However, identifying interactions that lead to increased risk of disease may be difficult with traditional statistical methods. This difficulty is due, in part, to the fact that in many statistical methods, interactions must be identified a priori in order to be studied. If all interactions are included in a model, it quickly becomes computationally prohibitive. Decision trees eliminate the need to specifically state interactions a pri- ori and instead can search all possible interactions over a large sample space. 1 Introduction Classification and Regression Trees (CART), a common decision tree method, 2 can identify interactions to model outcome but it is biased toward the in- clusion of continuous variables as opposed to binary genetic data[5]. Logic regression is an alternative tree-based method specifically designed to find interactions in genetic data, but it was not designed for continuous variables [6]. Continuous variables must be dichotomized before use in the model. [6]. Continuous variables must be dichotomized before use in the mode This paper suggests an alternative algorithm for identifying continuous and binary interactions associated with disease. Section 2 of this paper will explore current methods for interaction identification as well as propose an alternative method. Section 3 will discuss a simulation designed to explore how this new methodology compares to current practices while section 4 will present the results. Section 5 will provide a discussion and review of the topics present as well as make recommendations for use of new methodology. 2.1 Current Methods Logistic Regression is a traditional statistical approach often used to model dichotomous outcome from continuous and binary data. When investigat- ing interactions, however, they must be added to the model a priori. When there are a small number of variables, all possible combinations of the vari- ables can be included in the model without difficulty. For example, if there are 4 main effects, the number of total terms to include in the model would be 24 −1 = 15. If the number of variables increases to 20, however, the number of model terms becomes 220 −1 = 1, 048, 575 which would be computation- ally prohibitive. Also, if the number of parameters exceeds the number of observations, which is common in genetic data, then logistic regression will fail. In Machine Learning techniques there is no need to identify interactions a priori. These techniques involve algorithms that search through an en- tire data space to find patterns that can predict outcome. Artificial Neural Networks (ANN) is a machine learning technique that can model complex data, but its models result in a ”Black Box” effect which is difficult to inter- pret. Another machine learning technique, Support Vector Machines (SVMs) classifies disease outcome. Like, ANN the results often lack interpretability. Also, if the number of variables exceeds the number of samples, the method will fail. 3 3 Decision Tree methods are an efficient way to identify possible interactions in the data that are associated with increased risk of disease. The resulting models are interpretable and the method can handle high dimensionality. CART, a common decision tree method, uses recursive partitioning to create homogeneous rectangular subsets in the data. As seen in Figure 1, the first split occurs at the parent node, Nparent, where the data space is divided into two regions Nleft and Nright. CART models response by majority vote meaning that if more observations in Nright are characterized by Y = 1, then the entire region is labeled as 1, or disease positive. Next, one or both of the regions are divided again and a vote is taken. This continuous until some stopping rule is applied. Figure 1: CART Splitting As stated earlier, CART is biased toward the inclusion of continuous variables meaning that if it has a choice of including a continuous variable or a binary variable into the model, it will choose the continuous [5]. Also, by design, CART is inflexible. Once a split is made on a variable, a branch is formed and certain combinations of variables are no longer viable. Logic regression is an alternative tree based method that uses Boolean logic to model a binary outcome. It is this use of Boolean logic that makes logic regression inherently more flexible than CART. For example, consider 4 he logic statement D = 1 : (X1and X2)or (X1and X3). As shown in figure 2, this can be modeled exactly with logic regression but not with CART. the logic statement D = 1 : (X1and X2)or (X1and X3). As shown in figure 2, this can be modeled exactly with logic regression but not with CART. Figure 2: CART Splitting Figure 2: CART Splitting 2.2 Methodology of C.Logic LR, however, is not designed for the inclusion of continuous variables. The splits that CART creates during its implementation must be done prior to implementation for LR. An important question, therefore, is the best way to make these dichotomizations while preserving the integrity of the data. This involves not dichotomizing interacting variables independent of each other. Thus, using simultaneous dichotomization [7] 2.3 Simultaneous Dichotomization What follows is a brief description of simulataneous dichotomization as de- veloped by Prince Nelson et al. [7]. Consider the possibility of two variables, 5 X1 and X2 with the following relationship with outcome: PY =1 = P(X1≥T1,X2≥T2)PY |(X1≥T1,X2≥T2) + P(X1<T1ORX2<T2)PY |(X1<T1ORX2<T2) (1) h ) (1) where where where P(X1<T1ORX2<T2) = P(X1≥T1,X2<T2) + P(X1<T1,X2≥T2) + P(X1<T1,X2<T2) and PY =1|X1<T1ORX2<T2 = PY |(P(X1≥T1,X2<T2)+P(X1<T1,X2≥T2)+P(X1<T1,X2<T2)) PY =1|X1<T1ORX2<T2 = PY |(P(X1≥T1,X2<T2)+P(X1<T1,X2≥T2)+P(X1<T1,X2<T2)) The probabilities corresponding to a 2 × 2 contingency table for the joint condition of (X1 ≥T1, X2 ≥T2) and Y = 1 or 0 are summarized in Table 1. Here, Y is associated with X1 and X2 through an interaction, and thus PY =1 is larger when the interaction is present. If Y is associated with X through an interaction, then PY =1 is larger when in the presence of the interaction. For this paper, an interaction between two or more variables means that there is an increased risk of PY =1 when both or all variables are present. 6 Y = 1 Y = 0 X1 ≥T1, X2 ≥T2 aJ = P(Y = 1, X1 ≥T1, X2 ≥T2) = PX1≥T1,X2≥T2PY |X1≥T1,X2≥T2 bJ = P(Y = 0, X1 ≥T1, X2 ≥T2) = PX1≥T1,X2≥T2 − PX1≥T1,X2≥T2PY |X1≥T1,X2≥T2 PX1≥T1,X2≥T2 X1 < T1 ∨X2 < T2 cJ = P(Y = 1, X1 < T1, X2 ≥T2) + P(Y = 1, X1 ≥T1, X2 < T2) + P(Y = 1, X1 < T1, X2 < T2) = (PX1<T1,X2≥T2 + PX1≥T1,X2<T2 + PX1<T1,X2<T2)PY |X1<T1∨X2<T2 dJ = P(Y = 0, X1 < T1, X2 ≥T2) + P(Y = 0, X1 ≥T1, X2 < T2) + P(Y = 0, X1 < T1, X2 < T2) = (PX1<T1,X2≥T2 + PX1≥T1,X2<T2 + PX1<T1,X2<T2) − (PX1<T1,X2≥T2 + PX1≥T1,X2<T2 + PX1<T1,X2<T2)PY =1|X1<T1∨X2<T2 PX1>T1,X2<T2 +PX1<T1,X2≥T2 +PX1<T1,X2<T2 PY =1 PY =0 Table 1: The 2x2 contingency table for continuous variables X1 and X2 and dichotomous outcome Y where X1 and X2 are jointly thresholded at T1 and T2 respectively 7 7 7 2.4 C.Logic Algorithm Algorithm for C.Logic Algorithm for C.Logic 1. Take t bootstrap samples of the data 2. For each continuous variables, X, in each sample order the values to create a variable oX 2. For each continuous variables, X, in each sample order the values to create a variable oX 3. For each continuous variable in each sample calculate the cell counts for a 2x2 contingency table as follows: 3. For each continuous variable in each sample calculate the cell counts for a 2x2 contingency table as follows: ak = t X k=1 I(Xk ≥oXk) ∧I(Yk = 1) bk = t X k=1 I(Xk ≥oXk) ∧I(Yk = 0) ck = t X k=1 I(Xk < oXk) ∧I(Yk = 1) dk = t X k=1 I(Xk < oXk) ∧I(Yk = 0) (2) (2) 4. At each value of Xt, use the corresponding contingency table to calculate the six statistics found in Table 2 5. Find the maximum value for each statistic for each variable in each bootstrap sample. 6. For each continuous variable, find the median of the bootstrap samples. These medians are the new thresholds. 7. Dichotomize the continuous variables of the original data set with the new thresholds. 8. Apply logic regression 8. Apply logic regression 8 8 Odds Ratio Youden’s Statistic Chi-Square ad bc a a+c + d b+d −1 (ad−bc)2 (a+b)(c+d)(b+d)(a+c) Kappa Statistic Relative Risk* Gini Index (a+d)−((a+b)(a+c)+(c+d)(b+d)) 1−((a+b)(a+c)+(c+d)(b+d)) a/(a+b) c/(c+d) (Py(1 −Py)) −( ab a+b + cd c+d) Table 2: Formulas for statistics for selecting a threshold for a continuous variable X to discriminate a binary outcome Y based on the probabilities in Table *For cohort study designs only Table 2: Formulas for statistics for selecting a threshold for a continuous variable X to discriminate a binary outcome Y based on the probabilities in Table *For cohort study designs only 3 Simulation Study In section 2 we developed an algorithm, C.Logic, for extending logic regre- sion to include continuous variables. In this section, we compare C.Logic to CART in order to investigate which method performs better in identifying interactions that are associated with the outcome. To compare C.Logic to CART, we first generate a data set as follows: 1. Two binary variables, X1 and X2, generated under a binomial distri- bution with probability of 0.3 1. Two binary variables, X1 and X2, generated under a binomial distri- bution with probability of 0.3 1. Two binary variables, X1 and X2, generated under a binomial distri- bution with probability of 0.3 2. A binary and continuous,X3 and X4, variable generated under a mul- tivariate distribution such that X ∼N2(0, 1 ρ ρ 1  ), where ρ = 0 2. A binary and continuous,X3 and X4, variable generated under a mul- tivariate distribution such that X ∼N2(0, 1 ρ ρ 1  ), where ρ = 0 3. Two continuous variables, X5 and X6, generated under a multivariate distribution such that X ∼N2(0, 1 ρ ρ 1  ), where ρ = 0 3. Two continuous variables, X5 and X6, generated under a multivariate distribution such that X ∼N2(0, 1 ρ ρ 1  ), where ρ = 0 3. Two continuous variables, X5 and X6, generated under a multivariate distribution such that X ∼N2(0, 1 ρ ρ 1  ), where ρ = 0 3. Two continuous variables, X5 and X6, generated under a multivariate distribution such that X ∼N2(0, 1 ρ ρ 1  ), where ρ = 0 4. Set true thresholds for continuous variables Tx4, Tx5, and Tx6, as the inverse normal value of 0.3, 0.524. 4. Set true thresholds for continuous variables Tx4, Tx5, and Tx6, as the inverse normal value of 0.3, 0.524. 4. Set true thresholds for continuous variables Tx4, Tx5, and Tx6, as the inverse normal value of 0.3, 0.524. 5. Generate an outcome variable Y so that Y = 1 when X1 = X2 = 1 or X3 = 1andX4 ≥Tx4 or X5 ≥Tx5andX6 ≥tx6 with PY |T such that PY |T > PY |F 5. Generate an outcome variable Y so that Y = 1 when X1 = X2 = 1 or X3 = 1andX4 ≥Tx4 or X5 ≥Tx5andX6 ≥tx6 with PY |T such that PY |T > PY |F 5. 3 Simulation Study Generate an outcome variable Y so that Y = 1 when X1 = X2 = 1 or X3 = 1andX4 ≥Tx4 or X5 ≥Tx5andX6 ≥tx6 with PY |T such that PY |T > PY |F 6. Generate 11 binary and 3 continuous noise variables. 6. Generate 11 binary and 3 continuous noise variables. For our simulation, we use sample sizs of 300, 500, 1000, 1500, and 2000. We also consider odds ratios of 2,4, and 8. Each simulation is run with 500 repititions using a bootstrap of 10 per sample. 9 4 Simulation Results 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X1*X2 OR=2 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X3*X4 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X5*X6 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X1*X2 OR=4 Proportion of times identified 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X3*X4 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X5*X6 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X1*X2 OR=8 X3*X4 500 1000 1500 2000 0.0 0.2 0.4 0.6 0.8 1.0 X5*X6 CART C.Logic Figure 3: Simulation results showing sample size by the proportion of times the interactions are identified under the case-control study design comparing C.Logic to CART 500 1000 2000 0.0 0.2 0.4 0.6 0.8 1.0 X3*X4 500 1000 2000 X3*X4 CART C.Logic Figure 3: Simulation results showing sample size by the proportion of times the interactions are identified under the case-control study design comparing C.Logic to CART Figure 3 shows the results from a cohort study design scenario. Each plot shows the sample size by the proportion of times the interaction is correctly identified exactly. The columns in the figure show the interactions: X1X2, X3X4, and X5X6, as described in section 3. The rows show the impact of increasing the strength of association with Y, represented by the odds ratio in the data. For each interaction, as the sample size increases both C.Logic and 10 CART improve in identifying the interactions. As the odds ratio increases so does the proportion of times the interactions are exactly identified in a C.Logic or CART tree. Both methods perform poorly when identifying the X5X6 interaction when the odds ratio is 2. As the odds ratio increases to 8, however, C.Logic improves and performs better than CART with C.Logic identifying the correct interactions 75% of the time compared to 37% with CART. C.Logic outperforms CART by the greatest margin for the X1X2 interaction when odds ratio is 8. 5 Conclusion Identifying interactions that lead to increased risk of disease is an impor- tant problem in both medicine and statistics. If a factor increases risk of disease only in the presence of another factor, it may go undiscovered with traditional statistical methods. CART is a tree-based method that can iden- tify interactions to model a binary outcome. Studies have shown, however, that CART can be biased toward the inclusion of continuous variables[5]. Logic regression is specifically designed to find binary interactions that are associated with a binary outcome. In this paper, we created a new algorithm, C.Logic, that allows for the inclusion of continuous variables in a logic regression framework. Simulation results show that C.Logic is superior to CART in identifying interactions of variables associated with outcome an improves as odds ratio increases. One limitation of this paper is that the data was simulated under the assumption of Equation 1. This implies that only an interaction between factors leads to increased risk of disease. Data that does not follow this framework could have less than optimal results. Yet, Prince et. al showed in their research that even when there are no interactions in the data, simultane- ous dichotomization, which is used in the C.Logic algorithm still determines more accurate cutpoints indicating that this algorithm will perform well in many types of data sets. 11 References [1] Guillaume Lettre and John D. Rioux. Autoimmune diseases: insights from genome-wide association studies. Human Molecular Genetics, 17(R2):R116–R121, 2008. [2] Mark I. McCarthy and Joel N. Hirschhorn. Genome-wide association studies: potential next steps on a genetic journey. Human Molecular Genetics, 17(R2):R156–R165, 2008. [3] MH van IJzendoorn, MJ Bakermans-Kranenburg, J Belsky, S Beach, G Brody, KA Dodge, M Greenberg, M Posner, and S Scott. Gene-by- environment experiments: a new approach to finding the missing heri- tability. Nat Rev Genet, 2011. [4] MJ Bakermans-Kranenburg, MH Van IJzendoorn, TA Pijlman, Femke, J Mesman, and F Juffer. Experimental evidence for differential suscep- tibility: Dopamine d4 receptor polymorphism (drd4 vntr) moderates in- tervention effects on toddlers’ externalizing behavior in a randomized controlled trial. Developmental Psychology, 44:293–300, 2008. [5] Wei-Yin Loh. Classification and regression tree methods. Encyclopedia of statistics in quality and reliability, 2008. [6] Ingo Ruczinski, Charles Kooperberg, and Michael LeBlanc. Logic regres- sion. Journal of Computational and Graphical Statistics, 12(3):475–511, 2003. [7] S. L. PrinceNelson, V. Ramakrishnan, P. J. Nietert, D. L. Kamen, P. S. Ramos, and B. J. Wolf. A comparison of joint dichotomization and single dichotomization of interacting variables to discriminate a disease out- come. International Journal of Biostatistics, 18(2):613–625, 2022. 12
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Mapping transmission foci to eliminate malaria in the People’s Republic of China, 2010–2015: a retrospective analysis
BMC infectious diseases
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Feng et al. BMC Infectious Diseases (2018) 18:115 https://doi.org/10.1186/s12879-018-3018-8 Feng et al. BMC Infectious Diseases (2018) 18:115 https://doi.org/10.1186/s12879-018-3018-8 Open Access Mapping transmission foci to eliminate malaria in the People’s Republic of China, 2010–2015: a retrospective analysis n Feng, Hong Tu, Li Zhang, Shaosen Zhang, Shan Jiang, Zhigui Xia and Shuisen Zhou* * Correspondence: shuisenzhou@126.com; zss163@hotmail.com National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Key Laboratory of Parasite and Vector Biology, Ministry of Health; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai 200025, People’s Republic of China Abstract Background: China has initiated the National Malaria Elimination Action Plan, which aims to eliminate malaria by 2020. However, the transmission of malaria occurs sporadically or in distinct foci, which greatly hampers progress toward elimination in China and other countries. The object of this study was to foci categorization and evaluates whether the response met the requirements issued by the nation or WHO. Methods: Residual transmissions were investigated and located with fine spatial resolution mapping from parasitological confirmed malaria cases by use of routine national surveillance data. The “1–3-7” timeframes were monitored for each focus between 2012 and 2015. Each focus was identified, and the application of appropriate measures was evaluated. Results: A total of 5996 indigenous cases were recorded between 2010 and 2015; during this period, the number of cases declined by 99.1% (2010, n = 4262; 2015, n = 39). Most indigenous cases (92.5%) were reported in Anhui (n = 2326), Yunnan (n = 1373), Henan (n = 930), Hubei (n = 459), and Guizhou (n = 458). The temporal distribution showed that the indigenous malaria cases were clustered during the period of May to August. A total of 320 foci were carefully investigated and analyzed: 24 were active foci; 72, residual non-active foci; and 224 cleared-up foci. For the foci response evaluation, all the active foci were investigated within 7 days, while 80.2% of the residual non-active foci were responded within 7 days. In addition, reactive case detection (RACD) was carried out with 92.9% of the active foci and vector investigation carried out with 75%. For residual non-active foci, RACD was carried out with 83.2% and vector investigation with 78.2% of the foci. Conclusions: This study used nationwide data to categorize foci in China and evaluate the response of these areas during the control and elimination phases. Our approach stratifies future control responses by identifying those locations where the elimination of endemic transmission is needed, such as in the counties at the China–Myanmar border and in Tibet. In addition, this study will help local CDC staff to reassess their needs and responses against different types of foci during the elimination and post-elimination phases. Abstract Keywords: Malaria elimination, Foci, China Keywords: Malaria elimination, Foci, China * Correspondence: shuisenzhou@126.com; zss163@hotmail.com National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Key Laboratory of Parasite and Vector Biology, Ministry of Health; WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai 200025, People’s Republic of China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 10 Feng et al. BMC Infectious Diseases (2018) 18:115 Background To interrupt malaria transmission, a two-pronged ap- proach is required: disease management (alleviation of symptoms and prevention of biting by mosquitoes) and disease prevention through vector control. China initiated the National Malaria Elimination Action Plan (NMEAP) in 2010, which aimed to eliminate indi- genous malaria in non-border areas by the end of 2015, and eliminate it nationwide by the end of 2020 [1]. All the counties in China were classified into 4 types, and each type has its own strategy and interventions [2] (Table 1). The object of this study is to categorize foci by using country-wide data collected under operational condi- tions during the important period from the control phase to the elimination phase in China, and to evaluate whether the responses meet the requirements set out by national or WHO guidelines. Many factors would be challenges in the performance of the case investigation, foci investigation and response. The findings are likely to reflect the real situation and would help local CDC staff to reassess their needs and responses against differ- ent types of foci during the elimination and post- elimination phases. To achieve the national goal of malaria elimination, China has made great strides in controlling indigenous malaria over the past several decades [3]. After imple- menting an integrated control strategy and interven- tions, along with socio-economic development, urbanization progress, and changes in the natural envir- onment and malaria vectors, the incidence of indigenous malaria in China has sharply declined, and the malaria- endemic areas have dramatically shrunk [4]. Since 2010, transmissions mainly occurred in the counties along the China–Myanmar border and in Motuo County of the Tibetan Autonomous Region of China. Study design y g A retrospective study was conducted to explore malaria- endemic characteristics from January 1, 2010 to December 31, 2015 at the Chinese Center for Disease Control and Prevention (CDC) [7–12]. All individual cases from the Infectious Diseases Information Reporting Management System (IDIRMS, http://chinacdc.cn) were carefully reviewed and analyzed [13]. The IDIRMS, which was set up in 2004 after the SARS outbreak, is a standardized platform that provides health care systems nationwide the ability to detect, analyze, prevent, and respond to any communicable dis- ease in the country. The data for the study were selected by use of the reporting data and reporting area, but the data from Hong Kong, Macao, and Taiwan were excluded from these statistics. The IDIRMS parameters consisted of the geographical distribution, species composition, gender, and age distribution of the cases. Both clinically diagnosed cases and laboratory-confirmed cases were included in this analysis (Table 2). Malaria tends to persist in “foci” or localized areas of self-sustaining transmission, due to spatially heteroge- neous influences, such as social and ecological factors that include Anopheline mosquito density, agricultural practices, human behavior, wealth, education, access to and utilization of health care, and urbanization [5]. In the elimination phase, cases occur sporadically or in dis- tinct foci. In our investigation, the foci were classified into 3 types (active, residual non-active and cleared-up) according to the guidelines issued by the World Health Organization (WHO) [6]. The elimination of residual foci is a dynamic process, taking place mainly during the late stage of malaria elimination. Monitoring the status of foci, with the precise identifi- cation of their functional status, is the cornerstone for success in interrupting malaria transmission and pre- venting the re-introduction of malaria where potential foci (foci with imported cases but without proof of local transmission) may be present. This step was important for China because there were many areas where trans- mission was sharply reduced but the vectors still exist. Another reporting system, the Parasitic Diseases Infor- mation Reporting Management System (PDIRMS, http://chinacdc.cn), was set up in 2012 to report on 3 Table 1 Classification of county types and strategy implemented according to NMEAP Type Classification Strategy I Local infections are detected in 3 consecutive years, and the incident rate is equal to or greater than 1 in 10,000. Study design Here, it refers to the patient who acquired the illness from a known malaria-prevalent region outside China. Induced case A case in which the origin of the illness can be traced to a blood transfusion or other form of parenteral inoculation of the parasite but not to transmission by a natural mosquito-borne inoculation. Introduced case A case contracted locally, with strong epidemiological evidence linking it directly to a known imported case (first-generation local transmission). Recrudescent case Recurrence of asexual parasitemia of the same genotype(s) that caused the original illness, due to incomplete clearance of asexual parasites after antimalarial treatment. Death from malaria Patient with signs and symptoms of complicated malaria, with confirmed diagnosis of P. falciparum (or P. vivax) or associated infection. Focus A defined and circumscribed area situated in a currently or formerly malarious area that contains the epidemiological and ecological factors necessary for malaria transmission. Reactive case detection A process that involves an active response after the identification of a local or imported case in a receptive area where the transmission intensity is low or assumed to be interrupted. further transmission within 7 days, was launched in 2012 to guide and monitor the elimination process na- tionwide [14]. Each case was investigated by staff at the county CDC to classify it as indigenous or imported; the definitions appear in Table 2 [15]. The PDIRMS contains the date of diagnosis, date of reporting, date of the case investigation, case classification (indigenous, imported, or other [induced, introduced, or recrudesce]), focus in- vestigation, and foci response, such as reactive case de- tection (RACD) and indoor residual spraying (IRS). Both the website for the IDIRMS and the PDIRMS are pri- vate. Malaria department in NIPD was responsible for national malaria data for these two systems and have permission to access them. Study design The integrated interventions of case management and vector control will be scaled up to reduce disease incidence because annual parasite incidence (API) < 1 compared to 2010. II Local infections are detected in 3 consecutive years, and the incident rate is lower than 1 in 10,000 in at least one of those 3 years. Response to any possible malaria cases and active foci is the main strategy to interrupt local transmissions. III No local infections reported for 3 years. Enhance monitoring and surveillance of imported cases to prevent secondary transmission. IV Areas without malaria epidemic. Sensitively detect and promptly respond to imported cases. Table 1 Classification of county types and strategy implemented according to NMEAP Type Classification Strategy I Local infections are detected in 3 consecutive years, and the incident rate is equal to or greater than 1 in 10,000. The integrated interventions of case management and vector control will be scaled up to reduce disease incidence because annual parasite incidence (API) < 1 compared to 2010. II Local infections are detected in 3 consecutive years, and the incident rate is lower than 1 in 10,000 in at least one of those 3 years. Response to any possible malaria cases and active foci is the main strategy to interrupt local transmissions. III No local infections reported for 3 years. Enhance monitoring and surveillance of imported cases to prevent secondary transmission. IV Areas without malaria epidemic. Sensitively detect and promptly respond to imported cases. IV Feng et al. BMC Infectious Diseases (2018) 18:115 Page 3 of 10 Page 3 of 10 diseases: malaria, schistosomiasis, and echinococcosis. Malaria was monitored and reported on within the time Table 2 Definitions used in this study Type of malaria Description Clinically diagnosed case An individual with malaria-related symptoms (fever [axillary temperature ≥37.5 °C], chills, severe malaise, headache, or vomiting) at the time of examination. Laboratory-diagnosed case A clinical case confirmed by microscopy, polymerase chain reaction, or rapid diagnostic tests in the laboratory. Indigenous case A case contracted locally with no evidence of importation and no direct link to transmission from an imported case. In this study, an indigenous case refers to malaria acquired by mosquito transmission in China. Imported case A malaria case or infection in which the infection was acquired outside the area in which it was diagnosed. Data extraction for foci identification and response evaluation A malaria case or infection in which the infection was acquired outside the area in which it was diagnosed. Here, it refers to the patient who acquired the illness from a known malaria-prevalent region outside China. In this study, a natural village is considered the smallest unit of focus. “Active focus” refers to a focus with on- going transmission; “residual non-active focus” refers to a transmission that was interrupted recently (1–3 years ago). A “cleared-up focus” is defined as a focus with no local transmission for more than 3 years. A case in which the origin of the illness can be traced to a blood transfusion or other form of parenteral inoculation of the parasite but not to transmission by a natural mosquito-borne inoculation. y For foci identification, the data of the indigenous cases from 2012 to 2015 in the IDIRMS were carefully reviewed and analyzed. Because it was not possible to distinguish the precise foci location for domestically mo- bile cases within the country (these cases are also de- fined as indigenous cases), only the foci with exact information were selected for categorization. If 2 or more cases were reported at 1 focus, they were consid- ered and recorded as only one focus because the aim of this study is to classify foci and evaluate the response strategy. For the foci response evaluation, only the indi- genous cases from the IDIRMS that matched the data in the PDIRMS were selected and analyzed. The population data for every county in China from 2010 to 2015 were obtained from the National Bureau of Statistics of China (http://data.stats.gov.cn/). All malaria cases reported were geo-coded and matched to the county-level layers of polygon and point using the software ArcGIS 10.1(Environmental Systems Research Institute, Inc., Redlands, CA). A case contracted locally, with strong epidemiological evidence linking it directly to a known imported case (first-generation local transmission). Recurrence of asexual parasitemia of the same genotype(s) that caused the original illness, due to incomplete clearance of asexual parasites after antimalarial treatment. Results Indigenous malaria in China, 2010–2015 From 2010 to 2015, 5996 indigenous cases were re- corded by the IDIRMS. During this period, the reported indigenous cases sharply declined by 99.1% (2010, n = 4262; 2015, n = 40). Since data of indigenous malaria cases were not available for 2010–2011, the epidemio- logical data for Plasmodium species from 2012 to 2015 were collected. These data were analyzed, and the results indicated that most of the indigenous cases (63.3%) were diseases: malaria, schistosomiasis, and echinococcosis. Malaria was monitored and reported on within the time frame indicators using the “1–3-7” strategy. The “1–3-7” strategy, which refers to the reporting of malaria cases within 1 day, case confirmation and investigation within 3 days, and foci investigation and response to prevent diseases: malaria, schistosomiasis, and echinococcosis. Malaria was monitored and reported on within the time frame indicators using the “1–3-7” strategy. The “1–3-7” strategy, which refers to the reporting of malaria cases within 1 day, case confirmation and investigation within 3 days, and foci investigation and response to prevent Page 4 of 10 Feng et al. BMC Infectious Diseases (2018) 18:115 attributed to P. vivax. Like P. vivax, the number of local P. falciparum cases declined from 97 in 2010 to 1 in 2015 (this strain only occurred in Yunnan Province). some unclear foci investigations (n = 60) and 2 or more cases reported in 1 focus (n = 46), 320 foci were finally obtained and used for foci identification (Fig. 5). Transmission largely decreased during the study period; indigenous cases occurred in 303 counties in 18 provinces in 2010, while in 2015, transmissions occurred in only 9 counties in 4 provinces (Table 3). The 320 foci were investigated and classified as 24 ac- tive foci, 72 residual non-active foci, and 224 cleared-up foci. The 24 active foci were distributed in 6 counties of Yunnan, 1 county of Hainan, 1 county of Liaoning, and 2 counties of Tibet (Table 4). The 72 residual non-active foci were distributed across 15 counties of Yunnan, 2 counties of Tibet, and 2 counties of Anhui. In this study, indigenous cases occurred in 93.6% of the Type I and Type II counties. Foci response evaluation For the foci response evaluation, 43.9% (n = 182) of the 426 indigenous cases selected from the IDIRMS were matched in the PDIRMS (Fig. 5). Further analysis found 32 of them were classified as 2 or more cases reported in 1 focus; therefore, 150 foci were evaluated for foci response. Most indigenous cases (92.5%) were reported in Anhui (n = 2326, 38.8%), Yunnan (n = 1373, 22.9%), Henan (n = 930, 15.5%), Hubei (n = 459, 7.7%), and Guizhou (n = 458, 7.6%). However, in Liaoning and Hainan, where local transmission was blocked for 4 years, officials re- ported 2 local P. vivax and 6 local P. malariae cases in 2015 (Fig. 2). p In 2015, 28 indigenous cases were reported and foci response was carried out. All the active foci were investi- gated within 7 days; 92.9% (n = 26) of foci carried out RACD, and 75% (n = 21) carried out vector investiga- tions (Table 5). Two positive patients were screened by RACD and found to have P. vivax (Yingjiang County) and P. malariae (Sanya County). A total of 101 residual non-active foci were evaluated, and most of them (n = 81, 80.2%) responded within 7 days (Table 5). In addition, 83.2% (n = 84) of the foci carried out RACD, and 78.2% (n = 79) carried out vector investigations. Four people were diagnosed as malaria-positive by RACD, and 83.2% (n = 84) of the foci carried out IRS. For the cleared-up foci, 21 foci were matched in both systems: only 1 focus investigated and responded within 7 days, while 16 carried out RACD and 19 conducted vector investigations (Table 5). Results From 2010 to 2015, transmissions in Type I and Type II counties signifi- cantly decreased, particularly for Type II counties, while in 2015 only 1 Type II county in Dandong city, Liaoning Province, reported indigenous cases (n = 2; Fig. 1). Temporal distribution of indigenous malaria The temporal distribution showed that indigenous mal- aria clustered between May and August. The highest number of transmissions was reported in June 2012, with 63 indigenous cases. The indigenous cases have sharply decreased since 2013: for example, only 12 indi- genous cases were reported between May and August 2015, a 92.7% reduction compared with the same months in 2012 (Fig. 3). The residual auto correlation function (ACF) and residual partial correlation function (PACF) for the indigenous model also proved that the summer season (May–August) was the time frame with the most reported cases (Fig. 4). y cNA indicates that data were not available in the annual reporting system ere not available in the annual reporting system to areas with local infections that are detected in 3 consecutive years with an incidence rate equal to or greater than 1 in 10,000 r to areas with local infections that are detected in 3 consecutive years with an incidence rate lower than 1 in 10,000 in at least 1 of aType I counties refer to areas with local infections that are detected in 3 consecutive years with an incidence rate equal to or greate bType II counties refer to areas with local infections that are detected in 3 consecutive years with an incidence rate lower than 1 in 1 those 3 years those 3 years cNA indicates that data were not available in the annual reporting system o 3 y a cNA indicates that data were not available in the annual reporting system Discussion A total of 426 indigenous cases that were reported be- tween 2012 and 2015 were carefully investigated and an- alyzed for foci identification and classification. Despite During the elimination phase, population-level measures become inefficient and inadequate, so as countries During the elimination phase, population-level measures become inefficient and inadequate, so as countries Table 3 Indigenous cases in China, 2010–2015 Year Total cases Indigenous (%) Local P. v (%) Local P. f (%) No. of counties with transmissions No. of Type Ia counties with transmissions No. of Type IIb counties with transmissions 2010 7855 4262 (54.3) NAc 97 (2.3) 303 71 203 2011 4498 1308 (29.1) NAc 32 (2.4) 155 60 92 2012 2718 244 (9.0) 228 (93.4) 16 (6.6) 41 30 9 2013 4128 86 (2.1) 77 (89.5) 9 (10.5) 12 9 3 2014 3078 56 (1.8) 45 (89.3) 6 (10.7) 10 9 1 2015 3116 39 (1.2) 38 (97.5) 1 (2.5) 9 8 1 Total 25,393 5995 (23.5) 393 32 530 187 309 a f h l l f h d d h d l h Table 3 Indigenous cases in China, 2010–2015 Year Total cases Indigenous (%) Local P v (%) Table 3 Indigenous cases in China, 2010–2015 Feng et al. BMC Infectious Diseases (2018) 18:115 Page 5 of 10 Fig. 1 Indigenous malaria in China, 2010–2015. a 2010, b 2011, c 2012, d 2013, e 2014, and f 2015. The red zones represent the areas where indigenous cases occur (county level) 10–2015. a 2010, b 2011, c 2012, d 2013, e 2014, and f 2015. The red zones represent the areas where Fig. 1 Indigenous malaria in China, 2010–2015. a 2010, b 2011, c 2012, d 2013, e 2014, and f 2015. The red zones represent the areas where indigenous cases occur (county level) b 2 3 4 5 6 7 8 c 9 B C A a 1 Myanmar DPRK Laos Vietnam India Fig. 2 The foci at county-level and its neighboring countries reported in 2015. Discussion a 1 represents Donggang County in Liaoning province neighboring with Democratic People’s Republic of Korea (DRPK), b 2 represents Motuo County in Tibet, 3, 4, 5, 6, 7, 8 represent counties of Lushui, Yingjiang, Mangshi, Zhengkang, Gengma, Cangyuan in Yunnan province, respectively, where neighboring with Myanmar, c 9 represents Sanya County in Hainan province a b b 2 3 4 5 6 7 8 Myanmar Laos Vietnam India c 9 c Fig. 2 The foci at county-level and its neighboring countries reported in 2015. a 1 represents Donggang County in Liaoning province neighboring with Democratic People’s Republic of Korea (DRPK), b 2 represents Motuo County in Tibet, 3, 4, 5, 6, 7, 8 represent counties of Lushui, Yingjiang, Mangshi, Zhengkang, Gengma, Cangyuan in Yunnan province, respectively, where neighboring with Myanmar, c 9 represents Sanya County in Hainan province Fig. 2 The foci at county-level and its neighboring countries reported in 2015. a 1 represents Donggang County in Liaoning province neighboring with Democratic People’s Republic of Korea (DRPK), b 2 represents Motuo County in Tibet, 3, 4, 5, 6, 7, 8 represent counties of Lushui, Yingjiang, Mangshi, Zhengkang, Gengma, Cangyuan in Yunnan province, respectively, where neighboring with Myanmar, c 9 represents Sanya County in Hainan province Feng et al. BMC Infectious Diseases (2018) 18:115 Page 6 of 10 Fig. 3 Temporal distribution of indigenous malaria, 2012–2015. Red zones represent the period from May to August, and blue zones represent the period from September to April (the next year) a single setting and have a high vector capacity for trans- mitting malaria [18]. In addition, there is a large mobile cross-border population since there is no natural barrier in this region, which makes management of imported malaria a significant challenge [19–21]. Poor transporta- tion also makes it difficult to conduct epidemiological studies and blood smear verification within 3 days [22]. In this study, the healthcare workers’ response in these foci was inefficient, although all the cases at the foci were treated within 7 days. First, not all the active foci carried out RACD and IRS, which are required by the national strategy. Second, only three-quarters of the ac- tive foci conducted a vector investigation; without favor- able entomological information, 2 potential risks remain. approach the goal of eliminating malaria, individual- based estimates of transmission must identify foci where resources should be targeted because transmission remains high [16]. Discussion Aggregate ratios of indigenous-to- imported cases in time (or in space) alone, could ob- scure localized transmission if, for example, most cases failed to transmit but some pockets of transmission remain. The border areas are a great challenge for malaria elimination in China because they harbor a mixed en- demic region with both P. falciparum and P. vivax mal- aria transmission [17]. The natural environment in these areas is complex and a variety of malaria vectors, such as Anopheles dirus and An. minimus, usually co-exist in Fig. 4 Residual ACF and residual PACF of indigenous malaria, 2012–2015 Fig. 4 Residual ACF and residual PACF of indigenous malaria, 2012–2015 Feng et al. BMC Infectious Diseases (2018) 18:115 Page 7 of 10 Fig. 5 Data flow for foci identification and response evaluation One is that the imported P. vivax can be re-introduced under conducive ecological conditions (transmission sea- son) if the area harbors an appropriate vector, although there is little evidence of re-introduction of imported malaria in China [23]. Another is the increasing import- ation of P. falciparum to China due to a lack of know- ledge about the efficient vector capacity to sustain imported P. falciparum for the re-establishment of transmission. A foci investigation consists of an assess- ment of potential Anopheles breeding sites, the collec- tion of adult mosquitoes to identify the species responsible for transmission, and the assessment of the vector’s susceptibility to insecticides; these steps are ab- sent in routine surveillance work, but they are required by national guidelines. In addition, 2 malaria-positive pa- tients who were screened by RACD revealed an insensi- tivity for passive case detection, this insensitivity is a particularly difficult issue for some regions where trans- mission was absent for several years, such as Hainan, where there was no transmission in 2011. However, in 2015, 7 indigenous cases were reported in this province [24]. This resurgence shows the significance of surveil- lance systems during the elimination stage, and espe- cially in the post-elimination stage, to monitor for the potential re-introduction of malaria into these areas. Discussion Table 4 Active foci in China (the foci that were investigated in each natural villagea) Foci No.b Province County Township Village Case 1 Yunnan Yingjiang Nabang Jiedao 2 2 Nabang 1 3 Lishu 1 4 Daonong 1 5 Qiaotou 1 6 Kachang Caobei 1 7 Gengma Mengding Hanhong 1 8 Qiushan 1 9 Mangshi Xuangan Qincaitang 1 10 Santaishan Yunqian 1 11 Zhengkang Mengdui Yakou 1 12 Lushui Pianma Pianma 1 13 Canyuan Banlao Banlao 1 14 Tibet Motuo Beibeng Gelin 1 15 Jiangxin 1 16 Beibeng 1 17 Motuo Yadong 1 18 Bodong 1 19 Dexing Dexing 1 20 Linzhi Bayi Yingbin 1 21 Hainan Sanya Fenghuang Lixin 4 22 Baolong 2 23 Nandao 1 24 Liaoning Donggang Jiangzi Zhangdao 2 aA natural village has approximately 50 households with a population of 200 to 250 people and is the lowest administrative level in China. In China, a natural village with a reported malaria case is considered a focus bIf 2 or more cases were reported at 1 focus, only 1 focus was recorded. In this study, more than 1 case was reported in Jiedao, Lixin, Baolong, and Zhangdao; therefore, those 4 sites were considered as 4 foci Table 4 Active foci in China (the foci that were investigated in each natural villagea) The active foci response required a strategy that com- bined RACD with vector control to clear the foci [25]. Given proof that asymptomatic people are present at the China–Myanmar border [26], a more sensitive technol- ogy is required to screen local residents and frequently mobile populations, including temporary workers and il- legal immigrants, who may not routinely use established health services. In China, RACD was performed in the households where a case was identified and neighboring households within a 300-m radius if the focus was con- sidered large (an entire village) [27]. The RDT was aA natural village has approximately 50 households with a population of 200 to 250 people and is the lowest administrative level in China. In China, a natural village with a reported malaria case is considered a focus bIf 2 or more cases were reported at 1 focus, only 1 focus was recorded. In this study, more than 1 case was reported in Jiedao, Lixin, Baolong, and Zhangdao; therefore, those 4 sites were considered as 4 foci Feng et al. Discussion BMC Infectious Diseases (2018) 18:115 Page 8 of 10 Table 5 Evaluation of foci response for the 3 types of foci in China Response Foci classification Activea Residual non-activeb Cleared-up Total cases 28 101 21 Population at riskc 6452 46,809 10,006 No. of foci responding within 7 days (%) 28 (100) 81 (80.2) 1 (4.8) No. of foci that carried out RACD (%) 26 (92.9) 84 (83.2) 16 (76.2) Screened population 1447 2985 712 No. of malaria-positive patients 2 4 0 No. of foci that carried out entomological investigations (%) 21 (75) 79 (78.2) 19 (90.5) No. of foci that carried out IRS 26 (92.9) 84 (83.2) 18 (85.7) aAll the indigenous cases reported were considered active foci. In 2015, 28 indigenous cases were reported, and foci response was carried out for all of them bA total of 101 cases reported from 2013 to 2014 were categorized as residual non-active foci cThe population at risk represents the recorded data for each type of foci Table 5 Evaluation of foci response for the 3 types of foci in China aAll the indigenous cases reported were considered active foci. In 2015, 28 indigenous cases were reported, and foci response was carried out for all of them bA total of 101 cases reported from 2013 to 2014 were categorized as residual non-active foci cThe population at risk represents the recorded data for each type of foci Myanmar) to conduct case diagnosis by use of PCR technology during the transmission season. adopted in the field and all blood samples were taken and sent to the provincial CDC or national CDC for PCR verification. Currently, the CDC staff at the China– Myanmar border use a high-throughput, low-cost, and highly sensitive screening method based on 18S riboso- mal RNA to detect asymptomatic sub-patient infections. In 96-well plates, the samples are quantified by the amount of ligated probes that bind continuously to the 18S rRNA of the genus Plasmodium; this method may offer an alternative for sensitive, large-scale molecular screening that can be used for RACD [28]. Despite a lack of indigenous cases occurring at these foci, there is still the possibility of relapse for P. vivax and P. ovale. A relapse would require that the local CDC staff emphasize the quality of treatment when visiting each house at the time of radical treatment. Discussion An attempt should be made to contact people who were absent dur- ing the healthcare visit. Although it is hard to carry out in the primary health care sectors, especially with minor- ity populations who were present at the China– Myanmar border and in Tibet, the use of glucose-6- phosphate dehydrogenase deficiency assays should also be considered. In China, people who relapsed with P. vivax and P. ovale should undergo directly observed therapy, taking primaquine for radical treatment [29]. After 1 or 2 years without evidence of transmission, the 72 residual non-active foci may be re-categorized as cleared-up foci. All the measures implemented in these foci, e.g., RACD, IRS, targeted mass drug administration (tMDA), and health education, should follow the national guide- lines because they significantly control the foci in the next year. Malaria elimination programs follow-up mal- aria cases reported by health facilities in order to carry out case investigations that will determine the origin of the infection, whether it has been imported or is due to local malaria transmission. All the active foci should carry out RACD and IRS to reduce the reservoir of asymptomatic and low-density infections, and prevent mosquitoes from biting to block transmission. Targeted interventions such as tMDA may be considered for pa- tients, tMDA was performed on a smaller scale, used for close contacts, households, and villages of index cases. In addition, we have also summarized the similarities and differences between the frameworks of the WHO and China. While both frameworks could guide classifi- cation of foci, WHO’s definition and identification of foci mainly classifies foci by tracking them over the last 3 years, and China investigates the response of foci to any current cases, including imported cases. A natural village with any reported cases was assumed to be a focus and was classified based on the Plasmodium spe- cies, season, and vectors. Once an imported case was re- ported, it was checked by microscopy or PCR in a reference lab to determine the Plasmodium species, and then the case was investigated to determine whether it occurred during transmission season. For example, if imported P. vivax was reported during transmission sea- son, the CDC staff would carry out a response appropri- ate for the type of focus, including RACD and IRS, combined with tMDA if it was an active focus. Competing interests Th h d l h The authors declare that they have no competing interests. Acknowledgements We thank all the staffs in the provincial CDC in China. We also thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. 12. Zhang L, Feng J, Zhang SS, Xia ZG, Zhou SS. Malaria situation in the People' s republic of China in 2015. Chin J Parasitol Parasit Dis. 2016;34(6):477–81. 13. Wang L, Wang Y, Jin S, Wu Z, Chin DP, Koplan JP, Wilson ME. Emergence and control of infectious diseases in China. Lancet. 2008;372(9649):1598–605. Authors’ contributions Firstly, not all indigenous cases were well recorded for the exact epidemiological information, especially for do- mestically mobile (within the country) cases; in this study we missed 60 indigenous cases information. Sec- ondly, the 2 web-based reporting systems were set up in different years, which led to a mismatch in the cases posted in these systems. This issue is also a problem with the PDIRMS, which was established in 2012. More time should be invested in integrating the IDIRMS data with the PDIRMS data and in training local CDC staff. Thirdly, we did not know the exact reasons for the delay in foci investigation and response though these are rela- tively minimal, they still need specific correction. Fourthly, because the ArcGis software used for foci mapping at village level is not presently available, and because the presentation of all foci in one map would give a low-resolution picture due to the number of foci (more than 200 foci in 2012 and dozens between 2013 and 2015), in this report we provided the indigenous cases at county level from 2010 to 2015. JF and HT conceived the study and drafted the manuscript. LZ, SSZ (Shaosen Zhang), and ZGX analyzed the data and provided suggestions for improving the quality of the data. HT undertook statistical analysis and model construction. JS carried out the ArcGIS software mapping and data exportation. SSZ (Shuisen Zhou) initiated the study and made major contributions to drafting the manuscript. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript. All authors read and approved the final manuscript. Availability of data and materials All individual cases in this study could be traced through the Infectious Diseases Information Reporting Management System (IDIRMS, http:// chinacdc.cn) or the Parasitic Diseases Information Reporting Management System (PDIRMS, http://chinacdc.cn). Abbreviations d l 6. World Health Organization: A framework for malaria elimination. Geneva: World Health Organization; 2017. ACF: Residual auto correlation function; CDC: Center for Disease Control and Prevention; IDIRMS: Infectious Diseases Information Reporting Management System; IRS: Indoor residual spraying; LLTN: Long-lasting insecticidal nets; NMEAP: National Malaria Elimination Action Plan; PACF: Residual partial correlation function; PCR: Polymerase chain reaction; PDIRMS: Parasitic Diseases Information Reporting Management System; RACD: Reactive case detection; tMDA: targeted mass drug administration; WHO: World Health Organization 7. Zhou SS, Wang Y, Li Y. Malaria situation in the People's Republic of China in 2010. Chin J Parasitol Parasit Dis. 2011;29(6):401–3. 8. Xia ZG, Yang MN, Zhou SS. Malaria situation in the People's Re China in 2011. Chin J Parasitol Parasit Dis. 2012;30(6):419–22. 10. Zhang L, Feng J, Xia ZG. Malaria situation in the People's Republic of China in 2013. Chin J Parasitol Parasit Dis. 2014;32(6):407–13. 11. Zhang L, Zhou SS, Feng J, Fang W, Xia ZG. Malaria situation in the People' s republic of China in 2014. Chin J Parasitol Parasit Dis. 2015;33(5):319–26. Consent for publication Not applicable. Consent for publication Not applicable. References This study used nationwide data to categorize foci in China and evaluate the response of these areas during the control and elimination phases. Our approach strati- fies future control responses by identifying those loca- tions where the elimination of endemic transmission is needed, such as in the counties at the China–Myanmar border and in Tibet. In addition, this study will help local CDC staff to reassess their needs and responses against different types of foci during the elimination and post-elimination phases. 1. Ministry of Health: National Malaria Elimination Action Plan. Beijing: Ministry of Health; 2010. 2. Zhou SS, Zhang SS, Zhang L, Rietveld AE, Ramsay AR, Zachariah R, et al. China's 1-3-7 surveillance and response strategy for malaria elimination: is case reporting, investigation and foci response happening according to plan? Infect Dis Poverty. 2015;4:55. 3. Feng J, Xiao H, Xia Z, Zhang L, Xiao N. Analysis of malaria epidemiological characteristics in the People's Republic of China, 2004-2013. Am J Trop Med Hyg. 2015;93(2):293–9. 4. Hu T, Liu YB, Zhang SS, Xia ZG, Zhou SS, Yan J, et al. Shrinking the malaria map in China: measuring the progress of the National Malaria Elimination Programme. Infect Dis Poverty. 2016;5(1):52. 5. Bousema T, Drakeley C, Gesase S, Hashim R, Magesa S, Mosha F, et al. Identification of hot spots of malaria transmission for targeted malaria control. J Infect Dis. 2010;201(11):1764–74. Ethics approval and consent to participate The data of this study was collected from web-source, so the ethics and participatory consent was not required, and this was approved by National Institute of Parasitic Diseases, Chinese Center for Diseases Control and Prevention. This study was supported National Natural Science Foundation of China (Grant No. 81602904). Discussion Unlike the WHO classification, the purpose of foci classification The residual non-active foci in China cover 19 coun- ties in 3 provinces. Not all the foci investigated and/or responded within 7 days because of poor transportation in Yunnan and Tibet, which caused delays for the CDC staff. To solve this problem, the government has im- proved road infrastructure; thus, the CDC staff could ar- rive at the foci in a timely manner. For remote areas, provincial CDC staff designated a central location at which they carry out to carry out case diagnosis, investi- gation, and treatment. For example, Yunnan Institute of Parasitic Diseases has set up a work station in Nabang village (one village of Yingjiang County, neighboring Page 9 of 10 Page 9 of 10 Feng et al. BMC Infectious Diseases (2018) 18:115 in China is to allow the CDC staff to determine the ap- propriate response so that each patient can be identified early and obtain appropriate treatment. in China is to allow the CDC staff to determine the ap- propriate response so that each patient can be identified early and obtain appropriate treatment. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 15 May 2017 Accepted: 28 February 2018 11. Zhang L, Zhou SS, Feng J, Fang W, Xia ZG. Malaria situation in the People' s republic of China in 2014. Chin J Parasitol Parasit Dis. 2015;33(5):319–26. 10. Zhang L, Feng J, Xia ZG. Malaria situation in the People's Republic of China in 2013. Chin J Parasitol Parasit Dis. 2014;32(6):407–13. Funding Thi d 14. Cao J, Sturrock HJ, Cotter C, Zhou S, Zhou H, Liu Y, et al. Communicating and monitoring surveillance and response activities for malaria elimination: China's "1-3-7" strategy. PLoS Med. 2014;11(5):e1001642. g This study was supported National Natural Science Foundation of China (Grant No. 81602904). Page 10 of 10 15. Feng J, Yan H, Feng XY, Zhang L, Li M, Xia ZG, et al. Imported malaria in Chi 2012 E I f t Di 2014 20(10) 1778 80 15. Feng J, Yan H, Feng XY, Zhang L, Li M, Xia ZG, et al. Imported malaria in China, 2012. Emerg Infect Dis. 2014;20(10):1778–80. China, 2012. Emerg Infect Dis. 2014;20(10):1778–80. 16. Hay SI, Smith DL, Snow RW. Measuring malaria endemicity from intense to interrupted transmission. Lancet Infect Dis. 2008;8(6):369–78. interrupted transmission. Lancet Infect Dis. 2008;8(6):369–78. 17. Clements AC, Barnett AG, Cheng ZW, Snow RW, Zhou HN. Space-time variation of malaria incidence in Yunnan province, China. Malar J. 2009; 8:180. 18. Huang JX, Xia ZG, Zhou SS, Pu XJ, Hu MG, Huang DC, et al. Spatio-temporal analysis of malaria vectors in national malaria surveillance sites in China. Parasit Vectors. 2015;8:146. 19. Moore SJ, Min X, Hill N, Jones C, Zaixing Z, Cameron MM. Border malaria in China: knowledge and use of personal protection by minority populations and implications for malaria control: a questionnaire-based survey. BMC Public Health. 2008;8:344. 20. Li S, Yin S, Wang J, Li X, Feng J. Shifting from control to elimination: analysis of malaria epidemiological characteristics in Tengchong County around China-Myanmar border, 2005-2014. Malar J. 2016;15:45. 21. Xu JW, Li Y, Yang HL, Zhang J, Zhang ZX, Yang YM, et al. Malaria control along China-Myanmar border during 2007-2013: an integrated impact evaluation. Infect Dis Poverty. 2016;5(1):75. 22. Feng J, Liu J, Feng X, Zhang L, Xiao H, Xia Z. Towards malaria elimination: monitoring and evaluation of the "1-3-7" approach at the China-Myanmar border. Am J Trop Med Hyg. 2016;95(4):806–10. 23. Duan YZ, Li SG, Kang XH, Yin SQ, XD S. A point-like outbreak caused by secondary transmission from an imported malaria vivax case. Int J Med Parasit Dis. 2013;40:57–9. 24. Lin C, Chen Z, Wang SQ, Luo PZ, Wu DL, Zhen AJ, et al. Investigation of a rare local epidemic of plasmodium malariae infection in Sanya City. Hainan Province Chin Trop Med. 2016;16(5):481–4. 25. Smith Gueye C, Sanders KC, Galappaththy GN, Rundi C, Tobgay T, Sovannaroth S, et al. Feng et al. BMC Infectious Diseases (2018) 18:115 Funding Thi d Active case detection for malaria elimination: a survey among Asia Pacific countries. Malar J. 2013;12:358. 26. Zhao Y, Zhao Y, Lv Y, Liu F, Wang Q, Li P, et al. Comparison of methods for detecting asymptomatic malaria infections in the China-Myanmar border area. Malar J. 2017;16(1):159. 27. Xiao HH, Liu J, Feng J, Zhang SS, Jiang WK, Xia ZG, et al. Screening radius of active case detection and the malaria parasite rate of carriers in China- Myanmar border. Chin J Parasitol Parasit Dis. 2015;33(2):86–90. 28. Cheng Z, Wang D, Tian X, Sun Y, Sun X, Xiao N, et al. Capture and ligation probe-PCR (CLIP-PCR) for molecular screening, with application to active malaria surveillance for elimination. Clin Chem. 2015;61(1):821–8. 29. National Health and Family Planning Commission: Technical regulations for application of antimalarials. Beijing: National Health and Family Planning Commission; 2016. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
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https://aacr.figshare.com/articles/journal_contribution/Supplementary_Methods_from_i_MLH1_i_Founder_Mutations_with_Moderate_Penetrance_in_Spanish_Lynch_Syndrome_Families/22383956/1/files/39829376.pdf
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Supplementary Methods from &lt;i&gt;MLH1&lt;/i&gt; Founder Mutations with Moderate Penetrance in Spanish Lynch Syndrome Families
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Supplementary Methods (LOH) Allele-specific expression (ASE) and loss of heterozygosity analysis Allele-specific expression (ASE) and loss of heterozygosity analysis PCR fragments encompassing the c.1865T>A change were generated from genomic DNA (gDNA) and cDNA obtained from peripheral blood lymphocytes, or tumor DNA (tDNA) from mutation carriers (Table S1). PCR products were purified using GFXTM PCR DNA and Gel Band Purification kit (GE Healthcare). Allele-specific expression (ASE) of the variant c.1865T>A of MLH1 at the cDNA level was analyzed by single-nucleotide primer extension (SNuPE) using the SNaPshot kit (Applied Biosystems) with primer 5’- TCTGAAGAAGAAGGCTGAGATGC-3’, according to the manufacturer’s instructions. Briefly, reactions were performed in a total volume of 10 µL containing 1.5 µL purified PCR product, 5 µL SNaPshot Ready Reaction Mix, and 0.2 µmol/L extension primer. Primer extension thermocycling conditions consisted of 25 cycles of 96ºC for 10 s, 50ºC for 5 s, and 60ºC for 30 s. SNaPshot reaction products were treated with 1 U shrimp alkaline phosphatase (usb) for 60 min at 37ºC and then 15 min at 75ºC. Products were run in an ABI Prism 3130 DNA sequencer and were analyzed by GeneMapper v4.0 (Applied Biosystems). Samples from c.1865T>A carriers showed a profile with two peaks (green and red, representing A and T alleles, respectively). ASE at cDNA level was calculated as the proportion of T allele between cDNA and gDNA (ASE = f(T)cDNA/ f(T)gDNA), were f(T) was obtained from peak intensities using the formula f(T) = [T allele/(T+A alleles)]. Loss of heterozygosity (LOH) at tumor DNA (tDNA) level was measured as the proportion of T allele between tDNA and gDNA (LOH = f(T)tDNA/ f(T)gDNA). We used gDNA samples from 8 carriers to establish a range for normal ASE and LOH between 0.89 and 1.11 (mean values ± 3·SD). Experiments were performed in duplicate. Estimating L from the count of recombination events The length of the genealogy of the sampled copies of a mutation (L) was obtained by dividing the number of recombination events by the per-generation probability of a recombination on the ancestral haplotype. The recombination map length of the c.306+5G>A haplotype was obtained using the Rutgers Map Interpolator (1). We supplied the interpolator with the position of D3S1298, which was included in the Rutgers smoothed map position file, and we interpolated the position of D3S2369, which was not included. For the interpolation we used the physical position of 36,472,209 bp from release 50 of the Ensembl database (2). We used the difference between the sex-averaged map position of D3S2369 and that of D3S1298 to obtain a map length of r=0.9796 cM for the haplotype. The procedure for the c.1865T>A mutation was similar except that both endpoints of the haplotype appeared in the smoothed map position file, and the haplotype length was r=2.2578 cM. Estimating the height of the genealogy of the sampled copies of each mutant allele Counting recombination events For the c.306+5G>A mutation we counted recombination events in the region spanning microsatellites D3S2369 and D3S1298; for c.1865T>A we chose the region spanning D3S1612 and D3S1298. We only counted recombinations on a subset of disease haplotypes corresponding to those in bold in Table 3 (for further discussion see the subsection below on estimating the genealogy 1 height). For each mutation, all diseased individuals had at least one allele in common in the immediate neighborhood of the disease allele, as would be expected if the mutation occurred on a single shared ancestral haplotype. We chose the most frequently occurring of the distinct disease haplotypes to be the ancestral one and counted the minimum number of recombination events necessary to obtain all of the other haplotypes. The choice of the ancestral disease haplotype does not affect the count of recombination events. When a haplotype was distinct from the ancestral disease haplotype we assumed that it resulted from a recombination, and we assumed that any two identical non- ancestral haplotypes arose from the same recombination event. Estimating the height of the genealogy of the sampled copies of each mutant allele To estimate TMRCA given the estimated genealogy length Lˆ , we simulated the joint probability density of genealogy heights and lengths, f(H,L;N,n). For a constant-sized population under the coalescent, this joint density is determined by the effective population size N and the number n of sampled copies of the mutation (3). Although N is unknown, the ratio of the most likely tree length Lm and the most likely height Hm given this length depends only on n and can be expressed as Lm/Hm=1/c(n) for some function c. Assuming that our genealogy 2 length estimate Lˆ is the most likely length under the true demographic history, we estimate TMRCA as Hˆ =c(n) Lˆ , where c(n) was determined from coalescent simulations. Our estimation procedure for c(n) used 5×107 genealogies simulated under a constant population model with n=17 sampled lineages for the c.306+5G>A mutation and n=12 lineages for c.1865T>A. Simulations were carried out using the program ms (4). We estimated Lm from a histogram of the simulated tree lengths with 1,000 bins. Using a moving window of width 5 bins and step-size 1, we smoothed the histogram by averaging the values within each window, producing a vector of length 996. We then found the maximal element in this vector and averaged the centers of the bins corresponding to that element in order to estimate the location of the maximum of the distribution. Using this method, the estimated most likely gene tree length was Lm(Ebro) = 2.8031 in units of 4N generations (Lm(Jaén)=2.3974). To find Hm, we selected all simulated trees whose lengths fell within a window around Lm, [2.8005, 2.8057] for n=17 and [2.3947, 2.4001] for n=12. Each window was obtained by incrementally increasing the size of a symmetric window around Lm until the number of simulated genealogies with lengths in the window reached or exceeded 1,000. For each mutation the size of the increment was 2×10-4 units of 4N generations (10-4 units of 4N generations in each direction). To estimate Hm from these trees, we employed the same procedure used for estimating Lm, except that because of the smaller number of trees, the histogram for H used 100 bins and the moving window had width 3 and step-size 1. The most likely gene tree height for the given gene tree length was estimated to be Hm(Ebro)=0.6514 in units of 4N generations (Hm(Jaén) = 0.6159). Confidence intervals for TMRCA To obtain a 95% confidence interval for TMRCA for the c.306+5G>A mutation we simulated 100,000 trees under a coalescent of constant size with n=17 lineages (n=12 for c.1865T>A) and recorded the height H and length L of each tree. The height of each tree was then estimated from its length by Hest= cL and the ratio h=H/Hest was computed for each tree. We found the 2.5 and 97.5 percentiles of these 100,000 ratios by ordering them from smallest to largest h(i) (i =1…100,000) and taking h(2,501) and h(97,500). The lower bound of the 95% confidence interval for the estimate of the TMRCA was computed as h(2,501)ĉLˆ+4 and the upper bound was computed as h(97,500)ĉ Lˆ+4. Estimating the height of the genealogy of the sampled copies of each mutant allele Dividing Hm by Lm yielded ĉEbro=0.2324 and ĉJaén=0.2569. Note that although the number of sampled copies of the c.306+5G>A mutation is 42 (40 for the c.1865T>A mutation), because no two of the 17 families (12 families for c.1865T>A) are believed to share a common individual within the last five generations, in the simulations we used n=17 (n=12 for c.1865T>A). Thus, to estimate the TMRCA of the sampled copies of a mutation we chose one haplotype per family to represent the haplotype in the fifth generation from that family, estimated the height of the genealogy that relates these disease 3 haplotypes, and added four generations to the estimated height. The haplotype chosen was the one common to all family members, except in the single case of an intrafamilial recombination in the region considered. In this case, all individuals had a common ancestor in the fourth generation and the disease haplotype of this individual, assumed to be that of her two children, was chosen. 1. Matise TC, Chen F, Chen W, et al. A second-generation combined linkage physical map of the human genome. Genome Res 2007;17:1783-6. 2 Fli k P Ak BL B l K t l E bl 2008 N l i A id R 1. Matise TC, Chen F, Chen W, et al. A second-generation combined linkage physical map of the human genome. Genome Res 2007;17:1783-6. 2. Flicek P, Aken BL, Beal K, et al. Ensembl 2008. Nucleic Acids Res 2008;36:D707-14. 3. Wakeley J. Coalescent Theory: An Introduction. Greenwood Village, Colorado: Roberts & Company Publishers; 2008. 4. Hudson RR. Generating samples under a Wright-Fisher neutral model of genetic variation. Bioinformatics 2002;18:337-8. g p y p g ; 2. Flicek P, Aken BL, Beal K, et al. Ensembl 2008. Nucleic Acids Res 2008;36:D707-14. 3. Wakeley J. Coalescent Theory: An Introduction. Greenwood Village, Colorado: Roberts & Company Publishers; 2008. Supplementary References 1. Matise TC, Chen F, Chen W, et al. A second-generation combine linkage physical map of the human genome. Genome Res 2007;17:1783-6. 1. Matise TC, Chen F, Chen W, et al. A second-generation combined linkage physical map of the human genome. Genome Res 2007;17:1783-6. 2. Flicek P, Aken BL, Beal K, et al. Ensembl 2008. Nucleic Acids Res 3. Wakeley J. Coalescent Theory: An Introduction. Greenwood Village Colorado: Roberts & Company Publishers; 2008. 4. Hudson RR. Generating samples under a Wright-Fisher neutral model o genetic variation. Bioinformatics 2002;18:337-8. 4. Hudson RR. Generating samples under a Wright-Fisher neutral model of genetic variation. Bioinformatics 2002;18:337-8. 4
https://openalex.org/W3002611649
https://revistaseletronicas.pucrs.br/index.php/iberoamericana/article/download/36317/19073
Portuguese
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Sobre a Mulher Escrava no Rio Grande do Sul
Estudos ibero-americanos/Estudos Ibero-Americanos
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* Autora do livro RS: escravlsmo e abolição, Porto Alegre, Mercado Aberto, 1982. Professora do curso de Graduação e Pôs-Graduação de História da Universidade Federal do Rio Grande do Sul. 47 47 F.8tudos Ibero-Amerkanos. PUCRS, XVI(1,2):47-56, jul. e dez., 1990 Sobre a Mulher Escrava no Rio Grande do Sul Margaret M Bakos* Margaret M Bakos* A versão original desta comunicação foi elaborada a partir de um convite da Secretaria Municipal de Cultura, na pessoa do organizador do Curso, professor Mário Maestri, para falar sobre a mulher negra escrava no Rio Grande do Sul. O tema proposto instigou um questionamento inicial: em quais aspectos o escravismo seria diferente para o homem e para a mulher? As relações sociais escravistas coisificam os indivíduos, impedin- do-os de se movimentarem livremente, embotando e inibindo os seus sentimentos e instintos. Homens e mulheres, na escravidão, tomam-se seres de propriedade de outrem, com vontade sujeita à autoridade do dono e seu trabalho ou serviços são obtidos através da coerção. Nesta linha de raciocínio, toma-se difícil delinear e conseqüen- temente falar sobre a situação específica da mulher no cativeiro, pois a base estrutural da sua existência não está em ser fêmea. Está em ser escrava. Será impossível identificarmos, a nível estrutural, uma posição masculina ou feminina na escravidão? Toda diferença entre o papel do homem e da mulher no escravismo será sempre conjuntural e relacionada ou com a atividade que o negro exerce, ou com o momento hist6rico que vivencia. Homens e mulheres negros escravizados no Rio Grande do Sul, ao longo de dois séculos, F.8tudos Ibero-Amerkanos. PUCRS, XVI(1,2):47-56, jul. e dez., 1990 Estudos Ibero-Americanos, XVl( 1,2) - 1990 Estudos Ibero-Americanos, XVl( 1,2) - 1990 48 sofreram, capitularam e reagiram ao cativeiro através de ações, mais ou menos cooperativas ou agressivas, independentemente do seu sexo, relacionadas basicamente com as variáveis apontadas. A minha proposta nesta apresentação não é, portanto, estabelecer um inventário, no meu entender forçadíssimo, das diferenças entre homens e mulheres escravizados. A tendência, nesta perspectiva, é a de situar aos primeiros como heróis em potencial, em constante enfren- tamento com o jugo senhoril, caracterizando as segundas como vítimas duplas, da passividade inerente ao sexo e da violência dos senhores, conforme as retrataram inúmeras vezes os historiadores positivistas. Impõe-se uma nova visão, pois não é assim que vejo as mulheres escravas, genericamente falando. Acredito que entre os escravos encontramos gente assumida, sensível e combativa, mas também homens e mulheres entregues aos desmandos dos senhores, interna e profundamente auto- coisificados, independente do seu sexo. Ao tentar resgatar aspectos do engajamento feminino na luta contra a opressão, analiso três momentos definidos na relação escravista: I - No Trabalho A idéia desta abordagem originou-se a partir de um debate que se estabeleceu, ao final de uma palestra que proferi, em 1988, em Porto Alegre. Eu falara sobre a mulher escrava na capital do Estado e, para ilustrar o encontro, li alguns anúncios, publicados em jornais do século passado, que ou solicitavam ou ofereciam escravas negras para serviços domésticos. Muitos dos anúncios solicitavam que a serviçal fosse real- mente habilitada como cozinheira. Neste momento, uma das assistentes, estranhando o fato, espontaneamente, comentou que sempre pensara que as negras fossem "naturalmente" boas cozinheiras e competentes domés- ticas, de um modo geral. O questionamento que me ocorreu em torno desta intervenção foi sobre as origens históricas deste pensamento que, possivelmente, ainda se haja presente na cosmovisão de muitos gaúchos. O escravo negro começou a ser introduzido no Rio Grande do Sul nos finais do século XVIII, com vistas a atender as necessidades das charqueadas sulinas. Aos poucos começou-se a utilizar o escravo para as atividades de peonagem e agricultura. Nestes primeiros tempos, o que mais importava era possuir um escravo negro, fisicamente apto para estas Sobre a mulher escrava no ... 49 atividades. Foi com o desenvolvimento dos centros urbanos, a partir principalmente da primeira metade do século XIX, que a mulher escrava passou a ser uma peça cobiçada e fundamental para a sociedade gaúcha. Uma das poucas estatísticas que temos, e que diferencia a presença numérica dos escravos homens e mulheres, data de 1859. Ela arrola 25 cidades sedes de municípios, salientando-se como localidades que mais possuem escravos as seguintes: atividades. Foi com o desenvolvimento dos centros urbanos, a partir principalmente da primeira metade do século XIX, que a mulher escrava passou a ser uma peça cobiçada e fundamental para a sociedade gaúcha. Uma das poucas estatísticas que temos, e que diferencia a presença numérica dos escravos homens e mulheres, data de 1859. Ela arrola 25 cidades sedes de municípios, salientando-se como localidades que mais possuem escravos as seguintes: Localidade Porto Alegre Pelotas Jaguarão Rio Grande Cruz Alta Homens 4.556 3.095 2.929 2.503 2.315 Mulheres 3.861 1.693 2.127 1.866 1.704 Localidade Porto Alegre Pelotas Jaguarão Rio Grande Cruz Alta Vemos assim que nestas localidades urbanas havia uma presença de escravos negros, homens e mulheres, significativa, o que revela a necessidade de ambos para diferentes trabalhos. I - No Trabalho Uma análise do seu valor de compra, no mesmo ano (1859), nos permite resgatar que a diferença de preço também não era muito significativa: Preços de escravos negros no Rio Grande do Sul em 1859 Preços de escravos negros no Rio Grande do Sul em 1859 Para a agricultura Homens 1.250$000 Mulheres 1.150$000 Para serviços domésticos Homens 1.400$000 Mulheres 1.250$000 Para a agricultura Homens 1.250$000 Para serviços domésticos Homens 1.400$000 Para serviços domésticos Se as mulheres negras eram numericamente importantes e econo- micamente valorizadas, resta perguntar para que atividades eram des- tinadas. Talvez uma das melhores fontes para obter esta resposta seja justamente a análise dos anúncios de jornais. O O Diário de Porto Alegre, de 1827, é uma das mais antigas fontes para este tema e aponta cerca de 21 atividades para as quais o trabalho escravo era requisitado, em Porto Estudos Ibero-Americanos, XVI( 1,2) - 1990 50 Alegre. As mulheres eram procuradas para trabalharem como cos- tureiras, doceiras, lavadeiras, engomadeiras, cozinheiras, amas de leite, arrumadeiras, fiadeiras e refinadoras de açúcar. Com vistas a responder ao questionamento inicial, é importante chamar atenção para alguns ângulos sob os quais esta série de anúncios pode ser analisada: Alegre. As mulheres eram procuradas para trabalharem como cos- tureiras, doceiras, lavadeiras, engomadeiras, cozinheiras, amas de leite, arrumadeiras, fiadeiras e refinadoras de açúcar. Com vistas a responder ao questionamento inicial, é importante chamar atenção para alguns ângulos sob os quais esta série de anúncios pode ser analisada: a) A mulher escrava foi utilizada como mão-de-obra em dezenas de atividades designadas, para fins públicos, como domésticas, ligando-se a estas remotas origens uma série de preconceitos, que pode ser resu- mida no ditado popular machista e racista que diz ter a mulher negra um lugar garantido: "no palco, na cama e na cozinha, sem fazer con- curso público e com estabilidade assegurada". (O Tição, Ano 1, out. 80, p.3) b) O que chama a atenção nesta coletânea de anúncios, em número de 46, é que 13 relacionam-se com a atividade de fazer comida. Assim, não fica difícil entender por que foi tão profundamente internalizada, nas pessoas, a relação escrava negra \ cozinha. A ponto de alguém, no ano de 1988, ainda considerar que todas as negras eram em princípio boas cozinheiras. Entretanto, é justamente aí que se pode buscar os aspectos da resistênciavcoerçâo ao trabalho, que, em última análise, assinalam a semelhança entre homemvmulher, na escravidão. I - No Trabalho Explicando melhor, tanto faz, num regime de trabalho compulsório, se se é escravovescrava; a diferença entre um e outra não se faz por aí. Ser senhor'(senhora isto é o que importa. Os pólos da relação escravista, independente do seu sexo, dominam ou ao chicote ou apenas ao seu próprio corpo. Sob esta ótica repensei os dados e, em base às fontes que enfatizavam a preparação para o trabalho doméstico, que se exigia das escravas, tentei recuperar o processo de ensino'(aprendizagem que muitas negras devem ter passado entre o fogão e a despensa, desesperadas por não saberem o que fazer com panelas e víveres, que não conheciam, dos quais não gostavam e que eram obrigadas a cheirar, cozinhar, servir e comer. O chicote, a senzala e o terror foram certamente os mestres mais eficientes do período e os responsáveis pelo nossa preconceituosa idéia de que todas as negras são "naturalmente" boas cozinheiras. Até este momento, não se tem uma análise exaustiva da documentação que evidencie tais conclusões. Entretanto, isto não justifica que se aceite a História que forma este- reótipos, tomando como verdadeiras afirmações geradas pela tradi- ção oral e pela leitura simplista dos documentos, sem críticas e questionamentos. A cozinheira, a copeira e a babá permitiram que ai Sobre a mulher escrava no ... 51 senhora se alimentasse e que a sua família se perfumasse, se banhasse, namorasse nas janelas e saraus e procriasse novas sinhazinhas. É através da análise do sistema escravista que se evidencia a formação, a ferro e fogo, das habilidades "naturais" da mulher negra, cuja excelência somente será questionada quando a mulher imigrante empobrecida vier a disputar com a negra o trabalho doméstico, principalmente o urbano. Entretanto, até os dias de hoje, ainda são comuns os ditados populares, que a tradição oral preservou, e que nos fazem olhar com simpatia ou resistência seres humanos, com relação à sua capacidade do trabalho. Quem nunca ouviu: "Negra de canela fina é que é boa trabalhadeira!"? 11- Na resistência Considero como resistência toda reação de inconformidade do negro quanto à escravidão. Isto inclui desde tentativas de fugas, formação de quilombos, agressões contra senhores e feitores, assassinato dos próprios filhos, suicídios até o "corpo mole" inocente e preguiçoso, o "dengue" doce que alicia e comove, influenciando o humor dos circun- dantes. Esta abrangência se justifica pela impossibilidade de saber, atualmente, se o sorriso de submissão não teria sido tão falso quanto o famoso e decantado riso do palhaço de circo. Não há nenhum trabalho sistemático sobre fugas, quilombos e criminalidade escrava no Rio Grande do Sul que permita uma compa- ração estatística entre o número de mulheres e homens que fugiram. Este estudo seria difícil pelas inúmeras variáveis que teriam de ser levadas em conta. Resta, pois, exemplificar a resistência feminina, relendo fontes, até agora, utilizadas para generalizações em torno da resistência escrava. Um dos documentos mais antigos sobre a revolta dos escravos no Rio Grande do Sul refere-se a um episódio ocorrido na Real Feitoria do Linho cânhamo, em 2 de agosto de 1822. Nesta ocasião, houve uma denúncia de que havia pretos desta fazenda que costumavam roubar na vizinhança, sendo destacado um grupo de soldados para prendê-los, porém eles nada puderam fazer, pois os negros não se entregaram à prisão e, instigados por suas mulheres, que gritavam para que eles matassem os soldados, entraram nas suas moradias, armaram-se e atacaram a guarda que terminou voltando para os seus quartéis. (Feitoria Nacional do Linho Cânhamo, lata 300 - AHRGS.) ~ Estudos !bero-Americanos, XVI(1,2) -1990 Estudos !bero-Americanos, XVI(1,2) -1990 52 Neste episódio, resgata-se a negra que grita e instiga, que nada teme na luta contra o branco armado; mas não é preciso ficar apenas nesta inferência, podemos ver, além disso,a companheira de uma luta maior contra a escravidão, que se expressa quando ela incita uma atitude agressiva. Leitura nesta direção pode ser feita sobre o significado da participação feminina na história do quilombismo gaúcho. No quilombo do Negro Lucas, em Rio Grande, em 1833, par- ticipavam seis homens e quatro mulheres, que viviam em uma casa grande, onde preparavam sebo, graxa e charque para vender. Nos quilombos de Rio Pardo foram indiciadas sete mulheres que, juntamente com treze homens, viviam de roubos nas plantações das vizinhanças. 11- Na resistência Para viver em um quilombo era, primeiramente, no mínimo, necessário que o escravo enfrentasse a problemática de sua fuga, que era mais ou menos difícil, dependendo das atividades que ele exercia, do grau de vigilância que sofria e, naturalmente, da sua constituição física, que devia ser a melhor possível, pois que ele deveria percorrer longos percursos, alimentar-se esporadicamente, quando encontrasse o que comer, e sofrer de muita ansiedade com a ameaça permanente de enfrentamento aberto com autoridades constituídas - como o fami- gerado capitão de mato - até chegar a um refúgio. Essa foi também a saga da mulher quilombola, naturalmente no caso de ela ter ido espontaneamente para o quilombo. Em tal caso, há necessidade de esboçar um quadro mais realista de um ato de resistência? Até mesmo os representantes do sistema, com melhores vestimentas, montarias e esquemas de sobrevivência, amargavam na incumbência de perseguir os negros fugidos, como é sobejamente ilustrado na documentação oficial. Então, porque não valorizarmos ao máximo a ação quilombola como um alto de extrema coragem e valentia? Se eles não tinham o objetivo de transformar a sociedade, como afirma Gorender, pelo menos, a hos- tilizavam. Se buscavam recompor a sociedade tribal africana, segundo pensamento do mesmo autor, homens e mulheres partilhavam do mesmo desejo. Naturezas femininas e masculinas, sob a escravidão, interagiam com o medo, planejavam atingir o estado utópico, o nirvana de qualquer cativo, em qualquer época histórica: a liberdade! Há alguns, verdadeiramente não muitos, relatos de casos em que as escravas resistiram ao assédio de negros, seus companheiros de cativeiro, como o incidente que culminou no assassinato da esposa de um feitor Sobre a mulher escrava no ... 53 negro - em fazenda do Alegrete - porque ela não cedeu a um escravo que queria forçá-Ia "para fins libidinosos". (Relatório do Presidente da Província do Rio Grande do Sul João Capistrano Miranda Castro, 1848, Porto Alegre, Tipografia Porto Alegrense.) O ato que ficou registrado no processo crime aberto contra o assassino nos permite, agora, rever a vítima no seu comportamento corajoso, que a levou à morte, desmis- tificando a idéia romântica da mulher por índole submissa. Em uma pesquisa sobre os cárceres de Porto Alegre, na primeira metade do século XIX, encontrei mulheres negras aprisionadas ou pelo simples arbítrio de seus senhores ou por terem cometido crimes. (Bakos, 1982: 209-221.) Apenas na cadeia de Justiça, foram encontradas 13 escravas negras presas. 11- Na resistência Impossível saber quais os sofrimentos físicos e mentais, as torturas, que elas sofreram na passagem de um espaço para o outro, pois o cenário maior dos quadros continuava a ser o escravismo. Havia dorvchicote, fedorvsujeira, em todos os locais destinados aos negros. Nos autos dos processos, ficou registrado: um machado foi desviado, da lenha para a senhora, que na visão da escrava, foi umajuíza injusta dos seus atos. Um punhado de veneno para ratos foi parar nas baixelas senhoris. Tudo por mãos de negras, tudo para exteriorizar sentimentos de raiva, medo, ódio: pura emoção. Com esta leitura, não pretendo desculpar ou minorar um ato agressivo, muito menos justificar o emprego da violência nas relações humanas. Ao contrário, vejo a capacidade de agredir física ou mentalmente ao seu semelhante como a pior das características humanas. Por esta razão, por ser constitutiva e essencial na luta pela vida, a violência pode ser reduzida a um viés de análise histórica. Entender o porquê da violência, na visão de quem a pratica e a sofre, pode ser mais um meio para o historiador resgatar o sentido do jogo social pela vida. As respostas de Felisberta e Florinda são muito eloqüentes: "agredi, porque fui agredi da sem motivo", "agredi, mas não queria machucar, só queria assustar". Poderíamos analisar dezenas de processos como estes, mas não estão aí resumidos, apenas nestes dois, os princípios básicos do ser humano no seu relacionamento em sociedade? Para isto, não foi necessário que as negras saissem às ruas, fazendo parte de grupos armados, praticando violências planejadas. As cenas protagonizadas por Felisberta e Florinda aconteceram entre as paredes de casas, onde viviam pessoas ordeiras. Naturalmente ali se reproduziam as relações de trabalho escravistas, com muita crueza, pois na privacidade do espaço íntimo, as autoridades são mais presentes, despóticas, viciosas, impunes. Entretanto, no esquema rotineiro, uma 'coisa' saiu do seu lugar. Tanto faz se era uma 'coisa' masculina ou feminina. Interessa que ela saiu; com isso, mexeu com a ordem, mostrou que não era só 'coisa', era gente e igual a toda gente: homem ou mulher. Felisberta e Florinda, protagonistas da mudança, nossos objetos de análise, conseguiram, à época, com suas atitudes, saírem do espaço privado da casa. Se elas queriam sair, nunca saberemos. Mas o fato aí está para ser analisado: elas foram conduzidas a um fórum, foram interrogadas, ouvidas. Suas vozes ficaram registradas nos autos dos processos, chegaram até nós. 11- Na resistência A seguir, discrimino dois crimes de que escravas foram acusadas, no Rio Grande do Sul, procurando com isto apenas exemplificar o pen- samento de que as mulheres reagiram, como os homens, à escravidão, agredindo os seus senhores com as armas de que dispunham. Julgo importante apontar a existência de centenas de processos crimes envolvendo negras escravas ou livres, nos arquivos públicos do Rio Grande do Sul, os quais ainda não foram analisados ou o foram precariamente. Importantes referenciais sobre o significado do ato de agressão da cativa podem e devem ser inferidos das leituras destes processos, que se constituem, fora de dúvida, na mais rica fonte disponível para a compreensão das atitudes do escravo de ambos os sexos, resgatando para a atualidade elementos constitucionais das naturezas feminina e masculina. Dados do Escravo Ano Motivo Felisberta 1821 Castigada S. Gabriel sem motivo Florinda 1828 queria só P. Alegre ameaçar 1828 P. Alegre Florinda 54 Estudos Ibero-Americanos, XVI(1,2) -1990 Estudos Ibero-Americanos, XVI(1,2) -1990 As respostas de Felisberta e Florinda são muito eloqüentes: "agredi, porque fui agredi da sem motivo", "agredi, mas não queria machucar, só queria assustar". Poderíamos analisar dezenas de processos como estes, mas não estão aí resumidos, apenas nestes dois, os princípios básicos do ser humano no seu relacionamento em sociedade? Para isto, não foi necessário que as negras saissem às ruas, fazendo parte de grupos armados, praticando violências planejadas. As cenas protagonizadas por Felisberta e Florinda aconteceram entre as paredes de casas, onde viviam pessoas ordeiras. Naturalmente ali se reproduziam as relações de trabalho escravistas, com muita crueza, pois na privacidade do espaço íntimo, as autoridades são mais presentes, despóticas, viciosas, impunes. Entretanto, no esquema rotineiro, uma 'coisa' saiu do seu lugar. Tanto faz se era uma 'coisa' masculina ou feminina. Interessa que ela saiu; com isso, mexeu com a ordem, mostrou que não era só 'coisa', era gente e igual a toda gente: homem ou mulher. Felisberta e Florinda, protagonistas da mudança, nossos objetos de análise, conseguiram, à época, com suas atitudes, saírem do espaço privado da casa. Se elas queriam sair, nunca saberemos. Mas o fato aí está para ser analisado: elas foram conduzidas a um fórum, foram interrogadas, ouvidas. Suas vozes ficaram registradas nos autos dos processos, chegaram até nós. 11- Na resistência Impossível saber quais os sofrimentos físicos e mentais, as torturas, que elas sofreram na passagem de um espaço para o outro, pois o cenário maior dos quadros continuava a ser o escravismo. Havia dorvchicote, fedorvsujeira, em todos os locais destinados aos negros. Nos autos dos processos, ficou registrado: um machado foi desviado, da lenha para a senhora, que na visão da escrava, foi umajuíza injusta dos seus atos. Um punhado de veneno para ratos foi parar nas baixelas senhoris. Tudo por mãos de negras, tudo para exteriorizar sentimentos de raiva, medo, ódio: pura emoção. Com esta leitura, não pretendo desculpar ou minorar um ato agressivo, muito menos justificar o emprego da violência nas relações humanas. Ao contrário, vejo a capacidade de agredir física ou mentalmente ao seu semelhante como a pior das características humanas. Por esta razão, por ser constitutiva e essencial na luta pela vida, a violência pode ser reduzida a um viés de análise histórica. Entender o porquê da violência, na visão de quem a pratica e a sofre, pode ser mais um meio para o historiador resgatar o sentido do jogo social pela vida. 55 Sobre a mulher escrava no ... Ill- No engajamento social Uma outra instância em que a mulher escrava pode ser analisada é no seu engajamento na sociedade. Um bom exemplo é a sua participação na Confraria de Nossa Senhora do Rosário, fundada em Porto Alegre, em 1786, que tinha como principal objetivo a educação religiosa e a benemerência, mas que também funcionava como um local de agru- pamento dos negros e, muitas vezes, para conspirações ou ataques espontâneos à ordem constituída. Assim, embora institucionalizadas, as confrarias eram muitas vezes perseguidas e as suas procissões e festas proibidas e os seus membros visados pela polícia local. Mesmo assim, a presença da mulher negra livre ou escrava era uma constante, prevista, inclusive no Regimento da Confraria, mas da qual ela participava com todo o seu engajamento, conspirando também, como o homem ... Conclusão A submissão da mulher foi uma invenção de um mundo comandado por homens que também inventaram a escravidão. (Mott, 1988: 21) Mas, subjacente a estes garrotes ideológicos e concretos, a mulher, cons- titucionalmente, tinha forças e meios para resistir, com garra e oportunis- mo, tal como o homem. Por isto ela não merece ser registrada historicamente como dupla vítima do sistema. O que realmente lhe é devido é uma revalorização de seus atos, concedendo-lhes a devida dimensão e mérito. Isto apenas será possível se entendermos que a mulher escrava podia resistir ao jugo senhoril, de uma forma diferente que o homem. Se aceitarmos como resistência coisas do cotidiano, muito simples, como a indolência no trabalho, a dificuldade no aprendizado de novas tarefas, o tratamento "dengoso" que dispensava ao senhor e à sua família. Ao lado destas atitudes que a livravam dos excessos de trabalho e dos castigos, a escrava negra, como vimos, também agrediu a sociedade escravista: fugiu, furtou, matou, como todas no país. Foram protagonistas da História da escravidão negra no Brasil, vítimas, mas também agres- sores, todos os seres humanos vindos da África e os seus descendentes, independentemente do seu sexo: impossível fazer um recorte e falar sobre a mulher escrava especificamente, sem desenvolver uma argumentação machista! Estudos Ibero-Americanos, XVI(1,2) -1990 Estudos Ibero-Americanos, XVI(1,2) -1990 56 Bibliografia citada BAKOS, M. Os escravos negros nos cárceres de Porto Alegre: Sugestões de uma documentação do século XIX. In Revista de Estudos Ibero Americanos. PUCRS, 8(2):209-23, 1982. -. RS: Escravismo e Abolição. Porto Alegre, Mercado Aberto, 1982. CARDOSO, C. Escravo ou Camponês? São Paulo, Brasiliense, 1987. GIACOMINI, S. Mulher e Escrava. Rio de Janeiro, Vozes, 1988. GORENDER, J. Questionamento sobre a teoria econômica do escravismo colonial. In Estudos Econômicos. 13(1):7-41, 1983. MAESfRI, M.J. O Escravo no Rio Grande do Sul. Caldas do Sul, Escola Superior São Lourenço dos Brindes, 1984. MOTI, Mário Lúcio. Submissão e Resistência. São Paulo, Contexto, 1988. MOTIA, F. Crime e rebeldia escrava no Rio Grande do Sul (1820-1845). Trabalho de conclusão de bacharelado. UFRGS, jan. 1985.p.47. [Mimeo1 REIS, J.R. & SILVA, E. Negociação e Conflito. Rio de Janeiro, Cia. das Letras, 1989. SCHWARTZ, S. Mocambos, quilombos e palmares: a resistência escrava no Brasil Colonial. In Estudos Econômicos. 17(especial), 1987.
W4291275963.txt
https://dergipark.org.tr/tr/download/article-file/1985787
de
Verbal Folklore About Death in Uzbeks
DergiPark (Istanbul University)
2,021
cc-by-sa
3,245
Kültürel Miras Araştırmaları – 2021: 2(2); 60-65 Kültürel Miras Araştırmaları https://dergipark.org.tr/tr/pub/kulmira e-ISSN 2687-6094 Özbeklerde Ölümle İlgili Sözel Folklör Shohkrukh Tuychibaev *1 1Erciyes Üniversitesi Sosyal Bilimler Enstitüsü Kültürel Çalışmaları Anabilim Dalı, Kayseri, Türkiye Anahtar Kelimeler Özbekler, Ölüm, Ağıt, Atasözü. ÖZ Çalışmamızda Orta Asya’daki en kalabalık Türk halkı olan Özbek Türklerindeki ölümle ilgili inanış ve uygulamaların sözel folklöre nasıl yansıdığı değerlendirilmiştir. Çalişmada nitel yöntem olarak yoğun bir şekilde etnografı çalışması gerçekleştirilmiştir. Bu kapsamda ölüm sırasında söylenen ağıtlar incelenmiş, çeşitleri ve halk arasında nasıl kullanıldığı araştırılmıştır. Makalede Özbekler arasında başsağlığı anlamında kullanılan ibare ve ifadeler de derlenmiş, ölüm olgusunun beddualara yansımasıyla ilgili örnekler sıralanmıştır. Ayrıca çalışmada ölüm olgusuyla ilgili Özbek atasözleri de ele alınmış ve değerlendirilmiştir. Verbal Folklore About Death in Uzbeks Keywords Uzbeks, Death, Lament, Proverb. ABSTRACT The study evaluates the beliefs and practices related to death and its reflection in verbal folklore of the Uzbeks, the most populous Turkic people in Central Asia. In the study, an intensive ethnography study was carried out as a qualitative method.In this context, the laments sung at the time of death were examined, its types and forms of use among the people were investigated. The article also compiles the phrases and expressions used to mean condolences among the Uzbeks, and it lists the examples of the reflection of the death phenomenon on curses. In addition, Uzbek proverbs related to the phenomenon of death were also discussed and evaluated in the study. *Sorumlu Yazar *(shohruhtuychi@gmail.com) ORCID ID 0000-0002-8793-2622 Araştırma Makalesi (Research Article) Kaynak Göster (APA); Tuychibaev S (2021). Özbeklerde Ölümle İlgili Sözel Folklör. Kültürel Miras Araştırmaları Dergisi, 2(2), 60-65. Geliş Tarihi: 21/09/2021; Kabul Tarihi: 22/10/2021 Kültürel Miras Araştırmaları – 2021; 2(2); 60-65 1. GİRİŞ 1. Merhum yakınlarının cesedin evi terk edene kadar gamla ve yüksek sesle ağıt yakarak ağlaması. 2. Başsağlığı için gelen kişiyle görüşme sırasında ağlamak. 3. Sabah ‘ses çıkartıp’ ağlamak. 4. Merhumun mezarı üzerinde ağlamak (bu çeşit ağlama ölen kişinin senesi geçene kadar devam eder). 5. Anma merasimlerindeki ağlama (Payziyeva, 2014). Kaynaklara göre kadınların yüksek sesle, saçlarını yayarak ve kendini vurarak ağlama âdeti eski Sakalar, Sogdlular, Harezemliler arasında da çok yaygındı (Yöldaşeva ve Payziyeva, 2002). Hâlihazırda başka halklarda olduğu gibi Özbeklerde de kadınların saçlarını yayarak gezmeleri bir anlam ifade etmeyip, aksine bazen tarz olarak görülmekte olsa da, anneler zaman zaman kızlarının saç yaymalarını eleştirmekte, “öldüğümde saçlarını yayacaksın” ya da “ben öldüm mü ki saçını yayıyorsun” ifadeleri kullanılmaktadır. Özbek halkında ölümle ilgili bir takım gelenek ve görenekler bulunmaktadır. Ölümün acısını ifade edebilme, matem ritüelleri sırasında sözel ağıtları kullanma, ölüm olayıyla ilgili düşünce ve kaygıları yakinen tanımakta yardımcı olabilecek alan Halk Bilimidir. Özbek Folklorundaki ölümle ilgili ağıtlar, atasözleri, deyim ve bedduaları değerlendirirken halkın bu konudaki bakış açısı, birikimi ve tasavvuru daha net anlaşacaktır. Özbeklerde de başka halklarda olduğu gibi ölüme yönelik ilk yankı ağlama olarak ifade edilmektedir. Ağlama sesini duyan komşular ilgili evde hoş olmayan bir olay yaşandığı düşüncesiyle yardıma koşmaktadırlar. Nitekim Özbeklerde özellikle kadınlar arasında birisinin vefatı haberini duyunca haber veren de haberi alan da beraber ağlamaktadırlar. Özbeklerde ağlama ve ağlama sırasında merhumu anarak söylenen şarkılar yani ağıtların faydaları hakkında belli inanışlar bulunmaktadır. Ona göre, ölen için ağıtın çok yararlı olduğu, ağıt söylenmeden defnedilen birisi için öteki dünyanın kapıları açılmadığı düşünülmektedir. İnanışa göre ağlama şarkıları aracılığıyla ruhlar dünyasına onların sayısı bir taneye daha çoğaldığı haberi verilmekte, bu haberi alan ruhlar da ağıtı söylenen merhumun yoluna çıkıp onu karşılamaktadırlar (Örayeva, 2004). Eski Türk halklarında kadınlar başta eşi olmak üzere diğer yakın akrabaları için de ağıtlar söyler, saçlarını yolar, yüz ve kulaklarını tırnaklarıyla delerlerdi (Aydarov, 1971). Çın kaynaklarına göre, eski Türkler yakın kişileri vefat ettiğinde kurbanlık için getirilen hayvanları özellikle at ya da koyunu yurt önüne getirip, at üzerine binerek yurt etrafını yedi kez dolaştırır, sonra yurda girmeden önce bıçakla yüzlerini deler, ses çıkarıp ağlarlardı (Biçürin 1950). Kadınların ağlamasına ilişkin saç yayma ve kendini acı yaşatmanın farklı şekilleri Panjikent’teki eserlerde, Tekkale’de bulunan çömleklere işlenen resimlerde de yansımıştır (Rempel, 1987). P Maev’in notlarında Taşkentli bayanların yas sırasında saçlarını yayıp, yüzlerini yolarak ağladıkları aktarmaktadır (Maev, 1876). S.S.Gubayeva da Fergana Özbeklerine göre ağlama ve kendine acı verme sırasında yüzlerinin kanamaması ayıp sayıldığı bilgisini vermektedir (Gubayeva, 1991). Aslında ağlayarak ağıt söyleme eyleminin de kendi düzeni bulunmaktadır. Ağıt; kime yönelik söylendiği, söyleyen ve söylenen arasındaki bağa göre değişmektedir. Nitekim babaya, anneye, kardeşlere, eşlere ve akrabalara göre söylenen ağıtların metni ve içeriği değişiklik göstermektedir. Ağıt söylenirken merhumun yaptığı iyilikler, onun erdemleri, özellikleri dile getirilmekte, ölmesinden ötürü duyulan acı dinleyicilerle paylaşılmaktadır. Ağıt söyleyen kadınlara Özbekçe de “aytuvchi” denir. Benzer görevli kişiler Fergana vadisindeki bölgelerde de bulunmakta, burada onlara “göyanda” denilmektedir (Aşirov, 2007). Fergana bölgesindeki matem merasimlerinde ses çıkartıp ağlamak sadece kadınlar için değil belki oğul çocuklar için de söz konusudur (Karmişeva, 1986). M. Payziyeva defin etme merasimlerinde ağlamakla bağlı aşamaları beşe ayırır: 1.1. Ağıtlardan Örnekler Ağıtı genelde kızlar, anneler ve kız kardeşler söyler. Söylerken her satrın sonunda ölen kişiye yönelik işaret verilir. Metinde “voy otam, voy onam” yani “vay atam, vay anam”, “voy bolam” yani “vay evladım”, “voy jigarim” yani “vay ciğerim” gibi sözler yer alır. Tablo 1. Yakınını kaybeden birisinin durumunu anlatan ağıt (Örayeva, 2004). Özbekçe Ağıt -Qop-qora sochim manim, -Ustumga yoydirgan o’lim. -Qip-qizil yuzim manim, Türkçe Tercümesi -Kapkara saçım benim, -Üstüme yaydıran ölümdür. - Kıpkızıl yüzüm benim, - Maydonda so’ldirgan o’lim. -Meydanda solduran ölümdür. Tablo 2. Baba için söylenen ağıt örneği Özbekçesi -Suv degan sharbat tutgan, voy otam, -Non degan novvot tutgan, voy otam, -Tomorqaga tollarni ekib ketgan, voy otam, - Tol hivichdek bellarimni bukib ketgan voy otam, -Tol bargidan xatlar qilay, jonim otam, - O’tirgan o’rinlaridan gul ungan, voy otam, -So’zlaridan bol to’kilgan, voy otam, -Mehribonim maslahatim, voy otam. 61 Türkçe Tercümesi -Su denen şerbet tutan, vay atam, - Nan denen navvat tutan, vay atam, -Tarlaya dallarını dikip giden, vay atam, - Dal kırbaç gibi bellerimi büküp giden, vay atam, -Ağaç yaprağından mektup yapıyım, atam, -Oturan yerinden çiçek çıkan, vay atam, -Sözlerinden bal dökülen, vay atam, -Mihribanım, maslahatım, vay atam. Kültürel Miras Araştırmaları – 2021; 2(2); 60-65 Tablo 3. Anne için söylenen ağıt örneği Özbekçesi -Kelar edim o’tday yonib, mehribonim, voy onam, -Ko’rar edim, mehrim qonib, mehribonim, voy onam, -O’tdi davron, ko’chdi karvon, ul davru davronlar qaydadur. -Shundayin mehribonimni sog’insam, Endi ko’rmoq qaydadir. -Dardim bilgan, dardkashim, voy onam, -G’amim bilgan g’amkashim, voy onam, -Uylarimning farishtasi, voy onam, -Hovlimizning sarishtasi, voy onam. Tablo 6. Fergana yöresine ait bit ağıt (Safarov ve Örayeva, 2007). Türkçe Tercümesi -Gelir idim ateş ışık saçıp, şefkatliyim, vay anam, -Görür idim, sevgim kanıp, şefkatliyim, vay anam, -Geçti devran, göçtü kervan, o devri devranlar nerededir, -Böyle şefkatlimi özlesem şimdi görmek nerededir, -Derdimi bilen dert paylaşanım, vay anam, -Gamımı bilen gam paylaşanım, vay anam, -Evlerimin meleği, vay anam, - Avlumuzun temizi, vay anam. -Mulki jahonim, voy bolam. -Mülki cihanım, vay balam. -Senin için anayı zar eda oldu, yar yar, -Yürek kana doldu ve kara oldu, yar yar, -Gelinliğin çeyizi kefen oldu, yar yar. Özbekçesi -Osmondagi oyim edingiz onama, -Jahondagi kunduzum onama, -Baland toğlar past boldimi, -G’amg’usorim onama, Türkçesi -Gökteki ayım idiniz anam ya, -Cihandaki gündüzüm anam ya, -Yüksek dağlar kısa oldu mu? -Öldüğünüz gerçek oldu mu? -Gamımı gideren anam ya, -Xaridorim onama, -Su desam choy bergan, -Non desam novvot bergan onama. -Müşterim anam ya, -Su desem çay veren, -Nan desem, şeker veren anama. -O’lganingiz rost bo’ldimi, Tablo 5. Evladı vefat ettiğinde annelerin söylediği ağıtlardan örnek (Payziyeva, 2014). Türkçe Tercümesi -Bahçecimin bahçıvanı, vay balam, -Evciğimin sultanı, vay balam, -Boz evimi boş bırakıp gittin, vay balam, -Annesine acı verip giden, vay balam, -Tahtı revanım, vay balam, -Sen uchun onayizor ado bo’ldi, yor yor, -Yurak qonga to’ldiyu qaro bo’ldi, yor yor. -Kelinligin sarpasi endi aza yor yor. Tablo 7. Andican yöresine ait bir ağıt (K.K, 17, 2019). Türkçe Tercümesi -Dar caddede koşan, vay ciğerim, -Ekranında şaşan, vay ciğerim, -Arığın kenarı vişnedir, vay ciğerim, -Seni özlesem yürek bağrım yanıyor, ciğerim, -Ak değil miydi bileklerin, vay ciğerim, -Allah’dan çok değil miydi dileklerin, vay ciğerim. Özbekçesi -Boqqinamning bog’boni, voy bolam, -Uyginamning sultoni, voy bolam, -Bo’z uyimni bo’shatib ketding, voy bolam, -Onasini qaqshatib ketgan, voy bolam, -Taxtı ravonım, voy bolam, Türkçesi -Bağlam bağlam saçların, toprak oldu, yar yar, Taşkent-Fergene bölgelerindeki Özbeklerde merhum defin edildikten sonra üç güne kadar sabah erkenden ağlamak, ses çıkarmak âdeti vardır. Genelde bu tür görenek merhumun kızı ya da kız kardeşleri tarafından yapılır. Günümüzde böyle alışkanlıklar Taşkent bölgesinde kaybolmaktayken Fergana vadisi Özbekleri, özellikle Andicanlılar bu âdeti yaşatıp gelmektedir. Andican Özbeklerinde ağıt yakma mutlaka yapılması gereken uygulamadır. Bu gelenek bugünlerde sıklıkla görülmektedir. Tablo 4. Kardeş için söylenen ağıt örneği Özbekçesi -Tor ko’chada talpingan, voy jigarim, -Tenglaridan adashgan, voy jigarim, -Ariq bo’yi chiyadir, voy jigarim, -Seni eslasam yurak bağrim kuyadir, jigarim. -Oq emasmidi bilaklaring, voy jigarim, -Ollohdan ko’pmasmidi tilaklaring, voy jigarim. Özbekçesi -O’ram o’ram sochlaring, tuproq bo’ldi, yor yor, Andican’daki çalışmalarımız sırasında kaynaklarımız yerel imamların bazen İslamiyet’e aykırı olan ağıtları eleştirdiği ve söyleyenlerden bunu durdurmalarını talep ettiklerini belirtti. Bunun neticesinde ağıt söyleyenler metinleri dinsel içerikle zenginleştirip İslam kurallarına daha yakın şekilde dönüştürüp söylemektedirler. Tablo 8. İslami kurallara uygun bir ağıt (K.K, 11, 2019). Özbekçesi -Jannatlardan joy bersin sizga onama, -Ollohimning bandasi, -Payg’ambarning ummati onama, -Ollohimga bandalar ko’paytirgan onama, -Payg’ambarga ummat kopaytırgan onama. Merhumun kızı, annesi ya da kardeşlerinin söylediği ağıt en etkili olarak değerlendirilir. Ağıt söyleyenler kendileriyle beraber cenaze törenine katılanları da etkilemekte ve ağlatmaktadır. Matem merasimlerinde ağıt söylemenin başka bir çeşidi gevrek görmeyenlere yöneliktir. Ona “yar-yar” ağıtı denilir. “Yar-yar” aslında bir düğün şarkısının adıdır. Ama böyle durumda düğün görmeyen gencin bu dünyadan “Yar-yar”sız geçmemesi niyetiyle, onun ağıtına bu sözler eklenip söylenir. Bu tür ağıtlar daha çok Fergana bölgesinde görülmektedir. Türkçesi -Cennetlerden yer versin size anam ya, -Allah’ımın bendesi, -Peygamberin ümmeti anam ya, -Allah’ıma bendeler çoğaltan anam ya, -Peygamber ümmetini çoğaltan anam ya. 1.2. Ölümle İlgili Deyimler, Beddualar ve Atasözleri Özbekler acılı aileleri teskin etme sırasında bazı özel ibare veya deyimleri kulannamaya özen gösterirler. Bununla beraber ölüm olayıyla ilgili yaygın kullanılan 62 Kültürel Miras Araştırmaları – 2021; 2(2); 60-65 deyimler de bulunmaktadır. Aşağıda bunların bazılarını değerlendirmeye çalışacağız. Allah’ın iradesi… Başsağlığı dilerken en çok kullanılan deyimlerden sayılır. Eve başsağlığı için gelenler çoğunlukla bu ibare kullanılır. Bu arada Türkçedeki “Başın sağ olsun” ibaresi Özbekçe biraz farklı anlam ifade etmektedir. Başın taştan olsun, başın sağ ve selamet olsun deyimleri daha çok genç yaşındakilere iyi ve sağlıklı ömür görmesini dileme sırasında kullanılmaktadır. Allah sabır versin. Bu da bir başsağlığı mesajını içeren deyimdir. Bendeçilik imiş… Özellikle Taşkent yöresinde akrabaların başsağlığı dilediği zaman kullanılan en etkili deyimdir. “Bende” sözü köle anlamına gelmekte olup, insanın kendi hayatı üzerinde karar veremediği, Büyük Yaradan’a bağlı olduğu gerçeğini hatırlatarak ölünün yakınlarını teselli etmeye çalışılmaktadır. Canı çıkmak. “Öldü” anlamında kullanan bir deyimdir. Bu da aslında ölüm süreci insanın canının bedeninden çıkmasıyla gerçekleştiği, dolayısıyla ruh hakkındaki düşünce Özbeklerin ibarelerine de yansıdığını ifade eder. Rızkı uzulmak. “Uzulmak” fiili Özbekçede “kesilmek”, “kopmak” anlamını verir. Bu deyim de “öldü” anlamında kullanılır. Deyim, herkesin Yaradan tarafından belirlediği rızkı bulunduğu inancını ifade etmektedir. Ona göre rızık tükendiği an ölüm gerçekleşmektedir. Kaza kıldı. Aslen Arapça olan çok anlam ifade eden “kaza” filinin manalarından biri de ölmektir. Özbekçede de birisinin öldüğü haberi verilirken “öldü” yerine daha kibar olan “kaza kıldı” fiili kullanılır. Gökerip çıkmak. Göz yummak. Ölmek anlamını ifade eder. Ölüm ile oynaşmak. Çok tehlikeli işlerle uğraşılan durumlarda kullanılır. Öz eceli ile ölmek. Ağır hastalık sonucu veya yaşlılık dolayısıyla önceden beklenen bir ölüm gerçekleştiğinde kullanılır. Ecelinden beş gün evvel ölmek. Genç birisinin veya ölümü beklenmeyen, sağ salim olduğu halde aniden ölen durumlarda kullanılır. Namusa ölmek. Birisi çok utanılacak bir duruma düştüğünü ifade eden deyimdir. Öleyim eğer. Bir yemin deyimidir. Muhatabı inandırmak istendiğinde genellikle cümle bu deyimle başlanır. Ölenin gününden. Normal bir durumda yapılmayan ve çok ihtiyaç duyulduğundan dolayı yapılan bir işte kullanılır. “Ölenin gününden bir şey yapmak” hiç yapmak istemediği şeyi yapmak zorunda kaldığını bildirir. Ölenin üstüne gömmek veya ölenin üstüne tepmek. Ardı ardına uğursuzluk olduğu zaman veya çok ağır durumda olan bir kişinin haline dikkat etmeden onu incitme gibi durumlarda kullanılır. Ölmeyen benim canım. Zorluklara bakmadan dayanabilme anlamında kullanılır. Bu kadar yıl boyunca ölmeden dayandım, bundan sonra da dayanacağım demektir. Bir ayağı torda, bir ayağı mezarda. Can çekişip ölüm döşeğinde yatanlara yönelik kullanılır (K.K, 5, 2019). 1.3. Beddualar Özbekler arasında beddua sırasında ölüm olayına işaret eden cümle ve ifadelerin kullanıldığı görülmektedir. Aşağıda bunların bazılarına değineceğiz. E öl! Özbekler arasında çok kullanılır. Aslında bir beddua olsa da, duruma ve söylem tarzına göre anlamı değişebilir. Şöyle ki, çok yakın ve samimi arkadaşlar şakalaştığı, birbiriyle alay ettiği zaman rahatsız olduğunu ifade etmek için “e öl” veya daha hafif şekilde “ölekal” sözlerini kullanır. Ama birisi cidden kırılınca öfkeyle “öl”, “ölüp git” gibi sözleri kullanması, tam anlamıyla beddua olarak değerlendirilebilir. Lahitte çürüyesin. Özbeklerdeki mezar şeklini ele aldığımızda eski Türklerde olduğu gibi mezarın yanında lahit denilen bir odacık kazıldığını öğrenmekteyiz. Bu bedduada da tam o lahit kastedilmekte ve insanın lahde düşüp çürümesine işaret edilmektedir. Çok ağır bedduadır. Çok öfkelenildiği zaman düşmanlara, çok zarar verenlere nispeten kullanılır. Ölüğün çıksın. Özbekler arasında çok ağır bir bedduadır (K.K, 12, 2019). Allah canını alsın. Bir önceki bedduayla aynı anlamı taşır. Normalde birisi yakın insanına yönelik bu bedduayı kullanmaz. Gûr çağırsın. Gûr, Özbekçede mezar anlamında kullanılmaktadır. Dolayısıyla bu beddua “mezar çağırsın” anlamını taşır. Önceki üç beddua gibi sert anlam ifade eder, düşmanlara ve acı verenlere yönelik kullanılır. Yer yutsun. Önceki beddualar gibi anlam ifade eder. Serttir. Yaşşamagur. Türkçede “yaşamayasın” anlamını vermektedir. Genelde yaramazlık yapan gençleri, çocukları uyarmak, azarlamak için kullanılır. Önceki dört beddua kadar sert anlamlı değildir (K.K, 8, 2019). Ağzından kanın gelsin. Çok sert anlamlı bedduadır. Genellikle büyükler tarafında çok öfkelenildiği zaman kullanılır. Karnın yarılsın. Çok sert bir bedduadır. Genellikle düşman kadınlara yönelik kullanılır (K.K, 5, 2019). 1.4. Atasözleri Araştırmalar Özbek atasözlerinde de ölümle ilgili ifade ve anlamların bulunduğunu göstermektedir. Aşağıda böyle atasözlerinin bazılarını incelemeye çalışacağız. Uzaktaki akrabadan yakınındaki komşu iyidir. Özbekler arasında komşulara ayrıca değer verilmekte ve bununla ilgili çok atasözü bulunmaktadır. Komşuların uzaktaki akrabalardan da daha yararlı olduğuna işaret eden bu atasözü de kişinin başına bir iş geldiğinde ve özellikle ailede acılı bir gün, ölüm gibi hadiseler yaşandığında duyan ve yardıma koşan ilk kişinin komşu olacağı, dolayısıyla komşularla daima iyi davranılması, onların değeri bilinmesi gerekmektedir. Öldü, aziz oldu. Genelde diriyken insanın değerli bilinmemekte, ölünce de artık onun dönmeyeceği gerçeği anlaşılınca kadri ve önemi fark edilmektedir. Öz evim, ölüm döşeğim. Başkalarının evinde misafir olmak iyidir, ama yine de en rahat olduğunuz yer kendi evinizdir. Çünkü burası sizin yuvanız, ölümünüze dek yaşayacağınız bir mekândır. 63 Kültürel Miras Araştırmaları – 2021; 2(2); 60-65 Çocuklu ev pazardır, çocuksuz ev mezardır. Çocuk olan evde her zaman gürültü, ses ve canlılık vardır. Çocuklar her şeyi merak etmekte, ebeveynlerine sormakta, bazen sıkıntı da çıkarabilmekte, rahat bırakmamaktadır. Buna rağmen rahat yaşayım diye çocuksuz olmayı tercih etmek isabetli değildir. Çünkü çocuksuz ev, sessizliği, üzüntüsü ve kederiyle tam da bir mezarı hatırlatmaktadır. Öldürse de pilav öldürsün. Burada pilav derken aslında lezzetiyle nam salan Özbek pilavı kastedilmektedir. Özbekler kendi pilavını çok sevmekte ve haftada birkaç kez evde pilav yapılıp tüketilmektedir. Pazar, çarşı ve lokantalardaki ana yemek de çoğunlukla Özbek pilavıdır. Dolayısıyla bu atasözüyle Özbeklerin değeri haline gelen pilav övülmekte, birisi gelip “öldüreceğini” söylese de pilavın değeri, önemi, itibarı hiç düşmeyeceği ifade edilmektedir. “Bir pulun kalsa da pilav ye, bir günün kalsa da pilav ye” atasözü de aynı anlamda kullanılır. Ölüm şahla fakire beraberdir. Ölüm insanın toplumdaki makamı, mevkiine bakmaksızın herkesin başında olan bir olaydır. Fakirle şahın hayattaki yaşam şartları çok farklı olabilir, ama ölünce her ikisi de aynı elbiseyi, kefeni giymekte, aynı araçta, tabutta yola çıkmakta ve aynı boyuttaki yere, mezara konulmaktadır. Ölüm kaş ila kavağın arasındadır. Normalde günlük hayatımızı sürdürürken ölüm denilen bir gerçeğin bulunduğunu çoğu kez unutabilmekteyiz. Oysa ölüm her beklenmedik an gelebilmekte. Ölüm eğer gerçekleşmesi Yaratan tarafından kararlaştırıldıysa kaş ile kavağın arasındaki mesafe kadar bizlere yakındır, hemen gelebilmektedir. Ölüm kapını çalarak gelmez. Yukarıdaki atasözüne benzer anlam taşır. Ölümün beklenmedik an gelebileceğini ifade eder. İhtiyar ölse, devlet; genç ölse, kıyamettir. Yaşlı birisinin, hayatta çok şeyi gören ve hayatın tadını çıkarıp ihtiyar durumuna gelen birisinin ölmesi normal bir durumdur. Zaten herkes yaşlanınca ölmektedir. Nitekim birisi evlenmiş, çoluk çocuk ve torunlarını görüp onları büyütmüş ve bundan sonra ölmüşse, bu aslında bir bahttır. Aksine genç birisinin ölmesi de çok acılı bir durumdur. Çünkü her bir ana baba genç çocuğunun büyümesini, mutlu ve saadetli bir hayat yaşamasını, yaşlandıklarında kendilerine yardımcı olmasını arzu etmektedir. Evde kalan eş seçmez, ölen kişi yer seçmez. Evde kalan kızların hayalleri kiminle olsa olsun bir an önce evlenmek iken ölen kişi aynı şekilde hızla toprağa kavuşması gerekir. Giden gelir, küreklenen (gömülen) gelmez. Birisi gittiyse dönebilir, ama ölen kişini beklemek mantıksızlıktır. Düğün şarkı, yas ağıt. Düğünler şarkılarla güzeldir, insanların eğlenmesine vesile olmaktadır; yaslarda da ağıtların insanların acısını hafifletmektedir. Batur ölse namı kalır, namert ölse neyi kalır. Hayatta fedakârlıklarla yaşayan, vatanı ve milleti için can verenlerin adı her zaman anılır, namı kalır; namertlik eden, adamları kıran birisini de öldükten sonra kimse hatırlamaz. Yamana ölüm yok, yahşiye tözüm. Yaman sözü Özbeklerde kötü, yahşi, sözü de iyi anlamını verir. Bu atasözü de çoğu kez hatta kötülükler yapan insanların cezasız kaldığı, iyi insanların da yaptıklarına rağmen hep acı içinde yaşadıkları, tözüm yani huzur bulamadıkları, başkalarının derdini paylaşadıklarını ifade etmektedir. Dert başka, ecel başkadır. İnsan bir derde yakalandığı zaman öleceğini düşünmemesi, yakınmaması, dertle güreşmesi gerekir. Çünkü ölüm ecelle bağlıdır. Ancak eceli gelen birisi ölmektedir. Çok ağır olan hastaların ayağa kalktığı ve sağ salim olan birisinin beklenmedir an yaşamını yitirdiği durumlarla hayatta karşılaşılmaktadır. Öldüğünde mezarın, diriyken gönlün geniş olsun. İnsanın öldüğünde huzur bulması yani mezarının “geniş” olması için hayattayken iyilikler yapması, herkese kızmaması, kıskanmaması, açık ve geniş gönüllü olması gerekmektedir. Düşünmeden yiyen, ağrımadan ölür. İnsan yemeye düşkün olmaması, yemeklerinin çeşidine, sağlıklılığına dikkat etmesi gerekir. Birisinin düşünmeden her şeyi yemesi, öğünlerinin arasına dikkat etmemesi ağır sonuçları getirebilmektedir (Mirzayev vd., 2005). 2. SONUÇLAR Sonuç itibariyle Özbeklerde ölümle ilgili tasavvurlar sözel folklöre de yansımıştır. Bunun başında ölüler için ağıt söyleme geleneği gelmektedir. Ağıt, halkın taziye merasimlerindeki ve uygulamaların belli bölümleri kuşaktan kuşağa aktarılan gelenek ve tecrübeyle yapılmış ve öğrenen kişilerin bazıları için bir meslek halına gelmiştir. Ağıt söylemlerinin metin içeriğine dikkat edilirse söyleyen kişinin vefat edenle helalleşme arzusu ortaya çıkmaktadır. Özbek atasözleri ve deyimleri incelendiğinde halkın ölüme yönelik bakış açısının ne kadar tedbirli ve ciddi olduğunu görmek mümkün. Ayrıca Özbekler arasında kullanılan çoğu beddua ifadelerinin de ölüm olayına işaret ettiği görülmüştür. Yazarların Katkısı Bu çalışmada tüm katkı yazara aittir. Çıkar Çatışması Beyanı Yazarlar arasında herhangi bir çıkar çatışması bulunmamaktadır. Araştırma ve Yayın Etiği Beyanı Yapılan çalışmada araştırma ve yayın etiğine uyulmuştur. KAYNAKÇA Aşirov A (2007). Özbek Halkıning Kadimiy Etikad ve Merasimleri. Taşkent: Alişer Nevaiy Namidegi Özbekistan Milliy Kütüphanesi Neşriyati, 112. Öreyeva D (2004). Özbek Matem Meresimleri Folklori. Taşkent: Fen Neşriyati, 15-27. Payziyeva M (2014). Özbeklerning An'anaviy ve Zamonoviy Matem Meresimleri. Taşkent: Yangi Naşr Neşriyati, 54-58. 64 Kültürel Miras Araştırmaları – 2021; 2(2); 60-65 Safarov O & Örayeva D (2007). Bözlardan uçgan ğazalay. Özbek Halk Yiği va Yöqlovlari. Buhara, 86. Sözlü Kaynaklar K.K, 5. Terzi. Taşkent şehri, Yunusabad ilçesi. Halkabağ mahallesi. 2019 K.K, 8. Ev hanımı. Fergana il, Beşarik ilçesi. 2019. Yöldoşeva G M, Payziyeva M (2002). Oilaviy Marosimlar. Toşkent Mahallalari: Ananalari va Zamonaviylik. Taşkent: Yangi Asr Avlodi, 205. K.K, 11. Ev hanımı. Andıcan il, İzbaskan ilçesi. 2019. Mirzayev T, Musoqulov A & Sarimsoqov B (2005). Özbek Xalq Maqollari. ‘Şarq’,241. K.K, 12. Öğretmen. Taşkent şehri, Şeyhantahur ilçesi. Çiğatay mahallesi. 2019. Aydarov G (1971). Yazik Orhonskih Pamyatnikov Drevnetyurskoy Pismennosti VIII v.15. K.K, 17. Ev hanımı. Andıcan il, İzbaskan ilçesi. Luğubey köyü 2019. Biçurin N (1950). Sobranie Svedeniy O Norodah, Obitavşih v Sredney Azii v Drevnie C1, 230. Gubaeva S (1991). Naselenie Ferganskoy Dolinı v Konse XIX Nachala XX v. Taşkent 54. Karmişeva B (1986). Arhaiçeskaya Simvolika v Pohoronno-Pominalnoy Obryadnosti Uzbekov Fergani // Drevnie Verovaniya i Kulti Narodov Sredney Azii. M: Nauka, 149. Maev P (1876). Aziatskiy Taşkent // Materiali Dlya Statistiki Turkestanskogo Kraya. 4,312. Rempel L (1987). Sep Vremeni. Vekovie Obryadi i Brodyaçie Syujeti v Tradisionnom, 163. © Author(s) 2021. This work is distributed under https://creativecommons.org/licenses/by-sa/4.0/ 65
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The work pattern of personal care workers in two Australian nursing homes: a time-motion study
BMC health services research
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University of Wollongong University of Wollongong Research Online Research Online Faculty of Engineering and Information Sciences - Papers: Part A Faculty of Engineering and Inform Sci 1-1-2012 The work pattern of personal care workers in two Australian nursing The work pattern of personal care workers in two Australian nursing homes: a time-motion study homes: a time-motion study Siyu Qian University of Wollongong, sq992@uowmail.edu.au Ping Yu University of Wollongong, ping@uow.edu.au Zhen-Yu Zhang University of Wollongong David M. Hailey University of Wollongong, dhailey@uow.edu.au Pamela J. Davy University of Wollongong, pjd@uow.edu.au See next page for additional authors University of Wollongong University of Wollongong Research Online Research Online Faculty of Engineering and Information Sciences Faculty of Engineering and Information Sciences - Papers: Part A Follow this and additional works at: https://ro.uow.edu.au/eispapers Publication Details Publication Details Qian, S. Y., Yu, P., Zhang, Z., Hailey, D. M., Davy, P. & Nelson, M. (2012). The work pattern of personal care workers in two Australian nursing homes: a time-motion study. BMC Health Services Research, 12 (305), 1-3. Keywords Keywords era2015 Keywords Keywords era2015 Disciplines Disciplines Engineering | Science and Technology Studies Abstract Abstract Background: Background: The aim of the study is to describe the work pattern of personal care workers (PCWs) in nursing homes. This knowledge is important for staff performance appraisal, task allocation and scheduling. It will also support funding allocation based on activities. Methods: Methods: A time-motion study was conducted in 2010 at two Australian nursing homes. The observation at Site 1 was between the hours of 7:00 and 14:00 or 15:00 for 14 days. One PCW was observed on each day. The observation at Site 2 was from 10:00 to 17:00 for 16 days. One PCW working on a morning shift and another one working on an afternoon shift were observed on each day. Fifty-eight work activities done by PCWs were grouped into eight categories. Activity time, frequency, duration and the switch between two consecutive activities were used as measurements to describe the work pattern. Results: Results: Personal care workers spent about 70.0% of their time on four types of activities consistently at both sites: direct care (30.7%), indirect care (17.6%), infection control (6.4%) and staff break (15.2%). Oral communication was the most frequently observed activity. It could occur independently or concurrently with other activities. At Site 2, PCWs spent significantly more time than their counterparts at Site 1 on oral communication (Site 1: 47.3% vs. Site 2: 63.5%, P = 0.003), transit (Site 1: 3.4% vs. Site 2: 5.5%, P < 0.001) and others (Site 1: 0.5% vs. Site 2: 1.8%, P < 0.001). They spent less time on documentation (Site 1: 4.1% vs. Site 2: 2.3%, P < 0.001). More than two-thirds of the observed activities had a very short duration (1 minute or less). Personal care workers frequently switched within or between oral communication, direct and indirect care activities. Conclusions: Conclusions: At both nursing homes, direct care, indirect care, infection control and staff break occupied the major part of a PCW’s work, however oral communication was the most time consuming activity. Personal care workers frequently switched between activities, suggesting that looking after the elderly in nursing homes is a busy and demanding job. Keywords Keywords era2015 Disciplines Disciplines Engineering | Science and Technology Studies * Correspondence: ping@uow.edu.au 1School of Information Systems and Technology, University of Wollongong, New South Wales 2522, Australia Full list of author information is available at the end of the article © 2012 Qian et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Authors Authors Siyu Qian, Ping Yu, Zhen-Yu Zhang, David M. Hailey, Pamela J. Davy, and Mark Nelson This journal article is available at Research Online: https://ro.uow.edu.au/eispapers/155 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Open Access The work pattern of personal care workers in two Australian nursing homes: a time-motion study Si-Yu Qian1, Ping Yu1*, Zhen-Yu Zhang1, David M Hailey1, Pamela J Davy2 and Mark I Nelson2 Abstract Background: The aim of the study is to describe the work pattern of personal care workers (PCWs) in nursing homes. This knowledge is important for staff performance appraisal, task allocation and scheduling. It will also support funding allocation based on activities. Methods: A time-motion study was conducted in 2010 at two Australian nursing homes. The observation at Site 1 was between the hours of 7:00 and 14:00 or 15:00 for 14 days. One PCW was observed on each day. The observation at Site 2 was from 10:00 to 17:00 for 16 days. One PCW working on a morning shift and another one working on an afternoon shift were observed on each day. Fifty-eight work activities done by PCWs were grouped into eight categories. Activity time, frequency, duration and the switch between two consecutive activities were used as measurements to describe the work pattern. Results: Personal care workers spent about 70.0% of their time on four types of activities consistently at both sites: direct care (30.7%), indirect care (17.6%), infection control (6.4%) and staff break (15.2%). Oral communication was the most frequently observed activity. It could occur independently or concurrently with other activities. At Site 2, PCWs spent significantly more time than their counterparts at Site 1 on oral communication (Site 1: 47.3% vs. Site 2: 63.5%, P = 0.003), transit (Site 1: 3.4% vs. Site 2: 5.5%, P < 0.001) and others (Site 1: 0.5% vs. Site 2: 1.8%, P < 0.001). They spent less time on documentation (Site 1: 4.1% vs. Site 2: 2.3%, P < 0.001). More than two-thirds of the observed activities had a very short duration (1 minute or less). Personal care workers frequently switched within or between oral communication, direct and indirect care activities. Conclusions: At both nursing homes, direct care, indirect care, infection control and staff break occupied the major part of a PCW’s work, however oral communication was the most time consuming activity. Personal care workers frequently switched between activities, suggesting that looking after the elderly in nursing homes is a busy and demanding job. Background aged care services. This requires a basic knowledge about which work activities are currently undertaken by direct care workers and how much time each activity ac- tually takes to meet a resident’s care needs. The growing ageing population has resulted in an in- creasing number of residents, especially the very old and frail, in residential aged care facilities (RACFs) [1]. This requires an increase in the number and intensity of the aged care services. The situation is worsened by a chronic shortage of direct care workers, on whom these people rely to live [2]. All of this represents a big chal- lenge to the delivery of aged care services in RACFs. Personal care workers (PCWs) make up the largest proportion (70%) of the direct care workers in RACFs. Because of the challenge of attracting registered nurses (RNs), the number of PCWs is increasing steadily in Australia [3]. Personal care workers have a minimum qualification of Certificate III in Aged Care awarded by the Technical and Further Education (TAFE) college sys- tem in Australia. They are the major providers of per- sonal care to residents, especially the activities of daily living (ADL) which are one of the important care needs supported by the Aged Care Funding Instrument (ACFI) An important strategy to address this challenge is to effectively design work activities to optimally deliver * Correspondence: ping@uow.edu.au 1School of Information Systems and Technology, University of Wollongong, New South Wales 2522, Australia Full list of author information is available at the end of the article Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Page 2 of 8 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 [4]. The ACFI assesses the day-to-day core care needs of a resident to determine the level of subsidy. a mean with standard deviation to indicate its variability. The switch between two consecutive activities includes the number of occurrence of a switch and the direction of this switch. These four measurements were used in this study to describe a PCW’s work pattern. Work sampling and time-motion techniques have been applied extensively to measure the work pattern in healthcare settings [5-12]. The former has a relatively small cost, but is not able to capture some important in- formation such as the duration of an activity because the observation is not continuous [13]. Background The latter allows precise time to be recorded for each activity, but this is labor-intensive and costly [14]. Ethical approval Ethical approval was granted by the Human Research Ethics Committee of the University of Wollongong based on written approval given by the two participant aged care organizations which run the two nursing homes. Settings g A time-motion observational study was conducted at two nursing homes. The first nursing home was located in Sydney and was owned by a not-for-profit organization which operates 23 RACFs. The observation was conducted in a 32-bed high-care wing (Site 1) staffed by one half-time and four full-time PCWs and one RN. The other nursing home was a stand-alone, not-for-profit facility in Newcastle with 108 beds. The observation was conducted in a 25-bed high-care wing (Site 2) in which three PCWs and one RN took care of 23 residents (two beds were empty at the time of the study). Previous studies have investigated the work pattern in hospital settings [6-8,10,11]. For example, Cornell et al. [7] inspected nurses’ workflow and their computer use in two acute care medical-surgical units in a general hospital in the USA. They found that nurses frequently switched between activities and the duration of most of the activities was very short and highly variable. A recent study described the work activities of bedside intensive care unit nurses in a private hospital in Australia [11]. Nurses spent most of their time on direct care and indir- ect care and they undertook two activities simultan- eously for almost half of their time. Classification of personal care workers’ activities Although there has been much research on the work pattern in hospital settings, few such studies have been undertaken in RACFs. Among the studies undertaken in RACFs, some only focused on particular activities (e.g. bathing-related care) [5,12,15]. Munyisia et al. [9] exam- ined the time expenditure on different types of activities performed by direct care workers by conducting a work sampling study in a high-care house and a low-care house of an RACF. They found that in both houses, oral communication was the most time-consuming activity (32.4%-51.9%). This study provides a comprehensive overview of what the direct care workers do and how they spend their working time, however it was confined to a single RACF and because it is a work sampling study, the duration of each activity or the switches which occur between activities could not be determined. The observational study requires a predefined classifica- tion of activities. Our research team has developed and applied an activity classification system of direct care workers in a longitudinal work sampling study con- ducted in an Australian nursing home [9,18-20]. This work activity classification system was further developed and revised through three focus group discussions with three researchers (including the researchers who devel- oped it) and three RNs who had extensive experience working in aged care. The final classification system contains 58 activities grouped into eight categories: direct care, indirect care, infection control, documentation, transit, staff break, oral communication and other activities not included in the previous categories. The activities in each category are presented in Table 1. This study aims to accurately describe the work pat- tern of PCWs in two high-care RACFs. These are similar to nursing homes in the USA in terms of the level of care provided to residents. Previous studies used differ- ent measurements to describe the work pattern [7,10,11,16,17]. The commonly used measurements are activity time, frequency, duration and the switch be- tween two consecutive activities. Inter-rater reliability Activity time is expressed by two parameters: (1) the time an activity takes over an eight-hour shift and (2) the percentage of time used to complete an activity in relation to the total amount of time for all activities. Ac- tivity frequency is the number of occurrences of an ac- tivity during a set period of time (e.g. an hour). Activity duration, usually assessed in seconds, is the length of time continuously spent on an activity. It is presented as Our observation was conducted by a single observer. To ensure the reliability of the observation process, our ob- server and a second observer, who has extensive experi- ence in conducting observational studies, independently observed and recorded the same activities of four PCWs for a period of four hours. Then a comparison of two hours of their records was conducted and discussed. A Page 3 of 8 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Table 1 Classification of personal care workers’ activities (Continued) Alcohol hand washing (related to toileting or pad change). Alcohol hand washing (unrelated to toileting or pad change). Water hand washing (related to toileting or pad change). Water hand washing (unrelated to toileting or pad change). Documentation Locating or collecting a resident's records. Taking a photo of a resident. Reviewing or writing resident's clinical information; reading notes; viewing results. Putting records back to filing area. Transit Standing or walking in the corridor between activities. Staff break Personal errands (off unit chores; meal break; making personal telephone call). Oral communication Asking for assistance from another personal care worker. Assisting another personal care worker to do his or her work. Participating in-service training. Communication of information about a resident (external). Communication of information about a resident (internal). Communicating with a resident. Communicating with a resident's family. Receiving a phone call; making a phone call. Others Other tasks not included. Table 1 Classification of personal care workers’ activities Category Activities Direct care Physical Assessment. Routine hygiene (e.g. daily shower or wash). Continence related hygiene (e.g. shower or wash following pad change). Oral Care. Shave or grooming. Toileting - prompted by a resident. Toileting - prompted by a personal care worker. Pad check. Pad change. Scheduled toileting. Dressing a resident. Resident mobility; passive & active exercises; turning a resident in bed. Medication administration. Specimen collection; urine collection. Assisting a resident with eating and drinking (include feeding systems). Inter-rater reliability Assisting a resident with food (e.g. cutting up food, uncovering food or delivery of food). Care of the deceased; laying out. Assisting a resident with hand washing following the use of toilet. Assisting a resident with transfer to and from a bed, a chair, etc. Transferring a resident to or from dining room or board room. Weighing a resident. Assisting a resident to receive a phone call. Attending to a resident call for assistance. Indirect care Equipment set up (e.g. sling set up, shower chair set up). Resident shower set up (e.g. preparing shampoo, towel or body lotion). Bed making routine. Changing a bed following an incontinent episode. Cleaning up spills following an incontinent episode. Re-stocking supplies to a trolley. Re-stocking supplies to a resident's cupboard. Transporting linen to and from laundry. Transporting clinical waste for disposal. Using or cleaning up bed pans. Emptying a resident's meal plate. Collecting pads from a storage cupboard. Collecting a resident's clothes from his or her cupboard; putting clothes back to the cupboard. Sorting and putting a resident's clothes to his or her room. Infection control Putting on personal protective equipment. Taking off personal protective equipment. Table 1 Classification of personal care workers’ activities (Continued) minimum agreement of more than 95% was achieved in the two records, suggesting the inter-rater reliability is adequate according to Pelletier and Duffield [21]. Data collection The observation was performed in 2010. Before the ob- servation, the nursing manager at each site introduced the observer to the RNs and the PCWs. On each obser- vational day, the observer arrived at the site 15 minutes before the start of the observation to identify one of the PCWs for observation, using convenience sampling. The observer tried to observe different PCWs on different days to maximize the number of participants. At the start, the observer explained the purpose and procedure of the observation to the PCW. Only after written con- sent was given by the participant, was the observation conducted. A clinical handheld was used to record the observational data on an Excel spreadsheet. For the first seven days of data collection at Site 1 only the start time was recorded for the observed activities. Page 4 of 8 Page 4 of 8 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Table 2 Time spent on each category of activities, combining the two sites Categories Time (%) 95% Confidence intervals Direct care 30.7 28.7 32.8 Indirect care 17.6 16.3 18.8 Infection control 6.4 5.8 7.1 Documentation 3.1 2.5 3.7 Transit 4.6 3.9 5.2 Staff break 15.2 11.8 18.6 Oral communication 59.2 53.7 64.6 Others 1.2 0.8 1.6 Table 2 Time spent on each category of activities, combining the two sites Table 2 Time spent on each category of activities, combining the two sites Because the start time of the current activity is the end time of the previous activity, it was not necessary to rec- ord the end time. The observer noticed that a PCW might only be speaking or might be performing some other activity concurrently. In order to correctly record oral communication time, the data collection protocol was modified to include both the start time and end time of an oral communication activity, and the concur- rently performed activity. At Site 2, both start and end times of oral communication activities were recorded from the beginning. At Site 1, a total of 11 PCWs were observed over a period of 14 days (three of the PCWs were observed twice). The observation was between the hours of 7:00 and 14:00 or 15:00 on each day, depending on the observed PCW’s finishing time. Activity time At Site 1, 81 hours of observation were recorded and at Site 2, 92 hours were recorded. Table 3 presents the time, frequency and duration by activity category at each site. Data analysis The data were analyzed in Microsoft Excel 2007, SPSS version 18.0 (SPSS inc., Chicago, IL, USA) and R version 2.12.1 [22]. The duration of each activity was calculated in Excel. Analysis concerning oral communication at Site 1 was based on the last seven days of observation since the end time was not recorded during the first seven ob- servational days. A Z test was used to compare the per- centage of time spent on each category of activities between the two sites. A Pearson’s chi square test was used to determine the difference between the two sites in the number of activities which fell into different dur- ation groups. Statistical significance was assumed when P < 0.05. At Site 1, the most time-consuming direct care activity was ‘assisting a resident with eating and drinking (in- clude feeding systems)’ at 35 minutes over an eight-hour shift. At Site 2, however, ‘assisting a resident with trans- fer to and from a bed, a chair, etc.’ was the most time- consuming direct care activity, taking 29 minutes over an eight-hour shift. At both sites, ‘equipment set up (e.g. sling set up, shower chair set up)’ took the most indirect care time (Site 1: 31 minutes, Site 2: 38 minutes). Most of the oral communication time was spent on ‘communi- cation of information about a resident (internal)’ (Site 1: 1 hour and 53 minutes, Site 2: 2 hours and 42 minutes) and ‘communicating with a resident’ (Site 1: 1 hour and 51 minutes, Site 2: 2 hours and 4 minutes). Results Fifty-one of the designated 58 activities were observed at Site 1. The seven activities which were not observed are: ‘care of the deceased or laying out’, ‘assisting a resident to receive a phone call’, ‘re-stocking supplies to a resident’s cupboard’, ‘using or cleaning up bed pans’, ‘sorting and putting a resident’s clothes to his or her room’, ‘partici- pating in-service training’ and ‘taking a photo of a resi- dent’. Fifty-five of the designated activities were observed at Site 2. The three activities which were not observed are: ‘cleaning up spills following an incontinent episode’, ‘re-stocking supplies to a resident’s cupboard’, and ‘taking a photo of a resident’. No statistically significant difference between the two sites was found in the time spent on direct care, indirect care, infection control or staff break, and these activities took approximately 70.0% of the working time (Site 1: 68.1%, Site 2: 71.6%). As shown in Table 3, PCWs at Site 2 spent significantly more time than their counterparts at Site 1 on oral communication (Site 1: 47.3% vs. Site 2: 63.5%, P = 0.003), transit (Site 1: 3.4% vs. Site 2: 5.5%, P < 0.001) and others (Site 1: 0.5% vs. Site 2: 1.8%, P < 0.001). They spent less time, however, on documen- tation (Site 1: 4.1% vs. Site 2: 2.3%, P < 0.001). A total of 173 hours of observation and 11,283 events were recorded. Table 2 shows the time spent on each category of activities, combining data from the two sites. The percentages do not sum to 100% because oral Data collection communication may occur either by itself or simultan- eously with an activity from one of the other seven cat- egories. This means that the percentage of time spent on oral communication has overlaps with the other categories. At Site 2, a total of 27 PCWs were observed over 16 days (five were observed twice). The observation was from 10:00 to 17:00. On each day, a morning shift PCW was observed first. After this individual finished work at 14:00 or 15:00, an afternoon shift PCW was observed. Discussion In this study 56 out of the 58 designated activities per- formed by PCWs at two Australian nursing homes were measured. The activities were classified into eight cat- egories: direct care, indirect care, infection control, documentation, transit, staff break, oral communication and others. In comparison with a previous study in a single nursing home [9], this study provides a much more accurate and complete picture of how PCWs spend their time on work activities described in the fol- lowing terms: actual time taken over an eight-hour shift, the time spent on it as a percentage of the time con- sumed by all of the observed activities, the activity fre- quency and duration. It also provides data on the switches between two consecutive activities. Our find- ings will be useful for nursing managers to understand how PCWs work and what the workload actually is in looking after residents with high-care needs in nursing homes. Although the care needs of the residents repre- sented an uncontrolled variable in what was a natural setting, it appeared that a PCW’s workload looking after residents and meeting their care needs was high in both nursing homes. Activity frequency I h 56 In one hour, 56 events occurred at Site 1 and 73 events occurred at Site 2. The most frequently occurring Page 5 of 8 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Page 5 of 8 Table 3 Time, frequency and duration by activity category at Site 1 and Site 2 Category Site Time Frequency per hour Duration (seconds) % 95% Confidence intervals 8-hour shift (h:m:s) Mean Standard deviation 95% Confidence intervals Direct care 1 30.9 28.7 33.0 2:28:05 14.5 75.6 143.5 67.4 83.8 2 30.7 28.1 33.2 2:27:07 19.6 56.5 56.7 53.8 59.1 Indirect care 1 16.7 15.5 17.9 1:19:55 10.6 56.1 54.5 52.5 59.8 2 18.4 16.7 20.2 1:28:22 13.2 50.5 46.8 47.9 53.2 Infection control 1 5.9 5.2 6.6 0:28:25 5.3 40.2 61.5 34.3 46.1 2 6.8 6.1 7.6 0:32:46 8.8 28.2 24.5 26.5 29.9 Documentation 1 4.1 a 3.4 4.8 0:19:31 3.2 45.5 82.4 35.4 55.6 2 2.3 b 1.7 3.0 0:11:13 1.4 58.8 80.7 44.9 72.7 Transit 1 3.4 a 3.0 3.9 0:16:29 3.0 41.3 46.7 35.4 47.2 2 5.5 b 4.5 6.6 0:26:34 3.0 67.1 84.4 57.0 77.1 Staff break 1 14.6 10.9 18.4 1:10:17 0.6 880.1 817.5 642.7 1117.5 2 15.7 11.0 20.4 1:15:24 0.7 815.0 845.4 603.8 1026.2 Oral communication 1 47.3 a 39.3 55.3 3:47:00 18.8 90.7 140.9 80.0 101.5 2 63.5 b 56.6 70.4 5:04:40 25.8 88.9 173.4 81.9 95.8 Others 1 0.5 a 0.3 0.7 0:02:24 0.2 96.3 70.0 57.6 135.1 2 1.8 b 1.1 2.5 0:08:46 0.6 110.3 118.5 78.3 142.3 a,bIndicate significant difference between the two sites in the percentage of time spent on this category of activities (P < 0.05). Table 3 Time, frequency and duration by activity category at Site 1 and Site 2 a,bIndicate significant difference between the two sites in the percentage of time spent on this category of activities (P < 0.05). activity was oral communication, followed by direct care and indirect care (Table 3). activity was oral communication, followed by direct care and indirect care (Table 3). Activity duration h From the mean and standard deviation shown in Table 3, the activity duration was very short and varied dramatic- ally. The short activity duration is also shown in Figure 1. Of the 3,679 events recorded at Site 1 (excluding the 889 oral communication events which occurred in the first seven days), 9.0% were completed in less than 10 seconds, which was significantly less than at Site 2 (12.4% of 6,715 events, P < 0.001). 15.5% of the events recorded at Site 1 and 14.5% at Site 2 took between 10 and 19 seconds. Overall, more than two-thirds of the observed events at both sites had a duration of less than 1 minute. Switch between two consecutive activities A PCW frequently switched from one activity to an- other. On average, 49.8 switches between two consecu- tive activities were observed in an hour at Site 1 and 62.5 switches were observed at Site 2. A PCW switched from one activity to another at a rate of one per minute. Most of the switches were within or between oral com- munication activities, direct care activities and indirect care activities. The directions of the most frequently observed switches were similar, as were the number of these switches at both sites (Table 4). Personal care workers spent 30.7% of their time on direct care. This is less than the finding (40.2%) from a previous study by Munyisia et al. [9] which was also conducted in an Australian nursing home. Indirect care consumed 17.6% of PCWs’ time, which is almost twice as the time (8.9%) obtained in the study by Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Page 6 of 8 Figure 1 Distribution of duration at Site 1 and Site 2. Figure 1 Distribution of duration at Site 1 and Site 2. Further analysis needs to be conducted to understand how indirect care activities support direct care. It is also necessary to examine how direct care activities distribu- ted throughout a shift and whether the direct care activ- ities were spread out evenly over an hour or performed in quick succession, for example, at the beginning of the hour. This can make a significant difference to residen- tial care, as was also mentioned in a previous study [10]. Munyisia. The difference in time may be caused by the different study design. For example, our study used time-motion technique to collect data while their study used a work sampling technique. The other possibility is that the differences are due to differences in care systems and practices in different nursing homes. Fur- thermore, in this study the percentage of time was cal- culated from the actual duration of activities, whereas their results were based on the frequency of occur- rence of activities. Munyisia. The difference in time may be caused by the different study design. For example, our study used time-motion technique to collect data while their study used a work sampling technique. Conclusions We described the work pattern of PCWs in two Austra- lian nursing homes. The work activities were examined using the following measurements: activity time, fre- quency, duration, and the switch between two consecu- tive activities. Fifty-six out of 58 designated work activities grouped into eight categories were observed. We found that direct care, indirect care, infection con- trol and staff break were the major part (70.0%) of the work and there was no statistically significant difference between the two nursing homes in the time spent on these activities. More than two-thirds of the observed activities at both sites had a very short duration-- less than 1 minute. Personal care workers frequently switched within or between oral communication, direct care and indirect care activities. Personal care workers at Site 1 spent significantly less time on oral communication than their counterparts at Site 2. This may be associated with the age and ethnicity of the PCWs. Most of those at Site 1 were 20 to 30 years old and from a non-English speaking background, whereas PCWs at Site 2 were local and aged between 35 and 55. As the PCWs at Site 2 had the same language and cultural background as the residents, oral communi- cation was less of a challenge than it was for the PCWs at Site 1. Our findings are useful for nursing managers for staff performance appraisal, task allocation, scheduling and cost estimation. The information may also help to design effective aged care services and provide possible research directions in nursing homes. Furthermore, it provides evidence for the government in funding allocation by ac- curately measuring the amount of time needed in con- ducting each category of care activities to meet a resident’s relevant care needs. Further research on how indirect care activities support direct care and how oral communication supports other types of care are needed. The often short duration of activities and the quick and frequent switching between activities caused ex- treme busyness and some stress. The practical routine and familiarity with the residents and their individual needs help the PCWs arrange their work to cope with this. This routine and familiarity with the residents can facilitate the work. This was also found in a previous study [23]. Although routine and familiarity may support their work, a PCW does have to think about what to do next while performing the task at hand. Switch between two consecutive activities The other possibility is that the differences are due to differences in care systems and practices in different nursing homes. Fur- thermore, in this study the percentage of time was cal- culated from the actual duration of activities, whereas their results were based on the frequency of occur- rence of activities. Communication with a resident and communication of information about a resident are the prime oral Table 4 Direction and number of the most frequently observed switches between two consecutive activities First activity Second activity Number of switches per hour Communicating with a resident. Communication of information about a resident (internal). 1.6 Communication of information about a resident (internal). Communicating with a resident. 1.6 Equipment set up (e.g. sling set up, shower chair set up). Assisting a resident with transfer to and from a bed, a chair, etc. 1.1 Communicating with a resident. Communicating with a resident. 0.9 Taking off personal protective equipment. Water hand washing (related to toileting or pad change). 0.8 Communicating with a resident. Equipment set up (e.g. sling set up, shower chair set up). 0.6 Assisting a resident with transfer to and from a bed, a chair, etc. Equipment set up (e.g. sling set up, shower chair set up). 0.9 Table 4 Direction and number of the most frequently observed switches between two consecutive First activity Second activity Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Page 7 of 8 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 nursing homes and provides a good reference for other studies of work activities in nursing homes. communication activities. This may be an indication that the PCWs had made an effort to spend time interacting with residents (e.g. explaining the care to a resident in order to receive cooperation from the resident) and co- operate with the working partners to provide care. The content of the oral communication and the way it is conducted may be among the critical elements which most affect the quality of care. Limitations The benefit of using a single observer is the potential consistency of the observations [24], however it may also cause systematic errors in observation. We addressed this potential limitation through an inter-rater reliability comparison study, which provided satisfactory results. There may be a ‘Hawthorn effect’ [13] (the participants might change their work behavior under the observa- tion) from PCWs being observed continuously, however we found that in the busy nursing home working envir- onment, PCWs had to focus on their job and very soon ignored the existence of the observer. This was also found in previous studies [25,26]. Personal care workers not only spent a great deal of time on oral communication, but also frequently switched between oral communication, direct care and indirect care activities. This may indicate that oral com- munication is one of the important activities which sup- port direct care and indirect care. Although the observational time periods at the two nursing homes were different (Site 1: 7:00 to 14:00 or 15:00, Site 2: 10:00 to 17:00), no statistically significant difference was found in the time spent on direct care, in- direct care, infection control or staff break. These activ- ities account for about 70.0% of a PCW’s working time. This suggests that apart from the unavoidable breaks which all staff must take, these activities represent the core of PCWs' workload. Nursing managers need to consider this finding carefully when allocating tasks, staff number and skill mix on a shift. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Abbreviations RACFs: Residential aged care facilities; PCWs: Personal care workers; RNs: Registered nurses; TAFE: Technical and Further Education; ADL: Activities of daily living; ACFI: Aged Care Funding Instrument. Conclusions Working in such a busy environment may lead to a cognitive overload, which may cause job fatigue and contributing, in turn, to nursing burn out. Therefore, nursing managers may need to consider which level of workload is appropriate for a PCW working in a nursing home. Abbreviations Competing interests The authors declare that they have no competing interests. Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 Qian et al. BMC Health Services Research 2012, 12:305 http://www.biomedcentral.com/1472-6963/12/305 17. Zheng K, Haftel HM, Hirschl RB, O'Reilly M, Hanauer DA: Quantifying the impact of health IT implementations on clinical workflow: a new methodological perspective. J Am Med Inform Assoc 2010, 17:454–461. paper for important intellectual content. All authors read and approved the final manuscript. 18. Munyisia EN, Yu P, Hailey D: The changes in caregivers’ perceptions about the quality of information and benefits of nursing documentation associated with the introduction of an electronic documentation system in a nursing home. Int J Med Inform 2011, 80:116–126. Author details 1S h l f I f j g 23. Bowers BJ, Lauring C, Jacobson N: How nurses manage time and work in long-term care. J Adv Nurs 2001, 33:484–491. 1School of Information Systems and Technology, University of Wollongong, New South Wales 2522, Australia. 2School of Mathematics and Applied Statistics, University of Wollongong, New South Wales 2522, Australia. 24. McCrea BH, Wright ME, Murphy-Black T: Differences in midwives' approaches to pain relief in labour. Midwifery 1998, 14:174–180. 25. Westbrook JI, Ampt A: Design, application and testing of the Work Observation Method by Activity Timing (WOMBAT) to measure clinicians' patterns of work and communication. Int J Med Inform 2009, 78(Suppl 1):S25–S33. Received: 7 June 2012 Accepted: 3 September 2012 Published: 6 September 2012 Received: 7 June 2012 Accepted: 3 September 2012 Published: 6 September 2012 Published: 6 September 2012 26. Ampt A, Westbrook J, Creswick N, Mallock N: A comparison of self- reported and observational work sampling techniques for measuring time in nursing tasks. J Health Serv Res Policy 2007, 12:18–24. Acknowledgements The project is jointly funded by Australian Commonwealth Government Enterprise Connect and Simavita Pty Ltd. Australian Government Enterprise Connect provides funding support for the labor cost of a research assistant (RA). Simavita Pty Ltd. contributes to part of the labor cost of the RA and the cost of the RA to conduct the field data collection. Three registered nurses in Simavita Pty Ltd. with extensive experience working in aged care contributed to the development of work activity classification system in nursing homes. We are grateful to Esther Munyisia for her assistance in the inter-rater reliability study. The participating of the management and care staff at the two nursing homes BCS Shalom Centre and Maroba Nursing Home is highly appreciated. Dr. Madeleine Strong Cincotta's kind help in the editing of the final manuscript is gratefully acknowledged. 19. Munyisia EN, Yu P, Hailey D: Development and testing of a work measurement tool to assess caregivers' activities in residential aged care facilities. Stud Health Technol Inform 2010, 160:1226–1230. 20. Munyisia EN, Yu P, Hailey D: Does the introduction of an electronic nursing documentation system in a nursing home reduce time on documentation for the nursing staff? Int J Med Inform 2011, 80:782–792. 21. Pelletier D, Duffield C: Work sampling: valuable methodology to define nursing practice patterns. Nurs Health Sci 2003, 2003:31–38. 22. R Development Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2008. http://www.R-project.org. References l 1. Australian Institute of Health and Welfare: Residential aged care in Australia 2009–10: a statistical overview. Aged care statistics series no. 35. Cat. no. AGE 66. Canberra: AIHW; 2011. doi:10.1186/1472-6963-12-305 Cite this article as: Qian et al.: The work pattern of personal care workers in two Australian nursing homes: a time-motion study. BMC Health Services Research 2012 12:305. 2. Hussein S, Manthorpe J: An international review of the long-term care workforce: policies and shortages. J Aging Soc Policy 2005, 17:75–94. 3. Martin B, King D: Who cares for older Australians? A picture of the residential and community based aged care workforce 2007. Adelaide: National Institute of Labour Studies; 2008. 4. Australian Government Department of Health and Ageing: Aged care funding instrument (ACFI). 2009. http://www.health.gov.au/acfi g g 5. Pelletier D, Duffield C, Donoghue J: Documentation and the transfer of 5. Pelletier D, Duffield C, Donoghue J: Documentation and the transfer of clinical information in two aged care settings. Aust J Adv Nurs 2005, 22:40–45. clinical information in two aged care settings. Aust J Adv Nurs 2005, 22:40–45. 6. Becker G, Kempf DE, Xander CJ, Momm F, Olschewski M, Blum HE: Four minutes for a patient, twenty seconds for a relative - an observational study at a university hospital. BMC Health Serv Res 2010, 10:94. 7. Cornell P, Herrin-Griffith D, Keim C, Petschonek S, Sanders AM, D'Mello S, Golden TW, Shepherd G: Transforming nursing workflow, part 1: the chaotic nature of nurse activities. J Nurs Adm 2010, 40:366–373. 8. Tipping MD, Forth VE, O'Leary KJ, Malkenson DM, Magill DB, Englert K, Williams MV: Where did the day go?-a time-motion study of hospitalists. J Hosp Med 2010, 5:323–328. 9. Munyisia EN, Yu P, Hailey D: How nursing staff spend their time on activities in a nursing home: an observational study. J Adv Nurs 2011, 67:1908–1917. 10. Westbrook JI, Duffield C, Li L, Creswick NJ: How much time do nurses have for patients? A longitudinal study quantifying hospital nurses' patterns of task time distribution and interactions with health professionals. BMC Health Serv Res 2011, 11:319. Authors' contributions bl f h Among the designated 58 work activities of PCWs, 56 were observed, suggesting that our activity classification system reflects a PCW’s work activities in Australian PY was responsible for the study conception and design. ZYZ performed data collection. SYQ, PD and MN were responsible for data analysis. SYQ drafted the manuscript. PY, DH, PD and MN made critical revisions to the Page 8 of 8 References l Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 11. Abbey M, Chaboyer W, Mitchell M: Understanding the work of intensive care nurses: a time and motion study. Aust Crit Care 2012, 25:13–22. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 12. Kobayashi N, Yamamoto M: Impact of the stage of dementia on the time required for bathing-related care: a pilot study in a Japanese nursing home. Int J Nurs Stud 2004, 41:767–774. • Convenient online submission 13. Finkler SA, Knickman JR, Hendrickson G, Lipkin M Jr, Thompson WG: A comparison of work-sampling and time-and-motion techniques for studies in health services research. Health Serv Res 1993, 28:577–597. 14. Wirth P, Kahn L, Perkoff GT: Comparability of two methods of time and motion study used in a clinical setting. Med Care 1977, 15:953–960. 15. Holloway K, McConigley R: Descriptive, exploratory study of the role of nursing assistants in Australian residential aged care facilities: the example of pain management. Australas J Ageing 2009, 28:70–74. 16. Upenieks VV: Work sampling. Assessing nursing efficiency. Nurs Manage 1998, 29:27–29. 16. Upenieks VV: Work sampling. Assessing nursing efficiency. Nurs Manage 1998, 29:27–29.
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Perceptions and experiences of Latinx parents with language barriers in a pediatric emergency department: a qualitative study
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Zamor et al. BMC Health Services Research (2022) 22:1463 https://doi.org/10.1186/s12913-022-08839-w Zamor et al. BMC Health Services Research (2022) 22:1463 https://doi.org/10.1186/s12913-022-08839-w Open Access © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Perceptions and experiences of Latinx parents with language barriers in a pediatric emergency department: a qualitative study Ronine L. Zamor1,2,3*, Lisa M. Vaughn1,2, Erin McCann4, Luisanna Sanchez4, Erica M. Page1 an E. Melinda Mahabee‑Gittens1,2 Abstract Background:  Prior research has shown disparities exist among Latinx children who require treatment for respiratory illnesses within the pediatric emergency department (PED). Limited data exist regarding Latinx families’ experiences on the care they received at PEDs within non-traditional destination areas (NDA). Their experiences can identify areas of improvement to potentially reduce healthcare disparities among pediatric patients within this population. The purpose of this qualitative study was to explore the lived experiences of Latinx families with low English proficiency in the PED with a NDA. The broader purpose was to identify areas of improvement for reducing health care disparities among Latinx families. Methods:  We used qualitative methods to analyze semi-structured interviews among Latinx families who presented to the PED with their 0–2 year-old child for a respiratory illness from May 2019 through January 2020. All participants had low English proficiency and requested a Spanish interpreter during registration. All interviews were transcribed and reviewed using thematic analysis based on a phenomenology framework. Results:  Interviews were conducted with 16 Latinx parents. Thematic analysis revealed four major themes: (1) Uncertainty - Families expressed uncertainty regarding how to care for a child with distressing symptoms, (2) Commu‑ nication – Families favored in-person interpreters which enhanced communication and allowed families to feel more informed, (3) System Burden – Families reported that the unfamiliarity with the US health system and lack of resources are additional burdens, and (4) Emotional Support – The emergency department visits garnered confidence and reas‑ surance for families. Conclusions:  Our study identified four major themes among Latinx families within a PED of a NDA. Potential areas of interventions should focus on supporting access to an interpreter, improving information delivery, and enhancing education on community resources for families with low English proficiency. Keywords:  Latinx, Pediatric emergency department, Language barriers, Health disparities Background In 2016, the Census Bureau estimated that the His- panic population of the United States (U.S.) was 56.6 million, which constitutes 17% of the nation’s total population. This proportion is expected to increase to 28% by 2060 [1]. About one-third of this popula- tion are children, which is the greatest proportion of *Correspondence: Ronine.Zamor@emory.edu; RLZamor@gmail.com 3 Present Address: Division of Emergency Medicine, Children’s Healthcare of Atlanta, Emory University, 1547 Clifton Road, NE 2nd Floor, Atlanta, GA 30322, USA Full list of author information is available at the end of the article Recruitment We conducted purposive sampling of patients by screen- ing electronic health records (EHR) for potential par- ticipants 7 day a week from May 2019 through January 2020. Parents or legal guardians were considered eligible for the study if they: 1) presented to the PED with their 0–2-year-old child for a viral upper or lower respiratory illness, 2) affirmed that their preferred language was Spanish at triage, and 3) requested a Spanish interpreter during registration. At the time of this study, only one pediatric emergency medicine attending was considered a certified language interpreter in Spanish. All other phy- sicians were required to use an interpreter if the patient and/or family did not speak English. Prior literature has shown significant healthcare disparities among Latinx children whose families face language barriers within the emergency department (ED) [9–12]. For instance, there are differences in diagnostic testing and hospital charges in the pediat- ric emergency department (PED) among families with language barriers [9, 13, 14]. Some studies suggest that language barriers may be associated with poor health outcomes such as increased obesity and com- plications of appendicitis [10, 15, 16]. Compared to Latinx patients in traditional destination areas, Latinx patients in NDAs have different healthcare barriers, experiences, and needs because healthcare systems in NDAs are not properly equipped to work with popula- tions that have limited English proficiency [5, 6, 17– 19]. Latinx patients in NDAs may face more challenges such as social isolation, discrimination, and lack of social support [5, 6, 18, 20]. Given the rapid growth of Latinx population and the need for promoting greater equity in pediatric emergency care [21], exploring the experiences and perceptions of Latinx families sur- rounding their management in the PED can help target areas of improvement for reducing disparities within a NDA. Given the known disparities among pediatric patients with respiratory complaints [9], the purpose of this study is to explore the lived experiences of non- English speaking Latinx families with children with respiratory illnesses within a PED of a non-traditional destination area. The broader aim of this study is to identify areas of improvement for reducing health care disparities for Latinx families. Potentially eligible families were approached by study staff while patients were in the PED. The study staff and a Spanish interpreter explained the study, review informed consent, and scheduled a telephone interview once informed verbal consent was obtained. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zamor et al. BMC Health Services Research (2022) 22:1463 Zamor et al. BMC Health Services Research (2022) 22:1463 Page 2 of 8 children compared to proportions among families of Black, Asian or White origin [2, 3]. In the past, immigration of Latinx families to the U.S. has largely been concentrated in states with large urban areas, such as Texas, Florida, and New York [4]. However, recent growth among the Latinx population has been observed in areas of the U.S. that were not common destination areas in the past, such as areas within the Midwest, which are defined as nontraditional des- tination areas (NDAs) [5]. NDAs, such as Ohio and Nevada, have experienced at least between 100 and 200% growth in the past 2 decades compared to tra- ditional areas, which experienced at least a 20% drop [6, 7]. The rapid growth of the Latinx population has significant health implications because NDAs do not have the infrastructure and resources to support the changing immigration patterns [6, 8]. For instance, a prior study conducted in the Midwest showed that lin- guistically and culturally appropriate services to meet the need of the growing Latinx community are lacking [5]. However, literature describing Latinx healthcare experiences within NDAs remains limited. Study settingh The study was conducted at Cincinnati Children’s Hos- pital Medical Center (CCHMC), one of the largest aca- demic children’s hospitals in the United States. The hospital treats children with rare and complex conditions from over 60 different countries [22]. There are two dif- ferent PEDs which in combination have approximately 92,000 visits per year. Upon arrival to the PED, families are asked their preferred language and whether they need an interpreter for the remainder of the visit. The pre- ferred language is documented within the patient’s chart. An interpreter can be used at any time or for the entire visit depending on availability, which includes access to an interpreter in-person, via video call, or via telephone. Recruitment Study staff also conducted weekly screening of the EHRs to identify potentially missed eligible families who had already been discharged. If the family met the inclu- sion criteria, study staff called the family to inform them of the study, solicit participation, and obtain informed verbal consent. The patient’s age, medical history, main complaint, and type of interpreter involved with the fam- ily was reviewed and confirmed prior to proceeding with the interview. All participants were given a $20 gift card in exchange for their participation in the study. Methods Study settingh children compared to proportions among families of Black, Asian or White origin [2, 3]. In the past, immigration of Latinx families to the U.S. has largely been concentrated in states with large urban areas, such as Texas, Florida, and New York [4]. However, recent growth among the Latinx population has been observed in areas of the U.S. that were not common destination areas in the past, such as areas within the Midwest, which are defined as nontraditional des- tination areas (NDAs) [5]. NDAs, such as Ohio and Nevada, have experienced at least between 100 and 200% growth in the past 2 decades compared to tra- ditional areas, which experienced at least a 20% drop [6, 7]. The rapid growth of the Latinx population has significant health implications because NDAs do not have the infrastructure and resources to support the changing immigration patterns [6, 8]. For instance, a prior study conducted in the Midwest showed that lin- guistically and culturally appropriate services to meet the need of the growing Latinx community are lacking [5]. However, literature describing Latinx healthcare experiences within NDAs remains limited. Data analysis Thi d d This study drew from the phenomenology framework developed by Max Van Manen and Martin Heidegger, who described a hermeneutic approach to phenomenol- ogy that stresses how human experiences and “reali- ties” are influenced by the world in which they live. This framework provided a way to understand each family’s meaning that they constructed from their experiences in the PED [24–26]. The research team utilized a thematic analysis approach to systematically identify meaning- ful subthemes and themes that represented major topics discussed in the interviews [27, 28]. Thematic analysis involved an iterative process to further clarify the themes that could be presented in a cohesive narrative. First, the research team independently reviewed all interview transcripts and developed codes, which were key words that represented specific phrases, patterns, and com- mon features found in the transcripts. A codebook was developed to help guide the data analysis. All members of the research team collectively reviewed and coded all Characteristics of study participants Interviews were conducted with 16 Latinx parents (15 mothers, 1 father) who were seen in the PED with a child presenting with respiratory symptoms. The length of interviews ranged from 11 minutes to 26 minutes (median, 20 minutes). Most pediatric patients were male (66%). Among the families who were comfortable shar- ing their country of origin and length of time in the US, the range of time in the US ranged from 18 months to 13 years. Table 2 shows the proportions of patient triage levels, chief complaints, disposition, and final diagnoses. Thematic analysis h Four major themes emerged from the thematic analy- sis: (1) Uncertainty - fear and uncertainty regarding how to care for a child with distressing symptoms, (2) Com- munication – the presence of in-person interpreters enhanced communication, allowing parents to feel more informed, (3) System Burden- unfamiliarity with the U.S. healthcare system and lack of resources are additional sources of hardship and (4) Emotional Support - despite limited understanding, “successful” visits garnered con- fidence and provided parents with needed reassurance. Data collectionh Three authors conducted 16 semi-structured, one-on- one qualitative interviews from May 2019 through Janu- ary 2020. Two (L.S. and E.M.) of the three interviewers were fluent in Spanish and did not use an interpreter for the interviews. One interviewer (L.S.) was of Hispanic descent. The other two interviews (E.M and R.Z) were Zamor et al. BMC Health Services Research (2022) 22:1463 Page 3 of 8 not of Hispanic descent, however both have worked pri- marily with Latinx patients during their medical training. All interviewers received qualitative interview training from L.V. who has doctoral level training and research experience in qualitative research methods within the Latinx community. The interviewers used an interview guide (Table 1) that allowed them the flexibility to devi- ate from the guide and ask probing questions based on participants’ responses. transcripts. Three other researchers other than the inter- viewers were included in the analysis for investigator tri- angulation. The research team met regularly to review the transcripts and discuss emerging codes until code agreement and saturation were reached. Each code was collated into unifying themes along with subcategories within each theme. The software program Dedoose was used to highlight specific themes or common phrases that might have been missed when reviewing the tran- scripts as a group [29]. Code frequencies within Dedoose were also used to identify patterns in families’ responses [29]. All families who gave informed verbal consent while in the PED had their interview completed via phone within 30 days of the index PED visit. All interviews were audio- taped then translated and transcribed. Interviews were conducted until thematic saturation was reached, which occurred when the interviewers did not elicit any new information or themes [23]. 1. Where are you from? How long have you lived in the United States? 2. What resources do you use in your community when your child is sick? 3. If you remember, what brought you and your child to the emergency department? 4. What concerned you the most about your child’s symptoms when you visited the emergency department? 5. Do you remember if you took your child to his/her primary doctor before going the emergency department that day? Tell me about that. 6. Tell me what the several days before you took your child to the emer‑ gency department were for you. 7. Describe to me how your visit went. Ex: Testing, treatments, expecta‑ tions 1. Where are you from? How long have you lived in the United States? 8. Was there anything done that day that you think helped your child feel better? 9. Describe your understanding of why things were or were not done. 10. How did your doctor communicate with you when deciding what should or should not be done? 11. Describe how you communicated what concerned you regarding your child during your visit. 12. What things did you not tell your doctor that you wish you had told them? 13. How did you feel having an interpreter there helped or did not help with your visit that day? 14 How do you think communication about what was done during your visit could have been better? Table 1  Interview guide 8. Was there anything done that day that you think helped your child feel better? 9. Describe your understanding of why things were or were not done. 10. How did your doctor communicate with you when deciding what should or should not be done? 11. Describe how you communicated what concerned you regarding your child during your visit. 12. What things did you not tell your doctor that you wish you had told them? 13. How did you feel having an interpreter there helped or did not help with your visit that day? 14 How do you think communication about what was done during your visit could have been better? 8. Was there anything done that day that you think helped your child feel better? 9. Describe your understanding of why things were or were not done. 10. How did your doctor communicate with you when deciding what should or should not be done? 11. Describe how you communicated what concerned you regarding your child during your visit. 12. What things did you not tell your doctor that you wish you had told them? 13. How did you feel having an interpreter there helped or did not help with your visit that day? 14 How do you think communication about what was done during your visit could have been better? 1. Where are you from? How long have you lived in the United States? 2. What resources do you use in your community when your child is sick? 3. If you remember, what brought you and your child to the emergency department? 4. What concerned you the most about your child’s symptoms when you visited the emergency department? 5. Do you remember if you took your child to his/her primary doctor before going the emergency department that day? Tell me about that. 6. Tell me what the several days before you took your child to the emer‑ gency department were for you. 7. Describe to me how your visit went. Ex: Testing, treatments, expecta‑ tions 14 How do you think communication about what was done during your visit could have been better? Zamor et al. BMC Health Services Research (2022) 22:1463 Page 4 of 8 Table 2  Characteristics of 16 participating families Characteristic No. Table 1  Interview guide (%) Patient Gender Male 9 (56%) Family Country of Origin   Mexico 4 (25%)   Guatemala 5 (31%)   Puerto Rico 1 (6%)   No answer 6 (38%) Chief Complaint   Fever and/or difficulty breathing 7 (44%)   Fever and/or cough 6 (38%)   Nasal congestion 1 (6%)   Fever 2 (12%) Triage Acuity   2 (Most severe) 3 (19%)   3 7 (44%)   4 (Least severe) 6 (38%) Final ED Diagnosis   Bronchiolitis 9 (56%)   Viral illness/Cold/Fever 4 (25%)   Croup 1 (6%)   Pneumonia 1 (6%)   Bilateral Otitis Media 1 (6%) Method of Interpreter Used   In-Person 6 (38%)   Video 5 (31%)   Phone 5 (31%) Admitted 6 (37%) Table 2  Characteristics of 16 participating families with medical professionals. Nevertheless, their child’s distressing symptoms prompted parents to seek help anyway. 2. Communication: The presence of in-person inter- preters enhanced communication, allowing parents to feel more informed: 2. Communication: The presence of in-person inter- preters enhanced communication, allowing parents to feel more informed: “They explained how everything was going to be done and I was okay with that.” “Yes, having the in-person interpreter over there was better” “It did help me a lot because that way I was able to communicate through this person. I was able to explain everything. I was able to explain and say everything – every- thing that was going on with my son. And everything the doctor and the nurse wanted to tell me I was able to understand all of it through this person” Participants reported that providers attempted to explain their child’s diagnosis and treatment plan; how- ever, limited understanding of medical terminology was a barrier. Despite this issue, in-person interpreters less- ened parents’ confusion and improved communication by translating parental concerns and describing proce- dures and tests discussed by the provider. The frequency of reported understanding was higher with in-person and video interpreters. To facilitate understanding, one participant reported that their provider showed a video depicting symptoms that would require medical atten- tion. These measures (i.e., videos, pictures, infographics) made most parents feel informed of the plan of care and few reported feeling ignored and uninformed. Table  3 lists illustrative quotations representative of major themes and subthemes. 3. System Burden: Unfamiliarity with the U.S. health- care system and lack of resources are additional sources of hardship: 3. System Burden: Unfamiliarity with the U.S. health- care system and lack of resources are additional sources of hardship: 1. Table 1  Interview guide BMC Health Services Research (2022) 22:1463 Page 5 of 8 Table 3  Themes and subthemes with illustrative quotations Themes and Subthemes, With Illustrative Quotations Theme or Subtheme Illustrative Quotation Uncertainty   Complexity “But that was just the fever but the thing I was worried the most about was his chest”   Concern “Oh, I was worried about my little girl dying of the disease, so I wanted to get her to the emergency room quickly”   Confidence in one’s ability “When he was home I couldn’t give him anything because if I gave him something I thought I could make things worse. I couldn’t give him just anything.” Communication   Communication during visit (intake, history) “They examined him and they told me that he was very sick so they gave him oxygen and they told me that the doctor would come to examine him and tell me if he was going to be hospitalized or not. Half an hour later the doctor came and he told us that he needed to stay”   Informed “Yes, they explained how everything was going to be done and I was ok with that”   Method of Communication “Yes, having the in-person interpreter over there was better” “I felt comfortable because they had a face-to-face interpreter”   Understanding of diagnosis/Treatment plan “I don’t speak English, I speak Spanish. I did understand everything. They had a, what’s it called? A translator, an interpreter. Something like that. “ System Burden   Community resources “Yeah, he has a social worker”   Familiarity with healthcare system “So, it’s very different. I have been living here for like 2 years but the way they treat you here is very different compared to the way they treat you over there when you come to the emergency room with a child. In Puerto Rico, they would run a CBC test, and they would run lab tests, and ultrasound. And that’s not how it is here”   Navigating the System “I haven’t found a way to get into health insurance”   Access “Yes, she’s going to a clinic, well I take her to a clinic called Price Hill. It’s about 5 minutes from here” “We have to ask a guy to give a us a ride every day. Table 1  Interview guide We just call and they come take us”   Family member “My sister-in-law told the girl to find a girl who spoke English so I could explain things to her” Emotional Support   Reassurance “Thanks to God, everything was going good” “I’m just grateful for everything they did”   Physician’s authority “I did expect something else, but I was fine with it, they’re the doctors, of course”   Trust “They did everything my baby needed” “I felt confident in my doctors and they took away that doubt and that anguish” Table 3  Themes and subthemes with illustrative quotations Themes and Subthemes, With Illustrative Quotations “When he was home I couldn’t give him anything because if I gave him something I thought I could make things worse. I couldn’t give him just anything.” “Yeah, he has a social worker” “So, it’s very different. I have been living here for like 2 years but the way they treat you here is very different compared to the way they treat you over there when you come to the emergency room with a child. In Puerto Rico, they would run a CBC test, and they would run lab tests, and ultrasound. And that’s not how it is here” “Yes, she’s going to a clinic, well I take her to a clinic called Price Hill. It’s about 5 minutes from here” “We have to ask a guy to give a us a ride every day. We just call and they come take us” “My sister-in-law told the girl to find a girl who spoke English so I could explain things to her” “Thanks to God, everything was going good” “I’m just grateful for everything they did” “I did expect something else, but I was fine with it, they’re the doctors, of course” “They did everything my baby needed” “I felt confident in my doctors and they took away that doubt and that anguish” coordinating care and accessing treatment. However, many participants reported social isolation and few community resources. rushed and their concerns were not taken seriously due to the mild nature of their child’s illness. Overall, par- ticipants trusted their providers and were calmed by their provider’s assurance about their child’s prognosis. 4. Emotional Support: Despite limited understanding, “Successful” visits garnered confidence and provided parents with needed reassurance: Table 1  Interview guide Uncertainty: Fear and concern regarding how to care for a child with distressing symptoms: 1. Uncertainty: Fear and concern regarding how to care for a child with distressing symptoms: “I was worried about my little girl dying of this disease.” “Maybe if it was another case, my baby might have died, because we don’t know how to take care of the baby in the house.” “I was worried about my little girl dying of this disease.” “Maybe if it was another case, my baby might have died, because we don’t know how to take care of the baby in the house.” “The way you are treated here is very different.” “I mean, for them to do something it has to be a life or death situation.” “For me yes; I haven’t found a way to get into health insurance” “I live alone and I am alone with my baby and so technically it is all me” “The way you are treated here is very different.” “I mean, for them to do something it has to be a life or death situation.” “For me yes; I haven’t found a way to get into health insurance” “I live alone and I am alone with my baby and so technically it is all me” Participants reported that their children were expe- riencing various symptoms that were disconcerting. Parents were frightened by the symptoms and were unsure of their ability to care for their child at home. Some participants reported that they did not provide any treatment at home because they were inexperi- enced as a parent and thus not sure of the appropriate course of action. Others believed that if they provided interventions, they might make the situation worse. Furthermore, parents were fearful that if they did seek medical treatment, their limited English proficiency would affect their ability to effectively communicate Parental expectations for care in the U.S. were affected by care they had previously received in their home country. Some parents believed that in order to receive the standard of care they are accustomed to in the PED, their child must be severely ill. Participants also reported having trouble making appointments with primary care pediatricians and limited accessibil- ity to clinics that provide interpreters. Some parents listed social workers and family members accustomed to the system as sources of advice and assistance in Zamor et al. Discussionh Nonetheless, families were reassured and more confident at the end of the encounter, regardless of whether the patient needed numerous tests or interven- tions in the PED. families’ uncertainty about the child’s clinical status, the importance of using in-person interpreters, unfamiliarity with the healthcare system, and the shift in perspective from lack of confidence to confidence in their ability to care for their child after the healthcare provider’s diagno- sis and management in the PED. families’ uncertainty about the child’s clinical status, the importance of using in-person interpreters, unfamiliarity with the healthcare system, and the shift in perspective from lack of confidence to confidence in their ability to care for their child after the healthcare provider’s diagno- sis and management in the PED. This study reinforced the importance of having in-per- son and/or video interpreters, which is a relatively simple but vital aspect of a family’s experience when presenting to a PED for any health concern while facing a language barrier. Families felt more informed and reassured when a video or a combination of an in-person and video inter- preter were used during the PED visit. It is known that the use of professional interpreters is associated with overall improvement in care for patients with limited English proficiency, with in-person or video interpreta- tion leading to higher patient satisfaction compared to telephone interpretation [30, 31]. Prior research examin- ing the Latinx population has shown that a lack of staff members of the same ethnic or language group is associ- ated with barriers to health care [18, 32]. Although sev- eral prior research studies show that families may still have a degree of mistrust regardless of having access to an interpreter [33, 34], families in this study reaffirmed that they remained informed and reassured when they were given the opportunity to interact with an interpreter in-person or through a video. There have been a few studies exploring Latino car- egivers’ experiences within the PED [32, 35, 36]. How- ever, no studies of similar methodology have been done within a NDA. A study within a PED in Texas was done with similar methodology to our study, and the researchers focused on children who presented to the PED with asthma symptoms [35]. Similar to our study, the researchers found themes that included uncertainty, importance of interpreter availability, and access to care. Discussionh This study explored Latinx families’ lived experiences regarding the treatment of their children when present- ing to a PED with respiratory symptoms within a NDA. This is extremely important for PED providers because they are treating patients at one point in time, without much, if any, foreknowledge of what led the families to their visit. Obtaining the clinical information, such as the child’s symptoms and clinical severity, is a streamlined process. However, exploring how families experienced their visit in the ED can be more difficult to assess, but can still be important information for providers. In this study, the major themes that emerged for all 3 interview- ers included the fear of progression of illness due to the “I felt confident in my doctors and they took away that doubt and that anguish.” “They did everything my baby needed.” “I loved how they [the inter- preter] explained the results of the x-ray.” “I felt confident in my doctors and they took away that doubt and that anguish.” “They did everything my baby needed.” “I loved how they [the inter- preter] explained the results of the x-ray.” Although parents did not completely understand their provider, many were satisfied with the visit. Most par- ticipants believed their providers adequately addressed their concerns and provided adequate care to their chil- dren. However, a few parents felt the appointment was Page 6 of 8 Page 6 of 8 Zamor et al. BMC Health Services Research (2022) 22:1463 if the intervention was minimal such as nasal suctioning or providing an antipyretic. Although the need for exten- sive testing and interventions was low in this participant cohort, parents were very thankful and reassured for the care they received in the PED. This finding is interesting in the light of prior quantitative findings showing that Latinx patients facing language barriers receive increased PED charges and increased testing compared to patients without language barriers [13, 14]. Furthermore, it was not clear if parents always understood their child’s diag- nosis or return precautions because of language barriers or health literacy levels. One family expressed apprecia- tion for a short video that was shared with them before discharge that demonstrated an infant in respiratory dis- tress as this ensured they understood appropriate return precautions. Discussionh However, in our study, families reported the increased reliance of family members in their access to care. We also found that families valued the emotional support provided by the providers and staff, which may be impor- tant in a NDA in which there is typically not a large Latinx population.h Navigating the healthcare system within a NDA was another major theme among participants. Disparities in pediatric care may be due to a lack of access to primary care for Spanish-speakers [7]. Families often reported trying to visit primary care clinics first, but they were unable to get a visit with a Spanish-speaking provider or they did not know what to do if it was after regular busi- ness hours. These experiences support the literature that suggests increased PED utilization for routine sick visits among Latinx children, resulting in fewer interventions in the PED due to the higher frequency of low acuity vis- its [12, 33]. In contrast, patients among families within this study who reported difficulty with navigating the healthcare system had lower triage levels (higher acuity). Navigating the healthcare system can be more difficult among Latinx families within a NDA, which could lead to sicker patients going to the PED. Lower acculturation among ethnic groups has been shown to be associated with higher numbers of PED visits [32]. The increased difficulty for Latinx families within an NDA can lead to even higher numbers of PED visits and may influence the level of care needed in the emergency setting. There were several limitations that should be noted. Two of the three interviewers were not from the Latinx community, which could have impacted participants’ willingness to be open and honest. One interviewer did have to use an interpreter; however, the emergent themes did not differ among the 3 interviewers. Although data reached thematic saturation [23], the majority of the interviewed families were from the same country (e.g., Guatemala). Thus, findings should be interpreted with caution and may not apply to other Latinx populations. Lastly, although intentional in qualitative research, the study was conducted at a single site with non-probabil- ity sampling which limits the applicability of findings to other clinical settings. Abbreviations PED P di t i E PED: Pediatric Emergency Department; ED: Emergency Department; NDA: Nontraditional Destination Area; CCHMC: Cincinnati Children’s Hospital Medi‑ cal Center; EHR: Electronic Health Record. 5. Jacquez F, Vaughn L, Zhen-Duan J, Graham C. Health care use and barri‑ ers to care among Latino immigrants in a new migration area. J Health Care Poor Underserved. 2016;27(4):1761–78. 6. Jacquez F, Vaughn LM, Pelley T, Topmiller M. Healthcare experiences of Latinos in a nontraditional destination area. J Community Pract. 2015;23(1):76–101. Funding This work was funded, in part, by the Eunice Kennedy Shriver National Institute of Child Health and Human Development: NIH Grant Number R01HD083354. This funding source had no involvement in the study design, data collection, analysis, or interpretation of data. 11. Valet RS, Gebretsadik T, Carroll KN, Minton PA, Woodward KB, Liu Z, et al. Increased healthcare resource utilization for acute respiratory illness among Latino infants. J Pediatr. 2013;163(4):1186–91. 12. Lassetter JH, Baldwin JH. Health care barriers for latino children and provi‑ sion of culturally competent care. J Pediatr Nurs. 2004;19(3):184–92. Received: 1 December 2021 Accepted: 17 October 2022 informed via an interpreter and felt reassured by the end of the PED visit. However, families did have dif- ficulty navigating the healthcare system. Qualitative studies such as this study can help identify specific factors from the parents’ experiences that could be improved to reduce disparities related to language bar- riers. Further areas of intervention should focus on support beyond providing access to an interpreter, such as improving information delivery, and enhancing edu- cation on community resources for families with low English proficiency in the PED. Acknowledgements Not Applicable. 7. Roan Gresenz C, Derose KP, Ruder T, Escarce JJ. Health Care Experiences of Hispanics in New and Traditional U.S. Destinations. Med Care Res Rev 2022;69(6):663–78. Available from: http://​mcr.​sagep​ub.​com 7. Roan Gresenz C, Derose KP, Ruder T, Escarce JJ. Health Care Experiences of Hispanics in New and Traditional U.S. Destinations. Med Care Res Rev 2022;69(6):663–78. Available from: http://​mcr.​sagep​ub.​com Availability of data and materials Data cannot be shared publicly because of potentially identifying patient information. Data are available from the Division of Emergency Medicine at Cincinnati Children’s Hospital Medical Center (contact Dr. Mahabee-Gittens via melin​da.​mahab​ee-​gitte​ns@​cchmc.​org) for researchers who meet the criteria for access to confidential data. 13. Hampers LC, Cha S, Gutglass DJ, Binns HJ, Krug SE. Language barriers and resource utilization in a pediatric emergency department. Pediatrics. 1999;103(6 I):1253–6. 13. Hampers LC, Cha S, Gutglass DJ, Binns HJ, Krug SE. Language barriers and resource utilization in a pediatric emergency department. Pediatrics. 1999;103(6 I):1253–6. 14. Payne NR, Puumala SE. Racial disparities in ordering laboratory and radiol‑ ogy tests for pediatric patients in the emergency department. Pediatr Emerg Care. 2013;29(5):598–606. 14. Payne NR, Puumala SE. Racial disparities in ordering laboratory and radiol‑ ogy tests for pediatric patients in the emergency department. Pediatr Emerg Care. 2013;29(5):598–606. for access to confidential data. 15. Lara M, Gamboa C, Kahramanian MI, Morales LS, Hayes Bautista DE. ACCULTURATION AND LATINO HEALTH IN THE UNITED STATES: A Review of the Literature and its Sociopolitical Context. Annu Rev Public Heal 2005;26:367–97. Available from: www.​annua​lrevi​ews.​org 15. Lara M, Gamboa C, Kahramanian MI, Morales LS, Hayes Bautista DE. ACCULTURATION AND LATINO HEALTH IN THE UNITED STATES: A Review of the Literature and its Sociopolitical Context. Annu Rev Public Heal 2005;26:367–97. Available from: www.​annua​lrevi​ews.​org Consent for publication Not Applicable. Not Applicable. 18. Topmiller M, Zhen-Duan J, Jacquez FJ, Vaughn LM. Place matters in non- traditional migration areas: exploring barriers to healthcare for Latino immigrants by region, neighborhood, and community health center. J Racial Ethn Heal Disparities. 2017;4(6):1214–23. Conclusions Another key issue highlighted in this study was the importance of educating the family about their child’s diagnosis and management in the PED. Families were often very grateful for the treatment they received even Our study identified four major themes among Latinx families within a PED of a NDA. All families were fear- ful of their child’s illness, but they appreciated being Page 7 of 8 Zamor et al. BMC Health Services Research (2022) 22:1463 Page 7 of 8 Received: 1 December 2021 Accepted: 17 October 2022 Received: 1 December 2021 Accepted: 17 October 2022 References 1. Cortez R. Hispanic Heritage Month. Not Am Math Soc. 2018;65(08):1. 2. PEW Research Center. The nation’s Latino population is defined by its youth. 2016 [cited 2022 Sep 7]; Available from: http://​www.​pewhi​span-​ic.​ org/​2016/​04/​20/​the-​natio​ns-​latino-​popul​ation-​is-​defin​ed-​by-​its-​youth/ 1. Cortez R. Hispanic Heritage Month. Not Am Math Soc. 2018;65(08):1. 2. PEW Research Center. The nation’s Latino population is defined by its youth. 2016 [cited 2022 Sep 7]; Available from: http://​www.​pewhi​span-​ic.​ org/​2016/​04/​20/​the-​natio​ns-​latino-​popul​ation-​is-​defin​ed-​by-​its-​youth/ 1. Cortez R. Hispanic Heritage Month. Not Am Math Soc. 2018;65(08):1. 2. PEW Research Center. The nation’s Latino population is defined by its youth. 2016 [cited 2022 Sep 7]; Available from: http://​www.​pewhi​span-​ic.​ org/​2016/​04/​20/​the-​natio​ns-​latino-​popul​ation-​is-​defin​ed-​by-​its-​youth/ 3. Connors KM. Barriers Latino Patients Face in the Pediatric Emergency Department. Pediatr Nurs [Internet]. 2019;45(1):37–41,46. Available from: https://​www.​census.​gov/ 3. Connors KM. Barriers Latino Patients Face in the Pediatric Emergency Department. Pediatr Nurs [Internet]. 2019;45(1):37–41,46. Available from: https://​www.​census.​gov/ 4. Passel JS, Cohn D, Lopez MH. Hispanics Account for More than Half of Nation’s Growth in Past Decade | Pew Research Center. Pew Hisp Cent [Internet]. 2011; Available from: http://​www.​pewhi​spanic.​ org/​2011/​03/​24/​hispa​nics-​accou​nt-​for-​more-​than-​half-​of-​natio​ ns-​growth-​in-​past-​decade/ Authors’ contributions RZ, LV and EMM-G conceived the study and the overall study design. LV and EMM-G supervised the conduct of the study. RZ, EM, and LS recruited participants and conducted the interviews. RZ, EM, LS and EP analyzed the data. All authors reviewed the data and results. RZ, EM, LS, and EP drafted the manuscript and all authors contributed substantially to its revision. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. 8. DeJonckheere MJ, Vaughn LM, Jacquez F. Latino immigrant youth living in a nontraditional Migration City: a social-ecological examination of the complexities of stress and resilience. Urban Educ. 2017;52(3):399–426. 8. DeJonckheere MJ, Vaughn LM, Jacquez F. Latino immigrant youth living in a nontraditional Migration City: a social-ecological examination of the complexities of stress and resilience. Urban Educ. 2017;52(3):399–426. 9. Zamor R, Byczkowski T, Zhang Y, Vaughn L, Mahabee-Gittens EM. Language barriers and the Management of Bronchiolitis in a pediatric emergency department. Acad Pediatr. 2020;20(3):356–63. 10. Levas MN, Dayan PS, Mittal MK, Stevenson MD, Bachur RG, Dudley NC, et al. Effect of hispanic ethnicity and language barriers on appendiceal perforation rates and imaging in children. J Pediatr. 2014;164(6). Ethics approval and consent to participate Informed verbal consent was obtained from all participants. The study, includ‑ ing verbal consent, was approved by the Institutional Review Board at CCHMC and the research team followed ethical guidelines corresponding to research with human subjects. 16. Ogden CL, Carroll MD, Kit BK, Flegal KM. Prevalence of obesity and trends in body mass index among US children and adolescents, 1999–2010. JAMA - J Am Med Assoc 2012;307(5):483–90. Available from: http://​www.​ jama.​com. 17. Jacquez F, Vaughn LM, Suarez-Cano G. Implementation of a stress intervention with Latino immigrants in a non-traditional Migration City. J Immigr Minor Health. 2019;21(2):372–82. Competing interests Competing interests The authors declare that they have no competing interests. Author details 19. Zhen-Duan J, Jacquez F, Vaughn L. Demographic characteristics associ‑ ated with barriers to health care among Mexican and Guatemalan immigrants in a nontraditional destination area. Fam Community Heal. 2017;40(2):101–11. 1 Division of Emergency Medicine, Cincinnati Children’s Hospital Medi‑ cal Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. 2 Department of Pediatrics, University of Cincinnati College of Medicine, 3230 Eden Avenue, Cincinnati, OH 45267, USA. 3 Present Address: Division of Emergency Medicine, Children’s Healthcare of Atlanta, Emory University, 1547 Clifton Road, NE 2nd Floor, Atlanta, GA 30322, USA. 4 Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. 20. Biolsi R. Our Diverse Society: Race and Ethnicity—Implications for 21 st Century American Society, by David W. Engstrom and Lissette M. Piedra, (eds.) . Adm Soc Work. 2009 Jan 5;33(1):129–31. Available from: https://​ doi.​org/​10.​1080/​03643​10080​25087​00. 20. Biolsi R. Our Diverse Society: Race and Ethnicity—Implications for 21 st Century American Society, by David W. Engstrom and Lissette M. Piedra, (eds.) . Adm Soc Work. 2009 Jan 5;33(1):129–31. Available from: https://​ doi.​org/​10.​1080/​03643​10080​25087​00. Page 8 of 8 Zamor et al. BMC Health Services Research (2022) 22:1463 Zamor et al. BMC Health Services Research (2022) 22:1463 21. McDermott KW, Stocks C, Freeman WJ. Overview of Pediatric Emergency Department Visits, 2015: Statistical Brief #242. Healthcare Cost and Utiliza‑ tion Project (HCUP) Statistical Briefs. Rockville, MD; 2006 [cited 2020 Aug 31]. Available from: http://​www.​ncbi.​nlm.​nih.​gov/​pubmed/​30277​692 31]. Available from: http://​www.​ncbi.​nlm.​nih.​gov/​pubmed/​30277​6 22. Cincinnati Children’s Hospitals. Cincinnati Children’s: International Patients and Families [Internet]. 2016 [cited 2018 Oct 8]. Available from: https://​www.​cinci​nnati​child​rens.​org/​patie​nts/​visit/​inter​natio​nal 23. Bowen GA. Naturalistic inquiry and the saturation concept: a research note. Qual Res. 2008;8(1):137–52. 24. Moustakas C. Phenomenology and human science inquiry. SAGE Publica‑ tions, Inc., 1994. https://​doi.​org/​10.​4135/​97814​12995​658. 25. Crotty M. The foundations of social research: meaning and perspective in the research process. London Thousand Oaks, Calif: Sage Publications; 1998. 26. Sundler AJ, Lindberg E, Nilsson C, Palmér L. Qualitative thematic analysis based on descriptive phenomenology. Nurs Open. 2019;6(3):733–9. 27. Hsieh HF, Shannon SE. Three approaches to qualitative content analysis. Qual Health Res. 2005;15(9):1277–88. 28. Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol. 2006;3(2):77–101. 29. Dedoose Version 8.0.35. Web application for managing, analyzing, and presenting qualitative and mixed method research data. 2018. Los Ange‑ les, CA: SocioCultural Research Consultants, LLC www.dedoose.com. 30. Karliner LS, Jacobs EA, Chen AH, Mutha S. Author details Do professional interpreters improve clinical care for patients with limited english proficiency? A systematic review of the literature. Health Serv Res. 2007;42(2):727–54. 31. Casey Lion K, Brown JC, Ebel BE, Klein EJ, Strelitz B, Gutman CK, et al. Effect of telephone vs video interpretation on parent comprehension, communication, and utilization in the pediatric emergency department a randomized clinical trial. JAMA Pediatr. 2015;169(12):1117–25. 32. Vaughn LM, Jacquez F. Characteristics of newly immigrated, Spanish- speaking Latinos who use the pediatric emergency department: preliminary findings in a secondary migration city. Pediatr Emerg Care. 2012;28(4):345–50. 33. Fields A, Abraham M, Gaughan J, Haines C, Hoehn KS. Language matters: race, trust, and outcomes in the pediatric emergency department. Pedi‑ atr Emerg Care. 2016;32(4):222–6. 33. Fields A, Abraham M, Gaughan J, Haines C, Hoehn KS. Language matters: race, trust, and outcomes in the pediatric emergency department. Pedi‑ atr Emerg Care. 2016;32(4):222–6. 34. Mayo RM, Sherrill WW, Sundareswaran P, Crew L. Attitudes and percep‑ tions of hispanic patients and health care providers in the treatment of hispanic patients: a review of the literature. Hisp Health Care Int. 2007;5:64–72. 34. Mayo RM, Sherrill WW, Sundareswaran P, Crew L. Attitudes and percep‑ tions of hispanic patients and health care providers in the treatment of hispanic patients: a review of the literature. Hisp Health Care Int. 2007;5:64–72. 35. Santos Malavé C, Diggs D, Sampayo EM. Spanish-speaking caregivers’ experience with an emergency department pediatric asthma-care bundle quality initiative. J Racial Ethn Heal Disparities. 2019;6(4):660–7. https://​doi.​org/​10.​1007/​s40615-​019-​00564-1. g 36. Grigg A, Shetgiri R, Michel E, Rafton S, Ebel BE. Factors associated with nonurgent use of pediatric emergency care among Latino families. J Natl Med Assoc. 2013;105(1):77–84. Zamor et al. BMC Health Services Research (2022) 22:1463 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 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Synthesis and characterization of the first mononuclear glycolate-aryloxide derivatives of antimony(III)
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Sunita Goyala and Anirudh Singhb* aDepartment of Chemistry, Jaipur Engineering College, Kukas, Jaipur-303 101, Rajasthan, India bDepartment of Chemistry, University of Rajasthan, Jaipur-302 004, Rajasthan, India Manuscript received 01 April 2013 accepted 08 May 2013 aDepartment of Chemistry, Jaipur Engineering College, Kukas, Jaipur-303 101, Rajasthan, India bDepartment of Chemistry, University of Rajasthan, Jaipur-302 004, Rajasthan, India Manuscript received 01 April 2013, accepted 08 May 2013 Abstract : Equimolar reactions of OGOSbOPri with ArOH (substituted phenols) in benzene after usual work-up afford hydrocarbon soluble, monomeric derivatives of the type OGOSbOAr, where G = CHMeCH2CMe2 : Ar = C6H3Me2-2,6 (1), C6H3Pri-2-Me-5 (2), C6H3Pri-2,6 (3) and G = CMe2CH2CH2CMe2 : Ar = C6H3Me2-2,6 (4), C6H3Pri 2-2,6 (5). The derivative (1) has also been prepared by in situ by equimolar reaction of Sb(OPri)3, HOCHMeCH2CMe2OH and HOC6H3Me2-2,6 in refluxing benzene. All of these new derivatives have been characterized by elemental analyses, molecular weight measurements and spectroscopic (IR and 1H NMR) studies. Keywords : Antimony(III), glycolates, aryloxides, spectroscopy. adjacent fragments, [Sb(2-OCH2CH2O)(OAc)]n. Reac- tions of Sb(OEt)3 and glycols in different molar ratios were investigated10 as early as in 1965, followed by syn- thesis of another type of mixed-ligand derivatives of anti- mony containing glycolate and a bidentate moiety with phenoxo functionality11. Reactions of SbCl3 with alkali metal (Na or K) aryloxides in 1 : 1 and 1 : 2 molar ratios afford antimony aryloxide-chloride derivatives, which are reported12 to be monomeric in refluxing benzene. It is worthmentioning that many of the antimony ethoxide-gly- colate derivatives10 are either insoluble or poorly soluble even in hot benzene, except the derivatives derived from hexylene glycol or pinacol, which are sufficiently soluble. J. Indian Chem. Soc., J. Indian Chem. Soc., NOTE Vol. 91, February 2014, pp. 339-342 Keywords : Antimony(III), glycolates, aryloxides, spectroscopy. *Present Address : M-2/213, Sector-H, L.D.A. Colony, Kanpur Road, Lucknow-226 012, Uttar Pradesh, India Table 2. Salient IR (cm–1) and 1H NMR (, ppm) spectral data for antimony(III) glycolate-aryloxide derivatives (1)-(5) (1) IR : 1073m (C-O) glycolate, 1220s (C-O) aryloxide, 852m (C-C) glycolate, 576w (Sb-O), 750s (ring pulsation), 458w (ring vibration). 1H NMR : 1.57 (2H, m, CH2), 2.18 (6H, s, CMe2), 1.22 (3H, d, J 6.2 Hz, CHMe), 4.45 (1H, br, CHMe) glycolate; 2.28 (6H, s, Me2-2,6), 6.81–7.35 (3H, m, Ar-H) aryloxide. (2) IR : 1073m (C-O) glycolate, 1279s (C-O) aryloxide, 867s (C-C), 720m (ring pulsation), 577s (Sb-O), 473m (ring vibration). 1H NMR : 1.63 (2H, m, CH2), 2.29 (6H, s, CMe2), 1.25 (3H, d, J 6.2 Hz, CHMe), 472 (1H, br, CHMe) glycolate; 2.21 (3H, s, Me-5), l.l8 (6H, d, J 6.2 Hz, CHMe2), 3.29 (1H, sept, J 6.2 Hz, CHMe2), 6.68–7.44 (3H, m, Ar-H) aryloxide. (3) IR : 1088s (C-O) glycolate, 1250m (C-O) aryloxide, 823s (C-C), 706m (ring pulsation), 547s (Sb-O), 466s (ring vibration). 1H NMR : 1.30 (3H, d, J 6.2 Hz, CHMe), 1.47 (2H, m, CH2), 2.28 (6H, s, CMe2), 4.82 (1H, br, CHMe) glycolate; 1.17 (12H, d, J 6.2 Hz, CHMe2), 3.34 (2H, sept, J 6.2 Hz, CHMe2), 6.78–7.45 (3H, m, Ar-H) aryloxide. (4) IR : 1044s (C-O) glycolate, 1227s (C-O) aryloxide, 868m (C-C), 757s (ring pulsation), 582m (Sb-O), 451s (ring vibration). 1H NMR : 1.36 (12H, s, CMe2), 1.48 (4H, m, CH2) glycolate; 2.37 (6H, s, Me2-2,6), 6.75–7.45 (3H, m, Ar-H) aryloxide. (5) IR : 1088s (C-O) glycolate, 1220m (C-O) aryloxide, 867s (C-C), 720w (ring pulsation), 548s (Sb-O), 466m (ring vibration). 1H NMR : 1.35 (12H, s, CMe2), 1.54 (4H, m, CH2) glycolate; 1.22 (12H, d, J 6.2 Hz, CHMe2), 3.45 (2H, sept, J 6.2 Hz, CHMe2), 6.91–7.44 (3H, m, Ar-H) aryloxide. IR : Abbreviations : s = strong, m = medium, w = weak. 1H NMR : Abbreviations : s = singlet d = doublet m = multiplet sept = septet br = broad J. Indian Chem. Soc., Vol. 91, February 2014 OAr represents an aryloxide moiety). Table 1. Analytical and some physical data for the compounds (1)-(5) Table 1. Analytical and some physical data for the compounds (1)-(5) Table 1. Analytical and some physical data for the compounds (1)-(5) Compd. Colour and Analysis (%) : Mol. wt. Empirical state Found (Calcd.) Found formula C H Sb (Calcd.) (1) White solid 46.78 5.84 33.85 350 Cl4H21SbO3 (46.81) (5.89) (33.91) (359) (2) White solid 49.52 6.45 31.38 382 Cl6H25SbO3 (49.64) (6.51) (31.45) (387) (3) Pale yellow solid 51.96 6.98 29.28 410 C18H29SbO3 (52.07) (7.04) (29.33) (415) (4) Pale yellow solid 49.58 6.48 31.40 380 Cl6H25SbO3 (49.64) (6.51) (31.45) (387) (5) Pale yellow solid 54.15 7.45 27.40 440 C20H33SbO3 (54.20) (7.50) (27.47) (443) 1H NMR : Abbreviations : s = singlet, d = doublet, m = multiplet, sept = septet, br = broad. Introduction The metal-aryloxide bond is one of the most promi- nent component of rapidly increasing number of inor- ganic and organometallic derivatives1,2 and is also present in nature in a large number of metalloproteins3. The chem- istry of metal-aryloxides2 and -glycolates4,5 has grown steadily over the last five decades. The X-ray crystallo- graphic structural elucidation of Sb(OPri)3 6 and Sb(OC6H3Me2-2,6)3 7 depict dimeric structure for the former involving isopropoxo bridging in tetrahedral en- vironment of isopropoxo groups around antimony, whereas later is a three coordinate monomer due to steric reasons, adopting a trigonal pyramidal architecture capped by a stereochemically active lone electron pair. Interestingly, analogous derivative derived from sterically compact methoxo groups forms a layer structure incorporating SbO6 moieties8, where the coordination number at antimony is six. The antimony ethylene glycolate-acetate complex [Sb(OC2H4O)(OAc)], which exhibits polycondensation catalytic activity is shown by X-ray diffraction studies9 to consist of a one-dimensional chain in which the oxygen atoms of the glycolate moiety of each [Sb(OCH2CH2O)- (OAc)] fragment bridge to the antimony centers of two In view of the above and our continuing interest in mixed-ligand derivatives of group 15 elements11,13, it was considered to be worthwhile to investigate the possibility of isolating hydrocarbon soluble and monomeric antimony(III) glycolate-aryloxide derivatives, which were not known prior to the present studies. We, therefore, report in this paper for the first time the synthesis, spec- tral (IR and 1H NMR) properties, and ebullioscopic mo- lecular weight measurements of five derivatives of the type OGOSbOAr (where OGO is a glycolate group and 339 J. Indian Chem. Soc., Vol. 91, February 2014 J. Indian Chem. Soc., Vol. 91, February 2014 IR : Abbreviations : s = strong, m = medium, w = weak. Table 2. Salient IR (cm–1) and 1H NMR (, ppm) spectral data for antimony(III) glycolate-aryloxide derivatives (1)-( Results and discussion Synthesis : Equimolar reactions of OGOSbOPri with substituted phenols (abbreviated as ArOH) in benzene afford mixed, glycolate-aryloxide derivatives of antimony(III) accord- ing to the following reaction : benzene OGOSbOPri + ArOH ————— reflux OGOSbOAr + PriOH benzene OGOSbOPri + ArOH ————— reflux OGOSbOAr + PriOH OGOSbOAr + PriOH (1) : G = CHMeCH2CMe2, Ar = C6H3Me2-2,6 (2) : G = CHMeCH2CMe2, Ar = C6H3Pri-2-Me-5 (3) : G = CHMeCH2CMe2, Ar = C6H3Pri 2-2,6 (4) : G = CMe2(CH2)2CMe2, Ar = C6H3Me2-2,6 (5) : G = CMe2(CH2)2CMe2, Ar = C6H3Pri 2-2,6 organic solvents (e.g. benzene, toluene, dichloromethane, etc.) and depict monomeric nature (ebullioscopically) in benzene solution. Spectroscopic studies : Spectroscopic studies : The derivative (1) has also been prepared in situ by equimo- lar reactions of Sb(OPri)3, HOCHMeCH2CMe2OH and HOC6H3Me2-2,6 under refluxing conditions in benzene : The IR and 1H NMR assignments11–14 (Table 2) have been made on the basis of published data about glycols, substituted phenols, and mixed-ligand derivatives of ele- ments. Sb(POri)3 + HOCHMeCH2Me2OH + Sb(POri)3 + HOCHMeCH2Me2OH + Sb(POri)3 + HOCHMeCH2Me2OH + benzene HOC6H3Me2-2,6 ————— (1) + 3PriOH reflux (a) IR spectra : Derivatives (1)-(5) exhibit medium to strong absorptions in the region 1220–1280 cm–1, due to (C- O) of aryloxide moiety. An increase of ~15 cm–1 in the (C-O) of aryloxo group, supports O-bonded All these derivatives (Table 1) are highly moisture-sensi- tive colourless to pale yellow solids, soluble in common (5) IR : 1088s (C-O) glycolate, 1220m (C-O) aryloxide, 867s (C-C), 720w (ring pulsation), 548s (Sb-O), 466m (ring vibration). 1H NMR : 1.35 (12H, s, CMe2), 1.54 (4H, m, CH2) glycolate; 1.22 (12H, d, J 6.2 Hz, CHMe2), 3.45 (2H, sept, J 6.2 Hz, CHMe2), 6.91–7.44 (3H, m, Ar-H) aryloxide. 340 Note Note aryloxo group to antimony(III). The absorptions due to (Sb-O) appear in the region 545–585 cm–1. Ab- sorptions observed in the regions 700–760 cm–1 and 450–475 cm–1 are due to ring pulsation and ring vi- brations, respectively. aryloxo group to antimony(III). The absorptions due to (Sb-O) appear in the region 545–585 cm–1. Ab- sorptions observed in the regions 700–760 cm–1 and 450–475 cm–1 are due to ring pulsation and ring vi- brations, respectively. sor derivatives, OGOSbOPri (G = CHMe2CH2CMe2 and CMe2CH2CH2CMe2) were prepared according to the pro- cedure described in the literature10,11. Isopropyl alcohol in the azeotrope was estimated oxidimetrically15 using 1 N K2Cr2O7 solution in 12.5% H2SO4. Antimony was de- termined volumetrically16. (b) 1H NMR spectra : The signal due to OH group of the substituted phenols in the region  4.70–4.90 is ab- sent in derivatives (1)-(5), supporting the metallation of the phenolic OH group proton and formation of Sb- OAr bond. All these derivatives show (Table 2) mul- tiplets due to aromatic ring protons in the region  6.70–7.50. Signals characteristic of a glycolate group appear in the regions :  1.20–1.30 (d, J 6.2 Hz, CHMe), 1.40–1.70 (br, CH2), 2.10–2.30 (s, CMe2), 4.40–4.90 (m, CHMe). The substituents present on an aryloxo group show signals for derivatives : (1) at  2.28 (s, Me2-2,6); (2) at  1.18 (d, J 6.2 Hz, CHMe2), 2.21 (s, Me-5), 3.29 (sept, J 6.2 Hz, CHMe2); (3) at  1.17 (d, J 6.2 HZ, CHMe2), 3.34 (sept, J 6.2 HZ, CHMe2); (4) at  2.37 (s, Me2-2,6); (5) at  1.22 (d, J 6.2 Hz, CHMe2), 3.45 (sept, J 6.2 Hz, CHMe2). Synthesis of antimony(III) glycoxide-aryloxides (1)-(5): These derivatives have been prepared by two routes : (i) reactions of OGOSbOPri with appropriate substituted phenols and (ii) in situ equimolar reaction of Sb(OPri)3 with a glycol and a substituted phenol, the former route was used for the synthesis of all the five compounds, whereas the latter route was used to prepare OCMe2CH2CH2CMe2OSbOC6H3Me2-2,6. The aforementioned findings along with the data avail- able for mixed-ligand derivatives of arsenic(III)13a and antimony(III)12 in the literature, tend to favour the struc- ture shown below for compounds (1)-(5) : Due to similarity in the preparative procedures, syn- thetic details of only one derivative for each route are described below : where G = CHMeCH2CMe2 and CMe2(CH2)2CMe2; Ar = C6H3Me2-2,6, C6H3Pri2-2,6 and C6H3Pri-2-Me-5 (i) OCHMeCH2CMe2OSbOC6H3Pri 2-2,6 (3) : (i) OCHMeCH2CMe2OSbOC6H3Pri 2-2,6 (3) : To a benzene (40 ml) solution of OCHMeCH2CMe2OSbOPri (1.54 g, 5.20 mmol) was added HOC6H3Pri 2-2,6 (0.928 g, 5.20 mmol) dissolved in benzene (20 ml), and the reaction mixture was re- fluxed over a period of ~10 h, during which time the liberated isopropyl alcohol was continuously fractionated out azeotropically and estimated periodically to check completion of the reaction. When the azeotrope showed negligible presence of an oxidisible material, the reflux- ing was stopped and the solution was allowed to cool to room temperature. Removal of the volatile components from the solution under reduced pressure afforded a white solid product (2.10 g, 92%), which was recrystallized in analytically pure state (1.80 g, 83.3%) from a 2 : 1 mix- ture of n-hexane and CH2Cl2 at –20 ºC. where G = CHMeCH2CMe2 and CMe2(CH2)2CMe2; Ar = C6H3Me2-2,6, C6H3Pri2-2,6 and C6H3Pri-2-Me-5 Sb(POri)3 + HOCHMeCH2Me2OH + (b) IR (4000–200 cm–1) spectra were recorded as Nujol mulls on a Nicolet Magna 550 spectrophotometer using CsI optics. 1H NMR (89.55 MHz, CDCl3) were recorded on a JEOL FX-90Q spectrometer using TMS (tetra- methylsilane) as an internal reference. C, H analyses were recorded on a Perkin-Elmer Series II CHNS/O analyzer- 2400. Molecular weights were determined (on a Gallenkamp ebulliometer using thermister sensing device) ebullioscopically in benzene. Conclusion Convenient and quantitative syntheses of five glyco- late-aryloxide derivatives of antimony(III), which are hy- drocarbon soluble and monomeric, are reported for the first time. 14. (a) S. Goyal and A. Singh, Synth. React. Inorg. Metal- Org. Chem., 2002, 32, 1433; (b) T. B. Brill and N. C. Campbell, Inorg. Chem., 1973, 12, 1884. 15. (a) C. Adams and J. R. Nichollas, Analyst, 1929, 50, 2; (b) R. C. Mehrotra, J. Indian Chem. Soc., 1953, 30, 585. J. Indian Chem. Soc., Vol. 91, February 2014 J. Indian Chem. Soc., Vol. 91, February 2014 pared. The amount of reactants actually used are shown in square brackets against each compound listed below : pared. The amount of reactants actually used are shown in square brackets against each compound listed below : istry, University of Rajasthan, Jaipur, for providing re- search facilities. istry, University of Rajasthan, Jaipur, for providing re- search facilities. (1) : OCHMeCH2CMe2OSbOPri [0.80 g, 2.69 mmol] and HOC6H3Me2-2,6 [0.33 g, 2.69 mmol]. Yield 0.94 g (90%). Isopropyl alcohol in the azeotrope (Calcd. 0.162 g, Found 0.16 g). (1) : OCHMeCH2CMe2OSbOPri [0.80 g, 2.69 mmol] and HOC6H3Me2-2,6 [0.33 g, 2.69 mmol]. Yield 0.94 g (90%). Isopropyl alcohol in the azeotrope (Calcd. 0.162 g, Found 0.16 g). (ii) Preparation of (1) by an alternative route : When azeotrope collected showed negligible presence of PriOH, The benzene (80 ml) solution containing equimolar amounts of Sb(OPri)3 (1.5 g, 5.02 mmol), 2-methyl- 2,4- pentanediol (0.58 g, 4.99 mmol) and 2,6-dimethyl phe- nol (0.61 g, 4.99 mmol) was refluxed for ~24 h, during which time the liberated isopropyl alcohol was continu- ously fractionated out and determined periodically to monitor the progress and completion of the reaction. When azeotrope collected showed negligible presence of PriOH, 6. H. Fleischer, H. Bayram, S. Elzner and N. W. Mitzel, J. Chem. Soc., Dalton Trans., 2001, 373. 6. H. Fleischer, H. Bayram, S. Elzner and N. W. Mitzel, J. Chem. Soc., Dalton Trans., 2001, 373. 7. G. A. Horley, M. F. Mahon, K. C. Molloy and M. M. Venter, Inorg. Chem., 2002, 41, 1652. 8. U. Ensinger, W. Schwarz, B. Schrutz, K. Sommer and A. Z. Schmidt, Anorg. Allg. Chem., 1987, 544, 181. 9. S. M. Biros, B. M. Bridgewater, A. V-Estrada, J. M. Tanski and G. Parkin, Inorg. Chem., 2002, 41, 4051. the refluxing was stopped and solution was allowed to cool to room temperature. The solvent was removed un- der reduced pressure to afford white solid, which was recrystallized from a 2 : 1 mixture of n-hexane- dichloromethane at –20 ºC to give 1.58 g of the desired product. Yield 88% (Found : C, 46.76; H, 5.82; Sb, 33.88. Calcd. for C14H21SbO3 : C, 46.83; H, 5.81; Sb, 33.91%). 10. R. C. Mehrotra and D. D. Bhatnagar, J. Indian Chem. Soc., 1965, 42, 327. 11. S. Goyal and A. Singh, Indian J. Chem., Sect. A, 2001, 40, 1302. 12. T. Athar, R. Bohra and R. C. Mehrotra, J. Indian Chem. Soc., 1989, 66, 189. 13. (a) S. Goyal and A. Singh, Synth. React. Inorg Metal- Org. Chem., 2002, 32, 1319; (b) S. Goyal and A. Singh, Phosphorus, Sulfur and Silicon, 2003, 1, 178; (c) S. Goyal, PhD Thesis, University of Rajasthan, Jaipur, India, 2003. (ii) Preparation of (1) by an alternative route : 5. A. Singh and R. C. Mehrotra, Coord. Chem. Rev., 2004, 248, 101. 5. A. Singh and R. C. Mehrotra, Coord. Chem. Rev., 2004, 248, 101. The benzene (80 ml) solution containing equimolar amounts of Sb(OPri)3 (1.5 g, 5.02 mmol), 2-methyl- 2,4- pentanediol (0.58 g, 4.99 mmol) and 2,6-dimethyl phe- nol (0.61 g, 4.99 mmol) was refluxed for ~24 h, during which time the liberated isopropyl alcohol was continu- ously fractionated out and determined periodically to monitor the progress and completion of the reaction. When azeotrope collected showed negligible presence of PriOH, the refluxing was stopped and solution was allowed to cool to room temperature. The solvent was removed un- der reduced pressure to afford white solid, which was recrystallized from a 2 : 1 mixture of n-hexane- dichloromethane at –20 ºC to give 1.58 g of the desired product. Yield 88% (Found : C, 46.76; H, 5.82; Sb, 33.88. Calcd. for C14H21SbO3 : C, 46.83; H, 5.81; Sb, 33.91%). The benzene (80 ml) solution containing equimolar amounts of Sb(OPri)3 (1.5 g, 5.02 mmol), 2-methyl- 2,4- pentanediol (0.58 g, 4.99 mmol) and 2,6-dimethyl phe- nol (0.61 g, 4.99 mmol) was refluxed for ~24 h, during which time the liberated isopropyl alcohol was continu- ously fractionated out and determined periodically to monitor the progress and completion of the reaction. When azeotrope collected showed negligible presence of PriOH, the refluxing was stopped and solution was allowed to cool to room temperature. The solvent was removed un- der reduced pressure to afford white solid, which was recrystallized from a 2 : 1 mixture of n-hexane- dichloromethane at –20 ºC to give 1.58 g of the desired product. Yield 88% (Found : C, 46.76; H, 5.82; Sb, 33.88. Calcd. for C14H21SbO3 : C, 46.83; H, 5.81; Sb, 33.91%). The benzene (80 ml) solution containing equimolar amounts of Sb(OPri)3 (1.5 g, 5.02 mmol), 2-methyl- 2,4- pentanediol (0.58 g, 4.99 mmol) and 2,6-dimethyl phe- nol (0.61 g, 4.99 mmol) was refluxed for ~24 h, during which time the liberated isopropyl alcohol was continu- ously fractionated out and determined periodically to monitor the progress and completion of the reaction. References 1. (a) K. C. Malhotra and R. L. Martin, J. Organomet. Chem., 1982, 239, 159; (b) M. H. Chisholm and I. P. Rothwell, in "Comprehensive Coordination Chemistry", eds. G. Wilkinson, R. D. Gillard and J. A. Me Cleverty, Pergamon Press, Ox- ford, 1987, Vol. 2, Chap. 15.3; (c) H. E. Bryndza and W. Tam, Chem. Rev., 1988, 88, 1163; (d) K. G. Caulton and L. G. Hubert-Pfalzgraf, Chem. Rev., 1990, 90, 969; (e) W. G. Vander Sluys and A. P. Sattelberger, Chem. Rev., 1990, 90, 1027; (f) R. C. Mehrotra, A. Singh and U. M. Tripathi, Chem. Rev., 1991, 91, 1287; (g) M. D. Healy, M. B. Power and A. R. Barron, Coord. Chem. Rev., 1994, 130, 63; (h) C. J. Carmalt and S. J. King, Coord. Chem. Rev., 2006, 250, 687; (i) T. J. Boyle and L. A. M. Ottley, Chem. Rev., 2008, 108, 1896. (2) : OCHMeCH2CMe2OSbOPri [2.21 g, 7.44 mmol] and HOC6H3Pri-2-Me-5 [1.11 g, 7.44 mmol]. Yield 2.80 g (91 %). Isopropyl alcohol in the azeotrope (Calcd. 0.447 g, Found 0.44 g). (4) : OCMe2CH2CH2CMe2OSbOPri [1.78 g, 5.47 mmol] and HOC6H3Me2-2,6 [0.669 g, 5.47 mmol]. Yield 1.98 g (94%). Isopropyl alcohol in the azeotrope (Calcd. 0.329 g, Found 0.32 g). (4) : OCMe2CH2CH2CMe2OSbOPri [1.78 g, 5.47 mmol] and HOC6H3Me2-2,6 [0.669 g, 5.47 mmol]. Yield 1.98 g (94%). Isopropyl alcohol in the azeotrope (Calcd. 0.329 g, Found 0.32 g). (5) : OCMe2CH2CH2CMe2OSbOPri [0.826 g, 2.54 mmol] and HOC6H3Pri 2-2,6 [0.453 g, 2.54 mmol]. Yield 1.18 g (97%). Isopropyl alcohol in the azeotrope (Calcd. 0.152 g, Found 0.15 g). 2. D. C. Bradley, R. C. Mehrotra, I. P. Rothwell and A. Singh, "Alkoxo and Aryloxo Derivatives of Metals", Academic Press, London, 2001. 2. D. C. Bradley, R. C. Mehrotra, I. P. Rothwell and A. Singh, "Alkoxo and Aryloxo Derivatives of Metals", Academic Press, London, 2001. 3. L. Que, Coord. Chem. Rev., 1983, 50, 73. 3. L. Que, Coord. Chem. Rev., 1983, 50, 73. Analytical details and some physical properties of all the new derivatives (1)-(5) are summarized in Table 1. 4. R. C. Mehrotra and A. Singh, Prog. Inorg. Chem., 1997, 46, 239. 4. R. C. Mehrotra and A. Singh, Prog. Inorg. Chem., 1997, 46, 239. (ii) Preparation of (1) by an alternative route : Experimental All preparations and manipulations were performed under strictly moisture-free conditions as already described in some of our earlier publications10–12. Benzene and n- hexane were dried and purified under anhydrous condi- tions by refluxing over Na/benzophenone, followed by distillation prior to use. Dichloromethane was dried by refluxing over P2O5, followed by distillation (b.p. 39 ºC). 2-Methylpentane-2,4-diol (b.p. 197 ºC/756 mm) (BDH) and 2,5-dimethylhexane-2,5-diol (b.p. 214 ºC/ 756 mm) (Aldrich) were distilled prior to use. Sb(OPri)3 was prepared by the literature method10,14b. The precur- All preparations and manipulations were performed under strictly moisture-free conditions as already described in some of our earlier publications10–12. Benzene and n- hexane were dried and purified under anhydrous condi- tions by refluxing over Na/benzophenone, followed by distillation prior to use. Dichloromethane was dried by refluxing over P2O5, followed by distillation (b.p. 39 ºC). 2-Methylpentane-2,4-diol (b.p. 197 ºC/756 mm) (BDH) and 2,5-dimethylhexane-2,5-diol (b.p. 214 ºC/ 756 mm) (Aldrich) were distilled prior to use. Sb(OPri)3 was prepared by the literature method10,14b. The precur- Using a procedure similar to that employed for (3), the other derivatives (1), (2), (4) and (5) were also pre- 341 Acknowledgement We thank the UGC, New Delhi for financial support. Our sincere thanks are to the Head, Department of Chem- 16. A. I. Vogel, "A Text Book of Quantitative Inorganic Analysis", 5th ed., Longmans, London, 1978. 342
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Microdecompression for lumbar synovial cysts: an independent assessment of long term outcomes
Journal of orthopaedic surgery and research
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3,567
Received: 10 October 2006 Accepted: 3 April 2007 © 2007 Weiner et al; licensee BioMed Central Ltd. © 2007 Weiner et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Journal of Orthopaedic Surgery and Research BioMed Central Open Access Abstract Background: Outcomes of surgical intervention for lumbar synovial cysts have been evaluated in the short and intermediate term. Concerns regarding cyst recurrence, the development of late instability at the involved level, and instability/stenosis at adjacent levels (when concomitant) fusion is performed suggest that long term follow-up is needed. This study aims to fill that void. Methods: Forty-six patients operated by a single surgeon not involved in the study were followed up long term at an average of 9.7 years (range 5 to 22 years) post-operatively. All patients underwent decompression (+/- concomitant arthrodesis in the presence of associated degenerative spondylolisthesis) using the operative microscope for magnification/illumination. Outcomes were assessed using a customized questionnaire evaluating: relief of pain/claudicant symptoms, numbness/parasthesias, and weakness; as well as late onset low back pain, new radicular symptoms, need for additional surgery, and patient satisfaction. Outcomes in patients with or without fusion were compared as well. Results: 87% of patients noted resolution of their pre-operative pain, numbness, and weakness. 28% of patients developed late onset low back pain. 17% developed late onset radicular symptoms in a new nerve root distribution. 15% required subsequent additional surgery. 89% of patients were satisfied with the surgical outcome. No differences were found for any outcome measure between patients undergoing concomitant fusion and those undergoing decompression alone using the two- sample t-test. Conclusion: This study provides outcome data at an average of nearly ten years post-operative. This information should allow surgeons to provide realistic expectations for their patients regarding outcomes and should enhance the informed consent and surgical decision-making process. ture [3,4] and were first well-delineated in English by Kao in the late 1960's/early 1970's [5,6]. Since then, CT and MRI scanning (Figure 1) have afforded highly sensitive and specific diagnosis of the cysts and the oft-associated Open Research article Microdecompression for lumbar synovial cysts: an independent assessment of long term outcomes Bradley K Weiner*1, Joel Torretti2 and Michael Stauff3 ddress: 1Division of Spinal Surgery The Methodist Hospital 6550 Fannin, Suite 2500 Houston, Texas 77030, USA, 2D artmouth Hitchcock Medical Center Hanover, New Hampshire, USA and 3Penn State College of Medicine Hershey Email: Bradley K Weiner* - bkweiner@tmh.tmc.edu; Joel Torretti - jtorretti@psu.edu; Michael Stauff - mstauff@psu.edu * Corresponding author l: Bradley K Weiner* - bkweiner@tmh.tmc.edu; Joel Torretti - jtorretti@psu.edu; Michael Stauff - mstauff@psu.edu responding author Received: 10 October 2006 Accepted: 3 April 2007 Surgical Technique g q Patients were placed under general endotracheal anesthe- sia and placed in a kneeling position on a standard frame. The involved level(s) was marked preoperatively using c- arm imaging. A midline skin incision was made and the dorsolumbar fascia incised just lateral to the midline ipsi- lateral to the synovial cyst. Unilateral laminae were exposed using the Cobb elevator to the mid-portion of the facet joint. An intraoperative radiograph was used to con- firm the level. A laminotomy on the undersurface of the cephalad lamina was undertaken to mirror the cephalad extent of the cyst as determined by pre-operative MRI. A similar caudal laminotomy was performed, again to mir- ror the extent of the cyst. Ligmantum flavum was then excised and the subarticular and foraminal zones decom- pressed via complete excision of soft-tissue/bony stenos- ing lesions to include extirpation of the synovial cyst. If the cyst was adherent to the dura (a common finding), it was carefully teased free so that no cyst pseudocapsule remained. The facet joint was opened and residual syno- vial tissue excised. If the patient had neurogenic claudica- tion, a contralateral microdecompression as previously described[24] was undertaken. If the patient had an asso- ciated degenerative spondylolisthesis, bilateral uninstru- mented intertransverse fusion as well as facet joint fusion was undertaken as previously described[25]. Magnifica- tion/illumination was provided by the operative micro- scope in all cases. The wound was irrigated, hemostasis obtained, and closure carried out in standard fashion. While multiple non-operative therapies have been imple- mented [12-16], few have demonstrated significant or lasting efficacy when used to treat patients with moderate or severe symptoms such as intractable pain or neurolog- ical deficit[12,14,16,17]. Accordingly, surgical interven- tion is commonly performed on patients in this group and several studies have demonstrated reasonable out- comes at short to intermediate term follow-up [7,11,15,18-23]. The longest follow-up published prior to the current study has been forty months and concerns about recurrence of the cysts, instability at the involved level (when isolated decompression is undertaken), or instability at adjacent levels (when concomitant fusion is performed) suggest that a longer-term look is needed to better understand the implications of our interventions. The purpose of this study was to independently evaluate the long-term clinical outcomes in patients who under- went microdecompression with or without concomitant arthrodesis for symptomatic lumbar synovial cysts unre- sponsive to non-operative measures. Methods compression of neurological structures. Such compres- sion can result in radiculopathy, neurogenic claudication, and, rarely, cauda equina syndrome [7-11]. Surgical Technique The average follow- up of 9.7 years (range five to 22 years) represents the long- est follow-up to date; the minimum follow-up for inclu- sion of five years being greater than the previously reported maximum follow-up of 3.25 years. A typical case of synovial cyst at L5-S1 Figure 1 A typical case of synovial cyst at L5-S1. Patients (Table 1) Forty-six patients operated between 1984 and 2001 were available for follow-up. All surgeries were performed by a single surgeon who was not involved in the study. Age at surgery ranged from 25 to 96 years with a mean of 73 years. Twenty-nine were females and seventeen males. Twenty-eight cysts were at the L4-L5 level, eight at L5-S1, six at L3-L4, and one each at L1-L2 and L2-L3. Clinical syndromes included unilateral monoradiculopathy in eighteen patients and neurogenic claudication in twenty- eight. Radiographically, twenty-three had an associated degenerative spondylolisthesis and underwent concomi- tant arthrodesis. This was the only indication for fusion in the study population. This follow-up study was approved by the institutional review board and oral consent was obtained from all participants. Methods Surgical Technique compression of neurological structures. Such compres- sion can result in radiculopathy, neurogenic claudication, and, rarely, cauda equina syndrome [7-11]. Background g Although originally recognized in peripheral joints by Baker in 1877[1,2], synovial cysts of the lumbar facet joints were not described until 1950 in the German litera- Page 1 of 5 (page number not for citation purposes) Page 1 of 5 (page number not for citation purposes) http://www.josr-online.com/content/2/1/5 Journal of Orthopaedic Surgery and Research 2007, 2:5 http://www.josr-online.com/content/2/1/5 http://www.josr-online.com/content/2/1/5 Patient Satisfaction Forty-one patients (89%) reported overall satisfaction with the outcome of their initial procedure and would rec- ommend it to a friend with the same problem. Same-site Numbness Twenty-three patients (50%) reported preoperative numbness. Of these, at follow-up, twenty (87%) reported complete or near-complete resolution, two (9%) were the same, and one (4%) was worse. New Leg Pain Forty of the forty-six patients (88%) reported relief of their preoperative pain/symptoms. Six (12%) had persisting complaints ranked in severity at an average of 5.5 on the visual analog scale (VAS: range 2–10) versus an average of 9 on VAS preoperatively. All patients had pain or claudi- cation preoperatively. Eight patients (17%) reported the eventual onset of new radicular leg pain (different root involved) with a mean VAS severity of 7.4. Additional Surgery Seven patients (15%) reported the need for additional lumbar spine surgery. Three patients who had not under- gone fusion at the initial surgery required eventual revi- sion decompression and fusion to include the operated levels due to instablility. Four patients who had under- gone concomitant arthrodesis at the primary surgery due to presence of a degenerative spondylolisthesis eventually developed juxtafusional stenosis/instability requiring sec- ondary decompression and fusion at involved adjacent levels. Results (Table 3) After initially doing well, thirteen patients (28%) reported the eventual development of new back pain ranked on average at 7.5 on the VAS. Follow-up averaged 9.7 years with a range of five to 22 years. Same-Site Weakness Nineteen (41%) had complained of weakness prior to sur- gery. Of these, at follow-up, sixteen (84%) reported com- plete resolution, two (11%) reported no significant change in strength, and one (5%) was worse than preop- eratively. 1. Do you have numbness or tingling in your leg(s) similar to what you had before surgery? (Better, Same, Worse) 2. Do you have weakness in your leg(s) similar to what you had before surgery? (Better, Same, Worse). 3. Do you still have pain/symptoms in the same site that made you have surgery in the first place? (Rated on Visual Analog Scale) 4. Have you developed back pain over the years that is new/different than before surgery? (Rated on Visual Analog Scale) 5. Have you developed leg pain over the years that is new/different than before your surgery? (Rated on Visual Analog Scale) 6. Have you had additional surgery on your back? (Type of surgery, Reason for surgery) 7. Are you happy with the results of the surgery and would you recommend it to a friend with the same problem? Positive responses on questionnaire were then followed up for specific details via telephone interview. Page 3 of 5 (page number not for citation purposes) Journal of Orthopaedic Surgery and Research 2007, 2:5 http://www.josr-online.com/content/2/1/5 Table 1: Patient Characteristics Number 46 Age in years 73 (range 25–96) Sex 29 Females 17 Males Anatomic Level of Cyst L4-L5 28 L5-S1 8 L3-L4 6 L2-L3 1 L1-L2 1 Clinical Syndromes Neurogenic Claudication 28 Monoradiculopathy 18 Associated Degenerative Spondylolisthesis 23 Associated Degenerative Spondylolisthesis Data Clinical outcomes and patient satisfaction were assessed by two independent spine surgeons using the question- naire in Table 2. All forty-six patients responded. A typical case of synovial cyst at L5-S1 Figure 1 A typical case of synovial cyst at L5-S1. A typical c Figure 1 Page 2 of 5 (page number not for citation purposes) Page 2 of 5 (page number not for citation purposes) Journal of Orthopaedic Surgery and Research 2007, 2:5 http://www.josr-online.com/content/2/1/5 Journal of Orthopaedic Surgery and Research 2007, 2:5 Did Presence of a Spondylolisthesis/Need for Fusion Alter Outcomes? There were no statistically significant differences for any of the outcome measures above between patients presenting without a degenerative spondylolisthesis (decompression alone) and those presenting with one (decompression with concomitant fusion) using the two-sample t-test. The potential weaknesses of long term studies such as this are two-fold. First, over a period of ten to twenty years patients may go on to further degeneration or develop new medical comorbidities such that their overall health status (SF-36) or disease specific status (ODI) may actu- ally appear worse than their pre-operative status – despite the fact that their specific reasons for surgery (e.g.; severe L5 root pain) may well have been relieved by the interven- tion. Long term studies are one of the rare cases where very specific outcome measures are indicated to ferret out this information, hence the choice of custom questionnaire used. Second, over that ten to twenty year period of time, the standards of care and the evidence base may have changed such that the information provided by the study is no longer relevant. This is a common problem in the total hip/knee replacement literature where long term outcomes are provided for prostheses no longer manufac- tured and surgical approaches no longer used. Just as the first potential weakness was avoided by intention, this second potential weakness was avoided by good fortune. Twenty-two years down the road, the standard of care, commensurate with the current evidence base, remains decompression of involved neurological tissue by com- plete excision of the cyst (including residual synovial tis- sue to avoid recurrence), excision of ligamentum flavum and other soft tissue and bony compressive pathology, and concomitant arthodesis in the presence of a degener- ative spondylolisthesis. Discussion and Conclusion This study demonstrates that at an average of nearly ten years following decompressive surgery for symptomatic lumbar synovial cysts (with associated fusion if a degener- ative spondylolisthesis is present), patients can anticipate: (a) about an 85% likelihood that their preoperative pain/ claudication, numbness, and weakness will be resolved, (b) about a 25% likelihood of developing later onset back pain, (c) about 15% likelihood of developing later onset radicular symptoms in a new nerve root distribution, (d) that they have a 15% likelihood of needing additional lumbar surgery, and that (e) about nine out of ten patients are happy with the results of surgery. These findings are generally commensurate with those of other studies having evaluated outcomes at much shorter intervals. Howington[7] achieved 88% good/excellent results at 40 months and Lyons[15] found similar results but with much shorter term follow-up. Khan[23] reported about 80% success rates at 26 months. At the extremes are Sandhu[20], Metellus[19], Pirotte[22], and Trummer[18] who reported between 95% and 100% success rates; and Epstein[21] who reported 60% good/excellent results at 24 months. The former studies likely representing snap- shots commonly encountered in short-term retrospective studies, the latter probably representing cases associated with the need for more extensive laminectomies given that 16 of 66 patients in this study went onto develop sig- nificant and progressive instability at the operated level. Journal of Orthopaedic Surgery and Research 2007, 2:5 Journal of Orthopaedic Surgery and Research 2007, 2:5 http://www.josr-online.com/content/2/1/5 Table 3: Summary of Results Complaint % of patients having; % of patients symptom-free Same Site Pain/Symptoms 12% averaging 5.5 on VAS; 88% resolved Same Site Numbness/Tingling 9% same, 4% worse; 87% resolved Same Site Weakness 11% same, 5% worse; 84% resolved New Back Pain 28% averaging 7.5 on VAS; 72% pain free New Radiculopathy 17% averaging 7.4 on VAS; 83% pain free Additional Surgery 15% Table 3: Summary of Results % of patients having; % of patients symptom-free 12% averaging 5.5 on VAS; 88% resolved 9% same, 4% worse; 87% resolved 11% same, 5% worse; 84% resolved 28% averaging 7.5 on VAS; 72% pain free 17% averaging 7.4 on VAS; 83% pain free 15% Same Site Pain/Symptoms Same Site Numbness/Tingling Same Site Weakness New Back Pain New Radiculopathy Additional Surgery need additional surgery long term. This additional infor- mation should allow surgeons to provide realistic expec- tations for their patients regarding outcomes and should enhance the informed consent and surgical decision-mak- ing process. 4. Schollner D: Ganglion on a vertebral joint. Z Orthop Ihre Gren- zgeb 1967, 102:619-620. Table 2: Questionnaire Page 3 of 5 (page number not for citation purposes) 1. Do you have numbness or tingling in your leg(s) similar to what you had before surgery? (Better, Same, Worse) 2. Do you have weakness in your leg(s) similar to what you had before surgery? (Better, Same, Worse). 3. Do you still have pain/symptoms in the same site that made you have surgery in the first place? (Rated on Visual Analog Scale) 4. Have you developed back pain over the years that is new/different than before surgery? (Rated on Visual Analog Scale) 5. Have you developed leg pain over the years that is new/different than before your surgery? (Rated on Visual Analog Scale) 6. Have you had additional surgery on your back? (Type of surgery, Reason for surgery) 7. Are you happy with the results of the surgery and would you recommend it to a friend with the same problem? Positive responses on questionnaire were then followed up for specific details via telephone interview. 1. Do you have numbness or tingling in your leg(s) similar to what you had before surgery? (Better, Same, Worse) 2. Do you have weakness in your leg(s) similar to what you had before surgery? (Better, Same, Worse). 3. Do you still have pain/symptoms in the same site that made you have surgery in the first place? (Rated on Visual Analog Scale) 4. Have you developed back pain over the years that is new/different than before surgery? (Rated on Visual Analog Scale) 5. Have you developed leg pain over the years that is new/different than before your surgery? (Rated on Visual Analog Scale) 6. Have you had additional surgery on your back? (Type of surgery, Reason for surgery) 7. Are you happy with the results of the surgery and would you recommend it to a friend with the same problem? Journal of Orthopaedic Surgery and Research 2007, 2:5 References 1. Baker WM: Formation of synovial cysts in connection with joints. St Bartholomews Hosp Rep 1885, 21:177-190. j p p 2. Baker WM: On the formation of synovial cysts in the leg in connection with disease of the knee joint. 1877. Clin Orthop 1994, 299:2-10. The value of the current study, given that its follow-up is dramatically longer than any previously published, is that it demonstrates, generally, that the beneficial effects of surgical intervention seen at shorter and intermediate time frames appear to persist, however some patients will develop late-onset low back pain, radicular pain, and may 3. Vosschulte K, Borger G: Anatomische and funktinonelle unter- suchugen iiber ben bandsheibenprolaps. Langenbecks Arch Klin Chir 1950, 265:329-355. Page 4 of 5 (page number not for citation purposes) Page 4 of 5 (page number not for citation purposes) (page number not for citation purposes) Journal of Orthopaedic Surgery and Research 2007, 2:5 http://www.josr-online.com/content/2/1/5 http://www.josr-online.com/content/2/1/5 5. Kao CC, Uilein A, Bickel WH, Turner JH: Lumbar intraspinal extradural ganglion cyst. J neurosurg 1968, 29:168-172. g g J g 6. Kao CC, Winkler SS, Turner JH: Synovial cyst of the spinal facet. J Neurosurg 1974, 41:372-376. 7. Howington JU, Connolly ES, Voorhies RM: Intraspinal synovial cysts. J neurosurg 1999, 91:193-199. y J g 8. Hsu KY: Lumbar intraspinal synovial and ganglion cysts. Spine 1995, 20:80-89. 9. Yarde WL, Arnold PM, Kepes JJ, Wilkinson SP, Batnitski S: Synovial cysts of the lumbar spine. Surg Neurol 1995, 43:459-464. 10. Baum JA, Hanley EN Jr: Intraspinal synovial cyst simulating spi- nal stenosis. Spine 1986, 11:487-489. 11. Kurz LT, Garfin SR, Ungar AS, Thorne RP, Rothman RH: Intraspinal synovial cyst causing sciatica. J Bone Joint Surg 1985, 67A:865-871. 12. Abrahams JJ, Wood GW, Eames FA: CT-guided needle aspiration biopsy of an intraspinal synovial cyst. Am J Neuroradiol 1988, 9:398-400. 13. Lemish W, Apsimon T, Chakera T: Lumbar intraspinal synovial cysts. Spine 1989, 14:1378-1383. y p 14. Parlier-Cuau C, Wybier M, Nizard R, Champsaur P, Laredo JD: Symptomatic lumbar synovial facet cysts. Radiology 1999, 210:509-513. 15. Lyons MK, Atkinson JL, Wharen RE, Dean HD, Zimmerman RS: Sur- gical evaluation and management of lumbar synovial cysts. J neurosurg 2000, 93:53-57. g 16. Bjorkgren AG, Kurz LT, Resnick D, Sartorius DJ, Garfin SR: Symp- tomatic intraspinal synovial cysts: percutaneous injection. Am J Roent 1987, 149:105-107. 17. Shah RV, Lutz GE: Lumbar intraspinal synovial cysts:conserva- tive management and review of the world's literature. References Spine J 2003, 3:479-488. 18. Trummer M, Laschka F, Tillich M, Homann CN, Unger F: Diagnosis and surgical management of intraspinal synovial cysts. J Neu- rol Neurosurg Psych 2001, 70:74-77. 19. Metellus P, Fuentes S, Adetchessi T, Levrier O, Flores-Parra I, Taliano D: Retrospective study of 77 patients harbouring lumbar syn- ovial cysts. Acta Neurochir (Wien) 2006, 148:4754. y ( ) 20. Sandhu FA, Santiago P, Fessler RG, Palmer S: Minimally invasive surgical treatment of lumbar synovial cysts. Neurosurg 2003, 54:107-112. 21. Epstein NE: Lumbar laminectomy for resection of lumbar syn- ovial cysts: an outcome study. Spine 2004, 29:1049-1056. 21. Epstein NE: Lumbar laminectomy for resection of lumbar syn- ovial cysts: an outcome study. Spine 2004, 29:1049-1056. 22. Pirotte B, Gabrovsky N, Massager N, Levivier M, David P: Synovial cysts of the lumbar spine. J Neurosurg 2003, 99S:14-19. y y p 22. Pirotte B, Gabrovsky N, Massager N, Levivier M, David P: Synovial cysts of the lumbar spine. J Neurosurg 2003, 99S:14-19. 23. Khan AM, Synnot K, Cammissa FP, Girardi FP: Lumbar synovial cysts of the spine: and evaluation of surgical outcome. J Sp Disord Tech 2005, 18:127-131. 24. Weiner BK, Walker M, Brower RS, McCulloch JA: Microdecom- pression for lumbar spinal canal stenosis. Spine 1999, 24:2268-2272. 25. McCulloch JA: Microdecompression and uninstrumented sin- gle-level fusion for spinal canal stenosis and degenerative spondylolisthesis. Spine 1998, 23:2243-2252. Journal of Orthopaedic Surgery and Research 2007, 2:5 References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 5 of 5 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge
https://openalex.org/W1505353042
https://europepmc.org/articles/pmc4472358?pdf=render
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Comparison of 15oxygen positron emission tomography and near-infrared spectroscopy for measurement of cerebral physiology
Critical care
2,015
cc-by
275,305
Introduction To assess cerebral hemodynamics in an experimental sepsis model. Methods The study was a prospective, observational pilot study conducted in our hospital. Consecutive adult patients with severe sepsis, on a mechanical ventilator with an IL-6 blood concentration ≥100 pg/ml in the acute phase, defi ned as being up to the 28th day of illness in the ICU, were entered in this study between June 2011 and December 2012. Subjects were divided into those who were treated with steroids (steroid treatment group) and those who were not (no- steroids group) during the target period, because steroids strongly aff ect IL-6 blood levels. Methods Nineteen juvenile female Hungahib pigs were subjected into control group (n = 9) or septic group (n = 10). Under general anesthesia in animals of the sepsis group, Escherichia coli culture (2.5 × 105/ml; strain: ATCC 25922) was intravenously administrated in a continuously increasing manner as follows: 2 ml in the fi rst 30 minutes, then 4 ml in 30  minutes and afterwards 16  ml/hour for 2  hours (so a total of 9.5 × 106 E. coli within 3 hours). In the control group the anesthesia was maintained for 8  hours, infusion was administered as a similar volume of isotonic saline solution and no other intervention was made. Hemodynamic monitoring of all animals was performed by PiCCo monitoring system. The middle cerebral artery of the pigs was insonated through the transorbital window and cerebral blood fl ow velocity (MCAV) and pulsatility index was registered. Results The subjects were fi ve adult patients in the acute phase of severe sepsis on a mechanical ventilator. Gastrointestinal motility was measured for a total of 62,399 minutes: 31,544 minutes in three subjects in the no-steroids group and 30,855 minutes in two subjects in the steroid treatment group. In the no-steroids group, the bowel sound counts were negatively correlated with IL-6 blood concentration (r  = –0.76, P  <0.01), suggesting that gastrointestinal motility was suppressed as IL-6 blood concentration increased. However, in the steroid treatment group, gastrointestinal motility showed no correlation with IL-6 blood concentration (r = –0.25, P = 0.27). The IL-6 blood concentration appears to have decreased with steroid treatment irrespective of changes in the state of sepsis, whereas bowel sound counts with the monitoring system refl ected the changes in the state of sepsis, resulting in no correlation. MEETING ABSTRACTS MEETING ABSTRACTS P1 but long-term observation and objective evaluation of gastrointestinal motility are diffi cult. We developed a non-invasive monitoring system capable of quantifying and visualizing gastrointestinal motility in real time. In the study gastrointestinal motility was performed in patients with severe sepsis using this developed bowel sound analysis system, and the correlation between bowel sounds and changes over time in blood concentrations of IL-6, which is associated with sepsis severity, was evaluated. P1 Cerebral autoregulation testing in a porcine model of intravenously administrated E. coli induced fulminant sepsis L Molnar1, M Berhes1, L Papp1, N Nemeth2, B Fulesdi1 1Deoec Aneszteziologia, Debrecen, Hungary; 2University of Debrecen, Hungary Critical Care 2015, 19(Suppl 1):P1 (doi: 10.1186/cc14081) P1 Cerebral autoregulation testing in a porcine model of intravenously administrated E. coli induced fulminant sepsis L Molnar1, M Berhes1, L Papp1, N Nemeth2, B Fulesdi1 1Deoec Aneszteziologia, Debrecen, Hungary; 2University of Debrecen, Hungary Critical Care 2015, 19(Suppl 1):P1 (doi: 10.1186/cc14081) Introduction To assess cerebral hemodynamics in an experimental sepsis model. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Introduction To assess cerebral hemodynamics in an experimental sepsis model. Results In the septic group, as expected, all animals developed fulminant sepsis and died within 3 to 7 hours two animals in 3 to 4 hours, and three in 6 to 7 hours). In the septic animals the heart rate rose and mean arterial pressure dropped, their ratio increased signifi cantly compared with both the base values (at the 6th hour: P <0.001) and the control group (P  =  0.004). The control animals showed stable condition over the 8-hour anesthesia. MCAV signifi cantly decreased during the development of sepsis (from 23.6 ± 6.6 cm/s to 16.0 ± 3.9 cm/s, P <0.01) and pulsatility indices increased (from 0.68  ±  0.22 to 1.37  ±  0.58, P  <0.01), indicating vasoconstriction of the resistance vessels. A signifi cant relationship was fund between percent change of the MAP and the pulsatility index in septic animals (R2 = 0.32) referring to maintained cerebral autoregulation. Conclusion The new real-time bowel sound analysis system provides a useful method of continuously, quantitatively, and non-invasively evaluating gastrointestinal motility in severe patients. Furthermore, this analysis may predict disease severity in septic patients. Conclusion Cerebral autoregulation is preserved in the pig model of experimentally induced fulminant sepsis. P3 Usefulness of sepsis screening tools and education in recognising the burden of sepsis on hospital wards EJ Galtrey, C Moss, H Cahill Guy’s and St Thomas’ Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P3 (doi: 10.1186/cc14083) P2 New real-time bowel sound analysis may predict disease severity in septic patients J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) 35th International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 17-20 March 2015 Publication charges for this supplement were funded by ISICEM. p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 1University of Cambridge, UK; 2Ipswich Hospital, Ipswich, UK Critical Care 2015, 19(Suppl 1):P5 (doi: 10.1186/cc14085) 1University of Cambridge, UK; 2Ipswich Hospital, Ipswich, UK Critical Care 2015, 19(Suppl 1):P5 (doi: 10.1186/cc14085) fi Methods All referrals to our critical care response team with a diagnosis of sepsis over a 3-month period (September to November 2014) were investigated to determine how many had an EPR sepsis alert comprising a prompt for blood cultures, serum lactate measurement, fl uid challenge if hypotensive, and antibiotics within 1 hour. Introduction Severe sepsis results in ~36,800 UK deaths each year [1]. Prior studies demonstrate the benefi t of early recognition and treatment of sepsis in reducing mortality [2]. The Sepsis Six [1] bundle aims to optimise the fi rst hour of sepsis management. We assessed the proportion of emergency department (ED) patients with severe sepsis receiving the Sepsis Six bundle and whether this was improved by a combination of staff education and use of Sepsis Six management stickers in patient notes. l g yp Results Only 25/174 (14%) patients with a diagnosis of sepsis had an EPR sepsis alert. There was no signifi cant diff erence between acute and nonacute ward areas in their likelihood of using the screening tool or alert, in contrast to previous audits of the alerted population which showed that acute areas such as A&E and medical acute admission wards had higher utilisation and bundle completion rates. Methods A closed loop audit was completed in the ED at Ipswich Hospital, UK. Each cycle was 14  days with interventions made in a 4-week period between the two cycles. The interventions consisted of: Sepsis Six management stickers and posters placed in the ED; two training sessions for all ED nurses on sepsis recognition and management; a teaching session for all middle-grade doctors; and a trolley in the ED with equipment required for the Sepsis Six. The notes of all patients with lactate ≥2 mmol/l were retrospectively reviewed. Those with ≥2 systemic infl ammatory response syndrome criteria and a documented suspicion of infection were deemed to have severe sepsis. The times at which these patients had each of the Sepsis Six completed were recorded, as were the fi nal diagnosis and 7/28 day mortality. P5 tool and electronic order set (EPR alert) alongside an education programme to improve delivery of the SSC bundle. Previous audits showed only 43% full bundle compliance in those that were alerted, and this raised concerns regarding the burden of unalerted sepsis. We sought to estimate the number of unalerted sepsis episodes to assess the effi cacy of our screening tool and improve early recognition. p CE Thakker1, P Crook1, B Davies1, L Mawer1, T Tomouk1, E Williams1, C Gray2, K Turner2 Results In Cycle 1, 31/106 patients met the criteria for severe sepsis, compared with 36/120 in Cycle 2. The delivery of the Sepsis Six interventions was highly variable. In Cycle 1 lactate levels and i.v. access had the highest 60-minute completion rates (90.3%, 83.9% respectively). Blood cultures and i.v. fl uid resuscitation were completed for 61.3% and 64.5% of patients within 60 minutes. Only 38.7% of septic patients were given i.v. antibiotics within 60 minutes. In total, 58.9% of patients received antibiotics in accordance with trust guidelines. High fl ow oxygen, catheters and fl uid balance charts had the lowest 60-minute completion rates (35.5%, 6.5%, 6.5% respectively). In Cycle 2, post intervention, there was no signifi cant change in the percentage of patients receiving the Sepsis Six bundle. Methods A closed loop audit was completed in the ED at Ipswich Hospital, UK. Each cycle was 14  days with interventions made in a 4-week period between the two cycles. The interventions consisted of: Sepsis Six management stickers and posters placed in the ED; two training sessions for all ED nurses on sepsis recognition and management; a teaching session for all middle-grade doctors; and a trolley in the ED with equipment required for the Sepsis Six. The notes of all patients with lactate ≥2 mmol/l were retrospectively reviewed. Those with ≥2 systemic infl ammatory response syndrome criteria and a documented suspicion of infection were deemed to have severe sepsis. The times at which these patients had each of the Sepsis Six completed were recorded, as were the fi nal diagnosis and 7/28 day mortality. Conclusion Despite these interventions, most patients still do not receive the full recommended treatment bundle. These fi ndings have prompted a point prevalence audit at ward level, which will examine all patients’ notes for the preceding 24 hours to ascertain if sepsis is truly unrecognised or whether it is simply that our current tool is not a helpful adjunct to care. With national guidelines expected within the year, we will redesign and re-launch our screening tools and education programme to improve awareness and management of this common medical emergency. Continuous versus intermittent temperature measurement in the detection of fever in critically ill patients p g p Conclusion The low rates of Sepsis Six completion require improvement to meet the targets set out by the College of Emergency Medicine. Our results suggest that simple interventions are ineff ective in increasing Sepsis Six completion and thus lend support to the case for integrated interventions such as electronic recording and alert systems. References Introduction An elevated body temperature is one of the four criteria in diagnosing systemic infl ammatory response syndrome (SIRS), and is often used at the bedside to trigger diagnostic investigations for infection. Standard intermittent temperature measurement may, however, delay the detection of an elevated temperature or miss this altogether. The aim of the study is to compare intermittent non- invasive versus continuous invasive body temperature measurement as a tool to detect an elevated body temperature. P4 P4 Continuous versus intermittent temperature measurement in the detection of fever in critically ill patients A Heyneman, V Bosschem, N Mauws, D Van Der Jeught, E Hoste, J Decruyenaere, J De Waele Ghent University Hospital, Ghent, Belgium Critical Care 2015, 19(Suppl 1):P4 (doi: 10.1186/cc14084) New real-time bowel sound analysis may predict disease severity in septic patients J G t 1 K M t d 1 N H ii1 T M i hi1 M Y i 1 D H d 1 p p J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) J Goto1, K Matsuda1, N Harii1, T Moriguchi1, M Yanagisawa1, D Harada1, H Sugawara1, O Sakata2 1University of Yamanashi School of Medicine, Chuo, Japan; 2University of Yamanashi, Kofe, Japan Critical Care 2015, 19(Suppl 1):P2 (doi: 10.1186/cc14082) Introduction Sepsis is defi ned as the presence of infection with systemic signs of infection, and severe sepsis as sepsis plus sepsis-induced organ dysfunction or tissue hypoperfusion [1]. Since the Surviving Sepsis Campaign (SSC) in 2002, the Health Service Ombudsman for England published recommendations for improving recognition and treatment of sepsis [2], the Royal College of Physicians issued a toolkit for the management of sepsis in the acute medical unit [3], and NHS England released a patient safety alert to support prompt recognition and treatment of sepsis [4]. In 2012 our Trust introduced a sepsis screening Introduction Healthy bowel function is an important factor when judging the advisability of early enteral nutrition in critically ill patients, © 2015 BioMed Central Ltd © 2015 BioMed Central Ltd © 2015 BioMed Central Ltd S2 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 interventions References 1. Daniels et al. Emerg Med J. 2011;28:507-12. 1. Daniels et al. Emerg Med J. 2011;28:507-12. 1. Daniels et al. Emerg Med J. 2011;28:507-12. 2. Kumar et al. Crit Care Med. 2006;34:1589-96. 2. Kumar et al. Crit Care Med. 2006;34:1589-96. References 1. Dellinger R, et al. Crit Care Med. 2013;41:580-637. 2. http://bit.ly/1nzV3Kp. 3. http://bit.ly/12Y3KHq. 4. http://bit.ly/10PYi8G. 1. Dellinger R, et al. Crit Care Med. 2013;41:580-637. 2. http://bit.ly/1nzV3Kp. 3. http://bit.ly/12Y3KHq. 4. http://bit.ly/10PYi8G. ,i g y y Results In Cycle 1, 31/106 patients met the criteria for severe sepsis, compared with 36/120 in Cycle 2. The delivery of the Sepsis Six interventions was highly variable. In Cycle 1 lactate levels and i.v. access had the highest 60-minute completion rates (90.3%, 83.9% respectively). Blood cultures and i.v. fl uid resuscitation were completed for 61.3% and 64.5% of patients within 60 minutes. Only 38.7% of septic patients were given i.v. antibiotics within 60 minutes. In total, 58.9% of patients received antibiotics in accordance with trust guidelines. High fl ow oxygen, catheters and fl uid balance charts had the lowest 60-minute completion rates (35.5%, 6.5%, 6.5% respectively). In Cycle 2, post intervention, there was no signifi cant change in the percentage of patients receiving the Sepsis Six bundle. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P5 Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia y q p C Joya-Montosa, MD Delgado-Amaya, E Trujillo-García, E Curiel-Balsera Hospital Regional de Málaga, Spain C Joya-Montosa, MD Delgado-Amaya, E Trujillo-García, E Curiel-Balsera Hospital Regional de Málaga, Spain p g g p ritical Care 2015, 19(Suppl 1):P9 (doi: 10.1186/cc14089) Introduction The aim of the study is to evaluate the calibration and discrimination of two specifi c risk scores for community-acquired pneumonia (CAP) in patients with this illness who required ICU admission. Results Severe sepsis criteria were fulfi lled in 31% (n  = 91) of the patients. These were older (median age: 79  years vs. 71  years) and experienced more symptoms (mean: 2.2, SD 0.9 vs. mean: 1.4, SD 0.7) than patients without severe sepsis, while there was no diff erence in C-reactive protein levels (OR per 50 mg/l: 1.07, 95% CI: 0.96 to 1.20). Among individual symptoms, altered mental status (OR: 4.4, 95% CI: 2.2 to 9.0), dyspnea (OR: 3.5, 95% CI: 2.1 to 5.9), and muscle weakness (OR: 2.2, 95% CI: 1.0 to 4.4) were signifi cantly related to severe sepsis. Adjusting for age and sex, altered mental status (adj. OR: 4.1, 95% CI: 2.0 to 8.4) and dyspnea (adj. OR: 3.1, 05% CI: 1.8 to 5.3) remained signifi cant. Conclusion General symptoms, especially altered mental status and dyspnea, appear to be more common in severe sepsis than in milder infections. These symptoms might be utilized as a diagnostic aid for severe sepsis in the clinical setting, complementing vital signs and laboratory tests. Methods A retrospective descriptive study of patients with severe CAP admitted to the ICU between January 2008 and September 2013. We analyzed clinical and epidemiological variables and APACHE II, SAPS III, CURB-65 and Pneumonia Severity Index (PSI) that were recorded in the fi rst 24 hours. We used the Student t test to compare means and the chi-square test for univariate analysis. The standardized mortality ratio (SMR) and Hosmer–Lemershow test were calculated to analyze the calibration and ROC curve analysis for discrimination of diff erent scores. Results We analyzed 111 patients aged 57.5 ± 17.7 years, with 63.1% (70) males. The APACHE II score at admission was 19.8 ± 17.7 and SAPS III was 60.6 ± 16.7. ICU mortality was 29.7% (33). There was association between the four scores and mortality. P9 weakness, gastrointestinal symptoms, localized pain, altered mental status) that were part of the reason the patient sought medical care were registered. Additionally, age, sex, vital signs, C-reactive protein, and blood cultures were registered. Patients that within 48 hours from admission fulfi lled any criteria for severe sepsis were compared with patients that did not. Odds ratios for severe sepsis were computed using univariable as well as multivariable logistic regression, controlling for age and gender as confounders. 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. P6 Systemic symptoms as markers for severity in sepsis J Edman-Wallér1, L Ljungström2, R Andersson3, G Jacobsson2, M Werner1 1Södra Älvsborg Hospital, Borås, Sweden; 2Skaraborg Hospital, Skövde, Sweden; 3Institute of Biomedicine, University of Gothenburg, Sweden Critical Care 2015, 19(Suppl 1):P6 (doi: 10.1186/cc14086) Methods This was a secondary analysis of a prospective study in 25 critically ill patients comparing diff erent measurement techniques. Temperature was measured intermittently with an axillary digital thermometer every 4 hours, and a urinary bladder thermistor catheter was used for continuous temperature measurement; the latter was considered the reference method. Fever (core temperature ≥38.3°C) episodes occurring within 60 minutes after each other were classifi ed as one episode. We compared the fever detection rate of both methods and calculated the diff erence in timing between both techniques. Introduction The objective of this study was to evaluate six general symptoms as markers for severe sepsis in patients with suspected bacterial infections. Severe sepsis is a common cause of death and morbidity. Early detection and treatment is critical for outcome. Clinical presentation varies widely and no single test is able to discriminate severe sepsis from uncomplicated infections or non-infectious emergencies. Apart from local symptoms of infection, the systemic infl ammatory reaction itself may give rise to general symptoms such as muscle weakness and vomiting. f Results Twenty-nine episodes of fever were detected in 10 patients (seven male) with a median APACHE II score of 10 (IQR 3 to 24) and a median SOFA score of 10 (IQR 8 to 11). Median duration of a fever episode was 1 hour 24 minutes (IQR 47 minutes to 2 hours 59 minutes) and median maximum temperature was 38.4°C (IQR 38.3 to 38.7). Median axillary temperature was 0.7°C (IQR 0.3 to 0.9) lower than core temperature. Using intermittent, non-invasive measurement, 27 out of 29 fever episodes (93.1%) remained undetected. Methods We present an observational, consecutive study. Data from ambulance and hospital medical records were analyzed. The survey included 290 patients (mean age: 70.6  years; median age: 74  years; male: 47%) who were admitted to a 550-bed secondary care hospital, receiving intravenous antibiotics for suspected community-acquired infections. General symptoms (fever/shivering, dyspnea, muscle Conclusion Intermittent, non-invasive temperature measurement failed to detect most of the fever episodes as measured by a bladder thermistor catheter and should not be used to screen for elevated body temperature in critically ill patients. Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study g y S Laukemann1, N Kasper1, N Kasper1, A Kutz1, S Felder1, S Haubitz2, B Müller1, P Schuetz1 Figure 1 (abstract P9). 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) Introduction Collection of blood cultures is routinely performed in patients with suspicion of infection in the emergency department (ED) despite a low yield of positive culture results. To increase sensitivity, diff erent clinical prediction rules and blood biomarkers have been put forward. Herein, we validated the performance of diff erent promising clinical prediction rules alone and in combination with novel blood biomarkers to predict blood culture positivity. Methods This is an observational cohort study including consecutive medical patients with suspected infection and collection of ED admission blood cultures. Five clinical prediction rules were calculated and admission concentrations of procalcitonin (PCT), C-reactive protein, neutrophil–lymphocyte count ratio (NLCR), lymphocyte count, white blood cell count, and red blood cell distribution width were measured. True blood culture positivity was assessed by two independent physicians. We used logistic regression models with area under the curve (AUC) to establish associations between clinical prediction rules and blood culture positivity. Figure 1 (abstract P9). y Results Of 1,083 included patients, 106 (9.8%) cultures were positive. Of the clinical prediction rules, the Shapiro rule performed best (AUC 0.733) followed by the Metersky rule (AUC 0.609). The best biomarkers were PCT (AUC 0.796), NLCR (0.692) and lymphocyte count (AUC 0.671). Combination of the Shapiro rule and PCT showed the best combination result (AUC of combined model 0.822). Limiting blood cultures to either the Shapiro rule ≥4 points or PCT >0.11 μg/l would reduce negative sampling by 25.6% while still identifying 100% of positive cultures. Using a Shapiro rule ≥3 points or PCT >0.25 μg/l limit would reduce negative sampling by 42.1% while still identifying 96.2% of positive cultures. Conclusion The four analyzed scores presented good calibration, but discrimination seems better in SAPS III. Given the diffi culty of calculating PSI, and its low discrimination (similar to CURB-65), we prefer to use the CURB-65 score in routine clinical practice. 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. P6 S3 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Assessment of specifi c risk scores for patients admitted to the ICU for severe community-acquired pneumonia The SMR for APACHE II was 0.87 and 0.85 for the SAPS III. Figure 1 shows the ROC curve for the four scores, the best observed discrimination obtained was for SAPS III score (AuC 0.79) and the worst was obtained for CURB-65 score (AuC 0.7). The Hosmer–Lemeshow test showed acceptable calibration for the four predictive systems (P > 0.05). Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study S Laukemann1, N Kasper1, N Kasper1, A Kutz1, S Felder1, S Haubitz2, B Müller1, P Schuetz1 1Kantonsspital Aarau, Switzerland; 2University Hospital, Bern, Switzerland Critical Care 2015, 19(Suppl 1):P8 (doi: 10.1186/cc14088) Clinical scores and blood biomarkers for prediction of bacteremia in emergency department patients: Bacteremia Assessment in Clinical Triage (BACT) study Pre-exposure to mechanical ventilation and endotoxin infl uence bacterial growth and immune response during experimental ventilator-associated pneumonia Table 1 (abstract P10) Sternal infection P value Insulin 9/85 0.001 Current smoker 4/272 0.037 COPD 11/197 <0.001 Transfusion >3 19/302 0.001 J Sperber1, A Nyberg1, M Lipcsey2, A Larsson2, J Sjölin2, M Castegren2 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Uppsala University, Uppsala, Sweden Introduction Overproduction of nitric oxide (NO) is correlated with adverse outcomes in sepsis. NO is additionally a central part of the innate immune system defense against pathogens causing ventilator- associated pneumonia (VAP), which can complicate the clinical course during mechanical ventilation (MV). We hypothesized that pre-exposure to MV and systemic infl ammation from endotoxin each would infl uence bacterial growth in lung tissue, based on an altered immune response in experimental pneumonia. We used a porcine Pseudomonas aeruginosa VAP model with ventilatory and infl ammatory pre-exposures before inoculation to evaluate bacterial growth, development of lung damage, total NO production and infl ammatory cytokine response. Conclusion Postoperative deep sternal wound infections have statistical signifi cant correlation with the following parameters: transfusion with >3 red blood cell units, history of COPD, insulin dependence and when the patient is a current smoker. Also there is a tendency for correlation with CBP time >120 minutes (P = 0.056). References 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg 2007;2:23 1. Diez C, et al. Risk factors for mediastinitis after cardiac surgery – a retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 2. Schimmer C, et al. Prevention of sternal dehiscence and infection in high-risk patients: a prospective randomized multicenter trial. Ann Thorac Surg. 2008;86:1897-904. y Methods Three groups of mechanically ventilated pigs were subjected to experimental VAP for 6  hours with intrapulmonary 1  ×  1011 CFU P. aeruginosa at baseline. Two groups were pre-exposed to MV for 24  hours before bacterial inoculation: MV  + Etx (n  = 6, received endotoxin 0.063 μg × kg–1 × hour–1) and MV (n = 6, received saline in equivalent volume). One group, Un (n  = 8), started the experiment unexposed to both MV and endotoxin, directly from the initiation of VAP. Postmortem lung tissue samples rendered bacterial cultures. References 1. Hoenig M, et al. Procalcitonin fails to predict bacteremia in SIRS patients: a cohort study. Int J Clin Pract. 2014;68:1278-81. y 2. Hoeboer SH, et al. Old and new biomarkers for predicting high and low risk microbial infection in critically ill patients with new onset fever: a case for procalcitonin. J Infect. 2012;64:484-93. y 2. Hoeboer SH, et al. Old and new biomarkers for predicting high and low risk microbial infection in critically ill patients with new onset fever: a case for procalcitonin. J Infect. 2012;64:484-93. q y Results A total of 35 patients (3.44%) were complicated by deep sternal wound infections. No statistical correlation was found with age >75, gender, DM, BMI >30, steroids, emergent operation, prolonged ventilation, CBP time >120 minutes, reintubation and NIV. Factors with statistical signifi cant correlation are presented in Table 1. P10 Predisposing factors for deep sternal wound infection after cardiac surgery Predisposing factors for deep sternal wound infection after cardiac surgery F Ampatzidou, M Sileli, A Madesis, K Antoniou, A Baddur, G Kechagioglou, T Asteri, G Drossos General Hospital G. Papanikolaou Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P10 (doi: 10.1186/cc14090) Conclusion Combination of clinical parameters combined in the Shapiro rule together with admission levels of PCT allows reduction of unnecessary blood cultures with minimal false negative rates. References 1. Shapiro NI, Wolfe RE, Wright SB, Moore R, Bates DW. Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med. 2008;35:255-64. Introduction The aim of our study was to investigate perioperative risk factors associated with deep sternal wound infections in complicated cardiac surgery. 2. Müller F, Christ-Crain M, Bregenzer T, Krause M, Zimmerli W, Mueller B, et al. Procalcitonin levels predict bacteremia in patients with community-acquired pneumonia: a prospective cohort trial. Chest. 2010;138:121-9. y Methods A total of 1,017 patients underwent cardiac surgery in a 2-year period. We investigate the correlation between deep sternal S4 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 factors might be useful to know whether or not adult febrile patients have bacteremia. wound infection with the following 14 preoperative characteristics and perioperative parameters: age >75, female gender, diabetes mellitus (DM), insulin dependence, body mass index (BMI) >30, current smokers, COPD, cardiopulmonary bypass time (CBP) >120 minutes, use of steroids, emergency operation, prolonged mechanical ventilation (>48 hours), reintubation, transfusion with more than 3 units of red blood cells, and the postoperative use of non-invasive ventilation (NIV). The chi-square test was used for statistical analysis. wound infection with the following 14 preoperative characteristics and perioperative parameters: age >75, female gender, diabetes mellitus (DM), insulin dependence, body mass index (BMI) >30, current smokers, COPD, cardiopulmonary bypass time (CBP) >120 minutes, use of steroids, emergency operation, prolonged mechanical ventilation (>48 hours), reintubation, transfusion with more than 3 units of red blood cells, and the postoperative use of non-invasive ventilation (NIV). The chi-square test was used for statistical analysis. P11 Risk factors for bacteremia in adult febrile patients in emergency settings g A Mikami1, Y Natori1, F Omata2, S Ishimatsu1 1St Luke’s International Hospital, Tokyo, Japan; 2St Luke’s Life Science Institute, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P11 (doi: 10.1186/cc14091) Results The animals pre-exposed to endotoxin (MV + Etx) displayed higher bacterial growth (CFU × g–1) (P <0.05), lower PaO2/FiO2 (P <0.05) and lower nitrate levels (P <0.01) than the unexposed animals (Un). Plasma TNFα levels were higher in Un than in both pre-exposed groups MV  + Etx and MV (P  <0.01). There were no signifi cant diff erences between the two pre-exposed groups. Critical Care 2015, 19(Suppl 1):P11 (doi: 10.1186/cc14091) Introduction Blood culture is a critical procedure for detecting potentially life-threatening bloodstream infections (BSI). At the same time, early diagnosis and appropriate treatment of BSI are the key factors in order to improve prognosis. The purpose of the current analysis was to identify risk factors for bacteremia in adult febrile patients in emergency settings. g Conclusion Mechanical ventilation for 24  hours with concomitant endotoxin exposure enhances bacterial growth and lung damage during P. aeruginosa VAP, compared with inoculation without any pre- exposure to MV or endotoxin. The greater bacterial clearance in the unexposed animals was associated with higher NO production and higher levels of pro-infl ammatory cytokines. Methods We conducted a retrospective case–control study within a population of adult patients visiting the emergency department at a community hospital (St Luke’s International Hospital, Tokyo, Japan) and who underwent two sets of blood culture testing between 2003 and 2012. Among a total of 13,582 patients, 1,322 (10%) were detected as bacteremia. We included in this study 179 randomly selected patients from the bacteremia group and 321 randomly selected patients from the negative blood culture group to serve as the comparison group. Multivariate logistic regression was used to evaluate the relationship between clinical characteristics factors and bacteremia. Pre-exposure to mechanical ventilation and endotoxin infl uence bacterial growth and immune response during experimental ventilator-associated pneumonia NO production was measured with urinary nitrate levels over 6 hours of VAP. retrospective analysis of 1700 patients J Cardiothor Surg. 2007;2:23. 2. Schimmer C, et al. Prevention of sternal dehiscence and infection in high-risk patients: a prospective randomized multicenter trial. Ann Thorac Surg. 2008;86:1897-904. P15 ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign g p p g J Melville, S Ranjan, P Morgan East Surrey Hospital, Redhill, UK Critical Care 2015, 19(Suppl 1):P15 (doi: 10.1186/cc14095) Introduction The aim of this study was to evaluate the eff ect of the Surviving Sepsis Campaigns on mortality rates, before and after the second surviving sepsis publication, and to assess whether patients with sepsis being admitted to the ICU had a lower APACHE II score on admission. Patients with sepsis, who require ICU care, have an extremely poor prognosis. It has been shown that the mortality rates range from 20.7% (severe sepsis) to 45.7% (septic shock) [1]. The surviving sepsis campaign was initiated in 2002. The fi rst, second and third publications were published in 2004, 2008 and 2012 respectively [2]. g Results According to elevation of CT grades, severity of illness was signifi cantly associated with high score (APACHE II: 10.5 to 4.0, 12.8 to 4.2, 16 to 4.2, SOFA: 2.6 to 1.5, 2.9 to 1.9, 6.8 to 3.7, CRP: 17.8 to 10.6, 22.4 to 10.1, 33.3 to 11.9) and also duration of mechanical ventilation and length of hospital stay were longer (duration of mechanical ventilation: 10.9 to 6.6, 11.5 to 6.7, 15.8 to 7.2, length of hospital stay: 23.4 to 10.6, 27.9 to 21.4, 48.7 to 36.2). y Methods A retrospective case note review was performed, looking at a sample of 5,954 patients who were 18 years or older who had been admitted to East Surrey Hospital (ESH) ICU between 1 January 2005 and 31 October 2014. The total number of patients with sepsis was 941. We compared results before and after the second publication of the surviving sepsis campaign, looking at mortality rates, age of patients, admission length prior to ICU transfer, APACHE II score and the length of stay on the ICU. Conclusion Novel classifi cation of CNF based on CT fi ndings showing the extension of fl uid collection is a useful indicator of the disease severity and predicting clinical outcome. These fi ndings may infl uence the strategy for the success of percutaneous catheter drainage. y Results From the beginning of 2005 to the end of 2008, the mortality rates for septic patients was 51.9% compared with 41.3% from the beginning of 2009 to end of October 2014. P15 this study. Cervical spaces were subdivided into three components according to the concept of interfascial planes. The extension of acute fl uid collection in cervical spaces was classifi ed into three grades: Grade I, fl uid collection confi ned to one component; Grade II, fl uid collection spreading into two or three components; and Grade III, fl uid collection spreading into four components or mediastinum. We analyzed association with CT grades and severity of illness (SOFA score, APACHE II score, CRP). All patients underwent percutaneous catheter drainage either ultrasonography guided or CT guided. We compared treatment outcome of CNF with CT grades. 1. Estaban A, et al. Crit Care Med. 2007;35:1284-9. 2. Alberti C, et al. Intensive Care Med. 2002;28:108-21. ICU mortality rates in patients with sepsis compared with patients without sepsis p J Melville, S Ranjan, P Morgan East Surrey Hospital, Redhill, UK Critical Care 2015, 19(Suppl 1):P14 (doi: 10.1186/cc14094) Introduction The aim of the study was to evaluate the diff erence in mortality rates between those admitted to the ICU with and without sepsis, and to assess the proportion of patients who had sepsis. Septic patients are one of the key groups of patients admitted to ICUs around the world. Septic patients have an extremely poor prognosis with published mortality rates ranging from 20.7% (severe sepsis) to 45.7% (septic shock) [1]. With septic patients making up roughly 21% of patients admitted to ICUs, it is important to assess whether these rates of mortality hold true to a district general ICU and to assess the extent of the diff erence in prognosis between patients with and without sepsis [2]. g y p y Conclusion Patients with sepsis admitted to ESH ICU had a 20% relative decrease in mortality after the second publication of surviving sepsis guidelines. The original aim of the campaign was to reduce mortality from sepsis by 25% in 5 years [3]. This decrease was not due to a signifi cant diff erence between the sets of patients. The decreased time to admittance to ICU may be due to improved recognition of the need for ICU care. Overall the surviving sepsis campaign has had a signifi cantly benefi cial eff ect on mortality rates in patients with sepsis. References 1. Estaban A, et al. Crit Care Med. 2007;35:1284-9. Methods We performed a retrospective case note review, looking at a sample of 5,954 patients 18 years or older who were admitted to East Surrey Hospital (ESH) ICU, which has an elective admissions rate of 3%, between 1 January 2005 and 31 October 2014. The total number of patients with sepsis was 941 compared with 5,013 without sepsis. We looked at mortality rates, APACHE II scores and length of stay on the unit. 2. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 3. Slade E, et al. Crit Care. 2003;7:1-2. Independent risk factors for long-term mortality in patients with severe infection Results From the beginning of 2005 to the end of October 2014, mortality rates in septic patients were 44.6% compared with 26.2% in nonseptic patients. Fisher’s two-tailed test showed a signifi cant diff erence (P <0.0001) between the mortality in septic and nonseptic patients. There was a signifi cant diff erence (Mann–Whitney) between APACHE II scores, with median scores of 18 and 13 in septic and nonseptic patients respectively. Septic patients had longer lengths of stay, with the mean and median 8.73 and 3.89  days respectively, compared with 4.90 and 2.5 in nonseptic patients. Septic patients made up 15.8% of all patients admitted to the ICU. J Francisco, I Aragão, T Cardoso J Francisco, I Aragão, T Cardoso Centro Hospitalar do Porto – Hospital Geral de Santo António, Porto, Portugal Critical Care 2015, 19(Suppl 1):P16 (doi: 10.1186/cc14096) Introduction The purpose of this study was to examine long-term mortality, 5 years after severe infection, and to identify independent risk factors associated with it. Methods A prospective cohort study developed at a tertiary care university-affi liated 600-bed hospital including all patients with severe infection admitted into intensive care, medical, surgical, haematology and nephrology wards, over a 1-year period (2008/2009). The outcome of interest was mortality 5  years following hospitalisation and its association with specifi c risk factors was studied through logistic regression. Conclusion Patients with sepsis admitted to ESH ICU made up a signifi cant minority of patients admitted to the ICU. Septic patients had a 70% relative higher mortality rate compared with nonseptic patients. The mortality rate of 44.6% fi ts with previously quoted mortality rates in septic shock. Patients with sepsis had a signifi cantly higher predicted mortality, recorded by their APACHE II score, which was statistically signifi cant. This also meant they needed longer ICU care, with the average length of stay almost doubled. Results There were 1,013 patients included in the study. Hospital mortality rate was 14% (n = 137) and 5-year mortality was 37% (n = 379). Factors independently associated with 5-year mortality were (adjusted odds ratio (95% confi dence interval)): age = 1.04 per year (1.03 to 1.05), cancer = 8.00 (3.06 to 20.88), chronic hepatic disease = 3.06 (1.06 to 8.87), chronic respiratory disease = 2.21 (1.06 to 4.62), haematologic Percutaneous drainage for patients with cervical necrotizing fasciitis with novel CT classifi cation based on extension of fl uid collection along the deep cervical space T Kiguchi, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P13 (doi: 10.1186/cc14093) Percutaneous drainage for patients with cervical necrotizing fasciitis with novel CT classifi cation based on extension of fl uid collection along the deep cervical space T Kiguchi, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P13 (doi: 10.1186/cc14093) Results In a multivariate logistic regression model, a statistically signifi cant independent eff ect was found for body temperature (BT) >38°C (OR = 2.58, 95% CI, 1.76 to 3.79, P <0.001), systolic blood pressure (SBP) <100 mmHg (OR = 1.72, 95% CI, 1.11 to 2.65, P = 0.01), CRP >10 mg/dl (OR = 3.03, 95% CI, 2.05 to 4.49, P <0.001) and PaCO2 <32 mmHg (OR = 2.3, 95% CI, 1.57 to 3.37, P <0.001). Receiver operating characteristic curve analysis revealed an area under the curve value of 0.725 for diff erentiating patients with bacteremia from negative culture. Introduction Cervical necrotizing fasciitis (CNF) is a rapidly evolving and life-threatening condition. Therefore, it is important for physicians to evaluate the severity of illness and to predict clinical outcome exactly in the early phase. We focused on extension of acute fl uid collection along the deep cervical space by CT fi ndings. The purpose of this study was to produce the CT grade and to analyze whether our CT grade is related to the clinical features and the responses to treatment of CNF. Methods Between June 2004 and December 2012, 42 patients diagnosed and treated for CNF in two institutions were included in p Methods Between June 2004 and December 2012, 42 patients diagnosed and treated for CNF in two institutions were included in Conclusion BT >38°C, SBP  <100  mmHg, CRP >10  mg/dl and PaCO2 <32  mmHg are independently associated with bacteremia. These S5 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P15 ICU mortality rates in patients with sepsis before and after the Surviving Sepsis Campaign Fisher’s two-tailed test showed a signifi cant diff erence (P = 0.003) between the mortality before and after the second publication. The median ages before and after 2009 were 63.9 and 64.8 years. The time in hospital before admission to the ICU was greater before 2009 (6.15 days) compared with after 2009 (5.53 days). There was no signifi cant diff erence (Mann–Whitney test) between the APACHE II scores, with the mean and median score the same at 17.6 and 18 for both groups. The mean length of stay was 1 day longer after 2009 (8.07 days compared with 9.07 days). P17 Methods A prospective study design was used in order to investigate all sepsis patients either presenting to the emergency department or admitted to the ICU of a regional trauma centre. A total of 106 patients were recruited and all patients were considered eligible as per the SIRS and sepsis criteria [1]. The Sepsis-related Organ Failure Assessment score was determined over the fi rst 24 hours to assess organ function. Patients were assigned to groups as follows: sterile SIRS; uncomplicated sepsis; severe sepsis or septic shock as per the criteria. Assignment into groups was blinded and performed by an intensive care specialist independent of the study. Baseline demographics, clinical characteristics and outcomes were collected and surviving patients were sent a SF-12v2 survey at between 6 months and 2  years post hospital discharge. P18 Long-term health-related quality of life in survivors of sepsis: an epidemiological study P18 Long-term health-related quality of life in survivors of sepsis: an epidemiological study CE Battle1,2,3, G Davies1, M Vijayakumar3, PA Evans1 1NISCHR HBRU Morriston Hospital, Swansea, UK; 2College of Medicine, Swansea University, Swansea, UK; 3Ed Major Critical Care Unit, Morriston Hospital, Swansea, UK Critical Care 2015, 19(Suppl 1):P18 (doi: 10.1186/cc14098) Figure 1 (abstract P16). disease = 3.40 (1.64 to 7.04), Karnovsky Index <70 = 2.56 (1.63 to 3.71), infection by an ESKAPE pathogen = 1.65 (1.02 to 2.66) and severity of infection (reference is infection without SIRS): sepsis = 1.14 (0.7 to 1.83), severe sepsis = 1.18 (0.73 to 1.93), septic shock = 3.69 (1.78 to 7.65). The fi nal model had a very good discrimination for long-term mortality with an area under the ROC curve of 0.78 (Figure 1).ii disease = 3.40 (1.64 to 7.04), Karnovsky Index <70 = 2.56 (1.63 to 3.71), infection by an ESKAPE pathogen = 1.65 (1.02 to 2.66) and severity of infection (reference is infection without SIRS): sepsis = 1.14 (0.7 to 1.83), severe sepsis = 1.18 (0.73 to 1.93), septic shock = 3.69 (1.78 to 7.65). The fi nal model had a very good discrimination for long-term mortality with an area under the ROC curve of 0.78 (Figure 1). Conclusion The authors identifi ed several factors that were signifi cantly associated with increased long-term mortality in patients with severe infection. This information will help clinicians in the discussion of individual prognosis and clinical decision-making. Introduction Survivors of sepsis report persistent problems that can last years after hospital discharge. The main aim of this study was to investigate long-term health-related quality of life in survivors of SIRS and sepsis compared with Welsh normative data, controlling for age, length of stay and pre-existing conditions. The second aim was to investigate any diff erences in long-term health-related quality of life specifi cally with the patients categorised into three groups: SIRS, uncomplicated sepsis, and severe sepsis/septic shock. Conclusion The authors identifi ed several factors that were signifi cantly associated with increased long-term mortality in patients with severe infection. This information will help clinicians in the discussion of individual prognosis and clinical decision-making. References Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S6 Figure 1 (abstract P16). Figure 1 (abstract P17). Median ICU costs per patients and ICU cost per day according to study years. Figure 1 (abstract P16). Figure 1 (abstract P17). Median ICU costs per patients and ICU cost per day according to study years. intensivists to contribute a high standard of care within a restricted budget. The cost-eff ectiveness analysis should be evaluated in sepsis care cases. Direct intensive care costs of severe sepsis and septic shock patients in Thailand B Khwannimit, R Bhurayanontachai Songklanagarind Hospital, Hat Yai, Thailand Critical Care 2015, 19(Suppl 1):P17 (doi: 10.1186/cc14097) Introduction Costs of severe sepsis care from middle-income countries are lacking. This study investigated direct ICU costs and factors that could aff ect the fi nancial outcomes. fi Methods A prospective cohort study was conducted in the medical ICU of a tertiary referral university teaching hospital in Thailand over a 4-year period. y p Results A total of 897 patients, with 683 (76.1%) having septic shock. Overall ICU mortality was 38.3%. The median (interquartile range) ICU length of stay (LOS) was 4 (2 to 9) days. Community, nosocomial and ICU-acquired infection were documented in 574, 282 and 41 patients, respectively. The median ICU costs were €2,067.2 (986.3 to 4.084.6) per patient and €456.6 (315.3 to 721.8) per day. The ICU costs accounted for 64.7% of the hospital costs. In 2008 to 2011, the ICU costs signifi cantly decreased by 40% from €2,695.7 to €1,617, whereas the daily ICU costs decreased only 3.3% from €463.9 to €448.7 (Figure  1). The average ICU costs of patient with nosocomial and ICU-acquired infection were signifi cantly higher than patients with community-acquired infection. By multivariate logistic regression analysis, age, nosocomial or ICU infection, admission from emergency department, number of organ failures, ICU LOS, and fl uid balance in the fi rst 72 hours were independently associated with total ICU costs.i Results A total of 106 patients were included in the study. A mortality rate of 34% was recorded, leading to a fi nal response rate of 72% by the end of the data collection period. Quality of life was signifi cantly reduced in all patients when compared with local normative data (all P <0.0001). Reductions in the physical components of health-related quality of life were more pronounced in severe sepsis/septic shock patients when compared with uncomplicated sepsis and SIRS patients. Conclusion This is the fi rst observational study to specifi cally focus on the diff erent groups of SIRS and sepsis patients to assess long-term quality of life. Local population norms were used for comparison, rather than wide geographical norms that fail to refl ect the intricacies of a country’s population. Signifi cant reductions in quality of life were found in severe sepsis/septic shock patients compared with in uncomplicated sepsis and SIRS patients, when controlling for age, pre- existing conditions, hospital and ICU length of stay. Reference g Reference P21 P21 Global burden of sepsis: a systematic review C Fleischmann1, A Scherag1, NK Adhikari2, CS Hartog1, T Tsaganos3, P Schlattmann1, DC Angus4, K Reinhart1 1Jena University Hospital, Jena, Germany; 2University of Toronto, ON, Canada; 3University of Athens, Greece; 4University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P21 (doi: 10.1186/cc14101) Introduction Sepsis is a global healthcare challenge. However, comprehensive information on sepsis morbidity and mortality across the world is scarce. We aimed to estimate the global burden of sepsis and to identify knowledge gaps based on available evidence from observational epidemiological studies. g Methods We searched 15 international and national citation databases for population-level estimates on incidence rates of sepsis or severe sepsis per 100,000 person-years and case fatality rates in adult populations using consensus criteria and published in the last 40 years. No language or publication restrictions were applied. Studies were stratifi ed into four subgroups (setting: hospital or ICU for sepsis and severe sepsis) and meta-analyzed using metaprop of the R 3.0.2 package. Heterogeneity of the underlying eff ects across studies was expressed by the estimated τ, the square root of the between-study variance. y Conclusion Patients admitted to the ICU with severe CAP and immunosuppressive therapy have higher mortality, with no diff erences between HCAP and CAP. The delay in intubation as well as bacterial and inappropriate antibiotic treatment are factors that increase mortality. Results The search yielded 1,553 reports from 1979 to 2013, of which 37 met our criteria and 33 provided data for meta-analysis. The included studies were from 15 high-income countries in North America, Europe, Asia, and Australia. For these countries, the population incidence rate was 256 (95% CI, 182 to 360, τ = 0.43) hospital-treated sepsis cases and 151 (95% CI, 94 to 242, τ = 0.98) hospital-treated severe sepsis cases per 100,000 person-years, with large between-study heterogeneity. Restricted to the last decade, the incidence rate was 427 (95% CI, 281 to 648, τ = 0.24) sepsis cases and 331 (95% CI, 207 to 530, τ = 0.59) severe sepsis cases per 100,000 person-years. Hospital mortality was 15% for sepsis and 25% for severe sepsis during this period of time. There were no population-level sepsis incidence estimates from lower income countries. A tentative extrapolation from high-income-country data suggests global estimates of 30.7 million sepsis and 23.8 million severe sepsis cases, with potentially six million deaths each year. Analysis of the mortality rate in patients admitted to the ICU for severe community-acquired pneumonia Conclusion Severe sepsis and septic shock are conditions that consume large amounts of resources. Nonsurvivors had higher average spending than survivors. Patients admitted with septic shock had higher mortality than patients with severe sepsis with high mortality in relation to the prognostic indices adopted. The beginning of the antibiotics was longer in the nonsurvivors. We should adopt measures aimed at recognizing and earlier treatment of sepsis. If we improve our treatment, especially in septic shock, we will prevent deaths and decrement costs. Introduction The aim of the study was to analyze the factors associated with hospital mortality in patients with severe community-acquired pneumonia (CAP) who required ICU admission. Methods An observational, retrospective study of patients with severe CAP admitted to the ICU between January 2008 and September 2013. We analyzed clinical, epidemiological and outcome variables. Quantitative variables were expressed as the mean and standard deviation. Qualitative variables are expressed as the percentage and absolute value. We applied the Mann–Whitney and Fisher’s exact test, as needed, with an alpha error of 5%. P21 p Results We analyzed 111 patients, 57.5 ± 17.7 years old, with 63.1% (70) males and APACHE II score on admission of 19.8 ± 17.7. ICU mortality was 29.7% (33) and in-hospital mortality was 32.4% (36). Ten percent of patients met criteria for medical care-associated pneumonia (HCAP); there were no signifi cant diff erences in mortality between HCAP and CAP (P = 0.075). Patients chronically taking immunosuppressive therapy had a signifi cantly higher mortality compared with the rest of the patients (47.8% vs. 28.4%, P = 0.07). The mortality rate was also higher in patients in whom NIV fail in the fi rst 24  hours (42.9% vs. 17.6% with P = 0.09). Patients who required intubation and mechanical ventilation in the fi rst 24 hours had a higher mortality rate (47.2% vs. 19%, P = 0.002). Regarding the etiology of pneumonia, in 11 patients the viral origin of infection was confi rmed (10 patients had H1N1 pneumonia and one patient CMV pneumonia), with a mortality rate signifi cantly lower than in patients with bacterial pneumonia (3.6% vs. 35.3%, P = 0.06). The use of the right antibiotic therapy at admission was associated with mortality (P = 0.0001). M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil Introduction Sepsis is a high-prevalence disease in ICUs, associated with high mortality and high costs, mainly in developing countries. The aim of this study is to demonstrate the ICU costs, in a private hospital, in patients admitted with severe sepsis and septic shock. p p p Methods A retrospective, observational, single-center study of patients admitted from November 2013 to March 2014 with severe sepsis and septic shock. The records data were taken from the Software Epimed, MV System, and IBM SPSS Statistics 21. The classifi cation was based on the Surviving Sepsis Campaign 2012. We included all 50 beds of an adult ICU, clinical and surgical. All patients older than 18 years with severe sepsis and septic shock were included. We evaluated the costs of patients during their ICU stay, and its relation to clinical presentation (severe sepsis and septic shock), antibiotic start time, permanence of ICU stay, and mortality. Only the fi rst episode per patient was recorded. Results From November 2013 to March 2014 were included 82 patients with criteria for severe sepsis and septic shock. The mean age of patients was 62.5 ± 21.8 years, divided equally between the genres. The overall mortality rate was 34.15%. The SAPS 3 was 56.43, with death probability set to Latin America 38.83%. Patients with severe sepsis had a mortality of 23.2% and those with septic shock had a mortality rate of 58%. The average total cost during ICU admission per patient was US$17,834 and the average daily cost was US$1,641. The daily cost in patients with severe sepsis and septic shock was US$1,263 and US$2,465 (P = 0.002), respectively, and in survivors and nonsurvivors was US$1,189 and US$2,512 (P = 0.001). The length of stay of patients Conclusion Our analyses underline the urgent need to implement global strategies to monitor sepsis morbidity and mortality  – especially in low-income and middle-income countries. For further epidemiological studies, more consistent and standardized methodological approaches are needed to reduce between-study heterogeneity. In particular, further research on sepsis coding using administrative data seems necessary to derive sensitive and specifi c sepsis case identifi cations. P19 in the ICU was 11.09 days, being 11.3 days in patients with severe sepsis and 10.7 days in patients with septic shock (P = 0.785). The beginning of the antibiotics in nonsurvivors was 73.7 minutes and in survivors was 64.7 minutes (P = 0.757), with the earliest onset in patients with septic shock than in patients with severe sepsis (38.5 vs. 81.5 minutes, P = 0.141). P19 Analysis of the mortality rate in patients admitted to the ICU for severe community-acquired pneumonia C Joya-Montosa, MD Delgado-Amaya, H Molina-Diaz, E Curiel Balsera Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P19 (doi: 10.1186/cc14099) Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil Evaluation of the cost of severe sepsis and septic shock in a private ICU in Brazil M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil Critical Care 2015, 19(Suppl 1):P20 (doi: 10.1186/cc14100) M Borges Velasco, MA Leitão, MB Leitão, DM Dalcomune, LP Massete Hospital Meridional S.A., Vila Velha, Brazil g Reference Conclusion The ICU costs of severe sepsis management signifi cantly declined in Thailand. However, the ICU costs were a fi nancial burden accounting for two-thirds of the hospital costs. It is essential for Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. Levy MM, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS. International Sepsis Defi nitions Conference. Crit Care Med. 2003;31:1250-6. S7 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We included adult (age >18  years) ED patients presenting with severe sepsis/septic shock (sepsis with elevated lactate (>4  mmol/l)) or hypotension) from the prospective clinical ProCESS trial. We studied a subset of patients with microcirculatory videos obtained along with non-infected control patients. Using a sidestream dark-fi eld videomicroscope, we obtained image sequences from the sublingual mucosa and used video stabilization and frame averaging techniques to visualize slowly-moving leukocytes. We quantifi ed the number of rolling and adhered leukocytes present per 1 mm × 1 mm visual fi eld in a standardized 3-second clip. Furthermore, we extracted the total length of vessels candidate for counting of rolling/adhered leukocytes (vessels with an adequate focus). We report sample means with standard deviation and compare them with Student’s t test. how these occur and how we might prevent them. Our objective is to identify disparities by race, language, gender, socioeconomic status, insurance status and geography in acute sepsis care in emergency department (ED) or ICU settings in the published literature. p g p Methods We performed a systematic review of disparities in sepsis care. The search strategy and inclusion and exclusion criteria were defi ned a priori. A medical librarian searched the entire MEDLINE (PubMed), EMBASE and Cinahl databases prior to 2013. One author reviewed all abstracts and a second author reviewed 10% of all abstracts for agreement. Both reviewers independently reviewed each included article using an explicit study review tool. We included studies that met the following inclusion criteria: ED or ICU setting; disparities due to race, language, gender, socioeconomic status, insurance status or geography; process of care measures (antibiotics, lactate, i.v. fl uid resuscitation, central line placement, vasopressor use) or outcome measures (mortality, length of stay, complications, costs). We excluded studies involving organ-specifi c infectious conditions, pediatric populations, case reports, and review articles.i p Results We included a total of 64 patients with severe sepsis/septic shock and 32 non-infected controls. The mean number of adhered leukocytes per fi eld in the sepsis group was 2.1 (SD 2.3) compared with 0.4 (SD 0.8) in the non-infected group (P <0.001). This corresponded to a mean number of adhered leukocytes per unit vessel length of 0.16/mm (SD 0.22) and 0.03/mm (SD 0.06) for sepsis and non-infected groups, respectively (P <0.001). P23 S bli g y Methods This is a prospective observational study in patients scheduled for elective cardiac surgery. Serum samples were drawn prior to surgery, after connection to cardiopulmonary bypass (ischemia), after opening of cross-clamp (reperfusion) and after termination of surgery. The redox status of patients was measured using the bedside point of care RedoxSYS Diagnostic System™ (Luoxis, USA). Simultaneously the antioxidant capacity in serum samples were calculated in all perioperatively obtained serum samples. P24 P24 Time course of redox potential and antioxidant capacity in patients undergoing cardiac surgery C Stoppe1, G Schaelte1, S Kraemer2, C Benstoem2, D Bar-Or3, A Goetzenich2 1RWTH Aachen University, Aachen, Germany; 2RWTH Aachen University, University Hospital, Aachen, Germany; 3Swedish Medical Center, Trauma Research, Engelwood, CO, USA Critical Care 2015, 19(Suppl 1):P24 (doi: 10.1186/cc14104) References 1. Lagu T, Rothberg MB, Nathanson BH, et al. Variation in the care of septic shock: the impact of patient and hospital characteristics. J Crit Care. 2012;27:329-36. Lagu T, Rothberg MB, Nathanson BH, et al. Variation in the care of se 2. Barnato AE, Alexander SL, Linde-zwirble WT, Angus DC. Racial variation in the incidence, care, and outcomes of severe sepsis: analysis of population, patient, and hospital characteristics. Am J Respir Crit Care Med. 2008;177:279-84. 3. Dombrovskiy VY, Martin AA, Sunderram J, Paz HL. Occurrence and outcomes of sepsis: infl uence of race. Crit Care Med. 2007;35:763-8. Introduction Cardiac surgery regularly provokes infl ammation and oxidative stress which contribute to the development of organ failure and mortality of patients. While the assessment of single markers does not refl ect a comprehensive investigation of redox status, the measurement of oxidation–reduction potential (ORP) provides a reliable measure to assess the balance between total prooxidant and antioxidant balance in the blood. The aim of the present study was to investigate the overall redox potential in patients undergoing cardiac surgery. 4. Mayr FB, Yende S, Linde-zwirble WT, et al. Infection rate and acute organ dysfunction risk as explanations for racial diff erences in severe sepsis. JAMA. 2010;303:2495-503. 5. Plurad DS, Lustenberger T, Kilday P, et al. The association of race and survival from sepsis after injury. Am Surg. 2010;76:43-7. 5. Plurad DS, Lustenberger T, Kilday P, et al. The association of race and survival from sepsis after injury. Am Surg. 2010;76:43-7. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 For the rolling leukocytes, we observed a mean number of 27.8 (SD 19.4) in the sepsis group and 12.0 (SD 8.7) in the non-infected group (P <0.001) per fi eld. This corresponded to a mean number of rolling leukocytes per unit vessel length of 2.00/mm (SD 1.67) and 0.75/mm (SD 0.55), respectively (P <0.001). Results We identifi ed 778 abstracts; yielding 31 for inclusion (k = 0.95), 26 of 31 studies were excluded due to quality issues. Five articles met our inclusion criteria. Only one of the studies [1] contained data on process of care measures, showing that central venous monitoring was less likely to occur in older patients. Three studies [2-4] showed that Black patients had a higher incidence of sepsis, a higher hospitalization rate, and higher mortality rate. Plurad and colleagues [5] reported that Asian patients had increased incidence of post-traumatic sepsis. Overall, Black patients with sepsis were younger, had lower socioeconomic status and were more likely to be cared for in urban settings compared with their cohorts. p y Conclusion Our results show a higher number of rolling and adhered leukocytes in patients with severe sepsis/septic shock when compared with non-infected controls. This also applies when taking the total vessel length in the fi eld of view into consideration. This may hold potential as a useful tool in sepsis assessment. Conclusion We found little published data addressing whether disparities due to race, language, gender, socioeconomic status, insurance status or geography exist in the acute care of sepsis. As sepsis is a leading cause of in-hospital mortality, future research should determine whether such disparities exist. Specifi cally, prospective studies of the process of care in sepsis management may further elucidate additional factors that may contribute to these disparities. References P22 P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) P22 Disparities in acute sepsis care: a systematic review D Yamane, N Huancahuari, P Hou, J Schuur Brigham and Women’s Hospital, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P22 (doi: 10.1186/cc14102) Introduction Disparities in the incidence and outcomes of sepsis have been documented in observational studies but little is known about S8 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Fatty acid composition of erythrocytes in multiple organ dysfunction syndrome y y A Osipenko1, A Marochkov2 A Osipenko1, A Marochkov2 1A. Kuleshov Mogilev State University, Mogilev, Belarus; 2Mogilev Regional Hospital, Mogilev, Belarus Critical Care 2015, 19(Suppl 1):P25 (doi: 10.1186/cc14105) Results We found that severe septic patients showed lower CIV activity/ protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. Survivor severe septic patients (n  = 130) showed lower CIV activity/protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. In addition, nonsurvivor severe septic patients (n = 68) showed lower CIV activity/protein quantity than controls at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis. Besides, nonsurvivor severe septic patients showed lower CIV activity/protein quantity than survivor ones at day 1 (P <0.001), day 4 (P <0.001) and day 8 (P <0.001) of severe sepsis diagnosis.i p , g , Critical Care 2015, 19(Suppl 1):P25 (doi: 10.1186/cc14105) Introduction Change in fatty acid composition of erythrocytes and blood plasma in cases of various pathological conditions is evidence of lipid metabolism disorder and can indicate the reasons for and the degree of these disorders [1]. The aim of this study was to assess the FA composition of plasma and erythrocytes in patients with multiple organ dysfunction syndrome (MODS). g y y Methods The objects of study were 19 people with MODS (37.6  ±  8.3  years) of various etiologies. The blood of 17 healthy volunteers aged 38.4 ± 3.3 years served as control. The FA analysis was conducted using capillary gas–liquid chromatography. Quantitative evaluation of individual FA content was made as a mass percentage of their total (C14:0 to C22:6). Statistical analysis was performed using the Mann–Whitney U test (P <0.05). y p g Conclusion The major fi nding of our work, that represents the largest series of severe septic patients with data on OXPHOS function, was that survivor and nonsurvivor severe septic patients showed lower platelet CIV activity than healthy controls during the fi rst week of severe sepsis diagnosis. y Results Our data indicate that changes in blood plasma FA composition in patients with MODS are mainly caused by activation of lipolysis in fat depots and are accompanied by an increase of monounsaturated fatty acids, a decrease in saturated stearic acid and polyunsaturated fatty acids in the ratio. Sublingual leukocyte activation in patients with severe sepsis or septic shock BK Fabian-Jessing1, MJ Massey1, MR Filbin2, PC Hou3, H Kirkegaard4, HE Wang5, DM Yealy6, JA Kellum6, DC Angus6, NI Shapiro1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3Brigham and Women’s Hospital, Boston, MA, USA; 4Aarhus University Hospital, Aarhus, Denmark; 5University of Alabama at Birmingham, AL, USA; 6University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P23 (doi: 10.1186/cc14103) y Results All patients’ sera (n = 17) demonstrated a signifi cant increase of ORP upon start of myocardial ischemia (141.0 ± 4.8 mV vs. 157.9 ± 4.9 mV; P  =  0.002) and compared with reperfusion (141.0  ±  4.8 mV vs. 158.6  ±  4.9mV; P  <0.001, Figure  1A). In parallel, the antioxidant capacity signifi cantly decreased during surgery (0.505 ± 0.190 μC vs. 0.384 ± 0.120 μC; P = 0.022) corresponding to the increase of oxidative stress (Figure 1B). Introduction The objective of this study was to compare the number of rolling and adhered leukocytes in patients with severe sepsis/septic shock with non-infected controls. Microcirculatory fl ow alterations and endothelial cell dysfunction are elements of sepsis pathophysiology. Traditionally, microcirculatory emphasis has been on red blood cell vessel perfusion. However, assessment of interactions between white blood cells and endothelial cells may be another early diagnostic modality. Introduction The objective of this study was to compare the number of rolling and adhered leukocytes in patients with severe sepsis/septic shock with non-infected controls. Microcirculatory fl ow alterations and endothelial cell dysfunction are elements of sepsis pathophysiology. Traditionally, microcirculatory emphasis has been on red blood cell vessel perfusion. However, assessment of interactions between white blood cells and endothelial cells may be another early diagnostic modality. Conclusion This preliminary study is the fi rst to highlight the time course of overall redox potential and antioxidant capacity in cardiac surgery patients. Further studies are underway to evaluate the clinical signifi cance on outcome in cardiac surgery patients. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S9 Figure 1 (abstract P24). (A), (B) Perioperative time course of oxidative stress and antioxidant capacity. acids. In the test group of patients, as compared with the control group there was an elevated level (27.12 ± 0.78% vs. 25.80 ±0.77%, P <0.05) of saturated palmitic (C16:0) acid combined with the reduced (11.46 ± 0.52% vs. 13.95 ± 1.09%, P <0.001) level of linoleic (C18:2) acid. Reference 1. Kremmyda LS, et al. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155:195-218. P26 Lower platelet mitochondrial function in severe septic patients than in controls L Lorente1, M Martin2, J Blanquer3, J Solé-Violán4, L Labarta5, C Díaz6, A Jiménez1, E López-Gallardo7, J Montoya7, E Ruiz-Pesini7 1Hospital Universitario de Canarias, La Laguna, Tenerife, Spain; 2Hospital Universitario Nuestra Señora Candelaria, Santa Cruz, Tenerife, Spain; 3Hospital Clínico Universitario de Valencia, Spain; 4Hospital Universitario Dr Negrín, Las Palmas de Gran Canaria, Spain; 5Hospital San Jorge, Huesca, Spain; 6Hospital Insular, Las Palmas de Gran Canaria, Spain; 7Universidad de Zaragoza, Spain Critical Care 2015, 19(Suppl 1):P26 (doi: 10.1186/cc14106) Introduction The oxidative phosphorylation system (OXPHOS) in septic patients has been scarcely analyzed in studies of small sample size and the results are apparently inconsistent. Previously, including 96 severe septic patients, we found that nonsurviving severe septic patients showed lower platelet respiratory complex IV (CIV) activity than surviving patients at the moment of severe sepsis diagnosis and during the fi rst week of sepsis diagnosis. However, we did not examine this enzyme activity in normal individuals. Thus, the objective of this study was to compare the CIV activity between severe septic patients and healthy control individuals in a larger series of patients (including 198 severe septic patients). Introduction The oxidative phosphorylation system (OXPHOS) in septic patients has been scarcely analyzed in studies of small sample size and the results are apparently inconsistent. Previously, including 96 severe septic patients, we found that nonsurviving severe septic patients showed lower platelet respiratory complex IV (CIV) activity than surviving patients at the moment of severe sepsis diagnosis and during the fi rst week of sepsis diagnosis. However, we did not examine this enzyme activity in normal individuals. Thus, the objective of this study was to compare the CIV activity between severe septic patients and healthy control individuals in a larger series of patients (including 198 severe septic patients). Figure 1 (abstract P24). (A), (B) Perioperative time course of oxidative stress and antioxidant capacity. Methods This was a prospective, multicenter, observational study in six Spanish ICUs. We obtained blood samples from 198 severe septic patients at days 1, 4 and 8 of the severe sepsis diagnosis and from 96 sex-matched and age-matched healthy control individuals and determined platelet CIV activity/protein quantity. The endpoint of the study was 30-day mortality. Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection J Sole-Violan1, M López-Rodríguez1, E Herrera-Ramos1, J Ruiz-Hernández1, J Horcajada2, L Borderías3, J Blanquer4, J Ferrer1, O Rajas5, J Aspa5, F Rodríguez de Castro1, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital del Mar, Barcelona, Spain; 3Hospital San Jorge, Huesca, Spain; 4Hospital Clínico, Valencia, Spain; 5Hospital de la Princesa, Madrid, Spain Critical Care 2015, 19(Suppl 1):P27 (doi: 10.1186/cc14107) 1. Kremmyda LS, et al. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155:195-218. Fatty acid composition of erythrocytes in multiple organ dysfunction syndrome In conditions of increased level of monounsaturated palmitoleic (C16:1) and oleic (C18:1) FA in blood plasma (2.53 ± 0.40% vs. 1.55 ± 0.29%, P <0.001 and 25.18 ± 2.15% vs. 16.55 ± 1.17%, P <0.001, respectively), only the level of palmitoleic (C16:1) acid is increased in erythrocytes (0.56  ±  0.12% vs. 0.16  ±  0.12%, P  <0.001). Despite the high content of oleic (C18:1) acid in blood plasma in case of MODS, in erythrocytes its relative level is not changed as compared with the control group. The disorder of lipid composition constancy in erythrocyte membranes is also manifested by change in the content of saturated palmitic (C16:0) and polyunsaturated linoleic (C18:2) fatty Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection J Sole-Violan1, M López-Rodríguez1, E Herrera-Ramos1, J Ruiz-Hernández1, J Horcajada2, L Borderías3, J Blanquer4, J Ferrer1, O Rajas5, J Aspa5, F Rodríguez de Castro1, C Rodríguez-Gallego1 1Hospital GC Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital del Mar, Barcelona, Spain; 3Hospital San Jorge, Huesca, Spain; 4Hospital Clínico, Valencia, Spain; 5Hospital de la Princesa, Madrid, Spain Critical Care 2015, 19(Suppl 1):P27 (doi: 10.1186/cc14107) Sublingual leukocyte activation in patients with severe sepsis or septic shock Conclusion The changes revealed in fatty acid composition of erythrocytes may indicate systemic modifi cations of cell membranes in MODS. Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort Results Patients who developed sepsis (n  = 18) presented with signifi cantly higher platelet fi brinogen binding at T1 compared with patients who did not get infected (basal: P = 0.0014, upon stimulation: P <0.0035). At T1, ROC AUC for association of basal fi brinogen binding with the occurrence of sepsis was 0.79 (95% CI: 0.68 to 0.89). Elevated basal CD62P expression level was associated with increased 90-day mortality (P  =  0.042, ROC AUC  = 0.78 (0.64 to 0.88)). Kaplan–Meier survival curves illustrated that mortality was signifi cantly higher after stratifi cation based on T1 basal CD62P level (cutoff MFI >31.56, HR = 13.6, P  =  8.23  ×  10–6). Multivariate logistic regression analysis using clinical scores (SOFA, APACHE II, SAPS II, SAPS III) indicated that addition of CD62P level or of bound fi brinogen level signifi cantly improved prediction of mortality (odds ratio 1.078, P = 0.003) and sepsis (odds ratio 1.033, P = 0.0012), respectively. A Tridente, G Clarke, A Walden, A Gordon, P Hutton, J Chiche, P Holloway, G Mills, J Bion, F Stuber, C Garrard, C Hinds, GenOSept Investigators St Helens and Knowsley, Liverpool, UK Introduction Patients admitted to intensive care following surgery for faecal peritonitis present particular challenges in terms of clinical management and risk assessment that require close collaboration between surgical and intensive care teams [1]. We aimed at establishing whether dynamic assessment of trends in selected variables may be associated with outcomes, and therefore inform medical decision-making. g Methods We analysed trends in all 35 variables available for the fi rst week of ICU stay in 977 patients from 102 centres across 17 countries. The primary study outcome was 6-month mortality. Secondary outcomes were ICU, hospital and 28-day mortality. For each trend, Cox proportional hazards (PH) regression analyses, adjusted for age and gender, were performed for each endpoint. Trends found to be signifi cant in these analyses, after Bonferroni correction for multiple testing, were entered into a multivariate Cox PH model, to determine independent associations with mortality.i Conclusion Predisposition to severe infection in selected critically ill medico-surgical adults can be identifi ed on day 1 of admission based on circulating basally activated platelets. Levels of activated platelets may add incremental prognostic information to clinical scoring. Reference 1. de Stoppelaar SF, van ‘t Veer C, van der Poll T. The role of platelets in sepsis. Thromb Haemost. 2014;11:666-77. Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort p y Results The trends over the fi rst 7 days of ICU stay (primary analysis) retained as independently associated with 6-month outcome were worsening thrombocytopaenia (mortality HR = 1.02, 95% CI = 1.01 to 1.03, P <0.001) and changes in renal function (total daily urine output HR = 1.02, 95% CI = 1.01 to 1.03, P <0.001; renal SOFA subscore HR = 0.87, 95% CI = 0.75 to 0.99, P = 0.047), highest recorded level of bilirubin (HR = 0.99, 95% CI = 0.99 to 0.99, P = 0.02) and GCS SOFA subscore (HR = 0.81, 95% CI = 0.68 to 0.98, P = 0.028). Changes in renal function (total daily urine output and renal component of the SOFA score), GCS component of the SOFA score, total SOFA and worsening thrombocytopaenia were also independently associated with secondary outcomes. Dynamic trends over the fi rst 7 days of ICU stay in all other measured laboratory variables, physiological parameters or radiological fi ndings failed to be retained as independently associated with outcome on multivariate analyses. Furthermore, changes in respiratory support, renal replacement therapy and inotropic and/or vasopressor requirements appeared not to be independently associated with any of the primary or secondary outcomes. Secondary post hoc analyses on trends over the fi rst 3 and 5 days corroborated these fi ndings. P29 P29 Elevated basal levels of circulating activated platelets predict ICU-acquired sepsis and mortality: a prospective study N Layios CHU Sart Tilman, Liège, Belgium Critical Care 2015, 19(Suppl 1):P29 (doi: 10.1186/cc14109) P28 P28 Phenotypic factors associated with outcome in 977 intensive care patients with faecal peritonitis: analysis of trends in the GenOSept cohort A Tridente, G Clarke, A Walden, A Gordon, P Hutton, J Chiche, P Holloway, G Mills, J Bion, F Stuber, C Garrard, C Hinds, GenOSept Investigators St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P28 (doi: 10.1186/cc14108) P30 Antiplatelet therapy does not infl uence outcome or host response biomarkers during sepsis: a propensity-matched analysis MA Wiewel1, SF De Stoppelaar1, LA Van Vught1, JF Frencken2, AJ Hoogendijk1, PM Klein Klouwenberg2, J Horn1, MJ Bonten2, MJ Schultz1, AH Zwinderman1, OL Cremer2, T Van der Poll1 1Academic Medical Center, University of Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P30 (doi: 10.1186/cc14110) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 have previously demonstrated that variants at SFTPA2 infl uence the severity of H1N1pdm infection. We have now studied genetic variants at diff erent genes, some of them previously associated with infections by infl uenza and/or other viruses. The purpose of this study was to analyze the role of genetic variants in the susceptibility and outcome of IVI. Conclusion Only deterioration in renal function, thrombocytopaenia and hyperbilirubinaemia over the fi rst 7  days of ICU stay were consistently associated with mortality at all endpoints. Reference 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. Methods In total, 136 white Spanish patients developed IVI (80.3% of them by H1N1pdm virus). The general population group consisted of 1,466 unrelated healthy volunteers. Patients and controls were analyzed for diff erent polymorphisms at 13 genes (FCGR2A, FCGR3A, FCGR3B, IL1RN, IL6, LTA, TIRAP, TLR1, TLR2, TLR3, TLR4, CCR5, IGHG2). Infl uence of genetic variants in the susceptibility and outcome of infl uenza virus infection Introduction The role of genetic variability in the susceptibility and outcome of infl uenza virus infection (IVI) remains largely unknown. We Introduction The role of genetic variability in the susceptibility and outcome of infl uenza virus infection (IVI) remains largely unknown. We S10 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 9 Elevated basal levels of circulating activated platelets predict ICU-acquired sepsis and mortality: a prospective study N Layios CHU Sart Tilman, Liège, Belgium Critical Care 2015, 19(Suppl 1):P29 (doi: 10.1186/cc14109) IVI was detected in nasopharyngeal swabs using real-time PCR. The Hardy–Weinberg equilibrium was analyzed by Haploview v. 4.2. The comparisons of genotypes distribution based on susceptibility and severity were performed using the chi-squared test or Fisher’s exact test when needed. The relationship between severity in hospitalized patients and genotypes was evaluated by binary logistic regression models. Introduction Platelets are now considered to be immune and infl am- matory agents as well as key cells in coagulation, and as such have been implicated in the pathophysiology of sepsis [1]. Thrombocytopenia is associated with sepsis severity and poor prognosis, and hyperactivated platelets probably contribute to microvascular thrombosis and organ failure. In the present study, we evaluated platelet activation markers as potential predictive markers of sepsis and of mortality among four commonly encountered populations of patients admitted to ICUs. Results No associations were found between the diff erent genetic variants and susceptibility or severity of IVI. Variants at LTA, FCGR2A, IGHG2, TLR3 and CCR5, previously associated with severity of IVI were not replicated in our study. Methods Ninety-nine non-infected ICU patients were prospectively screened at day 1 (T1) and day 3 (T2) of admission after elective cardiac surgery, trauma, acute neurologic dysfunction or prolonged ventilation (>48 hours). A third sample was drawn when infection was diagnosed (Tx). We evaluated platelet activation by measuring the expression of P-selectin (CD62P) and fi brinogen binding on the cell surface before and after stimulation with major platelet agonists (ADP, collagen, and TRAP) through fl ow cytometry. Clinical scores were obtained at admission. Conclusion Our study does not suggest that polymorphisms at LTA, FCGR2A, IGHG2, TLR3 and CCR5 genes are associated with susceptibility or severity of IVI. 1. de Stoppelaar SF, van ‘t Veer C, van der Poll T. The role of platelets in sepsis. Thromb Haemost. 2014;11:666-77. P32 Methods We performed a prospective observational study in patients admitted with sepsis to the mixed ICUs of two hospitals in the Netherlands between January 2011 and July 2013. Cox proportional hazards regression was used to estimate the eff ect of antiplatelet therapy on mortality. To account for indication bias, a propensity score was constructed, and used to match antiplatelet therapy users to nonusers. Plasma biomarker levels, providing insight into hallmark host responses to sepsis, including activation of endothelial cells and the cytokine network, were determined during the fi rst 4 days after ICU admission. Mitochondrial dysfunction and ischemia in critical illness: an adipose tissue microdialysis study in 203 ICU patients M Theodorakopoulou, S Apollonatou, N Nikitas, D Vassiliadi, A Diamantakis, V Tsagkari, F Frantzeskaki, I Dimopoulou University Hospital of Athens, Greece Critical Care 2015, 19(Suppl 1):P32 (doi: 10.1186/cc14112) Introduction Ischemia and mitochondrial dysfunction have been implicated in critical illness. The potential of MD to diagnose and separate ischemia and mitochondrial dysfunction in ICU patients remains currently unknown. Introduction Ischemia and mitochondrial dysfunction have been implicated in critical illness. The potential of MD to diagnose and separate ischemia and mitochondrial dysfunction in ICU patients remains currently unknown. Results Of 1,070 sepsis patients, 297 (27.8%) were on antiplatelet therapy, including acetylsalicylic acid, clopidogrel and dipyridamole, prior to ICU admission. Antiplatelet users and nonusers diff ered signifi cantly with regard to several baseline characteristics, such as age, gender and cardiovascular disease. Antiplatelet therapy was not related to sepsis severity at presentation, the primary source of infection, causative pathogens, the development of organ failure or shock during ICU stay, or mortality up to 90 days after admission, in either the unmatched or propensity-matched analyses. Antiplatelet therapy did also not modify plasma concentrations of biomarkers.l Methods A retrospective, observational study of 203 mechanically ventilated patients studied over a 6-year period with MD including medical, surgical and trauma patients. Sepsis stages: SIRS (n  = 24), severe sepsis (n = 46) and septic shock (n = 133). Median age 67 years (range: 17 to 92  years). Mortality was 53%. All subjects had a MD catheter placed in femoral adipose tissue upon admission to the ICU. Interstitial fl uid samples were collected six times per day, for 3 consecutive days, and were analyzed for glucose, lactate, pyruvate, and glycerol levels. The lactate to pyruvate (LP) ratio was calculated. Blood lactate was measured. P31 Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery S Ono1, T Ikeda1, T Kubo2, H Tsujimoto2, M Kinoshita2, T Ueno1 1Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan; 2National Defense Medical College, Tokorozawa, Saitama, Japan Critical Care 2015, 19(Suppl 1):P31 (doi: 10.1186/cc14111) Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery Antiplatelet therapy does not infl uence outcome or host response biomarkers during sepsis: a propensity-matched analysis l1 l 1 h 1 k 2 MA Wiewel1, SF De Stoppelaar1, LA Van Vught1, JF Frencken2, AJ Hoogendijk1, PM Klein Klouwenberg2, J Horn1, MJ Bonten2, MJ Schultz1, AH Zwinderman1, OL Cremer2, T Van der Poll1 1Academic Medical Center, University of Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P30 (doi: 10.1186/cc14110) Introduction Sepsis is a life-threatening condition, during which triggering of infl ammatory and coagulation cascades, together with endothelial damage, invariably leads to activation of platelets. Although platelets are essential components of primary hemostasis, uncontrolled platelet activation during sepsis may contribute to organ failure. The aim of this study was to investigate whether chronic antiplatelet therapy impacts on the presentation and outcome of, and the host response to, sepsis. S11 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Perioperative programmed death-1 expression on CD4+ T cells predicts the incidence of postoperative infectious complications following gastrointestinal surgery g g g y S Ono1, T Ikeda1, T Kubo2, H Tsujimoto2, M Kinoshita2, T Ueno1 1Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan; 2National Defense Medical College, Tokorozawa, Saitama, Japan Critical Care 2015, 19(Suppl 1):P31 (doi: 10.1186/cc14111) Introduction Programmed death-1 (PD-1) has been reported to be an immunoinhibitory receptor expressed by chronically stimulated T cells after T-cell activation. The present study was designed to evaluate the relationship between perioperative PD-1 expression on CD4+ T cells and the incidence of postoperative infectious complications in patients undergoing gastroenterological surgery. Conclusion Bedside subcutaneous adipose tissue MD is possible to diagnose and separate ischemia and mitochondrial dysfunction in general ICU patients. These two conditions are not so common; however, mitochondrial dysfunction seems to be associated with higher mortality rates. Methods This was a prospective observational study. The subjects of this study included 101 patients with gastroenterological disease who underwent elective abdominal surgery via laparotomy at the National Defense Medical College Hospital. Blood samples were taken on the preoperative day (Pre) and the fi rst postoperative day (POD1). We calculated CD4+ T-cell count and PD-1 expression on CD4+ T cells by fl ow cytometer. The occurrence of postoperative infectious complications was defi ned according to a combination of clinical fi ndings and the results of laboratory and other tests. The postoperative infectious complications in this study included incisional surgical site infections (SSIs), organ/space SSIs, enterocolitis, urinary tract infections, and pneumonia. Incisional and organ/space SSIs were diagnosed according to the defi nitions stated in the guidelines issued by the Center for Disease Control and Prevention. P32 Ischemia was defi ned as LP ratio >30 and pyruvate level <70 mmol, while mitochondrial dysfunction was defi ned as LP ratio >30 and pyruvate >70 mmol. y y Conclusion Pre-existing antiplatelet therapy does not infl uence clinical disease severity at presentation, nor the host response or outcome following sepsis. Acknowledgement This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (http://www. ctmm.nl), project MARS (grant 04I-201). py Results Analysis during the course of the 3-day period revealed three distinct patterns: no ischemia/mitochondrial dysfunction (n = 150 or 74%), ischemia (n = 27 or 13%) and mitochondrial dysfunction (n = 26 or 13%). On day 1, median blood lactate was higher in mitochondrial dysfunction (2.2 mmol/l) compared with both ischemia (1.3 mmol/l) and with no ischemia/mitochondrial dysfunction (1.3  mmol/l) (P = 0.004). Again on day 1, median interstitial fl uid lactate was higher in mitochondrial dysfunction (8.4 mmol/l), in comparison with ischemia (1.4  mmol/l) and with the group without ischemia/mitochondrial dysfunction (2.5 mmol/l) (P <0.001). Similar results were obtained with interstitial fl uid glycerol levels (P = 0.009). Median LP ratio was higher in ischemia (LP = 36), and mitochondrial dysfunction (LP = 33) compared with those without ischemia/mitochondrial dysfunction (LP  =  17) (P  <0.001). Median interstitial fl uid glucose was lower in ischemia (2 mmol/l) compared with both mitochondrial dysfunction (4 mmol/l) and with no ischemia/mitochondrial dysfunction (5 mmol/l) (P <0.001). ICU mortality was 77% in mitochondrial dysfunction, 52% in ischemia and 49% in the group without ischemia/mitochondrial dysfunction (P = 0.033). Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Beth Israel Deaconess Medical Center, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P33 (doi: 10.1186/cc14113) Introduction Pyruvate dehydrogenase (PDH) is a key component of aerobic metabolism. Multiple rodent studies have shown that PDH levels are low in sepsis. This leads to a shift to anaerobic metabolism, resulting in increased lactic acid. Alteration in PDH levels during sepsis, however, has never been studied in humans. The aim of this study was to identify whether PDH levels (activity and quantity) were altered in humans in sepsis. Results Postoperative infectious complications occurred in 30 of the 101 patients. CD4+ T-cell count was signifi cantly lower in the patients who developed postoperative infectious complications at POD1 compared with those from the patients who did not. In addition, PD-1 expression on CD4+ T cells was signifi cantly higher at Pre or POD1 in patients who developed postoperative infectious complications. Those results were similar for the incidence of organ/space surgical site infection. Preoperative PD-1 expression on CD4+ T cells tended to be higher in males than in females. We found there was a signifi cant negative correlation between preoperative PD-1 expression on CD4+ T cells and CD4+ T-cell count. Methods We conducted a case–control study at a single urban tertiary care center. We compared PDH levels between sepsis and healthy control subjects by measuring PDH levels in peripheral blood mononuclear cells via a novel assay. We measured PDH levels in control subjects at baseline and in sepsis subjects at 0, 24, 48 and 72 hours. Results There were 39 sepsis (age 67 ± 14 years, M ± SD) and 19 control (age 50 ± 12 years) subjects of similar gender (56% and 63% female, respectively) and race (79% and 68% Caucasian, respectively). PDH levels in the sepsis group were signifi cantly lower than the control Conclusion Perioperative CD4+ T-cell count or PD-1 expression on CD4+ T cells could be an early predictive marker for the development of postoperative infectious complications. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S12 group at all time points (Figures  1 and 2). After controlling for age, gender, race, and assay plate via multivariable linear regression, the eff ect of treatment group remained signifi cant. We were unable to control for comorbid illness, which was exclusively concentrated in the sepsis group. Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Figure 1 (abstract P33). Figure 2 (abstract P33). Figure 2 (abstract P33). Conclusion In the nonsurvivor or the severe patient with sepsis requiring steroid administration, the enhancement of C1INH activity was not observed, and the C1INH quantitative values were low. Further evaluation of the serial change of C1INH and the validity of C1INH replacement therapy in patients with septic shock may lead to a new strategy for management in sepsis. Expression of apolipoproteins L in neutrophils during sepsis I Akl1, C Lelubre1, M Piagnerelli1, P Biston1, P Uzureau1, H Fayyad Kazan2, B Badran3, M Ezzedine3, K Zouaoui Boudjeltia1, L Vanhamme4 1CHU de Charleroi-Hopital Andre Vesale, Montigny-Le-Tilleul, Belgium; 2Institut Jules Bordet, Université Libre de Bruxelles, Belgium; 3Doctoral School of Sciences and Technology, Platform of Research and Environmental Sciences, Beirut, Lebanon; 4Institute of Medicine and Molecular Biology IBMM, Charleroi, Belgium Critical Care 2015, 19(Suppl 1):P35 (doi: 10.1186/cc14115) Figure 2 (abstract P33). Figure 2 (abstract P33). group at all time points (Figures  1 and 2). After controlling for age, gender, race, and assay plate via multivariable linear regression, the eff ect of treatment group remained signifi cant. We were unable to control for comorbid illness, which was exclusively concentrated in the sepsis group.i Introduction Sepsis is characterized by a strong systemic infl ammatory reaction. The pathogenesis is driven by alterations in the immune system and is associated with high neutrophil counts related to a specifi c delay in apoptosis [1]. The apolipoproteins L (ApoLs) family comprises six members in humans (ApoL1 to ApoL6). In light of their deregulated expression in several pathologies, they are likely to be important molecular players of programmed cell death [2]. We analyzed ApoL expression in cohorts of septic and nonseptic ICU patients and healthy volunteers in order to test whether ApoLs could be involved in the neutrophil apoptotic program. Introduction Sepsis is characterized by a strong systemic infl ammatory reaction. The pathogenesis is driven by alterations in the immune system and is associated with high neutrophil counts related to a specifi c delay in apoptosis [1]. The apolipoproteins L (ApoLs) family comprises six members in humans (ApoL1 to ApoL6). In light of their deregulated expression in several pathologies, they are likely to be important molecular players of programmed cell death [2]. Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno We analyzed ApoL expression in cohorts of septic and nonseptic ICU patients and healthy volunteers in order to test whether ApoLs could be involved in the neutrophil apoptotic program. Conclusion PDH levels are signifi cantly lowered in humans during sepsis when compared with healthy controls, even when controlling for age, race and gender. Further research is needed to determine whether this fi nding persists after adjustment for comorbid disease, and whether lower PDH levels are associated with clinical outcomes. Methods By means of magnetic cell sorting, peripheral neutrophils were purifi ed from 20 healthy volunteers and 40 ICU patients with (n = 20) or without sepsis (n = 20). ApoL expression was analyzed at the mRNA and protein levels by real-time PCR and western blot analysis respectively. Apoptosis of purifi ed neutrophils was assessed using fl ow cytometry following 4 and 24  hours of in vitro incubation. We monitored the expression of C-reactive protein (CRP), an infl ammatory marker, and its correlation with ApoL expression in PMNs was studied by linear regression analysis.i Pyruvate dehydrogenase levels are low in sepsis E Nuzzo, X Liu, K Berg, L Andersen, M Doninno Conclusion PDH levels are signifi cantly lowered in humans during sepsis when compared with healthy controls, even when controlling for age, race and gender. Further research is needed to determine whether this fi nding persists after adjustment for comorbid disease, and whether lower PDH levels are associated with clinical outcomes. P34 S i l h f C1 i hibi i i i h i li i Figure 1 (abstract P33). Figure 2 (abstract P33). Figure 1 (abstract P33). but also the plasma kallikrein–kinin system, fi brinolytic system and coagulation system. The biologic activities of C1INH can be divided into the regulation of vascular permeability and anti-infl ammatory functions. In recent years, hereditary angioedema (HAE), caused by an inherited defi ciency of C1INH, has been focused. During HAE attacks, vascular permeability was markedly increased, which leads to angioedema. In sepsis, signifi cant endothelial hyperpermeability is similarly observed systemically, but the role of C1INH has not been clarifi ed in the pathogenesis. The serial change of C1INH in patients with sepsis is not clear. The objective of this study was to clarify the serial change in C1INH in patients with sepsis and evaluate the impact of C1INH on their clinical course. Figure 1 (abstract P33). Methods We serially examined C1INH activity values (normal range 70 to 130%) and quantitative values (normal range 160 to 330 μg/ml) in patients with sepsis during the period between December 2012 and February 2013. We also analyzed their clinical course: prognosis, volume of infusion, body weight, urine volume, catecholamine administration, and steroid administration. Results The serial change of C1INH was evaluated in fi ve patients with sepsis (three male and two female; four survivors and one nonsurvivor; mean age, 68  ±  11  years). In the nonsurvivor, C1INH activity on admission value was 97.2% (normal range), and quantitative value was 133.1  μg/ml (below normal). In the patient with severe sepsis requiring fl uid resuscitation, catecholamine and steroid administration to maintain hemodynamics, C1INH activity value on admission was 94.4% (normal range), and quantitative value was 126.7 μg/ml (below normal range). His general condition was improved on day 6, and C1INH activity value and quantitative value increased (139.9%; above normal range, 250.1 μg/ml; normal range). In the other three patients with sepsis not requiring steroid administration, C1INH activity value on admission was 130.6 ± 8.7% (above normal range), and quantitative value was 215 ± 26.5 μg/ml (normal range). P34 Serial change of C1 inhibitor in patients with sepsis: a preliminary report T Hirose1, H Ogura1, K Jinkoo1, Y Nakamura1, H Hosotsubo1, T Shimazu1, E Kitano2, M Hatanaka2 1Osaka University Graduate School of Medicine, Suita, Japan; 2Kobe Tokiwa University, Kobe, Japan Critical Care 2015, 19(Suppl 1):P34 (doi: 10.1186/cc14114) Introduction C1 inhibitor (C1INH), belonging to the superfamily of serine protease inhibitors, regulates not only complement system, Results Our results showed a signifi cant downregulation in mRNA expression of ApoL1 (P <0.0001), ApoL2 (P = 0.0009), ApoL3 (P <0.0001) S13 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 correlates inversely with outcome. The aim of the study was to identify phenotypic and functional early markers of T cells and NK cells related to prognosis in the septic patient population. and ApoL6 (P = 0.0003) in purifi ed PMNs from ICU patients as compared with the healthy individuals. This downregulation was also validated at the protein level for ApoL1 and ApoL2, whereas ApoL 6 was upregulated in septic patients. We could not detect ApoL3 protein in any of the cohorts. This was accompanied by a signifi cant delay in PMN apoptosis in septic patients as compared with healthy volunteers (P <0.05) at 4 and 24 hours. We also showed a strong negative correlation in the three mixed groups between CRP and ApoL1 (R = –0.607), ApoL2 (R = –0.651), ApoL3 (R = –0.578) and ApoL6 (R = –0.506). and ApoL6 (P = 0.0003) in purifi ed PMNs from ICU patients as compared with the healthy individuals. This downregulation was also validated at the protein level for ApoL1 and ApoL2, whereas ApoL 6 was upregulated in septic patients. We could not detect ApoL3 protein in any of the cohorts. This was accompanied by a signifi cant delay in PMN apoptosis in septic patients as compared with healthy volunteers (P <0.05) at 4 and 24 hours. We also showed a strong negative correlation in the three mixed groups between CRP and ApoL1 (R = –0.607), ApoL2 (R = –0.651), ApoL3 (R = –0.578) and ApoL6 (R = –0.506). p g p p p p Methods We collected peripheral blood mononuclear cells from 47 patients with severe sepsis or septic shock at ICU admission (T0) and from 50 healthy controls. P34 Serial change of C1 inhibitor in patients with sepsis: a preliminary report On these subjects we evaluated frequency and absolute numbers of CD4+ and CD8+ T cells and of NK and B lymphocytes, the rates of regulatory CD4+CD25+Foxp3+ T cells (Tregs), the cytotoxic potential of CD4+, CD8+ T cells and of NK cells by evaluation of perforin (PER) and granzyme (GRA) expression and production of eff ector cytokines (namely IL-2, IL-17, IL-4, TNFα, IFNγ) by CD4+, CD8+ T cells and NK cells upon polyclonal stimulation. The markers were compared in patients with diff erent outcome. p p Conclusion The altered apoptotic fate of neutrophils in sepsis was correlated with the modifi cation of the expression profi le of ApoLs, a family of proteins thought to be involved in the apoptotic process. The role of these proteins in the sepsis-associated phenotype of neutrophils remains to be further elucidated. p pf Results Septic patients, compared with healthy donors, were characterized by global lymphopenia; we found increased frequencies of CD4+ T cells producing IL-2 (P = 0.0000000003), increased percentage of CD8 T cells producing IFNγ (P = 0.03), and reduced proportion of CD4+ T cells (P = 0.00007) and NK cells (P = 0.002) producing IFNγ. We also noticed an increased frequency of CD8+ T cells expressing PER (P = 0.00000025) and GRA (P = 0.01); moreover, the proportion of NK cells expressing GRA was also signifi cantly increased (P = 0.000019). To establish the prognostic value of these biological markers, we compared the cytokine expression by lymphocytes in septic patients that survived with those that died (D). We found that CD4+ and CD8+ TNFα-producing T cells were signifi cantly increased in D (P  =  0.01 and P = 0.0001 respectively); similarly the percentage of CD8+ T cells producing IFNγ was more elevated in D (P  =  0.006). The same was observed for IL-17 production by CD4+ T cells (P = 0.03) in D. On the contrary we observed a tendency to the reduction of circulating CD4+CD25+foxp3 (Tregs) in D (P = 0.08). References 1. Görgülü P, et al. Crit Care. 2011;15:R20. g 2. Vanhollebeke B, et al. Cell Mol Life Sci. 2006;63:1937-44. 2. Vanhollebeke B, et al. Cell Mol Life Sci. 2006;63:1937-44. P37 Results Seventy-three critically ill patients were included, 10 of whom developed an ICU-acquired infection. Compared with healthy subjects, whole blood leukocytes of patients were less responsive to ex vivo stimulation with LPS, as refl ected by strongly reduced TNFα, IL-1β and IL-6 levels in culture supernatants. However, results were not diff erent between patients who did and those who did not develop an ICU- acquired infection (Figure 1). Ex vivo and in vivo generation of neutrophil extracellular traps by neutrophils from septic patients N Takeyama, MH Huq, M Ando, T Gocho, MH Hashiba, H Miyabe, H Kano, A Tomino, M Tsuda, T Hattori, A Hirakawa N Takeyama, MH Huq, M Ando, T Goc Fujita Health University, Aichi, Japan Critical Care 2015, 19(Suppl 1):P36 (doi: 10.1186/cc14116) Introduction The primary aim of this study was to determine the diff erences in ex vivo generation of neutrophil extracellular traps (NETs) by neutrophils from septic and nonseptic patients. We further sought to examine plasma levels of cell-free DNA (cf-DNA) and histones to assess in vivo NET formation. p g Conclusion Septic patients are characterized by a peculiar immunophenotype which includes global lymphopenia and a specifi c pattern of cytokines. Some of the evaluated markers seem to individuate those with worse outcome; in particular, this group showed an infl ammatory phenotype with a higher expression of IFNγ, TNFα, IL- 17 and a tendency to a reduction of Tregs. Methods We isolated neutrophils from consecutive patients with sepsis (n = 17) and without sepsis (n = 18) admitted to the ICU. Neutrophils were activated by incubation with phorbol myristate acetate to induce release of NETs and NET formation was assessed by measuring the extracellular DNA level. Immunolabeling and fl uorescence imaging were also performed. Extracellular killing of bacteria by NETs was studied by co-culture of Escherichia coli and neutrophils in the presence of the phagocytosis inhibitor cytochalasin D. To assess in vivo NET formation, plasma levels of cf-DNA and histones were measured.i Reduced responsiveness of blood leukocytes to lipopolysaccharide does not predict nosocomial infections in critically ill patients LA Van Vught, MA Wiewel, AJ Hoogendijk, BP Scicluna, H Belkasim, J Horn, MJ Schultz, T Van der Poll Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P38 (doi: 10.1186/cc14118) Reduced responsiveness of blood leukocytes to lipopolysaccharide does not predict nosocomial infections in critically ill patients LA Van Vught, MA Wiewel, AJ Hoogendijk, BP Scicluna, H Belkasim, J Horn, MJ Schultz, T Van der Poll Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P38 (doi: 10.1186/cc14118) Results The condition of the nonseptic patients was signifi cantly less severe than that of the septic patients. The SOFA score of septic patients and the nonseptic patients was 6 (3 to 18) and 2.5 (1 to 8), respectively (median (IQR), P  =  0.02). The overall mortality rate was 29%. After stimulation with PMA, neutrophils isolated from septic patients released 4.08 ± 1.02% of their total DNA, whereas neutrophils from nonseptic patients released 29.06  ±  2.94% (P  <0.0001). Immunofl uorescent staining of released DNA, elastase, and myeloperoxidase also revealed similar results. Neutrophils from nonseptic patients showed eff ective extracellular killing of E. coli through NETs, whereas neutrophils from septic patients did not (P <0.001). Plasma levels of cf-DNA and histones were higher in septic patients than in nonseptic patients (P <0.001). Introduction Critically ill patients show signs of immune suppression, which is considered to increase vulnerability to nosocomial infections. Whole blood stimulation is a frequently used functional test for immune suppression. We here aimed to assess the association between whole blood leukocyte responsiveness to lipopolysaccharide (LPS) and the subsequent occurrence of nosocomial infections in critically ill patients admitted to the ICU. g Conclusion The increase of the immature PMN count and immature/ total PMN ratio confi rmed recruitment of immature neutrophils from the bone marrow into the circulation. The ex vivo generation of NETs is downregulated in neutrophils isolated from patients with sepsis. However, it is unclear whether in vivo NET formation is also impaired during sepsis, so further investigation is necessary. Methods All consecutive critically ill patients admitted to the ICU between April 2012 and June 2013 with two or more systemic infl ammatory response syndrome criteria and an expected length of ICU stay of more than 24 hours were enrolled. Age-matched and gender-matched healthy individuals were included as controls. Blood was drawn the fi rst morning after ICU admission and stimulated ex vivo with 100 ng/ml ultrapure LPS for 3 hours. Tumor necrosis factor (TNF)α, interleukin (IL)-1β and IL-6 were measured in supernatants. Alarming levels of heat shock proteins 72 and 90α in critically ill children The stimulated blood or plasma from septic patients was treated in vitro with 10 vol% polystyrene– divinylbenzene (PS-DVB)-based polymers (CG161, mean pore size 16 nm; CG300, mean pore size 30 nm) or left untreated. After adsorption, the plasma was separated and diluted with cell culture medium. The resulting conditioned medium was used to stimulate human umbilical vein endothelial cells (HUVEC) for 16  hours. HUVEC activation was assessed by the release of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)α, plasminogen activator inhibitor-1 (PAI- 1), as well as the expression of intercellular adhesion molecule (ICAM)- 1 and E-selectin. HUVEC were cultured at a shear stress of 5 dyne/ cm2 using the Ibidi perfusion system. Adhesion of monocytic THP-1 cells to HUVEC was studied after 4 hours of HUVEC stimulation with conditioned media. THP-1 cells were perfused over HUVEC at 1 dyne/ cm2 for 15 minutes, and adhering THP-1 were quantifi ed over time. Results The adsorbents CG161 and CG300 substantially decreased levels of TNFα, IL-1β, IL-6, IL-8 and IL-10 in LPS-stimulated blood. TNFα, a key stimulus for HUVEC, was reduced to 12% and 8% of the initial concentration by CG161 and CG300, respectively. Stimulation of HUVEC with the adsorbent-treated plasma resulted in signifi cantly diminished release of IL-6, IL-8, PAI-1 and decreased ICAM-1 and E-selectin expression, indicating reduced HUVEC activation. THP-1 adhesion was substantially decreased when HUVEC were stimulated with CG300-treated plasma as compared with untreated controls. Conclusion The fl ow model allows to study the eff ect of cytokine modulation on endothelial activation and to assess the interaction of activated endothelial cells with blood cells. Modulation of infl ammatory Methods Human whole blood was stimulated with LPS (100 ng/ ml) from Escherichia coli for 4 hours. The stimulated blood or plasma from septic patients was treated in vitro with 10 vol% polystyrene– divinylbenzene (PS-DVB)-based polymers (CG161, mean pore size 16 nm; CG300, mean pore size 30 nm) or left untreated. After adsorption, the plasma was separated and diluted with cell culture medium. The resulting conditioned medium was used to stimulate human umbilical vein endothelial cells (HUVEC) for 16  hours. HUVEC activation was assessed by the release of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)α, plasminogen activator inhibitor-1 (PAI- 1), as well as the expression of intercellular adhesion molecule (ICAM)- 1 and E-selectin. Alarming levels of heat shock proteins 72 and 90α in critically ill children HUVEC were cultured at a shear stress of 5 dyne/ cm2 using the Ibidi perfusion system. Adhesion of monocytic THP-1 cells to HUVEC was studied after 4 hours of HUVEC stimulation with conditioned media. THP-1 cells were perfused over HUVEC at 1 dyne/ cm2 for 15 minutes, and adhering THP-1 were quantifi ed over time. p j y y Methods Critically ill children with S (n = 16), SS (n = 15) or SIRS (n = 18) and H (n = 21) were enrolled in the study. ELISA was used to evaluate HSPs, chemiluminescence to measure ILs, and fl ow cytometry to evaluate nCD64 expression (IRB approved). p pp Results Patients in both septic groups had elevated HSP90α (P <0.0001), HSP72 (P  <0.05), IL-6 (P  <0.0001), IL-8 (P  <0.02) and IL-10 (P  <0.05) levels compared with H, whereas SS had increased HSP72, IL6 and TNFα compared with SIRS (P <0.05). SIRS patients presented increased HSP90α, IL-6 and IL-8 compared with H (P  <0.05). Both HSPs were dramatically increased among nonsurvivors. In a logistic regression model, only HSP90α was independently associated with mortality (P <0.0001). HSP90α related positively (P <0.001) to nCD64, IL-8, IL-10, CRP, PRISM, PELOD, TISS, and LOS and negatively to HDL (P <0.001) and LDL (P <0.02). HSP72 also related negatively to HDL (P <0.001). i Results The adsorbents CG161 and CG300 substantially decreased levels of TNFα, IL-1β, IL-6, IL-8 and IL-10 in LPS-stimulated blood. TNFα, a key stimulus for HUVEC, was reduced to 12% and 8% of the initial concentration by CG161 and CG300, respectively. Stimulation of HUVEC with the adsorbent-treated plasma resulted in signifi cantly diminished release of IL-6, IL-8, PAI-1 and decreased ICAM-1 and E-selectin expression, indicating reduced HUVEC activation. THP-1 adhesion was substantially decreased when HUVEC were stimulated with CG300-treated plasma as compared with untreated controls. Conclusion Extracellular HSP72 and HSP90α are alarmingly elevated in critically ill children, especially in severe sepsis. HSP90α levels are independently associated with mortality, related to CD64, IL-8, IL-10, severity of illness, and outcome. Both HSPs are inversely related to the low LDL/low HDL septic metabolic pattern [2].i Acknowledgements This research has been co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. Investing in knowledge society through the European Social Fund. P40 P40 Alarming levels of heat shock proteins 72 and 90α in critically ill children M Fitrolaki1, H Dimitriou2, M Venihaki2, M Katrinaki2, G Briassoulis1 1University Hospital, Heraklion, Greece; 2University of Crete, Medical School, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P40 (doi: 10.1186/cc14120) P37 Specifi c patterns of T-cell cytokines as an early marker of outcome in septic patients A Franci, A Peris, F Liotta, F Annunziato, P Ruggiano, M Ferraro A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P37 (doi: 10.1186/cc14117) A Franci, A Peris, F Liotta, F Annunziato, P Ruggiano, M Ferraro A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P37 (doi: 10.1186/cc14117) Introduction The infl ammatory response of sepsis is developed in two phases, an infl ammatory phase (SIRS) and a phase more variable in frequency and intensity (CARS): this balance has an important eff ect on morbidity and mortality. Lymphopenia aff ects particularly T cells, and Conclusion The extent of reduced LPS responsiveness of blood leukocytes in critically ill patients on the fi rst day after ICU admission does not relate to the subsequent development of ICU-acquired infections. S14 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P38). Similarly reduced responsiveness of whole blood leukocytes to lipopolysaccharide. Figure 1 (abstract P38). Similarly reduced responsiveness of whole blood leukocytes to lipopolysaccharide. mediators with porous polystyrene-based polymers attenuates endothelial activation and reduces monocyte adhesion. mediators with porous polystyrene-based polymers attenuates endothelial activation and reduces monocyte adhesion. 39 ell-culture model to study endothelial activation in sepsis Alarming levels of heat shock proteins 72 and 90α in critically ill children Introduction The endothelium is a complex organ infl uenced by circulating mediators, adjacent cells, physico-chemical factors, and shear stress. During systemic infl ammation and sepsis, excessive and sustained activation of the endothelium result in the loss of its anti- coagulant and anti-adhesive characteristics as well as in a loss of endothelial barrier function. We set up a cell-culture model to study endothelial activation induced by lipopolysaccharide (LPS) or by plasma from septic patients and studied the eff ect of adsorbent-based mediator modulation on endothelial activation. Introduction The endothelium is a complex organ infl uenced by circulating mediators, adjacent cells, physico-chemical factors, and shear stress. During systemic infl ammation and sepsis, excessive and sustained activation of the endothelium result in the loss of its anti- coagulant and anti-adhesive characteristics as well as in a loss of endothelial barrier function. We set up a cell-culture model to study endothelial activation induced by lipopolysaccharide (LPS) or by plasma from septic patients and studied the eff ect of adsorbent-based mediator modulation on endothelial activation. Introduction Extracellular heat shock proteins (HSP) act as inducers of interleukins (IL) and stimulants for immune cells during systemic infl ammatory response syndrome (SIRS). Little is known about the alarming roles of extracellular HSP72 and HSP90α in the acute phase [1] of sepsis (S) or severe sepsis (SS). We determined serum HSP90α, HSP72 and neutrophil CD64 expression, IL-6, IL-8, IL-10, and TNFα in children with S or SS compared with SIRS (brain injury) or healthy children (H). Introduction Extracellular heat shock proteins (HSP) act as inducers of interleukins (IL) and stimulants for immune cells during systemic infl ammatory response syndrome (SIRS). Little is known about the alarming roles of extracellular HSP72 and HSP90α in the acute phase [1] of sepsis (S) or severe sepsis (SS). We determined serum HSP90α, HSP72 and neutrophil CD64 expression, IL-6, IL-8, IL-10, and TNFα in children with S or SS compared with SIRS (brain injury) or healthy children (H). Methods Critically ill children with S (n = 16), SS (n = 15) or SIRS (n = 18) and H (n = 21) were enrolled in the study. ELISA was used to evaluate HSPs, chemiluminescence to measure ILs, and fl ow cytometry to evaluate nCD64 expression (IRB approved). mediator modulation on endothelial activation. Methods Human whole blood was stimulated with LPS (100 ng/ ml) from Escherichia coli for 4 hours. Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome p p g g p Results Nineteen controls, six SIRS patients and 25 severe sepsis patients were studied. The percent expression of HLADR on CD14+ monocytes was signifi cantly diff erent between the three groups showing progressive decrease from controls (mean 90.5  ±  3.8%) to SIRS (mean 61.2 ± 5.9%) to severe sepsis (mean 39.2 ± 5.5%) patients (controls vs. severe sepsis, P <0.001; controls vs. SIRS, P = 0.006; SIRS vs. severe sepsis, P  =  0.03). hsp70 and hsp90 MFI were signifi cantly diff erent between controls (mean 49.5 ± 4.9 and 33.5 ± 3.4 respectively), SIRS (mean 69.9 ± 16.5 and 46.5 ± 5.7 respectively) and severe sepsis patients (mean 33.3  ±  4.5 and 21.7  ±  2.7 respectively) (P  <0.05 for all comparisons). Notably, the hsp level rose from controls to SIRS and fell from SIRS to severe sepsis patients. APACHE score increased signifi cantly (P = 0.023) in septic patients compared with SIRS.if Introduction Heat shock proteins (HSPs) have intracellular cyto pro tec- tive actions, while they act extracellularly as inducers of cytokines and stimulants for immune cells during stress. Their induction constitutes a highly conserved cellular defense mechanism against all kinds of stress. Our objective was to determine the intracellular as well as extracellular levels of HSP72 and HSP90α in patients with severe sepsis (SS) or systemic infl ammatory response syndrome (SIRS) admitted to a general ICU, compared with those of healthy individuals; to correlate their expression with severity of illness. i Conclusion There were a signifi cant diff erence in CD14/HLADR, a marker of immune paralysis, between controls and patients with SIRS or severe sepsis. hsp70 and hsp90 showed an initial stimulation followed by exhaustion as sepsis progressed.i Methods Eighty-two consecutively admitted patients in the ICU (35 SIRS, 47 SS) as well as 35 healthy controls (H) were fi nally enrolled in the study. Patients’ demographic characteristics, laboratory examinations and Acute Physiology and Chronic Health Evaluation (APACHE II) score were recorded on admission. HSP levels were determined intracellularly using four-color fl ow cytometry. Mean fl uorescence intensity (MFI) values for each HSP were measured and analyzed. Extracellular levels of HSPs were determined via ELISA. Acknowledgements This research was co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. P42 P42 Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1, A Pistiki2, T Christodoulopoulou1, M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) P42 Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1, A Pistiki2, T Christodoulopoulou1, M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) Heat shock proteins 70/90 and associations with immunosuppression along with sepsis: preliminary data P Papadopoulos1 A Pistiki2 T Christodoulopoulou1 p p p M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) p p p M Theodorakopoulou1, V Tsagkari1, A Armaganidis1, S Tsiodras2, I Dimopoulou1, G Briassoulis3, G Briassoulis3 1University Hospital of Athens, Greece; 2University Hospital ATTIKON, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P42 (doi: 10.1186/cc14122) Results Ninety-nine patients were included in the fi nal analysis. Eighteen patients developed severe sepsis or septic shock. They presented with signifi cantly higher levels of intermediate (CD14++/16+) and CD62L– monocytes and lower IL-2 levels at T1 compared with patients who did not get septic. ROC AUC for association of these parameters with the occurrence of sepsis were 0.78 (95% CI: 0.63 to 0.91), 0.72 (0.62 to 0.82) and 0.73 (0.65 to 0.82), respectively. High counts of these monocytic cells were also associated with increased 90- day mortality (P <0.01, ROC AUC = 0.87 (0.77 to 0.95), 0.79 (0.66 to 0.9)). Kaplan–Meier survival curves showed signifi cantly higher mortality after stratifi cation based on these cell counts at T1 (CD14++CD16+: cutoff >236.8 cells/μl, HR = 23.6 (P = 1.24 × 10–5); CD62L–: cutoff >95.4 cells/μl, HR = 6.67 (P = 7.6 × 10–4)). Multivariate logistic regression analysis using Introduction CD14/HLADR is an index of immune suppression. Heat shock proteins (hsp) regulate cell response to oxidative stress. Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome This abstract is part of the study ‘Heat Shock Proteins and Glutamine Alterations Related to Hormonal, Immunological, Infl ammatory and Molecular Response to Sepsis: A Combined Clinical and Experimental Study’. Results HSP expression diff ered signifi cantly between groups (Kruskal–Wallis), both intracellularly (HSP72 lower in SS, P  <0.001), and extracellularly (higher levels of HSP90α (P  <0.001) and HSP72 (P  =  0.003) in SS). HSP72 and HSP90α intracellular expression was inversely correlated to severity of illness, as expressed by APACHE II score (Spearman’s, P = 0.003 and P = 0.025 respectively). Intracellular HSP72 was correlated to mortality when confounding factors were excluded from the analysis (logistic regression, P = 0.05). Extracellular HSP90α levels correlated with prolonged PT (P  =  0.021) and INR (P = 0.008). Finally, in the SIRS group, intracellular levels of HSP90α were higher in nonsurvivors (P <0.001). Alarming levels of heat shock proteins 72 and 90α in critically ill children Conclusion The fl ow model allows to study the eff ect of cytokine modulation on endothelial activation and to assess the interaction of activated endothelial cells with blood cells. Modulation of infl ammatory S15 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 To evaluate the %HLA-DR expression on monocytes, the fresh whole blood was stained with anti-CD14-FITC, anti-HLA-DR-PE and CD45- PC5 while staining with anti-CD33-PE, anti-CD45-PC7, anti-hsp70-FITC and anti-hsp90-PE allowed evaluation of the MFI expression of hsps on CD33+ monocytes. Cells were then analyzed using fl ow cytometry. ANOVA with post hoc tests was used to compare CD14/HLADR cell counts and hsp70 and hsp90 levels among the three groups. P41 Early heat shock protein 72 and 90α intracellular and extracellular responses in patients with severe sepsis or systemic infl ammatory response syndrome K Apostolou1, K Vardas1, E Briassouli1, K Psara1, D Goukos1, E Mageira1, S Nanas1, C Routsi1, G Briassoulis2 1Evangelismos Hospital, National and Kapodistrian University of Athens, Greece; 2University Hospital, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P41 (doi: 10.1186/cc14121) Prospective immune profi ling in critically ill adults: before, during and after severe sepsis and septic shock Introduction Rethinking the host’s defense mechanisms during severe infection has led to the use of fl ow cytometry (FCM) and to the current concept of sepsis-induced immunosuppression. However, organ dysfunctions that develop in the period preceding severe sepsis as a consequence of surgery, trauma or burn might also trigger immune reprogramming predisposing to overwhelming infection. Our aim was to look for correlation of specifi c phenotypes among four commonly encountered populations of patients and the later occurrence of severe sepsis and septic shock. g Conclusion SS is characterized by high levels of extracellular HSPs. Intracellular HSP72 is highly expressed during the acute phase of stress in SIRS, while being downregulated in SS. HSP72 and HSP90α intracellular expression and extracellular level variations correlate with severity of illness and mortality.i Acknowledgements This research was co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. p p Methods In total, 114 non-infected patients were prospectively screened via FCM on days 1 (T1) and 3 (T2) of elective cardiac surgery, trauma, acute neurologic dysfunction and prolonged ventilation (>48 hours). A third sample was drawn when infection was diagnosed (Tx) and 7  days later (Tx  + 7). Exclusion criteria included use of immunosuppressive agent(s). The broad panel of cell-specifi c antibodies focused on B, T lymphocytes (Tregs, Th17, NKT), NK cells, monocytes and neutrophils. Plasmatic levels of IL-2/IL-6/IL-7/TNFα/ IFNγ were also determined.i References References 1. Briassouli E, et al. Nutrition. 2014;30:1185-94. 2. Fitrolaki DM, et al. BMC Pediatr. 2013;13:31. References 1. Briassouli E, et al. Nutrition. 2014;30:1185-94. 2. Fitrolaki DM, et al. BMC Pediatr. 2013;13:31. P42 Macrophage phenotype in sepsis immunosuppression E Theodorakis, E Diamantaki, C Tsatsanis, D Georgopoulos, K Vap University of Crete, School of Medicine, Heraklion, Greece C l C (S l ) (d / ) Macrophage phenotype in sepsis immunosuppression E Theodorakis, E Diamantaki, C Tsatsanis, D Georgopoulos, K Vaporidi University of Crete, School of Medicine, Heraklion, Greece Critical Care 2015, 19(Suppl 1):P44 (doi: 10.1186/cc14124) expression. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Introduction Sepsis is followed by profound, yet poorly characterized, innate immune system suppression. While low monocyte HLA-DR expression is observed in septic patients, its clinical signifi cance has not been established [1]. In vitro, repeated LPS stimulation induces a tolerant or M2 macrophage phenotype, characterized by decreased cytokine production [2], which could contribute to sepsis immunosuppression. The present study examines macrophage phenotype in a mouse model and in patients with sepsis immunosuppression. p p pp Methods Sepsis was induced in C57Bl6 mice by cecal ligation and puncture (CLP) followed by intratracheal instillation of Pseudomonas aeruginosa. Bronchoalveolar lavage fl uid (BALF), cells and serum, collected 12 hours after lung infection, were analyzed for bacterial load, cytokine levels and the classical M1 marker, iNOS. Peripheral blood monocytes isolated from septic adult patients admitted to the ICU on the 1st and 7th day after admission were analyzed by fl ow cytometry for the expression of HLA-DR and CD86 (co-stimulatory molecule and M1 marker), and for the M2 markers, CD163 and CD206. Additional blood samples from patients and healthy volunteers were exposed ex vivo to LPS prior to isolation and analysis of monocyte markers. p y y Results CLP-induced sepsis resulted in immunosuppression in mice, indicated by higher BALF bacterial load after infection in CLP than in sham-operated mice, and more severe injury on histology. Serum cytokines TNF and MIP2 were greater in CLP than in sham-operated mice. Although recruitment of CD11c+ alveolar macrophages post infection was threefold greater in CLP than in sham-operated mice, those macrophages expressed 40% lower levels of iNOS. Evidence of sepsis immunosuppression was present in most patients on the 7th day after ICU admission. P42 We evaluated the relationship of CD14/HLADR and hsp70/90 in patients with SIRS and severe sepsis versus healthy volunteers. Methods We evaluated 31 patients with SIRS or severe sepsis against a group of sex-matched healthy volunteers. Demographic data were obtained for all patients. APACHE score was calculated upon admission. Blood samples were collected upon diagnosis of SIRS or severe sepsis. S16 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 clinical scores indicated that addition of IL-2 levels at T1 signifi cantly improved prediction of sepsis (OR = 0.834, P = 0.02). Conclusion Predisposition to sepsis in selected critically ill medico- surgical adults can be identifi ed on day 1 of admission based on high counts of circulating intermediate and CD62L– monocytes and low levels of IL-2 (the latter provide incremental prognostic information). High counts of these specifi c monocytes correlate with higher 90-day mortality. clinical scores indicated that addition of IL-2 levels at T1 signifi cantly improved prediction of sepsis (OR = 0.834, P = 0.02). that septic patients display a strong correlation between mHLA-DR and mRNA-levels of HLA-DRA in whole blood [1]. mRNA-based HLA- DR monitoring by PCR would improve the clinical usage and facilitate conduction of multicentre studies. The primary focus in this study was to evaluate the correlation between mHLA-DR and HLA-DRA at diff erent time points during sepsis. In addition, we assessed the dynamic expression of both mHLA-DR and HLA-DRA, in relation to sepsis severity. Conclusion Predisposition to sepsis in selected critically ill medico- surgical adults can be identifi ed on day 1 of admission based on high counts of circulating intermediate and CD62L– monocytes and low levels of IL-2 (the latter provide incremental prognostic information). High counts of these specifi c monocytes correlate with higher 90-day mortality. y Methods Study patients (n  = 54) were included at day 1 to 2 after hospital admission if blood cultures turned positive. Repeated sampling at days 1 to 2, 3, 7, 14 and 28 was performed. mHLA-DR was monitored by FCM and HLA-DRA by quantitative RT-PCR. Mixed models for longitudinal data were used after logarithmic transformation to calculate the interactional eff ects of time and severity on HLA-DR expression. P42 Low expression of CD86 and/or HLA-DR was observed in 71% of patients, and increased expression of M2 markers in 15% of patients. Upon LPS stimulation the normal decrease in M2 markers was absent in all patients on day 1, and partially restored in 50% of patients on day 7. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 1 (abstract P45). Box plots of mHLA-DR, measured by fl ow cytometry. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Figure 2 (abstract P45). Box plots of HLA-DRA, measured by qRT-PCR. Conclusion Sepsis is associated with decreased monocyte expression of M1 markers and increased expression of M2 markers in septic mice and critically ill patients. Therefore, in addition to decreased HLA-DR expression, M2 macrophage polarization appears to be a component of sepsis-induced monocyte dysfunction, and should be considered for immune monitoring and targeted intervention. g g Acknowledgement Supported by GSRT research grant ExcellenceII-4620. R f References 1. Gomez H, et al. Crit Care Med. 2014;42:771. 2. Porta C, et al. Proc Natl Acad Sci U S A. 2009;106:14978. 2. Porta C, et al. Proc Natl Acad Sci U S A. 2009;106:14978. P45 Expression of mRNA levels of HLA-DRA in relation to monocyte HLA-DR: a longitudinal sepsis study SC Cajander, ET Tina, AB Bäckman, AM Magnuson, KS Strålin, BS Söderquist, JK Källman Örebro University, Örebro, Sweden Critical Care 2015, 19(Suppl 1):P45 (doi: 10.1186/cc14125) Expression of mRNA levels of HLA-DRA in relation to monocyte HLA-DR: a longitudinal sepsis study SC Cajander, ET Tina, AB Bäckman, AM Magnuson, KS Strålin, BS Söderquist, JK Källman Örebro University, Örebro, Sweden Critical Care 2015, 19(Suppl 1):P45 (doi: 10.1186/cc14125) Introduction Decreased monocyte surface HLA-DR (mHLA-DR) measured by fl ow cytometry (FCM) is an independent marker of immunosuppression in sepsis. In a previous report we demonstrated S17 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Correlation between mHLA-DR(FCM) and HLA-DRA(PCR) at day 1 to 2 (R = 0.78) and day 14 (R = 0.27). Both HLA-DR markers increased linearly on a log scale over time. The linear association was signifi cantly diff erent between the severe (n = 16) and nonsevere septic patients (n = 38) when measuring either mHLA-DR(FCM) or HLA-DRA(PCR). By pairwise comparison of means between the two severity groups, at every time point, the diff erences between groups were shown to be signifi cant at days 1 to 2 and 3 when monitoring mHLA-DR(FCM) and at days 1 to 2, 3 and 7 for HLA-DRA(PCR) (Figures 1 and 2).l used as a diagnostic criterion of sepsis. There are no published data to defi ne the role of dynamic control of EC in the process of intensive therapy as a prognostic marker and indicator of severity condition in critically ill patients. The aim was to determine the informative value of EC in the development of SIRS as a biomarker of sepsis and indicator of the severity condition and prognosis of outcome in the pathological process. Methods A total of 143 patients were enrolled in this study who were admitted to the ICU and had SIRS. All patients were divided into a septic group – patients with community-acquired pneumonia, complicated by sepsis – and two SIRS groups of noninfectious genesis – patients who had an acute cerebrovascular accident (CVA) and an acute myocardial infarction (AMI). The absolute EC was measured at admission and in the dynamics on days 3 to 5 of stay. HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) y Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) Introduction The aim of the present study was to evaluate the prognostic value of HLA-DR antigen expression in monocytes, in patients with community-acquired infections presenting with fever, as possible markers for the patients’ fi nal outcome. Table 1 (abstract P47). Dynamics of eosinophil count depending on outcome in groups i Methods A total of 81 patients (males = 46; females = 35) presenting with fever >38°C to the emergency room (ER) of the Department of Internal Medicine of the Patras University Hospital were enrolled in the study during a period of 12 months. Sera for monocyte HLA-DR expression were obtained from the patients on admission (day 1) and on days 3, 7 or discharge/death. Results were expressed as percentages of HLA-DR-positive monocytes, calculated by the coexpression of CD14 and HLA-DR antigens in the total CD14+ population. Additionally, the patients were evaluated using the Simplifi ed Acute Physiology Score (SAPS-II), the Sequential Organ Failure Assessment (SOFA) and the Mortality in Emergency Department Sepsis (MEDS) score on the same days while all the indicated clinical, laboratory and imaging procedures as required for fever’s diff erential diagnosis were followed. A questionnaire regarding demographic characteristics, comorbidities, medications used and patients’ survival was also completed. All statistical analyses were performed using SPSS v.21. Table 2 (abstract P47). Informational value of eosinophil count in assessment of disease outcome Results Lower mean HLA-DR monocyte antigen expression percen- tages were signifi cantly correlated to lower Glasgow Scale scores on all days of measurement. HLA-DR expression was signifi cantly negatively correlated to MEDS, SOFA and SAPS-II scores whereas patients who developed sepsis, severe sepsis, septic shock and MODS had signifi cantly lower HLA-DR values compared with the ones who did not. HLA-DR expression on day 1 was lower in patients who would develop SIRS and/or sepsis on days 3 and 7 (P <0.01). Additionally, HLA- DR expression was signifi cantly decreased in nonsurvivors (n  = 33) compared with survivors (n = 48), whereas lower HLA-DR expression was correlated to longest duration of hospital stay at all time points (P <0.01). Conclusion EC may be an additional diagnostic marker which characterizes the nature of SIRS. Eosinopenia associated with prognosis of outcome in critical conditions. P45 y g Conclusion The correlation between fl ow cytometry and PCR-based HLA-DR monitoring is stronger in the early phase of sepsis. However, the linear associations over time, in relation to sepsis severity, display similar results for both HLA-DR markers. HLA-DRA(PCR) as a biomarker could be an alternative approach in monitoring immune status in sepsis but needs to be evaluated in relation to clinically relevant immunosuppression. Reference y y y Results The median EC was 75 cells/mm3 in septic patients on admission, which was signifi cantly lower than in patients with CVA (120 cells/mm3) and AMI (130 cells/mm3). Comparison of EC in septic patients between survivors and those who died showed signifi cant diff erences (Table 1). Receiver operating characteristic (ROC) analysis determined a value less than 80 cells/mm3 as the optimal diagnostic cutoff value with a high level of confi dence in the comparison of septic and noninfectious groups. Area under the ROC curves was 0.94, sensitivity of 80.8%, specifi city of 95.6%, P <0.0001. There was a signifi cant increase of EC in survivors, while the EC did not change signifi cantly among those who died in the dynamics. ROC analysis determined the cutoff values of EC, which indicated a high risk of an adverse outcome in septic patients (Table 2). 1. Cajander S, et al. Crit Care. 2013;17:R223. 1. Cajander S, et al. Crit Care. 2013;17:R223. HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) HLA-DR monocyte antigen expression as predictors of outcome in patients with community-acquired infections presenting with fever D Logothetis, E Giourou, I Starakis, A Lekkou, G Theodorou, M Leotsinidis, M Karakantza, C Gogos University of Patras, Patra, Greece Critical Care 2015, 19(Suppl 1):P46 (doi: 10.1186/cc14126) Conclusion Monocyte HLA-DR appears to be an early indicator for survival and infection progression and therefore it can be used as a predictive marker for the fi nal outcome of patients presenting in ER departments with fever. P48 Prevalence and clinical signifi cance of early endotoxin activity in septic shock patients M Bottiroli1, R Pinciroli1, G Monti2, M Mininni1, G Casella2, R Fumagalli2 1Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy; 2Anestesia Rianimazione 1, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P48 (doi: 10.1186/cc14128) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) Introduction The endotoxin activity (EA) assay is a useful test to risk stratify critically ill patients and assess for Gram-negative (GN) infection. However, the prevalence and signifi cance of early high levels of EA in patients with septic shock (SS) has yet to be elucidated. Introduction The idea of using the eosinophil count (EC) as a diagnostic marker for clarifying the nature of systemic infl ammatory response syndrome (SIRS) belongs to K Abidi, who showed that EC could be Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S18 Figure 1 (abstract P48). patients with systemic infl ammatory response syndrome (SIRS) and organ failure in ICU setting. Methods In total, 76 patients with SIRS and organ failure or who were suspected of sepsis during critical care were included. According to the levels of EAA, all patients were classifi ed into three groups (group L, EAA <0.4; group M, EAA ≥0.4 or EAA <0.6; group H, EAA ≥0.6). In order to evaluate the severity of illness, the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score and catecholamine (CA) index were recorded. Blood samples were obtained to measure EAA levels, infl ammatory markers (procalcitonin, C-reactive protein and white blood cells), serum lactate level as an indicator of tissue hypoxia, and for blood culture. All patients were followed up for 6 months. APACHE II score, SOFA score, CA index, infl ammatory markers, serum lactate levels and blood culture results were examined for diagnosis of sepsis, severe sepsis, septic shock and for prognosis of 30-day mortality. Each value was also compared with EAA levels. Results Patient age was 69 ± 9.9 years (male: n = 48, female: n = 25). The total number of samples was 106 (group L/group M/group H: 35/35/36). Twenty-seven specimens were obtained from nonseptic patients and 83 specimens were obtained from septic patients. APACHE II score was highly correlated with SOFA score (P <0.05). In group H, the APACHE II score was signifi cantly higher (22.2 ± 0.8) than that in group M (18.4 ± 0.87) (P = 0.01). 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) Results A total of 107 consecutive patients were included. The overall median EA was 0.56 (0.44 to 0.71), with 46/107 (43%) patients testing positive for elevated EA (≥0.6). GN species were identifi ed in microbial cultures as the infective etiology in 49/107 (46%) patients, of which 28 (57%) developed bacteremia. GN infections were associated with higher levels of EA compared with other microbial causatives (0.61 (0.52 to 0.77) vs. 0.52 (0.38 to 0.64), P = 0.021). Patients with EA ≥0.6 showed signifi cantly higher lactate levels (2 (1 to 3) vs. 3.8 (1.7 to 6.4), P = 0.01), Sequential Organ Failure Assessment (9 (6 to 12) vs. 10 (8 to 14), P = 0.04) and inotropic score (20 (5 to 50) vs. 50 (16 to 100), P = 0.003) at inclusion. See Figure 1. Introduction Sepsis is a common reason for admission to ICUs throughout the world. During the past two decades, the incidence of sepsis in the USA has tripled and is now the 10th leading cause of death. As sepsis continues to impact negatively on critically ill patients, it is clear that early diagnosis and eff ective management could improve patient morbidity and mortality. Numerous studies have attempted to examine biomarkers and their ability to diagnose and prognosticate septic patients. Despite multiple eff orts, currently there are no reliable markers that can eff ectively improve our clinical eff ectiveness in diagnosing and managing septic patients. The purpose of our systematic review was to evaluate the diagnostic and prognostic value of various biomarkers used in septic patients. g Conclusion Elevated EA is a common fi nding in SS patients. In patients developing SS from a GN infection, higher levels of endotoxin activity could be measured within 24  hours. Furthermore, in our study, EA ≥0.6 identifi ed a subgroup of subjects at greater risk for worse clinical outcomes. We therefore propose use of the EA assay for the early identifi cation and risk stratifi cation of SS patients. Methods A systematic search of the literature was performed with MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials databases using terminology selected for biomarkers (through to and including November 2013). All articles involving neonates and not in English were excluded. Inclusion was agreed on by two independent reviewers of abstracts or full text. Biomarkers in sepsis: a systematic review Biomarkers in sepsis: a systematic review F Morriello1, J Marshall2 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) P47 Eosinopenia as a marker of sepsis and mortality in critically ill patients A Savitskiy, V Rudnov, V Bagin City Clinical Hospital # 40, Yekaterinburg, Russia Critical Care 2015, 19(Suppl 1):P47 (doi: 10.1186/cc14127) The SOFA score in group H was signifi cantly higher (9.9  ±  0.5) than that in group M (7.5  ±  0.6) and group L (7.9 ± 0.6) (P = 0.006). EAA levels were signifi cantly increased in septic patients (septic patients: 0.56 ±0.03, nonseptic patients: 0.42 ±0.05) (P  =  0.011) and in the positive blood culture group (positive group: 0.66 ± 0.05, negative group: 0.48 ± 0.03) (P = 0.006). There was no relationship between EAA levels and other infl ammation markers or 30-day mortality. Conclusion In patients with suspected sepsis and positive blood culture, EAA levels were signifi cantly increased and had strong correlation with severity of disease. This result suggests that EAA indicates the state of sepsis regardless of the possibility of infection in patients with SIRS with organ failure. Figure 1 (abstract P48). Figure 1 (abstract P48). Methods We designed a prospective observational study including adult patients with clinically diagnosed SS. EA was measured on arterial blood by a chemiluminescent assay within the fi rst 24 hours from SS diagnosis. The fi nding of an EA value ≥0.6 was used as the cutoff for test positivity, as described elsewhere. In addition, laboratory, microbiological and clinical data were collected at inclusion. In-hospital follow-up was also conducted. Biomarkers in sepsis: a systematic review F Morriello1, J Marshall2 F Morriello1, J Marshall2 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) C-reactive protein and hemogram parameters for the nonsepsis SIRS and sepsis: what do they mean? p y B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) y gi Results Reduced BChE activity correlated with trauma severity and the resulting systemic infl ammation. Compared with serum levels of routinely measured infl ammatory biomarkers, changes in the BChE activity were detected signifi cantly earlier, suggesting that the BChE activity might serve as an early indicator of systemic infl ammation. yi Results Reduced BChE activity correlated with trauma severity and the resulting systemic infl ammation. Compared with serum levels of routinely measured infl ammatory biomarkers, changes in the BChE activity were detected signifi cantly earlier, suggesting that the BChE activity might serve as an early indicator of systemic infl ammation. Conclusion Our results suggest that BChE activity, measured using a POCT system, might play an important role in the early diagnosis of trauma-induced systemic infl ammation. Introduction The aim of this study was to investigate the laboratory parameters as an indicator of sepsis. Sepsis is one of the most common reasons of mortality and morbidity in the ICU [1]. Thus, it is important to distinguish sepsis from nonsepsis SIRS. CRP and hemogram parameters may be fast, easy and aff ordable alternatives in distinguishing sepsis from nonsepsis SIRS. Eosinophil count (EoC), lymphocyte count (LymC) and neutrophil–lymphocyte count ratio (NLCR) are used as sepsis indicators [1,2]. Conclusion Our results suggest that BChE activity, measured using a POCT system, might play an important role in the early diagnosis of trauma-induced systemic infl ammation. P53 Association between apoptosis and mortality in severe septic patients In the fully adjusted model, C C C C C d C d h d l L Lorente1, M Martín2, A González-Rivero1, J Ferreres3, J Solé-Violán4, L Labarta5, C Díaz6, A Jiménez1, J Borreguero-León1 1Hospital Universitario de Canarias, La Laguna, Tenerife, Spain; 2Hospital Universitario Nuestra Señora Candelaria, Santa Cruz, Tenerife, Spain; 3Hospital Clínico Universitario de Valencia, Spain; 4Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain; 5Hospital San Jorge, Huesca, Spain; 6Hospital Insular, Las Palmas de Gran Canaria, Spain Critical Care 2015, 19(Suppl 1):P53 (doi: 10.1186/cc14133) Introduction The apoptotic process, in which cells are actively elimi- nated by a programmed pathway, is increased in sepsis. Extrinsic and intrinsic apoptotic death cell pathways activate caspase-3, which leads to cell apoptosis. Cytokeratin 18 (CK-18), a protein present in most epithelial and parenchymal cells, is cleaved by the action of caspases and released into the blood as caspase-cleaved CK (CCCK)-18 during apoptotic death. The novel objectives of this study were to determine whether there are associations between serum caspase-3 levels, serum CK-18 levels and mortality in septic patients. WBC, CRP, LymC, NeuC, NLCR and EoC were entered into the model. Results A total of 1,302 patients were categorized as nonsepsis SIRS (816, 62.7%) and sepsis (486, 37.3%). In the sepsis group, age, APACHE II, SOFA, mortality, length of ICU stay, CRP, NLCR and EoC were higher; LymC was lower than in the nonsepsis SIRS group (P <0.001 for each). Likelihood of sepsis (reference to nonsepsis SIRS) increased 2.62 (2.05 to 3.34), 2.02 (1.42 to 2.88) and 1.88 (1.36 to 2.60) times (OR (95% CI)) by the values of CRP >4.4  mg/dl, LmyC <500/mm3 and NLCR >15.7 respectively in mutually adjusted multivariate logistic regression (P <0.001 for each). y p p Methods A prospective, multicenter, observational study in six Spanish ICUs, including 216 patients with severe sepsis. We collected blood samples at the severe sepsis diagnosis moment to determine serum levels of caspase-3 (to assess the main executor of apoptosis) and CCCK- 18 (to assess the apoptosis level). The endpoint was 30-day mortality. Results We found that nonsurvivor (n = 76) in comparison with survivor (n = 140) septic patients showed higher serum levels of caspase-3 (0.41 ng/ml (0.14 to 0.52) vs. 0.11 ng/ml (0.10 to 0.25); P <0.001) and CCCK- 18 (448 (310 to 723) vs. 319 (236 to 445); P <0.001). P53 Association between apoptosis and mortality in severe septic patients Methods A total of 2,777 patients admitted to the ICU of two centers between 2006 and 2013 were evaluated retrospectively. The patients were diagnosed as SIRS(–), nonsepsis SIRS or sepsis at ICU admission by the consensus of two doctors in accordance with 1992 sepsis guidelines [3]. The patients who were under 18 years old, readmitted, immunosuppressive, SIRS(–) and whose laboratory values and outcomes were unknown were excluded. In total, 1,302 patients were divided into two groups as the nonsepsis SIRS group and the sepsis group. The patient’s age, gender, diagnoses (medical, elective and urgent surgery), APACHE II, SOFA, CRP, WBC, neutrophil count (NeuC), LymC, NLCR, EoC, platelet, mean platelet volume, length of ICU stay and mortality were recorded by a third doctor. In the fully adjusted model, WBC, CRP, LymC, NeuC, NLCR and EoC were entered into the model. Results A total of 1,302 patients were categorized as nonsepsis SIRS (816, 62.7%) and sepsis (486, 37.3%). In the sepsis group, age, APACHE II, SOFA, mortality, length of ICU stay, CRP, NLCR and EoC were higher; LymC was lower than in the nonsepsis SIRS group (P <0.001 for each). Likelihood of sepsis (reference to nonsepsis SIRS) increased 2.62 (2.05 to 3.34), 2.02 (1.42 to 2.88) and 1.88 (1.36 to 2.60) times (OR (95% CI)) by the values of CRP >4.4  mg/dl, LmyC <500/mm3 and NLCR >15.7 respectively in mutually adjusted multivariate logistic regression (P <0.001 for each). Methods A total of 2,777 patients admitted to the ICU of two centers between 2006 and 2013 were evaluated retrospectively. The patients were diagnosed as SIRS(–), nonsepsis SIRS or sepsis at ICU admission by the consensus of two doctors in accordance with 1992 sepsis guidelines [3]. The patients who were under 18 years old, readmitted, immunosuppressive, SIRS(–) and whose laboratory values and outcomes were unknown were excluded. In total, 1,302 patients were divided into two groups as the nonsepsis SIRS group and the sepsis group. The patient’s age, gender, diagnoses (medical, elective and urgent surgery), APACHE II, SOFA, CRP, WBC, neutrophil count (NeuC), LymC, NLCR, EoC, platelet, mean platelet volume, length of ICU stay and mortality were recorded by a third doctor. P52 P52 Serum cholinesterase activity as an early indicator of systemic infl ammation References 1. Abidi K, et al. Crit Care. 2008;2:R59. 2. Castelli GP, et al. Minerva Anestesiol. 2006;1-2:69-80. 3. Bone RC, et al. Chest. 1992;101:1644-55. P53 Association between apoptosis and mortality in severe septic patients Multiple logistic regression showed that serum caspase-3 levels >0.25 ng/ml were associated with mortality at 30 days (odds ratio = 6.51; 95% confi dence interval = 3.32 to 12.77; P <0.001), controlling for SOFA score and age. Kaplan–Meier survival analysis showed a higher risk of death in septic patients with serum caspase-3 levels >0.25 ng/ml than in patients with lower levels (hazard ratio = 3.80; 95% CI = 2.35 to 6.15; P <0.001). We found a positive association between serum levels of caspase-3 and CCCK-18 (ρ = 0.32; P <0.001).i Methods A prospective, multicenter, observational study in six Spanish ICUs, including 216 patients with severe sepsis. We collected blood samples at the severe sepsis diagnosis moment to determine serum levels of caspase-3 (to assess the main executor of apoptosis) and CCCK- 18 (to assess the apoptosis level). The endpoint was 30-day mortality. Conclusion CRP, LymC and NLCR may distinguish sepsis from nonsepsis SIRS. Thus, CRP and hemogram parameters may contribute to early diagnosis of sepsis. Results We found that nonsurvivor (n = 76) in comparison with survivor (n = 140) septic patients showed higher serum levels of caspase-3 (0.41 ng/ml (0.14 to 0.52) vs. 0.11 ng/ml (0.10 to 0.25); P <0.001) and CCCK- 18 (448 (310 to 723) vs. 319 (236 to 445); P <0.001). Multiple logistic regression showed that serum caspase-3 levels >0.25 ng/ml were associated with mortality at 30 days (odds ratio = 6.51; 95% confi dence interval = 3.32 to 12.77; P <0.001), controlling for SOFA score and age. Kaplan–Meier survival analysis showed a higher risk of death in septic patients with serum caspase-3 levels >0.25 ng/ml than in patients with lower levels (hazard ratio = 3.80; 95% CI = 2.35 to 6.15; P <0.001). We found a positive association between serum levels of caspase-3 and CCCK-18 (ρ = 0.32; P <0.001). P51 P51 C-reactive protein and hemogram parameters for the nonsepsis SIRS and sepsis: what do they mean? B Gucyetmez1, HK Atalan2, M Berktas3, E Ozden4, N Cakar5 1International Hospital, Istanbul, Turkey; 2Atasehir Memorial Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey; 4Antalya Memorial Hospital, Antalya, Turkey; 5Acibadem University, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P51 (doi: 10.1186/cc14131) 1Institute of Medical Sciences, Toronto, ON, Canada; 2St. Michael’s Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P50 (doi: 10.1186/cc14130) Assessment was based on the biomarker’s ability to diagnose septic patients and its ability to predict mortality.i P49 Usefulness of endotoxin activity assay for early diagnosis of sepsis S Sekine, H Imaizumi, K Masumoto, H Uchino Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P49 (doi: 10.1186/cc14129) Results Of 5,257 articles identifi ed, all abstracts were screened, and 750 full-text articles were selected for review. These included primarily randomized controlled trials, cohort studies and postmortem studies. Of 49 biomarkers examined, 72% of the studies examined procalcitonin. Comparing the serum of septic patients with that of controls, most biomarkers were elevated in septic patients, even though only a few had high sensitivity (>85%) and high specifi city (>80%). It was often Introduction The purpose of this study was to evaluate the diagnostic and prognostic value of the endotoxin activity assay (EAA) of sepsis in S19 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 an important role in the infl ammatory response. Serum cholinesterase (butyrylcholinesterase (BChE)) is the major Ach hydrolyzing enzyme in plasma. The role of this enzyme during infl ammation has not yet been fully understood. Here, we describe a correlation between the BChE activity and the early systemic infl ammatory response upon traumatic injury. diffi cult to compare study group with control group as the control group patients were usually not healthy controls. Conclusion Overall the heterogeneity of studies, small sample size and the lack of true healthy controls infl uenced the ability to use the biomarker for prognostication of a septic patient. Furthermore, the lack of healthy control raises the question of redefi ning selection criteria in order to better study septic patients. Methods We measured BChE activity in patients with traumatic injury admitted to the emergency room using a point-of-care-test (POCT) system. In addition, we measured levels of routine infl ammation biomarkers during the initial treatment period. We used the Injury Severity Score to assess the trauma severity. Data were statistically analyzed using the Friedman test. Correlation analysis was performed using Spearman’s rank correlation test. P  <0.05 was considered statistically signifi cant. P56 P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial A Kutz1, P Hausfater2, M Oppert3, C Alonso4, C Wissmann4, B Mueller1, P Schuetz1 1Kantonsspital Aarau, Switzerland; 2Hôpital Pitié-Salpêtrière and Univ- Paris 06, Paris, France; 3Klinikum Ernst von Bergmann, Potsdam, Germany; 4BRAHMS GmbH, Hennigsdorf, Germany Critical Care 2015, 19(Suppl 1):P56 (doi: 10.1186/cc14136) P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial A Kutz1, P Hausfater2, M Oppert3, C Alonso4, C Wissmann4, B Mueller1, P Schuetz1 P56 Validation of B·R·A·H·M·S PCT direct, a new sensitive point-of- care testing device for rapid quantifi cation of procalcitonin in emergency department patients: a prospective multinational trial Results We included 87 patients (32 Group A, 25 Group B, 30 Group C). The serum concentration of S1P (mean ± SD) in the control group was 484.6  ±  152.6  μg/l and signifi cantly higher compared with Group A 239.4  ±  61.3  μg/l, Group B 248.6  ±  93.7  μg/l and Group C 141.6 ± 46.3 μg/l. We observed a negative correlation between S1P and SOFA score (Pearson r = –0.45, P <0.001, R2 = 0.2). The median SOFA score in our cohort was 6. We divided the cohort into two groups: SOFA score <6; and SOFA score >6. We tested the sensitivity and specifi city of S1P to indicate disease severity by ROC analysis. In our cohort the area under the curve (AUC) for S1P was 0.77 (CI 0.670 to 0.870) and therefore higher when compared with common markers of infl ammation (PCT, IL-6, CRP with AUC of 0.68 (0.560 to 0.796), 0.68 (0.554 to 0.786) and 0.67 (0.571 to 0.794), resp.).i 1Kantonsspital Aarau, Switzerland; 2Hôpital Pitié-Salpêtrière and Univ- Paris 06, Paris, France; 3Klinikum Ernst von Bergmann, Potsdam, Germany; 4BRAHMS GmbH, Hennigsdorf, Germany Critical Care 2015, 19(Suppl 1):P56 (doi: 10.1186/cc14136) Introduction Procalcitonin (PCT) is increasingly the standard in the emergency department (ED) for the diagnostic and prognostic workup of patients with suspected infections. Recently, B·R·A·H·M·S PCT direct, a new high-sensitive point-of-care test, has been developed for fast PCT measurement on capillary or venous blood samples with a measuring range of 0.1 to 10.0 μg/l. Methods This is a prospective, comparative international study conducted in three European EDs. P56 Consecutive patients with suspicion of bacterial infection were included. Duplicate determination of PCT was performed on two distinct B·R·A·H·M·S PCT direct test devices on capillary (fi ngertip) and venous whole blood (EDTA), and compared with the reference method (B·R·A·H·M·S PCT sensitive Kryptor or Elecsys B·R·A·H·M·S PCT, respectively). The diagnostic accuracy was evaluated by correlation and concordance analyses. Conclusion Our fi ndings suggest that S1P is a novel marker for severity of sepsis with severe sepsis and septic shock being associated with low levels of S1P. Moreover, blood concentrations of S1P might play a key role in sepsis pathophysiology. y gy Acknowledgements MSW and AN are equal contributors. Reference 1. Maceyka M, et al. Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol. 2012;22:50-60. y y Results A total of 303 patients were included over a 6-month period (60.4% male, median age 65.2 years). The correlation between capillary or venous whole blood and the reference method was excellent: r2 = 0.96 and 0.97, sensitivity 88.1% and 93.0%, specifi city 96.5% and 96.8%, concordance 93% and 95% respectively at a 0.25 μg/l threshold. No signifi cant bias was observed (–0.04 and –0.02 for capillary and venous whole blood) although there were 6.8% and 5.1% outliers, respectively. B·R·A·H·M·S PCT direct had a shorter time to result as compared with the reference method (25 vs. 147 minutes, diff erence 122 minutes, 95% CI = 110 to 134 minutes, P <0.0001). P55 Diagnostic accuracy and clinical relevance of an infl ammatory biomarker panel in early sepsis in adult critical care patients P Bauer, R Kashyap, S League, J Park, D Block, N Baumann, A Algeciras-Schimnich, S Jenkins, C Smith, O Gajic, R Abraham Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P55 (doi: 10.1186/cc14135) Diagnostic accuracy and clinical relevance of an infl ammatory biomarker panel in early sepsis in adult critical care patients Introduction Low awareness, late recognition and delayed treatment of sepsis are still common. CD64 is a marker of the innate immune response upregulated in sepsis. The primary goal of this prospective, double-blind study was to compare the diagnostic accuracy of neutrophil CD64 and other cellular markers, along with C-reactive protein (CRP) and procalcitonin (PCT) levels, in early sepsis. Conclusion This study found a high diagnostic accuracy and a faster time to result of the PCT direct in the ED setting. The B·R·A·H·M·S PCT direct may allow a more widespread use of PCT tests in outpatient clinics and smaller institutions. Methods Adult ICU patients, between 2012 and 2014 were eligible. The eight-color fl ow cytometric biomarker panel included CD64, CD163, HLA DR, CD15 and others. Diagnostic test results were compared with infection as the reference standard and sepsis as the target condition, using receiver operating characteristic curve analyses. Multivariable logistic regression was used to assess the relationship of sets of markers with the probability of sepsis, adjusting for other patient characteristics. Results A total of 219 patients were enrolled, 120 with sepsis, 99 served as controls. APACHE IV (median 70 vs. 57), SOFA (8 vs. 7), ICU (2 vs. 1) and hospital length of stay (6 vs. 4) were higher in the sepsis group. Serum cholinesterase activity as an early indicator of systemic infl ammation l AR Zivkovic, K Schmidt, J Bender, T Brenner, S Hofer Heidelberg University Hospital, Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P52 (doi: 10.1186/cc14132) l AR Zivkovic, K Schmidt, J Bender, T Brenner, S Hofer Heidelberg University Hospital, Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P52 (doi: 10.1186/cc14132) Conclusion The novel fi ndings of our study were that there is an association between serum caspase-3 levels, serum CK-18 levels and mortality in septic patients. There has been reported decreased apoptosis and increased survival in septic rats with the administration of caspase inhibitors; thus, it may be interesting to explore those agents in septic patients. Introduction Early diagnosis of systemic infl ammation, a generalised response to noxious stimuli, is fundamentally important for eff ective and goal-directed therapy. Various infl ammation biomarkers have been used in clinical and experimental practice. However, a defi nitive diagnostic tool for an early detection of systemic infl ammation remains to be identifi ed. Acetylcholine (Ach) has been shown to play Acknowledgements Funded by Grant FIS-PI-14-00220 from Instituto de Salud Carlos III (Madrid, Spain) and co-fi nanced by Fondo Europeo de Desarrollo Regional (FEDER). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S20 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Mortality was not diff erent. After adjustment for APACHE IV, CRP and PCT, CD64 molecules/neutrophil measure remained a signifi cant predictor of sepsis (OR = 1.852 for one-unit increase on the log scale, 95% CI = 1.083 to 3.168, P = 0.02, AUC = 0.90). See Table 1. P54 Sphingosine-1-phosphate is a new biomarker for severity in human sepsis MS Winkler1, A Nierhaus1, E Mudersbach1, M Holzmann1, A Bauer1, L Robbe1, C Zahrte1, G Daum2, S Kluge1, C Zoellner1 1University Medical Center Eppendorf, Hamburg, Germany; 2University Heart Center, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P54 (doi: 10.1186/cc14134) Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Table 1 (abstract P55). Area under the curve (AUC) for individual biomarkers Cutoff for Sensitivity Specifi city Measure N AUC sepsis (%) (%) C-reactive protein (mg/l) 208 0.86 43 76.9 76.9 CD64 molecules/neutrophil 196 0.83 1,040.5 76.4 76.7 Procalcitonin (ng/ml) 216 0.82 0.74 73.1 73.2 %CD64+ neutrophils 196 0.81 49.96 74.5 74.4 Conclusion Neutrophil CD64 expression is an accurate predictor of early sepsis. Measure Introduction During sepsis a leading symptom is capillary leakage caused by endothelial damage, followed by multiorgan dysfunction. Sphingosine-1-phosphate (S1P) is a bioactive lipid with multiple functions. Cellular reactions depend on the S1P concentration in the blood and its binding to fi ve specifi c G-protein coupled receptors. S1P-regulated functions include: control of endothelial permeability; lymphocyte migration across microvessels depending on an S1P gradient; and control of vascular tone [1]. This clinical study will address the question of whether S1P blood concentrations are associated with sepsis severity.i p y Methods Following ethical approval we enrolled patients fulfi lling the ACCP/SCCM sepsis criteria into three groups (Group A: sepsis; Group B: severe sepsis; Group C: septic shock). A group of 20 healthy donors served as controls. Serum blood samples, laboratory data and clinical parameters are presented for day 1. The primary outcome variable was serum S1P concentration (μg/l) quantifi ed by mass spectrometry (Agilent®). The SOFA score was used to describe disease severity. Serum procalcitonin level correlates with endotoxin activity in patients with septic shock Serum procalcitonin level correlates with endotoxin activity in patients with septic shock B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) B Adamik, J Smiechowicz, D Jakubczyk, B Adamiczka-Ciszewicz, T Kaiser, A Kübler Medical University, Wroclaw, Poland Critical Care 2015, 19(Suppl 1):P57 (doi: 10.1186/cc14137) Introduction Endotoxin, a key component on the outer membrane of Gram-negative bacteria, is considered to be the most important toxin Introduction Endotoxin, a key component on the outer membrane of Gram-negative bacteria, is considered to be the most important toxin S21 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Procalcitonin as prognostic marker in severe sepsis of abdominal origin g A Gonzalez-Lisorge1, C Garcia-Palenciano1, G Ercole1, T Sansano-Sanchez1, M Campos Aranda2, F Acosta Villegas1 1Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; 2Universidad de Murcia, Murcia, Spain Critical Care 2015, 19(Suppl 1):P58 (doi: 10.1186/cc14138) Introduction We evaluated the utility of procalcitonin (PCT) as a marker of outcome in severe sepsis of abdominal origin (SSAO). SSAO is one of the most prevalent pathologies in surgical ICUs (sICUs). Mortality from 19 to 70% has been reported. Biomarkers are basic tools for diagnosis, follow-up and outcome of sepsis. One of the most studied in last decades has been PCT. Its levels and kinetics could be useful to evaluate the outcome of septic patients. Conclusion Both biomarkers, procalcitonin and lactate provide diagnostic and prognostic information in ICU patients with sepsis, particularly when looking at biomarker kinetics. An evidence- based protocol incorporating both markers may further improve management of septic patients. p p Methods We studied all patients admitted to a sICU with SSAO, from 2007 to 2008. Data collected were: PCT levels on days 1, 3 and 7, gender, age, APACHE II on admission, positivity of surgical cultures, microorganisms isolated and sepsis origin (community or nosocomial). Results Sixty-nine patients were included. Mortality was 23%. Median age was 64.94  years. Median APACHE II was 16.43 points. At day 1, PCT levels were higher in survivors (S) than in exitus (E) (S: 29.22 ng/ ml vs. E: 14.93 ng/ml, P <0.05). PCT levels were infl uenced by gender (males: 27.74 ng/ml vs. females: 15.04 ng/ml, P <0.05), positive cultures (positive: 25.25 ng/ml vs. negative: 13.49 ng/ml, P <0.05) and isolation of Gram-negative microorganisms (Gram-negative: 27.53 ng/ml vs. Gram-positive: 14.77 ng/ml, P  <0.05). Patients with community- acquired sepsis had higher levels of PCT on admission (37.53 ng/ml vs. 13.29 ng/ml, P <0.02). None of these factors had an infl uence on mortality. On day 3 PCT levels where higher in S (S: 20.65 ng/ml vs. E: 16.23 ng/ml, P <0.05). On day 7 PCT levels were higher in E (S: 3.54 ng/ ml vs. E: 12.88 ng/ml, P <0.05). PCT kinetics was diff erent depending on outcome. E patients presented persistently higher levels, whereas PCT in S decreased over time (P <0.05). PCT on day 7 best identifi ed outcome (AUC ROC 0.768). PCT ≥3.5 ng/ml predicted mortality (sensitivity 55%, specifi city 73%). References involved in the development of septic shock, and procalcitonin (PCT) serum concentration has been strongly associated with the severity of sepsis. The eff ect of elevated endotoxin activity (EA) on PCT serum level, organ function and mortality of patients with septic shock was evaluated on the day of admission to the ICU. 1. Charles PE, et al. BMC Infect Dis. 2008;8:38. 2. Rau BM, et al. Arch Surg. 2007;142:134-42. 3. Dahaba AA, et al. Minerva Anestesiol. 2009;75:447-52. 4. Karlsson S, et al. Crit Care. 2010;14:R205. Methods ICU patients with diagnosis of septic shock were consecutively added to the study group within the fi rst 24 hours. Serum PCT level and whole blood EA was immediately measured in all patients on admission (n = 157). Endotoxemia was defi ned as EA >0.4 EAU. Critical Care 2015, 19(Suppl 1):P59 (doi: 10.1186/cc14139) Introduction Blood lactate level is a routinely used biomarker for management of patients in septic conditions in the ICU. There is a lack of clinical research data comparing lactate with novel sepsis biomarkers, such as procalcitonin, in regard to the diagnostic and prognostic potential. Herein, we investigated the diagnostic and prognostic value of initial lactate and procalcitonin levels and their kinetics within the ICU stay for prediction of positive blood cultures and fatal outcome in a well characterized cohort of sepsis patients in a US critical care setting. Methods This is a retrospective, observational cohort study of adult patients with confi rmed severe sepsis or septic shock and with at least one procalcitonin and lactate measurement on admission to the ICU of Morton Plant Hospital (Clearwater, FL, USA). Logistic regression models were calculated to assess the association of biomarkers with blood culture positivity and fatal ICU outcome with area under the curve (AUC) as a measure of discrimination. Conclusion Septic shock with endotoxemia was associated with biochemical and clinical consequences including a higher PCT level, higher frequency of bacteremia, kidney failure, and death. Results The in-hospital mortality rate of the 1,075 included patients (age 68 years) was 23.8% (95% CI = 21.2 to 26.3%) and 18.4% of patients had positive cultures. In regard to the diagnostic value for bacteremic disease, initial procalcitonin had a higher discriminatory value (AUC 0.71) compared with initial lactate levels (AUC 0.52). In regard to prognosis, initial lactate level was the better mortality predictor (AUC 0.69) compared with procalcitonin (AUC 0.55), although both initial levels were signifi cantly lower compared with APACHE III (P >0.05 for both comparisons). When looking at biomarker kinetics, procalcitonin decrease was more strongly associated with fatal outcome compared with initial levels alone (AUC 0.66), but still lower compared with lactate kinetics (AUC 0.73). Procalcitonin or lactate clearance, or both, for risk assessment in patients with sepsis? Results from a prospective US ICU patient cohort i Results Endotoxemia was present in 61% of patients (group 1, n = 95, age 66 (57 to 75)), and in 39% of patients EA was low (group 2, n = 62, age 63 (55 to 76)). Median EA was 0.57 EAU (0.46 to 0.67) in group 1 and 0.27 EAU (0.17 to 0.36) in group 2 (P <0.001). The PCT level was six times higher in group 1 than in group 2 (19.6 ng/ml vs. 3.1 ng/ml, P <0.001) and was correlated with EA (P <0.001, R = 0.5). Median APACHE II score was 23 points (16 to 29) in group 1 and 19 (16 to 25) in group 2; but observed diff erence was not signifi cant. The severity of clinical status estimated by SOFA score was similar in both groups (10 (7 to 13) in group 1 and 11 (8 to 12) in group 2; NS). Forty-six percent of patients in group 1 and 27% in group 2 required renal replacement therapy (P = 0.01). ICU mortality of patients was 41%. The mortality rate was higher in group 1, compared with group 2, and Kaplan–Meier survival analysis of time to death showed statistical signifi cance between the two groups (P = 0.001, log-rank test). A Gram-negative pathogen as the primary source of infection was identifi ed in 64% of patients in group 1 and in 44% in group 2 (P = 0.004); bacteremia was detected in 26% of cases in group 1 and in 12% in group 2 (P = 0.02). Diagnostic value of procalcitonin and IL-6 for sepsis of older patients and other patients in the emergency department SH Woo College of Medicine, The Catholic University of Korea, Incheon St. Mary’s Hospital, Incheon, South Korea College of Medicine, The Catholic University of Korea, Incheon St. Mary’s Hospital, Incheon, South Korea Critical Care 2015, 19(Suppl 1):P60 (doi: 10.1186/cc14140) Introduction Emergency physicians are able to identify severely ill older patients with SIRS in the emergency department (ED). A previous study showed that elevated procalcitonin (PCT) and IL-6 level is a known marker of sepsis and predicts bacteremia and mortality. We assessed the diagnostic value of PCT and IL-6 in older patients and other patients with SIRS and sepsis in the ED. Methods This retrospective cohort study was conducted from January 2013 to December 2013. We enrolled 122 patients with SIRS, 55 were classifi ed as the older age group (>65 years of age). Measurement of serum PCT, IL-6, and white blood cell count was performed on initial admission to the ED. We analyzed these markers in older patients and other patients groups with sepsis. Results Of the 55 patients in the older group 33 (60%) patients had sepsis, and 40 (59.7%) patients of the other group had sepsis. PCT and IL-6 levels were signifi cantly higher in other patients with sepsis (P <0.001, P <0.001). But PCT and IL-6 levels were not higher in old age i Conclusion PCT on day 7 and PCT kinetics can be useful to predict outcome in SSAO. PCT is higher in community-acquired sepsis, when surgical cultures are positive, in Gram-negative isolations and in males. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S22 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 activity; and enhancement of sensitivity to antibiotics. In the case of severe infectious disease, antibiotics are often supplemented with administration of IVIg. patients with sepsis (P = 0.400, P = 0.169). The area under the receiver operating characteristic curve (AUC) for diagnosis of sepsis according to PCT and IL-6 was 0.823, and 0.772 for the other patients group. The diagnostic sensitivity, specifi city, positive predictive value, and negative predictive value of PCT for sepsis in other patients group were 79.5%, 81.5%, 86.1%, and 73.3% respectively, with a PCT cutoff value of 0.18 ng/ml. Procalcitonin levels in patients undergoing left ventricular assist device implantation Results The patient APACHE II score were 24.9 ± 8.2, the SOFA score 9.1 ± 3.7, and the survival rate after 28 days 83.4%. The values before IVIg administration were: procalcitonin 36.0  ±  463.3 (median 110) ng/ml, presepsin 4,548 ± 4,250 (median 3,337) pg/ml, CRP 15.6 ± 9.6 (median 14.7)  mg/dl, and IL-6 13,860  ±  47,299 (median 630)  pg/ml. All values were thus elevated. On the days after the completion of IVIg administration and on day 7, the level of almost all mediators (procalcitonin, presepsin, CRP, IL-6) decreased signifi cantly. In patients with suspected severe sepsis and septic shock, presepsin reveals valuable diagnostic capacity to diff erentiate sepsis severity compared with procalcitonin, IL-6, CRP, and WBC. Additionally, presepsin and IL-6 reveal prognostic value with respect to 30 days and 6 months all-cause mortality throughout the fi rst week of ICU treatment [1]. p M Holek, J Kettner, J Franeková, A Jabor M Holek, J Kettner, J Franeková, A Jabor Institute for Clinical and Experimental Medicine, Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P61 (doi: 10.1186/cc14141) Introduction Procalcitonin (PCT) is used for diagnosis of a bacterial infection. Several works described nonspecifi c elevation of PCT after cardiac surgery with cardiopulmonary bypass (CPB) caused by systemic infl ammatory response syndrome (SIRS) with various cutoff values for the presence of the infection (0.47 to 2.47 μg/l). However, in patients undergoing left ventricular assist device (LVAD), implantation data about PCT dynamics are lacking. y gi Conclusion The results of the present study found signifi cant decreases of procalcitonin, presepsin and IL-6 resulting from 3 days of immunoglobulin administration, but evidence is still limited and this needs to be confi rmed in larger studies. Methods PCT levels in 25 patients indicated for LVAD were prospectively assessed before surgery and during the postoperative period (days 1, 2, 14 and 30). Values were compared according to the presence of infectious complications (IC) and non-infectious complications such as acute renal failure (ARF) defi ned as injury by RIFLE criteria or necessity of right ventricular assist device (RVAD). Data were also analyzed using combined endpoints A (ARF, RVAD) and B (IC, ARF, RVAD). Values are presented as median with interquartile range (in μg/l). Diagnostic value of procalcitonin and IL-6 for sepsis of older patients and other patients in the emergency department SH Woo The diagnostic sensitivity, specifi city, positive predictive value, and negative predictive value of IL-6 for sepsis in other patients group were 67.5%, 81.5%, 84.4%, and 62.9% respectively, with a IL-6 cutoff value of 74.43 pg/ml. Methods The changes in sepsis markers (procalcitonin, presepsin, interleukin-6, C-reactive protein) followed by IVIg administration were investigated in severe sepsis or septic shock patients. The subjects were 410 patients admitted to an ICU with a diagnosis of severe sepsis or septic shock and from whom informed consent had been obtained for the present study. IVIg was administered intravenously for 3 days (5.0 g/ day) and measurements were undertaken before administration (day 1), on the day after completion of administration (day 4), and on day 7. The items measured were procalcitonin, presepsin, IL-6, and CRP. The eff ect of IVIg administration on these markers was then studied. The IVIg studied was polyethylene glycol-treated human immunoglobulin injection fl uid (2.5 g, 50 ml, one vial). f Conclusion PCT and IL-6 is useful predictive markers for diagnosing sepsis in adult patients (<65 years of age) with SIRS in the ED. But these markers are not useful for identifi cation of sepsis in older patients. Diagnostic and prognostic performance of PATHFAST Presepsin in patients with SIRS and early sepsis E Spanuth1 R Carpio2 R Thomae3 Diagnostic and prognostic performance of PATHFAST Presepsin i patients with SIRS and early sepsis E Spanuth1, R Carpio2, R Thomae3 1DIAneering GmbH, Heidelberg, Germany; 2Hospital Nacional Edgardo Rebagliati Martins-EsSalud, Lima, Peru; 3Mitsubishi Chemical Europe, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P63 (doi: 10.1186/cc14143) p , p , 1DIAneering GmbH, Heidelberg, Germany; 2Hospital Nacional Edgardo Rebagliati Martins-EsSalud, Lima, Peru; 3Mitsubishi Chemical Europe, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P63 (doi: 10.1186/cc14143) Introduction Presepsin (sCD14-ST) serves as a mediator of the response to infectious agents. First evidence suggested that presepsin may be utilized as a sepsis marker. Introduction Presepsin (sCD14-ST) serves as a mediator of the response to infectious agents. First evidence suggested that presepsin may be utilized as a sepsis marker. Methods Presepsin was determined at presentation (T0), after 8, 24 and 72 hours in 123 individuals admitted with signs of SIRS and/ or infection. Primary endpoint was death within 30  days. Presepsin was determined using the POC assay PATHFAST Presepsin (Mitsubishi Chemical, Japan). Conclusion PCT levels in patients undergoing LVAD implantation rise signifi cantly in the fi rst 2  days after surgery. Interestingly, this elevation is much higher than after routine cardiac surgery with CPB. Recent works suggest that PCT concentrations are aff ected by SIRS caused by contact with a nonphysiological surface. In the case of LVAD this immunological stimulation is long lasting and even more potent with additional RVAD or ARF treated with renal replacement therapy. In accordance with this hypothesis, our data show that the ability of PCT to detect infectious complication in LVAD patients is limited and its concentrations more probably correlate with postoperative complications in general. p Results Mean presepsin concentrations of the patient group at presentation and of the control group were 1,945 and 130  pg/ml, respectively (P <0.0001). Baseline presepsin diff ered highly signifi cant between patients with SIRS, sepsis, severe sepsis and septic shock. Table 1 (abstract P63). Presepsin levels during the course of the disease in survivors and nonsurvivors Table 1 (abstract P63). Presepsin levels during the course of the disease in survivors and nonsurvivors PSEP, median (IQR) (pg/ml) T0 T8 hours T24 hours T72 hours Survivors 590 622 574 533 (345 to 1,396) (367 to 1,912) (336 to 1,610) (324 to 1,246) Nonsurvivors 1,763 1,859 1,731 2,056 (705 to 6,616) (1,001 to 5,744) (809 to 4,586) (811 to 5,540) P value 0.0046 0.0005 0.0003 0.0013 P61 P61 Procalcitonin levels in patients undergoing left ventricular assist device implantation M Holek, J Kettner, J Franeková, A Jabor Institute for Clinical and Experimental Medicine, Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P61 (doi: 10.1186/cc14141) Reference 1. Behnes M, Bertsch T, Lepiorz D, et al. Diagnostic and prognostic utility of soluble CD 14 subtype (presepsin) for severe sepsis and septic shock during the fi rst week of intensive care treatment. Crit Care. 2014;18:507. p q g μg Results PCT levels were low before surgery (0.16, 0.10 to 0.35), increased signifi cantly within the fi rst (5.72, 2.18 to 9.75; P <0.001) and second (5.94, 2.54 to 11.99; P <0.001) day after operation and decreased on the 14th (0.27, 0.11 to 0.74) and 30th (0.10, 0.06 to 0.19) day. There was no signifi cant diff erence in PCT values between patients with or without IC as well as with or without RVAD. ARF increased PCT level signifi cantly only 14 days after LVAD implantation (0.68, 0.37 to 1.65 vs. 0.15, 0.11 to 0.34; P = 0.015). Subjects with endpoint A had signifi cantly higher PCT values on the second (19.53, 5.66 to 63.12 vs. 3.95, 2.33 to 8.85; P = 0.033), 14th (0.55, 0.31 to 1.44 vs. 0.15, 0. to 0.34; P = 0.020) and 30th (0.19, 0.11 to 0.29 vs. 0.08, 0.05 to 0.13; P = 0.016) day after operation. Patients with endpoint B had signifi cantly elevated PCT levels 2 (11.99, 3.23 to 24.16 vs. 3.95, 2.54 to 7.39; P = 0.027) and 14 (0.55, 0.28 to 0.90 vs. 0.13, 0.09 to 0.23; P = 0.005) days after surgery. Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury Y Nakamura, H Ishikura, J Tanaka, T Nishida, M Mizunuma, D Ohta, N Matsumoto, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P64 (doi: 10.1186/cc14144) g p p Methods We analyzed data from 14 trials and 4,211 ARI patients to study associations of admission PCT levels and setting specifi c treatment failure and mortality alone at 30 days. We used multivariable hierarchical logistic regression and conducted sensitivity analyses stratifi ed by clinical settings and ARI diagnoses to assess the results’ consistency. Introduction At the moment, we have few reports about the diagnostic accuracy of procalcitonin (PCT), presepsin (P-SEP) and CD64 as a diagnostic marker of sepsis for patients with acute kidney injury (AKI). This study aimed to clarify which is a more useful diagnostic biomarker for sepsis using PCT, P-SEP and CD64 with or without AKI in ICU patients. Methods This study was a single-center observational retrospective study. Blood samples were collected from 334 patients admitted to our ICU between April 2013 and March 2014. Then, we classifi ed the patients with or without AKI. In this study, we adopted RIFLE criteria for AKI diagnosis. After that, the patients in each group were classifi ed into the sepsis group and the nonsepsis group. We measured PCT, P-SEP and CD64 levels at the time of ICU admission and subsequently investigated the diagnostic accuracy of these biomarkers for detecting sepsis. y Results Overall, 864 patients (20.5%) experienced treatment failure and 252 (6.0%) died. The ability of PCT to diff erentiate patients with and without treatment failure was highest in the emergency department setting (treatment failure; area under the curve (AUC): 0.64 (95% confi dence interval (CI): 0.61, 0.67), adjusted odds ratio (OR): 1.85 (95% CI: 1.61, 2.12), P <0.001 – mortality; AUC: 0.67 (95% CI: 0.63, 0.71), adjusted OR: 1.82 (95% CI: 1.45, 2.29), P <0.001). In lower respiratory tract infections, PCT was a good predictor of identifying patients at risk for mortality (AUC: 0.71 (95% CI: 0.68, 0.74), adjusted OR: 2.13 (95% CI: 1.82, 2.49), P <0.001). In primary care and ICU patients no signifi cant associations of initial PCT levels and outcome were found. See Figure 1. Conclusion Admission PCT levels are associated with setting specifi c treatment failure and provide most prognostic information in ARI in the emergency department setting. p Results In this study we met 225 patients with non-AKI and 109 patients with AKI. Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury We conducted ROC analysis for diagnosing sepsis. In non-AKI patients, the AUC of PCT, P-SEP and CD64 were 0.904 (95% CI: 0.824 to 0.950), 0.892 (95% CI: 0.794 to 0.947) and 0.917 (95% CI: 0.842 to 0.958), respectively. In AKI patients, the AUC were 0.933 (95% CI: 0.859 to 0.970), 0.755 (95% CI: 0.642 to 0.840) and 0.905 (95% CI: 0.803 to 0.957), respectively. P64 P64 Examination of the diagnostic accuracy of sepsis using procalcitonin, presepsin and CD64 for patients with or without acute kidney injury Y Nakamura, H Ishikura, J Tanaka, T Nishida, M Mizunuma, D Ohta, N Matsumoto, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P64 (doi: 10.1186/cc14144) Introduction Whether the infl ammatory biomarker procalcitonin (PCT) provides prognostic information across clinical settings and diff erent acute respiratory tract infections (ARI) is poorly understood. Herein, we investigated the prognostic value of admission PCT levels to predict adverse clinical outcome in a large ARI population. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion CD64 and PCT were a useful biomarker for detecting sepsis for ICU patients with AKI. Conclusion CD64 and PCT were a useful biomarker for detecting sepsis for ICU patients with AKI. Twenty-four patients died during 30 days. The 30-day mortality was 19.5% in total, ranging from 10 to 32% between the fi rst and the fourth quartile of presepsin concentration. Nonsurvivors showed high presepsin values with increasing tendency during the course of the disease while in surviving patients this tendency was decreasing. See Table 1. Conclusion Presepsin demonstrated a strong relationship with disease severity and outcome. Presepsin provided reliable discrimination between SIRS and sepsis as well as prognosis and early prediction of 30- day mortality already at admission. Presepsin showed close association with the course of the disease. Changes in procalcitonin and presepsin before and after immunoglobulin administration in septic patients Changes in procalcitonin and presepsin before and after immunoglobulin administration in septic patients T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) g p T Ikeda, S Ono, T Ueno, H Tanaka, S Suda T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) T Ikeda, S Ono, T Ueno, H Tanaka, S Suda Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) Hachiouji Medical Center, Tokyo Medical University, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P62 (doi: 10.1186/cc14142) Introduction The potentially envisaged actions of intravenous immunoglobulin (IVIg) on severe infectious disease include: virus or toxin neutralizing action; opsonic eff ect; complement bacteriolytic S23 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P65 Prognostic value of procalcitonin in respiratory tract infections across clinical settings A Kutz, B Mueller, P Schuetz, for the IPD Study Group Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P65 (doi: 10.1186/cc14145) P65 Prognostic value of procalcitonin in respiratory tract infections across clinical settings A Kutz, B Mueller, P Schuetz, for the IPD Study Group Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P65 (doi: 10.1186/cc14145) P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin The aim of this study was to investigate a possible association between the etiology of BSIs and the serum PCT levels. f g Methods We randomly chose 425 patients from a 9-month epidemiologic study on the incidence of community-onset severe sepsis and septic shock in western Sweden 2011 to 2012. In total, 207 had severe sepsis and 218 had sepsis, mean age 71.2 versus 64.2 years; males 51%. Sampling was made on arrival in the ED. The NLCR was analyzed immediately, PCT later on plasma frozen at –80°C. A total of 122/425 patients had bacteremia, 72 (35%) in the severe sepsis group versus 50 (23%) in the sepsis group. Most common fi ndings were Escherichia coli (n = 33), Staphylococcus aureus (n = 24), streptococcal spp. (n = 33) and other enterobacteriacae spp. (n = 17).i y Methods ICU patients with clinical and laboratory signs of sepsis or septic shock, with documented BSIs and with both serum PCT and CRP measurements on the day of the positive blood sample (±1 day), were included. Illness severity was assessed by SOFA score on both admission and BSI day. Demographic, clinical, and laboratory data including PCT and CRP levels, as well as the white blood cell (WBC) count on the day of the BSI were recorded. PCT was measured by an electrochemiluminescence analyzer and CRP by the tholosimetric method (Roche, Switzerland). Results The NLCR shows signifi cantly higher sensitivity than PCT at recommended cutoff levels for bacteremia. Interestingly, this is true even for all 207 patients with severe sepsis, irrespective of bacteremia or not. Sensitivity fi gures with 95% confi dence interval: bacteremia (n = 122): NLCR 80% (0.73 to 0.87) versus PCT 66% (0.58 to 0.75), P = 0.01; severe sepsis with bacteremia (n = 72): NLCR 85% (0.77 to 0.93) versus PCT 70% (0.59 to 0.81), P = 0.03; and severe sepsis but no bacteremia (n = 135): NLCR 71% (0.65 to 0.77) versus PCT 61% (0.54 to 0.68), P = 0.03. Conclusion The NLCR can be used in the ED as a biomarker for bacteremia as well as severe sepsis and seems to perform as well as or even better than PCT in this setting. Rapid response, low cost and no need for extra sampling make it useful as a screening tool. P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin Introduction The objective of this study was to evaluate the neutrophil to lymphocyte count ratio (NLCR) versus procalcitonin (PCT) in diagnosing bacteremia in the emergency department (ED). The NLCR is a biomarker that appears early in the course of the acute infl ammatory response and reaches maximum levels within 4 hours after onset. An elevated NLCR has been shown to correlate to bacteremia at a cutoff level of >10 [1]. It is rapidly analyzed on a full blood cell count at low cost. The lowest recommended cutoff level for PCT is <0.5 ng/ml. Methods We randomly chose 425 patients from a 9-month epidemiologic study on the incidence of community-onset severe sepsis and septic shock in western Sweden 2011 to 2012. In total, 207 had severe sepsis and 218 had sepsis, mean age 71.2 versus 64.2 years; males 51%. Sampling was made on arrival in the ED. The NLCR was analyzed immediately, PCT later on plasma frozen at –80°C. A total of 122/425 patients had bacteremia, 72 (35%) in the severe sepsis group versus 50 (23%) in the sepsis group. Most common fi ndings were Escherichia coli (n = 33), Staphylococcus aureus (n = 24), streptococcal spp. (n = 33) and other enterobacteriacae spp. (n = 17). R l Th NLCR h i ifi l hi h i i i h PCT Introduction The objective of this study was to evaluate the neutrophil to lymphocyte count ratio (NLCR) versus procalcitonin (PCT) in diagnosing bacteremia in the emergency department (ED). The NLCR is a biomarker that appears early in the course of the acute infl ammatory response and reaches maximum levels within 4 hours after onset. An elevated NLCR has been shown to correlate to bacteremia at a cutoff level of >10 [1]. It is rapidly analyzed on a full blood cell count at low cost. The lowest recommended cutoff level for PCT is <0.5 ng/ml. E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) Introduction Early diff erentiation of bacterial from candidal bloodstream infections (BSIs) in the presence of sepsis or septic shock is crucial because of the need for appropriate treatment initiation. Clinical data, although limited, suggest a role for procalcitonin (PCT) [1-3]. References Introduction The purpose of the study was to assess the prognosis value of pro-adrenomedullin (pADM), C-reactive protein (CRP) and procalcitonin (PCT), lactate (LT), albumin (ALB), cholesterol (CHOL), white blood cell (WBC) and severity score in patients with severe sepsis or septic shock. 1. Martini A, et al. J Infect. 2010;60:425. 2. Petrikkos GL, et al. Eur J Clin Microbiol Infect Dis. 2005;24:272. 3. Charles PE, et al. Intensive Care Med. 2006;32:1577. P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin R f ( , ) Results A total of 64 ICU patients (mean age 58 ± 18 years, 39 males) with BSIs were included. SOFA sore was 9 ± 4 on ICU admission and 8 ± 4 on the day of BSI. In 30 of these patients Candida spp. were isolated in blood culture (candidemia group) whereas the remaining 34 had a bacterial etiology of BSI (bacteremia group). Serum PCT concentrations remained within normal ranges in most patients with candidemia whereas a wide range was observed in patients with bacteremia. Mean values of PCT and CRP levels were higher in the bacterial than in the candidemia BSI group: 18.5 ± 33.2 versus 0.73 ± 1.40 ng/ml, P <0.001 and 17.7 ± 10.3 versus 8.9 ± 8.0 mg/dl, P = 0.001, respectively. There was a signifi cant diff erence in WBC count between the two groups: 19,460  ±  10.174 versus 11,000  ±  5,440, P <0.001 for the bacteremia and candidemia BSI group, respectively. A ROC curve analysis of the predictive ability of PCT showed an AUC of 0.79 (P <0.001). When a cutoff point of 0.40 ng/ml was selected using Youden’s J statistic, a low value of PCT had in our sample a negative predictive value of 0.76 and a likelihood ratio (negative) of 0.30. 1. de Jager et al. Crit Care. 2010;14:R192. 1. de Jager et al. Crit Care. 2010;14:R192. Prognosis value of biomarkers in severe sepsis and septic shock MV De La Torre-Prados1, A García-de la Torre2, R Escobar-Conesa1, Conclusion A low serum PCT value could be considered as a diagnostic marker in distinguishing between BSIs of candidal or bacterial origin in ICU patients with varying severity of sepsis. R f g y Reference P66 Neutrophil to lymphocyte count ratio performs better than procalcitonin as a biomarker for bacteremia and severe sepsis in the emergency department LL Ljungström1, D Karlsson1, A Pernestig2, R Andersson3, G Jacobsson1 1Skaraborg Hospital Skövde, Sweden; 2School of Biosciences, University of Skövde, Sweden; 3Institute of Biosciences, Sahlgrenska Academy at the University of Gothenburg, Sweden Critical Care 2015, 19(Suppl 1):P66 (doi: 10.1186/cc14146) P68f P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) P68 Diff erential diagnosis of bacterial from candidal bloodstream infections in ICU patients: the role of procalcitonin E Angelopoulos1, E Perivolioti1, S Kokkoris1, E Douka1, E Barbouti1, P Temperekidis1, C Vrettou1, C Psachoulia1, G Poulakou2, S Zakynthinos1, C Routsi1 1Evangelismos Hospital, Athens, Greece; 2Attikon Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P68 (doi: 10.1186/cc14148) g y Reference . Schuetz P, Briel M, Christ-Crain M, et al. Procalcitonin to guide initiation and duration of antibiotic treatment in acute respiratory infections: an individual patient data meta-analysis. Clin Infect Dis. 2012;55:651-62. Figure 1 (abstract P65). Multivariate regression analysis for estimation of predictive value of PCT levels on admission stratifi ed by adverse events and mortality in diff erent settings and diagnoses. *Treatment failure, ~mortality. ARI, acute respiratory tract infection; ECOPD, exacerbated chronic obstructive pulmonary disease; CAP, community-acquired pneumonia; VAP, ventilator-associated pneumonia; Adj., adjusted; OR, odds ratio; CI, confi dence interval. Figure 1 (abstract P65). Multivariate regression analysis for estimation of predictive value of PCT levels on admission stratifi ed by adverse events and mortality in diff erent settings and diagnoses. *Treatment failure, ~mortality. ARI, acute respiratory tract infection; ECOPD, exacerbated chronic obstructive pulmonary disease; CAP, community-acquired pneumonia; VAP, ventilator-associated pneumonia; Adj., adjusted; OR, odds ratio; CI, confi dence interval. S24 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 diff erences were signifi cant in pADM (2.46 (1.21 to 4.89) vs. 1.68 (0.94 to 3.32) nmol/l; P = 0.012), LT (2.6 (1.6 to 3.94) vs. 1.6 (1.2 to 2.43) mmol/l; P <0.001) and ALB (2 (1.55 to 2.38) vs. 2.22 (1.96 to 2.7) g/dl; P = 0.001). Conclusion The protein pADM, LT and ALB showed good prognosis accuracy when measured on admission of septic patients to the ICU. diff erences were signifi cant in pADM (2.46 (1.21 to 4.89) vs. 1.68 (0.94 to 3.32) nmol/l; P = 0.012), LT (2.6 (1.6 to 3.94) vs. 1.6 (1.2 to 2.43) mmol/l; P <0.001) and ALB (2 (1.55 to 2.38) vs. 2.22 (1.96 to 2.7) g/dl; P = 0.001). Conclusion The protein pADM, LT and ALB showed good prognosis accuracy when measured on admission of septic patients to the ICU. P67 Prognosis value of biomarkers in severe sepsis and septic shock MV De La Torre-Prados1, A García-de la Torre2, R Escobar-Conesa1, J Perez-Vacas1, A Puerto-Morlán1, E Cámara-Sola1, A García-Alcántara1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Puerto Real University Hospital, Puerto Real, Cádiz, Spain Critical Care 2015, 19(Suppl 1):P67 (doi: 10.1186/cc14147) P69 Methods A prospective, observational study in adult patients with severe sepsis or septic shock in a polyvalent ICU. Demographics, severity scores (APACHE II and SOFA) and all of the biomarkers were studied within 24  hours from septic shock onset. Descriptive and comparative statistical analysis was performed using the statistical software packages SPSS v.15 and MedCalc® 9.2.1.0. P69 Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA Nouer1, AL Colombo2, P Esteves2, T Guimaraes3, F Scapinello4, B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Estimating the duration of a central venous catheter at time of insertion g g Results A total of 2,148 patients were screened, and 85 (4%) fulfi lled entry criteria. The incidence of candidemia in these 85 patients was 8.2%, compared with 0.5% in the remaining 2,063 patients (relative risk 16.9%, 95% confi dence interval (CI) = 6.63 to 43.55). Baseline BDG was positive in 74 patients (87%), with a median number of positive tests of 3 (range 1 to 3) and a median value of 523 pg/ml (range 83 to 6,860). All seven patients with candidemia had positive baseline BDG (median value 523 pg/ml, range 203 to 3,660). The best cutoff of baseline BDG for the diagnosis of candidemia was 522 pg/ml (area under the ROC curve 0.883, 95% CI = 0.769 to 0.997), with sensitivity and specifi city of 86% and 88%, respectively. The cutoff value of 80 pg/ml had sensitivity and specifi city of 73% and 27%, respectively.f MJ Holmberg1, LW Andersen1, A Graver1, SB Wright1, D Yassa1, MD Howell2, MW Donnino1, MN Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2University of Chicago, IL, USA Introduction The aim of this study was to investigate whether clinicians can estimate, at the time of insertion, the length of time a central venous catheter (CVC) will remain in place, and to identify clinical variables which may predict CVC duration. CVC-related bloodstream infection is a known complication among critically ill patients. As infection rates may increase with duration of catheterization, more expensive antimicrobial-coated catheters may be used in patients with anticipated long duration of CVC use. i Conclusion This dynamic prediction rule was able to diff erentiate a group of ICU patients at high risk to develop candidemia, with a relative risk of 16.9. BDG is frequently positive in ICU patients. A cutoff value of 522 pg/ml was able to discriminate between candidemic and noncandidemic patients. A revision of the cutoff value for BDG in the ICU is needed. p g Methods We conducted a single-center, prospective study from January 2012 to November 2012. Clinicians prospectively estimated the anticipated duration of CVC at the time of line placement in an electronic procedure note. We collected demographics, past medical history, type of ICU, vital signs, laboratory values, SOFA score, mechanical ventilation and use of vasopressors at the time of placement. Continuous variables were compared with the Wilcoxon rank-sum test and categorical variables with the Fisher’s exact test. Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database y g S Fernandes1, D Bruno2, J Morgado3, C Calle4, C Ferreira5 1Centro Hospitalar Lisboa Norte CHLN, Lisbon, Portugal; 2Hospital Fernando Fonseca, Lisbon, Portugal; 3Centro Hospitalar de Lisboa Central, Lisbon, Portugal; 4Instituto Português de Oncologia, Lisbon, Portugal; 5National Institute of Legal Medicine and Forensic Sciences, Coimbra, Portugal Critical Care 2015, 19(Suppl 1):P70 (doi: 10.1186/cc14150) Results We enrolled 150 patients; median age was 65 (IQR: 52 to 74), 63 (42%) were female and mortality was 22%. Median time from CVC placement to removal was 5 (IQR: 3 to 8) days. The correlation between estimated CVC time and actual time was low (r = 0.36, P <0.001). Forty- eight (32%) patients had a long CVC duration. Clinician estimate had 46% sensitivity and 76% specifi city for predicting long duration of CVC. Of 30 variables tested, only temperature at the time of insertion was signifi cantly associated with long duration (OR: 1.30, 95% CI: 1.04 to 1.63, P = 0.02). The AUC for this model was 0.59 (95% CI: 0.49 to 0.69). Results We enrolled 150 patients; median age was 65 (IQR: 52 to 74), 63 (42%) were female and mortality was 22%. Median time from CVC placement to removal was 5 (IQR: 3 to 8) days. The correlation between estimated CVC time and actual time was low (r = 0.36, P <0.001). Forty- eight (32%) patients had a long CVC duration. Clinician estimate had 46% sensitivity and 76% specifi city for predicting long duration of CVC. Of 30 variables tested, only temperature at the time of insertion was signifi cantly associated with long duration (OR: 1.30, 95% CI: 1.04 to 1.63, P = 0.02). The AUC for this model was 0.59 (95% CI: 0.49 to 0.69). Conclusion Our results suggest a low correlation between clinician prediction at time of insertion and actual duration of CVC. We did not fi nd any good predictors of long duration of CVC. Given our relatively low sample size, we may have been underpowered. It may not be feasible to identify patients at the time of insertion who may benefi t from antimicrobial-coated catheters. Introduction Bloodstream infections in the ICU are a major trigger of morbidity and mortality. Several risk factors for bacteremia have been previously identifi ed, such as presence of a central venous catheter or invasive ventilation [1,2]. Iron is a key element for bacteria growth, and its metabolism is extensively altered by infl ammation. References following: dialysis, surgery, pancreatitis, and receipt of corticosteroids, other immunosuppressive agents or parenteral nutrition. Diff erent from the original description of the score which considered only the fi rst 7 days of ICU stay, we selected patients who fulfi lled these criteria at any time during the ICU stay. Once a patient fulfi lled these criteria, AFT (anidulafungin 200  mg followed by 100  mg daily) was initiated provided that the patients also presented with any of the following: fever, hypothermia, hypotension, leukocytosis, acidosis or elevated C-reactive protein. Blood cultures (days 1 to 2) and baseline serum BDG (days 1 to 3) were performed. Patients with candidemia were treated for ≥14 days, those without candidemia but ≥1 positive BDG (≥80 pg/ml) received AFT for ≥10 days, and patients with negative blood cultures and negative BDG discontinued anidulafungin.i 1. Tabah A, Koulenti D, Laupland K, et al. Characteristics and determinants of outcome of hospital-acquired bloodstream infections in intensive care units: the EUROBACT International Cohort Study. Intensive Care Med. 2012;38:1930-45. 2. Prowle JR, Echeverri JE, Ligabo EV, et al. Acquired bloodstream infection in the intensive care unit: incidence and attributable mortality. Crit Care. 2011;15:R100. Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA N 1 AL C l b 2 P E t 2 T G i 3 F S i ll 4 Performance of the beta-glucan test and a dynamic prediction rule to identify patients in the ICU at high risk to develop candidemia SA N 1 AL C l b 2 P E 2 T G i 3 F S i ll 4 SA Nouer , AL Colombo , P Esteves , T Guimaraes , F Scapinello , B Grassi de Miranda3, F Queiroz-Telles4, M Nucci1 1Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; 2Federal University of São Paulo, Brazil; 3Hospital Servidor Publico Estadual de São Paulo, Brazil; 4Federal University of Parana, Curitiba, Brazil Critical Care 2015, 19(Suppl 1):P69 (doi: 10.1186/cc14149) Results We analyzed 246 consecutive episodes of severe sepsis (38%) or septic shock (62%). The 28-day mortality was 36.2%. The profi le of dead patients had a signifi cantly higher average age (65 (IQR: 75.5 to 57.5) vs. 63 (47 to 72); P <0.06), APACHE II (27 (22 to 30) vs. 23 (18 to 27); P <0.001) and SOFA (11 (9 to 12.75) vs. 9 (7 to 10); P <0.001). CRP (168.4 (106 to 285) vs. 165.4 (87.8 to 275) mg/dl; P = NS), PCT (6.5 (0.94 to 23.8) vs. 5.8 (0.97 to 19.59) ng/ml; P = NS) and WBC 14.7 (9.5 to 21.4) vs. 12.9 (5.5 to 17.5); P = NS) were increased in those who died, but CHOL (102 (75 to 134) vs. 108 (86 to 141) mg/dl; P = NS) had lower values. These Introduction Early initiation of antifungal therapy (AFT) improves the outcome in candidemic patients, but empiric AFT is not considered the standard of care. Methods We used a scoring system based on the presence of a central venous catheter and receipt of antibiotics, plus at least two of the S25 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P70 P70 Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database S Fernandes1, D Bruno2, J Morgado3, C Calle4, C Ferreira5 1Centro Hospitalar Lisboa Norte CHLN, Lisbon, Portugal; 2Hospital Fernando Fonseca, Lisbon, Portugal; 3Centro Hospitalar de Lisboa Central, Lisbon, Portugal; 4Instituto Português de Oncologia, Lisbon, Portugal; 5National Institute of Legal Medicine and Forensic Sciences, Coimbra, Portugal Critical Care 2015, 19(Suppl 1):P70 (doi: 10.1186/cc14150) Estimating the duration of a central venous catheter at time of insertion Pearson’s correlation coeffi cient was used to assess the correlation between estimated CVC time and actual time. Duration of CVC use was dichotomized into long (≥7 days) or short (<7 days), based on previous literature, and sensitivity and specifi city for predicting long duration was calculated. We performed a logistic regression analysis to identify variables associated with long CVC duration and calculated the area under the ROC curve (AUC). P72 Preventing catheter-related infections in ICUs: comparing catheter care techniques Preventing catheter-related infections in ICUs: comparing catheter care techniques S Ozden, R Iscimen, H Akalin, N Kelebek Girgin, F Kahveci, M Sinirtas Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P72 (doi: 10.1186/cc14152) Low serum iron as a risk factor for ICU-acquired bacteremia: study of a large cohort database We aim to determine whether iron defi ciency is a risk or protective factor for bacteremia in the ICU. Methods We performed a retrospective analysis of patients included in the MIMIC-II database, an ICU database that collected data from patients admitted to the medical, surgical, coronary and cardiac surgery ICU of Boston’s Beth Israel Deaconess Medical Center during a period of 7 years. We performed logistic regression models to assess the association between iron and bloodstream infection. Results We included 3,980 patients, 2,988 with low serum iron (<60 ng/ ml) and 992 with normal/high serum iron (≥60 ng/ml). During their fi rst stay in the ICU, 351 (8.82%) patients developed bloodstream infections. Low serum iron was associated with increased odds of bloodstream infection (OR: 1.37; 95% CI: 1.04 to 1.80). After adjusting for age, gender, Simplifi ed Acute Physiology Score, presence of central venous catheter, ICU type, transfusions performed before iron measured, neoplastic disease, diabetes mellitus, hepatic disease, congestive heart failure and ferritin levels, low levels of iron were still associated with an increased odds of bacteremia (OR: 1.41; 95% CI: 1.03 to 1.9). In contrast, low serum iron was associated with a decreased risk of death in the hospital (adj OR: 0.73, CI: 0.57 to 0.95). P73 Results Among 1,746 ICU patients, CLA-BSI were diagnosed in 69 cases. The incidence index was 3.88/100 admissions to the ICU. CLA-BSI were diagnosed in 18% of the overall number (381) of device-associated healthcare-associated infections. Central line was used in 91.41 ± 4.4% patients during 19,819 patient-days and 18,155 central line-days. The median density of CLA-BSI/1,000 central line-days was 3.88/3.77/3.36 and 0.0 accordingly in years 2011/2012/2013 and 2014 (from January to November). The main pathogens of CLA-BSI were CN staphylococci (22%), Staphylococcus aureus (21%), and Enterobacteriaceae (29%). In this study, the density of CLA-BSI was about 50% lower (2.75 (2.0 to 6.06)) than in our previous study and in the INICC’s report (2014), but higher than in the CDC’s NHSN (2012) report. Comparison of three cutaneous antiseptic solutions for the prevention of catheter colonization in an ICU for adult patients: a multicenter prospective randomized controlled trial H Yasuda1, M Sanui2, T Komuro2, J Hatakeyama3, S Matsukubo4, S Kawano5, H Yamamoto6, K Andoh7, R Seo8, N Shime9, E Noda10, N Saito11 1Japanese Red Cross Musashino Hospital, Tokyo, Japan; 2Saitama Medical Center Jichi Medical University, Saitama, Japan; 3YokohamaCity Minato Red Cross Hospital, Kanagawa, Japan; 4Kurashiki Central Hospital, Okayama, Japan; 5The Jikei University School of Medicine, Tokyo, Japan; 6Toyonaka Municipal Hospital, Osaka, Japan; 7Sendai City Hospital, Miyagi, Japan; 8Kobe City Medical Center General Hospital, Hyogo, Japan; 9National Hospital Organization Kyoto Medical Center, Kyoto, Japan; 10Kyushu University Hospital, Fukuoka, Japan; 11Nippon Medical School Chiba Hokusoh Hospital, Tiba, Japan Conclusion The implementation of the infection control program and preventive interventions for patients with central venous catheters improved the safety and quality of healthcare in the ICU by reducing CLA-BSI rate. Critical Care 2015, 19(Suppl 1):P73 (doi: 10.1186/cc14153) Introduction The current CDC guideline for the prevention of intravascular catheter-related infections recommends skin preparation with a greater than 0.5% chlorhexidine with alcohol solution before central venous catheter (CVC) or peripheral arterial catheter (AC) placement. However, few studies investigated the superiority of 1% alcoholic chlorhexidine gluconate (1% CHG) over either 0.5% alcoholic chlorhexidine gluconate (0.5% CHG) or 10% aqueous povidone iodine (10% PVI) for the prevention of catheter colonization. The aim of this study is to compare the eff ectiveness of three skin antiseptic solutions for the prevention of intravascular catheter colonization. Reference 1. Kübler A, Duszyñska W, Rosenthal VD, et al. Device-associated infection rates and extra length of stay in an intensive care unit of a university hospital in Wroclaw, Poland: International Nosocomial Infection Control Consortium’s (INICC) fi ndings. J Crit Care. 2012;27:105.e5-10. Catheter-associated bloodstream infections in an ICU of a university hospital in Wroclaw, Poland: an international nosocomial infection control consortium’s fi ndings The main pathogens of CLA-BSI were CN staphylococci (22%), Staphylococcus aureus (21%), and Enterobacteriaceae (29%). In this study, the density of CLA-BSI was about 50% lower (2.75 (2.0 to 6.06)) than in our previous study and in the INICC’s report (2014), but higher than in the CDC’s NHSN (2012) report. 1. Safdar N, et al. Chlorhexidine-impregnated dressing for prevention of catheter-related bloodstream infection: a meta-analysis. Crit Care Med. 2014;42:1703-13. Preventing catheter-related infections in ICUs: comparing catheter care techniques S Ozden, R Iscimen, H Akalin, N Kelebek Girgin, F Kahveci, M Sinirtas Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P72 (doi: 10.1186/cc14152) Introduction Catheter-related bloodstream infections (CRBSI) are common and an important cause of morbidity and mortality in critical patients. Optimum approaches for preventing infections are presented in guidelines. This study aims to evaluate effi cacy of diff erent care techniques and education, to defi ne risk factors for decreasing ratio of CRBSIs and to analyze eff ects on morbidity and mortality. Introduction Catheter-related bloodstream infections (CRBSI) are common and an important cause of morbidity and mortality in critical patients. Optimum approaches for preventing infections are presented in guidelines. This study aims to evaluate effi cacy of diff erent care techniques and education, to defi ne risk factors for decreasing ratio of CRBSIs and to analyze eff ects on morbidity and mortality. Conclusion Low serum iron increases the risk of bloodstream infection in the ICU, and should be considered as a risk factor to stratify patients’ risk of bacteremia during ICU stay. S26 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods After ethics committee approval, patients admitted to the ICU, older than 18 years, who were thought to have a central venous catheter (CVC) for more than 48 hours, and whose fi rst catheter was inserted in the ICU were included in the study. Staff were educated before the study and periodically during the study. Catheter care and insertion were applied according to the guidelines. The study was planned as three sequences. In the fi rst group, catheter care was made with a sterile gauze pad. In the second and third groups, catheter care was made with chlorhexidine gluconate impregnated dressing. Also in the third group, a silver-coated needleless connector was inserted into the tip of venous catheters. median duration of catheter indwelling in the entire population was 3.7 days with an interquartile range of 2.0 to 6.7 days, with no signifi cant diff erence between the groups (P = 0.36). Thirteen catheters (5.1%) in the 10% PVI group were positive for catheter-tip colonization, whereas six catheters (2.2%) in the 1% CHG group and fi ve catheters (1.9%) in the 0.5% CHG group were positive (P = 0.07). P75 Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source B Civantos Martin, I Pozuelo Echegaray, C Guallar Espallargas, A Robles Caballero, C Briones, P Extremera Navas, P Millan Estañ, A Garcia de Lorenzo y Mateos Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P75 (doi: 10.1186/cc14155) Preventing catheter-related infections in ICUs: comparing catheter care techniques The probability of catheter colonization was signifi cantly higher in the 10% PVI group than each CHG groups (P  =  0.028, log-rank test). The incidence of catheter colonization and CRBSI is compared between the three groups. Conclusion In this randomized controlled trial comparing the eff ective- ness of three cutaneous antiseptic solutions for the prevention of catheter colonization, either 0.5% or 1.0% CHG was superior to 10% PVI. median duration of catheter indwelling in the entire population was 3.7 days with an interquartile range of 2.0 to 6.7 days, with no signifi cant diff erence between the groups (P = 0.36). Thirteen catheters (5.1%) in the 10% PVI group were positive for catheter-tip colonization, whereas six catheters (2.2%) in the 1% CHG group and fi ve catheters (1.9%) in the 0.5% CHG group were positive (P = 0.07). The probability of catheter colonization was signifi cantly higher in the 10% PVI group than each CHG groups (P  =  0.028, log-rank test). The incidence of catheter colonization and CRBSI is compared between the three groups. Conclusion In this randomized controlled trial comparing the eff ective- ness of three cutaneous antiseptic solutions for the prevention of catheter colonization, either 0.5% or 1.0% CHG was superior to 10% PVI. p Results Totally 105 patients were included in the study and every group included 35 patients. There was no diff erence between groups when evaluating reasons for catheter insertion. There was no statistically signifi cant diff erence according to emergent or elective catheterization, trying times, or catheter insertion side (P >0.05). CRBSI was determined in two patients in group 1, in one patient in group 2, and in no patient in group 3. In group 1 it was observed on the 4th and 11th days. In group 2 it was observed on the 18th day after catheterization. Before the study, a statistically signifi cant decrease was determined in CRBSI ratios before and after education (16.4/1,000, 12.9/1,000 catheter-days (P <0.001)). According to Group 1 a statistically meaningful decrease was assigned in CRBSI ratios in Groups 2 and 3 (4.84/1,000, 2.22/1,000, 0/1,000 catheter-days) (P <0.001, P <0.001, P <0.001). Catheter-associated bloodstream infections in an ICU of a university hospital in Wroclaw, Poland: an international nosocomial infection control consortium’s fi ndings i g W Duszynska1, VD Rosenthal2, A Litwin1, E Woznica1, A Kubler1 1University Hospital, Wroclaw, Poland; 2International Nosocomial Infection Control Consortium, Buenos Aires, Argentina Critical Care 2015, 19(Suppl 1):P74 (doi: 10.1186/cc14154) i g W Duszynska1, VD Rosenthal2, A Litwin1, E Woznica1, A Kubler1 1University Hospital, Wroclaw, Poland; 2International Nosocomial Infection Control Consortium, Buenos Aires, Argentina Critical Care 2015, 19(Suppl 1):P74 (doi: 10.1186/cc14154) Introduction Catheter-associated bloodstream infections are serious but potentially possible to reduce complication of treatment in the ICU. The aim of the study was to evaluate the frequency and etiology of central line-associated bloodstream infections (CLA-BSI) in ICU patients according to the International Nosocomial Infection Control Consortium (INICC) project. y Conclusion Continued education is important in preventing CRBSIs. Maximum precautions must be taken. Usage of antiseptic solutions with clorhexidine and chlorhexidine gluconate impregnated dressing decreased insertion side infections and usage of silver-coated needleless connectors reduced microorganism entry through the catheter lumen and provided a severe decrease in infection ratio. Reference j Methods A prospective, observational study was conducted in the 20-bed ICU of the University Hospital in Wroclaw from January 2011 to November 2014. CLA-BSI were diagnosed and evaluated according to protocols standardized by the INICC. The density of CLA-BSI/1,000 central line-days, the incidence index/100 admissions to the hospital, the central line utilization ratio (CL-UR) as well as the microbiological profi le of CLA-BSI were evaluated. The results were compared with our earlier published data and with the fi ndings of international reports. Methods A prospective, observational study was conducted in the 20-bed ICU of the University Hospital in Wroclaw from January 2011 to November 2014. CLA-BSI were diagnosed and evaluated according to protocols standardized by the INICC. The density of CLA-BSI/1,000 central line-days, the incidence index/100 admissions to the hospital, the central line utilization ratio (CL-UR) as well as the microbiological profi le of CLA-BSI were evaluated. The results were compared with our earlier published data and with the fi ndings of international reports. Results Among 1,746 ICU patients, CLA-BSI were diagnosed in 69 cases. The incidence index was 3.88/100 admissions to the ICU. CLA-BSI were diagnosed in 18% of the overall number (381) of device-associated healthcare-associated infections. Central line was used in 91.41 ± 4.4% patients during 19,819 patient-days and 18,155 central line-days. The median density of CLA-BSI/1,000 central line-days was 3.88/3.77/3.36 and 0.0 accordingly in years 2011/2012/2013 and 2014 (from January to November). Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source Methods This multicenter prospective randomized controlled trial was conducted in 15 Japanese ICUs from December 2012 to March 2014. Patients over 18 years of age undergoing CVC and AC placement in the ICU are randomized to have one of three skin antiseptic preparations before catheter insertion. After removal of the catheter, the distal tip is cultured using semiquantitative or quantitative techniques. The incidence of catheter colonization and catheter-related bloodstream infection (CRBSI) is compared between the three groups. Introduction Long experience in the treatment of neutropenic patients admitted to the ICU has taught us the importance of removing the permanent central venous catheter when infection is suspected, because of the great mortality associated. The problem usually comes when the origin of sepsis is not clear and we assume that mortality is Results A total of 997 catheters were placed, including 339 catheters using 1% CHG, 329 using 0.5% CHG, and 329 using 10% PVI. The S27 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 immune systems to suppress dissemination of infection by the netted chromatin decorated with antibacterial molecules. It was reported that NETs play an important role in various kinds of infections such as pneumonia. However, there is no report on NETs in patients of soft tissue infections. In this study, we evaluated NET production in pus and clarifi ed the role of NETs as a host defense in patients of soft tissue infections. not easy to avoid. It is important to know what happens to neutropenic patients admitted to the ICU because of sepsis from any source, in whom catheter infection cannot be excluded. Methods A retrospective, cohort, descriptive study was carried out between January 2013 and November 2014. Epidemiology data were collected from all neutropenic patients admitted to the ICU who came from hemato-oncology services and had an implanted central venous catheter. Microbiology results and data related to the catheter removal were described. Methods This study was conducted in the ICU of the Trauma and Acute Critical Care Center at Osaka University Hospital. We collected pus from the patients of soft tissue infections at the time when drainage or debridement was performed and when clinical improvement was observed. The smears of pus specimens were examined by immunohistochemistry to visualize the major NET components: DNA, neutrophil elastase, and histone H1. Use of nanotechnology-based surface antiseptic solutions in the ICU Y Kuplay, N Akgun, C Agalar, H Aydýn, O Alýcý, G Turan FSM Teaching and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P78 (doi: 10.1186/cc14158) Y Kuplay, N Akgun, C Agalar, H Aydýn, O Alýcý, G Turan FSM Teaching and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P78 (doi: 10.1186/cc14158) Introduction In our study, we aimed to compare the application of benzalkonium chloride (BC) – a nanotechnology-based product – for 24-hour periods and didecyl dimethyl ammonium chloride (DDAC) for 12-hour periods regarding effi ciency in application of surface antiseptics in the ICU.f Introduction In our study, we aimed to compare the application of benzalkonium chloride (BC) – a nanotechnology-based product – for 24-hour periods and didecyl dimethyl ammonium chloride (DDAC) for 12-hour periods regarding effi ciency in application of surface antiseptics in the ICU.f y Methods A retrospective observational study in a medical and surgical ICU in a tertiary care hospital on adult patients admitted from January 2010 to December 2013. The study enrolled 845 patients divided into 283 internal jugular CVC (IJC), 270 subclavian CVC (SCC) and 287 femoral CVC (FC) in which the catheters were inserted in the ICU by experienced physicians with at least 50 previously successful trials of central line insertion, using CVC bundle checklist. ICU length of stay, incidence of complications, APACHE II score adjusted severity and mortality were calculated for each group. Methods Two diff erent areas with eight beds at both sides of a common corridor in the ICU were named as areas A and B. BC was applied in area A with 24-hour periods and DDCA was applied in area B with 12- hour periods for surface cleaning. Samples were taken from a total of 20 diff erent surfaces including nurse-station desks, phones, keyboards, beds, bedside monitors and ventilators by the same infection control nurse every 24 hours from area A and every 12 hours from area B for 7  days. Swab samples were cultured on 5% sheep bloody agar and McConkey agar in the laboratory. Then the cultured mediums were incubated at 35 to 37°C in an aerobic environment for 18 to 24 hours. NCSS (Number Cruncher Statistical System) 2007 and PASS 2008 Statistical Software (UT, USA) programs were used for the statistical analysis.f Results Patient and catheter characteristics including the duration of catheterization were similar in all groups. Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source Concurrently, the patients’ clinical data and laboratory data of blood were recorded to analyze the relationship with NET production.i Results A total of 15 patients were included, mean age was 53 years old and 66% were male. The implanted catheter was removed in 80% of patients. Platelet transfusion was needed in 100% of patients before catheter removal and no complication was observed during catheter removal or in the insertion of a new one. In 53% of patients, catheter infection was confi rmed a posteriori. i p Conclusion Removal of an implanted central venous catheter from neutropenic patients admitted to the ICU due to sepsis from any source can be benefi cial for this kind of patient as it was found that in more than 50% of patients catheter infection was confi rmed a posteriori. Results A total of fi ve patients were included in this study and drainage of abscess or debridement of infection site was performed in all of the cases. Four patients of them were diagnosed as necrotizing soft tissue infections by Clostridium spp. (n = 1) and Bacteroides spp. (n = 3) and the other was diagnosed as cervical abscess by Streptococcus spp. In all cases, no NETs but neutrophils were identifi ed in the fi rst pus: however, NETs appeared in the later smears as the patients’ condition was getting better. Eff ect of insertion route on risk of central line-associated bloodstream infection in critically ill patients R Alhubail, N Hassan g g Conclusion These results suggested that NETs also worked as an immune system against soft tissue infections. Drainage or debridement of infection focus might promote NET production. KFSH-D, Dammam, Saudi Arabia Introduction Femoral, jugular or subclavian central venous catheterization (CVC) is routinely performed during the care of the critically ill. These invasive procedures contribute to additional morbidity, mortality, and costs derived from the interactions between traumatic, infectious and other complications. The aim of this study is to determine whether the subclavian, jugular or femoral central venous access (CVA) routes have an eff ect on the incidence of CLABSI in critically ill patients and to compare between these routes regarding major complications and ICU mortality. P78 Use of nanotechnology-based surface antiseptic solutions in the ICU The rate of CLABSI in the IJC, SCC and FC groups was 5.8 versus 7.2 versus 3.45 per 1,000 catheter- days respectively with P = 0.35. ICU mortality was 134 (47%) cases of the IJC group, 108 (39%) cases of the SCC group and 113 (39%) cases of the FC group. There was no signifi cant diff erence between the three groups of CVC in the incidence of CLABSI rate in the critically ill patients, and a slight increase in ICU mortality in the IJC group compared with the other two groups. Pneumothorax occurred in six (2.2%) cases of SCC and 11 (3.8%) cases of IJC with no signifi cant diff erence between the two groups as the P value was 0.3.f Results There were no statistical diff erences between two groups (Table 1). Conclusion Site of insertion of CVC does not appear to aff ect the rate of CLABSI among critically ill patients. Pneumothorax was recorded in SCC and IJC groups with no statistical preference to either group. Table 1 (abstract P78). Isolated pathogen ratio percentage A (BC) B (DDCA) P value First day 25 20 1.000 Second day 5 15 0.605 Third day 30 20 0.715 Fourth day 65 50 0.527 Fifth day 45 60 0.527 Sixth day 25 25 1.000 Seventh day 60 45 0.527 Table 1 (abstract P78). Isolated pathogen ratio percentage Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials h 1 k2 Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials E Choi1, J Park2 1Yeungnam University College of Medicine, Daegu, South Korea; 2Uijeongbu St. Mary’s Hospital, Uijeongbu, South Korea Critical Care 2015, 19(Suppl 1):P81 (doi: 10.1186/cc14161) Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials Results We observed a signifi cant reduction in DSSI rates in cardiac surgery following implementation of a multimodal ICP from 3.1% in 2010 down to 0.23% in November 2014 (Figure 1). Introduction Whole-body skin decolonization with chlorhexidine in critically ill patients reduces multidrug-resistant bacterial colonization, and catheter-related bloodstream infection (BSI). We performed a meta-analysis of randomized controlled trials to determine whether daily bathing with chlorhexidine decreased hospital-acquired BSIs in critically ill patients. Introduction Whole-body skin decolonization with chlorhexidine in critically ill patients reduces multidrug-resistant bacterial colonization, and catheter-related bloodstream infection (BSI). We performed a meta-analysis of randomized controlled trials to determine whether daily bathing with chlorhexidine decreased hospital-acquired BSIs in critically ill patients. Conclusion Implementing a multimodal ICP signifi cantly reduced the incidence of DSSI in our hospital but it remains diffi cult to identify which interventions were most eff ective. Reference 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. Figure 1 (abstract P79). Incidence of DSSI. Figure 1 (abstract P79). Incidence of DSSI. y Methods We searched the MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials databases to identify randomized controlled trials that compared daily bathing with chlorhexidine and a control (daily bathing with soap and water or nonantimicrobial washcloths, or implementation of MRSA screening and isolation) in critically ill patients. The primary outcome was hospital-acquired BSIs. Secondary outcomes were adverse eff ects of chlorhexidine and the incidence of identifi ed pathogens. i Results This meta-analysis included four studies. The overall incidence of hospital-acquired BSIs was signifi cantly lower in the chlorhexidine group compared with the control 0.80 (95% CI, 0.71 to 0.90; P <0.001; I2 = 29.4%). Gram-positive (RR = 0.59, 95% CI, 0.44 to 0.79, P = 0.000; I2 = 46.0%) and MRSA-induced (pooled RR = 0.64; 95% CI, 0.47 to 0.88, P  =  0.006; I2  = 0.0%) bacteremias were signifi cantly less common in the chlorhexidine group. Chlorhexidine did not aff ect Gram-negative bacteremia or fungemia. Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU y GM Plantier1, CE Bosso1, BN Azevedo2, AC Correa3, AL Silva3, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil; 3Santa Casa de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P80 (doi: 10.1186/cc14160) Introduction Urinary catheter insertion is a common procedure in ICUs and can be an important cause of infection in the hospital environment [1,2]. We aimed to analyze the eff ect of chlorhexidine on long-term urinary catheter insertion and urinary tract infection (UTI) during a 5-year period in patients admitted to a coronary ICU. y p p y Methods Analysis of patients admitted to a coronary ICU of a medium- sized hospital in Brazil from January 2010 to May 2014. The institutional protocol of periprocedural antisepsis was changed from iodine-based antiseptic to chlorhexidine in 2012. The UTI diagnosis was based on urine culture (>105 colony-forming units per ml of urine) associated with at least one clinical/laboratory abnormality (fever >38°C, urination urgency, increased urinary frequency, dysuria, or suprapubic or lumbar pain). The UTI rate represents the urinary tract infections associated with long-term urinary catheter (patient with UTI associated with long-term urinary catheter divided by patients with long-term urinary catheter × 1,000). Results The urinary tract infection rates were 4.8 (year 2010: patients·day–1 (n: 2,511), long-term urinary catheter·day–1 (n: 1,455), device usage rate (958%)), 4.4 (year 2011: patients·day–1 (n: 2,529), long- term urinary catheter·day–1 (n: 1,140), device usage rate (45%)), 0.0 (year 2012: patients·day–1 (n: 2,660), long-term urinary catheter·day–1 (n: 783), device usage rate (29%)), 0.0 (year 2013: patients·day–1 (n: 2,573), long- term urinary catheter·day–1 (n: 960), device usage rate (37%)), and 0.0 (year 2014: patients·day–1 (n: 1,070), long-term urinary catheter·day–1 (n: 444), device usage rate (42%)). y y Methods Analysis of patients admitted to a coronary ICU of a medium- sized hospital in Brazil from January 2010 to May 2014. The institutional protocol of periprocedural antisepsis was changed from iodine-based antiseptic to chlorhexidine in 2012. The UTI diagnosis was based on urine culture (>105 colony-forming units per ml of urine) associated with at least one clinical/laboratory abnormality (fever >38°C, urination urgency, increased urinary frequency, dysuria, or suprapubic or lumbar pain). P79 P79 Reduction of deep surgical site infections in cardiac surgery by introducing a multimodal infection control program A Rutten, JP Ory, L Jamaer, A Van Assche, J Dubois Jessa Ziekenhuis, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P79 (doi: 10.1186/cc14159) y g y Results The urinary tract infection rates were 4.8 (year 2010: patients·day–1 (n: 2,511), long-term urinary catheter·day–1 (n: 1,455), device usage rate (958%)), 4.4 (year 2011: patients·day–1 (n: 2,529), long- term urinary catheter·day–1 (n: 1,140), device usage rate (45%)), 0.0 (year 2012: patients·day–1 (n: 2,660), long-term urinary catheter·day–1 (n: 783), device usage rate (29%)), 0.0 (year 2013: patients·day–1 (n: 2,573), long- term urinary catheter·day–1 (n: 960), device usage rate (37%)), and 0.0 (year 2014: patients·day–1 (n: 1,070), long-term urinary catheter·day–1 (n: 444), device usage rate (42%)). Introduction Deep surgical site infections (DSSI) are a major compli- cation after cardiac surgery with a high mortality rate and reported incidences between 0.5 and 5%. Implementing a comprehensive infection control program (ICP) reduces this incidence [1]. The incidence in our hospital varied from 3.1 to 3.8%, which was considered too high. We evaluated the impact of introducing a multimodal ICP on the incidence of DSSI. Methods We noticed a too high incidence of DSSI after cardiac surgery during an observational 3-year period (Figure 1). In February 2013 we introduced a bundle of interdisciplinary infection control measures. Medical and nursing staff of all involved departments took part in developing and implementing these guidelines. Besides emphasizing the importance of existing guidelines (antiseptic shower, hair removal by clipper, strict hand hygiene, prophylactic antibiotics, limiting OR traffi c, tight glycemic control (80 to 110 mg/ dl), and so on), new strategies were introduced. The most important new strategies were nasal decolonization with mupirocin twice daily 48  hours perioperatively, preoperative antiseptic skin preparation twice (chlorhexidine gluconate 0.5%), applying topical skin adhesive to the sternal wound postoperatively and in the case of CABG procedures maintaining a strict barrier between the vein harvesting procedure and the chest procedure. Conclusion The use of chlorhexidine in the periprocedural antisepsis of urinary catheterization contributed to the decrease of urinary tract infections associated with long-term urinary catheter in patients admitted to the coronary ICU. P80 Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU GM Plantier1, CE Bosso1, BN Azevedo2, AC Correa3, AL Silva3, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil; 3Santa Casa de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P80 (doi: 10.1186/cc14160) References 1. Silva E, et al. Crit Care, 2004;8:R251-60. 2. Vincent JL, et al. JAMA. 2009;302:2323-9. Eff ect of chlorhexidine and urinary catheter infection prevention in a Brazilian coronary ICU The UTI rate represents the urinary tract infections associated with long-term urinary catheter (patient with UTI associated with long-term urinary catheter divided by patients with long-term urinary catheter × 1,000). 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. P77 P77 Role of neutrophil extracellular traps against soft tissue infections N Yamamoto1, M Ojima2, S Hamaguchi1, T Hirose2, R Takegawa2, N Matsumoto2, T Irisawa2, M Seki1, O Tasaki3, T Shimazu2, K Tomono1 1Division of Infection Control and Prevention, Osaka University Graduate School of Medicine, Suita, Japan; 2Osaka University Graduate School of Medicine, Suita, Japan; 3Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Critical Care 2015, 19(Suppl 1):P77 (doi: 10.1186/cc14157) Critical Care 2015, 19(Suppl 1):P77 (doi: 10.1186/cc14157) Introduction Neutrophils work as the frontline of defense against infections and neutrophil extracellular traps (NETs) are one of the S28 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The eff ect of a good surface disinfectant should begin immediately and it should have a long-lasting disinfecting eff ect on the surface. DDAC is an effi cient disinfectant used in medicine and the food industry to protect the surfaces. However, it may cause severe skin itching. BC, which is a nanotechnology-based product, leaves its active metabolites on the surface; it is applied by constituting a spongy layer. Since the effi ciency of BC lasts for 24 hours and it is applied to perform cleaning with 24-hour intervals, we think that it is preferable with regards to workforce gain and cost. Clinical validation of an electronic hand hygiene surveillance system y Methods A quasi-experimental study was done in three level III ICUs of a tertiary care hospital in Kolkata (January to April 2014). Data were collected on existing hand hygiene compliance rate, ventilator- associated pneumonia (VAP) rate, catheter-related bloodstream infection (CRBSI) rate, catheter-related urinary tract infection (CAUTI) rate, standardized mortality ratio (SMR) and average ICU length of stay in the abovementioned units. Root cause analysis was done and interventions were developed to improve hand hygiene compliance and was implemented (July to October 2014). Comparison was done between preintervention and postintervention periods. y P Levin, R Razon, MJ Cohen, CL Sprung, S Benenson Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2015, 19(Suppl 1):P84 (doi: 10.1186/cc14164) P Levin, R Razon, MJ Cohen, CL Sprung, S Benenson Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2015, 19(Suppl 1):P84 (doi: 10.1186/cc14164) Introduction Good hand hygiene (HH) is critical to infection control in the ICU. Electronic HH surveillance systems are purported to improve HH practices. Such a system was recently trialed in our ICU. The system is based on radiofrequency transponders in three locations: bracelets worn by ICU personnel; on all HH product dispensers; and above each patient’s bed. By correlating input from these three sources the system detects whether HH was performed before and after each patient contact. In the event that HH is not performed, the bracelet alerts the user (by vibration) in real time. This study represents a clinical validation of the system. Results In the preintervention period (January to April 2014) the hand hygiene compliance among the caregivers was found to be 40%, VAP rate (8.77), CRBSI rate (3.42), CAUTI rate (5.27), SMR (1.14) and average ICU length of stay was 6 days ± 5.85 SD (median 4.5). Interventions were developed and implemented as follows: education and awareness – road shows; positive reinforcement; secret watch nurse; e-ICU  – electronic surveillance; ring the bell once every hour – baseline hand hygiene; visual reminders; availability of alcohol-based hand rub, soap and water and sinks; random hand swabs; and compliance audits. In the postintervention period (July to October 2014) data showed a signifi cant improvement in the hand hygiene compliance (75%). Further analysis showed an association with decrease in the incidence of VAP rate (4.71), CAUTI rate (3.51), CRBSI rate (2.65), SMR (1.05) and average ICU LOS 5.05 days ± 4.03 SD (median 4). Improving hand hygiene compliance leads to improved health outcome: an analysis y V Rao, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India p Conclusion Introduction of infection control bundle in the ICU reduced the incidence of nosocomial MDRO transmission and infection, which resulted in the reduction of anti-MRSA antibiotics and carbapenems use in critically ill patients. p p y p Critical Care 2015, 19(Suppl 1):P82 (doi: 10.1186/cc14162) Introduction Hand hygiene is the single most eff ective but least practiced action in breaking the chain of transmission of microbes. Studies have shown a correlation between the compliance of hand hygiene and its impact on the health outcome. Eff ect of daily bathing with chlorhexidine on hospital-acquired bloodstream infection in critically ill patients: a meta-analysis of randomized controlled trials h 1 k2 The overall incidence of adverse events, such as skin rashes, was similar in both groups. Conclusion Implementing a multimodal ICP signifi cantly reduced the incidence of DSSI in our hospital but it remains diffi cult to identify which interventions were most eff ective. Reference 1. Guide for the prevention of mediastinitis surgical site infections following cardiac surgery. 2008. www.apic.org. S29 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Daily bathing with chlorhexidine was associated with a reduction in the rates of hospital-acquired BSI without signifi cant complications in critically ill patients. It also decreased the incidence of Gram-positive hospital-acquired BSIs, especially MRSA. Results Admission to the ICU comprised 363 patients in the fi rst period and 380 patients in the second period. The incidence of transmission was decreased from 48 (13.2%) to 21 (5.5%) in methicillin-resistant Staphylococcus aureus (MRSA), from 16 (4.4%) to zero (0%) in multidrug- resistant Acinetobacter baumannii. The incidence of nosocomial infection by MDRO was also decreased from 23 (6.3%) to 17 (4.5%) in pneumonia, from fi ve (1.4%) to two (0.3%) in urinary tract infection, and from 12 (3.3%) to one (0.3%) in surgical site infection. The incidence of antibiotic use for MDRO infection was decreased from 41 (11.3%) to 24 (6.3%) in anti-MRSA antibiotics, and from 19 (5.2%) to eight (2.1%) in carbapenems. Clinical validation of an electronic hand hygiene surveillance system y Methods ICU staff (nurses and physicians) were followed by a trained observer over 60-minute periods. Each movement and contact during the period was documented. HH opportunities were determined according to WHO criteria and actual HH performance recorded. Observer and electronic data were compared for number of opportunities, HH performance and compliance. A satisfaction questionnaire was distributed to all users. Paired Student’s t test was used for comparison of the observer and electronic data. Results Observations were made over 56 time periods that included 836 HH opportunities and 485 occasions when HH was performed. The observer recorded 10.9  ±  7.6 HH opportunities/hour compared with 6.8 ± 6.9 for the electronic system (P <0.001). HH performance occurred on 8.7 ± 3.9 occasions/hour versus 6.0 ± 3.1 occasions/hour as recorded by the electronic system (P <0.001). Overall HH compliance was 62.5  ±  17.7% versus 57.5  ±  21.0% respectively (P  =  0.523). On comparison of specifi c observation periods, there was poor correlation between compliance as recorded by the observer and electronic system (r = 0.03, P = 0.915). Satisfaction questionnaires were completed by 41 personnel. Satisfaction with the system was low or very low for 21/41 (61%). System inaccuracy (either bracelet alerts without cause, or lack of bracelet alerts when HH was required) was the most common reason for dissatisfaction (31/41, 76%), followed by physical discomfort from the bracelet (18/41, 44%). Conclusion Improved hand hygiene compliance can be attributed to decreased incidence of VAP, CRBSI, CAUTI, SMR and average ICU LOS. This does defi nitely impact the overall clinical outcome. However, continued surveillance of hand hygiene compliance and regular audits is of utmost importance to make the change sustainable. P83f Eff ects of infection control bundle to prevent nosocomial infection in the ICU A Matsushima, M Kawanami, S Fujimi, N Inadome, N Kubo, T Yoshioka Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P83 (doi: 10.1186/cc14163) Eff ects of infection control bundle to prevent nosocomial infection in the ICU in the ICU A Matsushima, M Kawanami, S Fujimi, N Inadome, N Kubo, T Yoshioka Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P83 (doi: 10.1186/cc14163) Introduction Multidrug-resistant organism (MDRO) infections in critically ill patients are often life-threatening. To prevent nosocomial infections of MDRO, we made an infection control bundle in our ICU in 2013. In this study we evaluated the eff ect of our infection control bundle to prevent nosocomial MDRO transmission and infection. Conclusion The electronic HH system consistently underestimated both HH opportunities and HH performance. The main reason for dissatisfaction with the system was inaccuracy of bracelet alerts. These data suggest that for an electronic system to be accepted by ICU staff , it has to be highly accurate and comfortable for the user. Methods Our infection control bundle consists of preemptive contact precaution to all care, active surveillance culture and isolation of patients with MDRO. This bundle was applied to all patients admitted to our ICU since 2013. The study period to evaluate the eff ects of the bundle was from April 2012 to March 2014, and we divided it into two periods; fi rst period (before introduction of the bundle) and second period (after introduction of the bundle). We compared the incidence of nosocomial transmission and infection of MDRO between the two periods. MDRO was defi ned as bacteria that were resistant to more than three kinds of antibiotics. Nosocomial transmission was defi ned when MDRO was detected later than 48  hours after admission. Nosocomial infection was diagnosed according to the National Nosocomial Infection Surveillance Manual. P86 A survey of UK acute clinicians’ knowledge of personal protective requirements for infectious diseases and chemical, biological, and radiological warfare agents AR Bond1, A Buckingham2, J Schumacher1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P86 (doi: 10.1186/cc14166) Evaluation of the microbial tightness of closed system transfer devices by simulating airborne and touch contamination University of Bonn, Germany Introduction The use of intravascular catheter devices is often associated with serious bloodstream infections due to microbial contaminations. To minimize risk of such infections NIOSH recommends S30 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 the use of closed system transfer devices (CSTDs). To evaluate the microbial tightness of CSTDs we developed two methods which simulate the bioburden in ambient air of operating rooms and ICUs. the use of closed system transfer devices (CSTDs). To evaluate the microbial tightness of CSTDs we developed two methods which simulate the bioburden in ambient air of operating rooms and ICUs. for clinicians treating patients exposed to infectious diseases and CBRN agents, ideally in a simulation setting. Further research into whether the required levels of PPE are readily available to clinicians would be pertinent. Methods The methods simulate airborne and touch contamination. We tested the microbial tightness of the integrated Safefl ow® valve of a Mini-Spike® which is used for drug admixture. The airborne contamination was done in an exposure chamber in which a nebulizer distributed defi ned B. subtilis spore aerosols [1]. A Mini-Spike® was inserted into a vial of 0.9% sodium chloride solution (NaCl). A nebulizer with a suspension of 4.8 × 105 CFU spores of B. subtilis per ml was used to generate an aerosol for 1 minute. The volume of B. subtilis suspension nebulized per minute was 0.278  ml. This corresponds to 1.34  ×  103 aerosolized spores in the exposure chamber, which has a volume of 0.24 m3 (5.6 × 103 CFU per m3 air). The used concentration was 100 times higher than the microbial burden found in hospitals [2]. After nebulization the valve was disinfected and NaCl was withdrawn into a syringe at certain time intervals. The NaCl was incubated on tryptic soy agar at 37°C for 48 hours. Results were documented as CFU. For touch contamination, a Mini-Spike® was attached to a vial of NaCl. The valve of the Mini-Spike® was contaminated with 105 CFU Staphylococcus aureus. The subsequent procedure was done as described above. g References 1. Dunkelberg H, Fleitmann-Glende F. Measurement of the microbial barrier eff ectiveness of sterilization containers in terms of the log reduction value for prevention of nosocomial infections. Am J Infect Control. 2006;34:285-9. 1. Dunkelberg H, Fleitmann-Glende F. Measurement of the microbial barrier eff ectiveness of sterilization containers in terms of the log reduction value for prevention of nosocomial infections. Am J Infect Control. 2006;34:285-9. 2. Qudiesat K. Assessment of airborne pathogens in healthcare settings. AJMR. 2009;3:66-76. Results During the study period, 40 patients with TB were admitted to the ICU; 75% male and median age of 52 years (IQR 37.5 to 62.8). Overall, 22 (55%) patients died in the hospital, of whom 16 (40%) died in the ICU. Comorbid illness was identifi ed in 32 (80%) patients, with HIV infection being the most common, present in 15 (37.5%) patients. The main reason for ICU admission was respiratory failure (70%), followed by sepsis/septic shock (22.5%). Twenty-eight (70%) patients had isolated pulmonary disease, four (10%) had isolated extrapulmonary disease and eight (20%) had association of pulmonary and extrapulmonary disease. Mycobacterial cultures were positive in 31 (77.5%) patients; three patients presented monoresistant strains. Twenty-nine (72.5%) patients required mechanical ventilation and 21 (52.5%) required vasopressor infusion in the ICU; two patients were treated with ECMO. Thirty-four (85%) patients received antituberculosis therapy. The median length of stay was 11.5 (IQR 3.25 to 28.5) days in the ICU and 40.5 (IQR 21.0 to 62.8) days in the hospital. The presence of at least one comorbidity, smoking, age, sepsis/septic shock on admission, high SAPS II and APACHE II score, positive direct examination and PCR in respiratory samples, the need for mechanical ventilation or vasopressor infusion were signifi cantly associated with mortality (P <0.05). There was no association between mortality and HIV status, site of TB disease, concomitant acute disease or development of hospital infections. 2. Qudiesat K. Assessment of airborne pathogens in healthcare settings. AJMR. 2009;3:66-76. R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento Introduction To describe the characteristics of the patients with tuberculosis (TB) requiring intensive care and to identify the factors associated with in-hospital mortality in an ICU in Portugal. Results Out of nine tested valves, none showed transmission of B. subtilis spores after airborne contamination. Three out of nine tested valves were contaminated with S. aureus after touch contamination. Conclusion Our study shows that both methods are suitable for evaluating the microbial tightness of CSTDs. References Methods A retrospective cohort study between January 2007 and July 2014 of all patients with TB admitted to the ICU of the Infectious Diseases Department of Centro Hospitalar de São João. Comorbid diagnoses, clinical features, radiological and laboratory investigations and outcomes were reviewed. The primary outcome was the in- hospital mortality. A univariate analysis was performed to identify risk factors for death. References References 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West Africa. http://www.cdc.gov/vhf/ebola/outbreaks/guinea/. 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West Africa http://wwwcdc gov/vhf/ebola/outbreaks/guinea/ 1. Centers for Disease Control and Prevention. 2014 ebola outbreak in West p g g 2. Brinker A, et al. Personal protection during resuscitation of CBW victims. A survey among medical fi rst receivers in the UK. Prehosp Disaster Med. 2009;24:525-8. A survey of UK acute clinicians’ knowledge of personal protective requirements for infectious diseases and chemical, biological, and radiological warfare agents AR Bond1, A Buckingham2, J Schumacher1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P86 (doi: 10.1186/cc14166) Introduction We conducted a survey to assess clinicians’ knowledge of personal protective equipment (PPE) requirements for infectious diseases and biochemical warfare agents. A safe level of PPE is essential when treating patients with highly infectious diseases or those contaminated with hazardous substances. The recent Ebola virus disease (EVD) outbreak in West Africa has highlighted that, although uncommon, contagious diseases with high mortality rates can be a threat to healthcare systems at local, national, and international levels [1]. Chemical, biological, radiological or nuclear (CBRN) contamination presents similar risks. Conclusion In this cohort we found a high mortality rate in the TB patients requiring intensive care. The risk factors for mortality due to severe TB are mainly related to the severity of organ failure, patient characteristics and burden of disease and not to HIV status or site of TB disease. Methods A validated, hand-delivered, multiple-choice questionnaire [2] was used to assess intensive care, emergency medicine, and anesthetics specialist registrars’ knowledge of respiratory and skin protection needed during a resuscitation scenario with advanced life support. Participants selected the PPE required for the biological hazards: EVD, severe acute respiratory syndrome (SARS), inhalational anthrax, plague and smallpox; and the biochemical hazards: sarin, hydrogen cyanide, phosgene and mustard gas (dichlordiethyl sulfi de). Responses were compared with UK national recommendations and a previous survey in 2009 [2]. Tuberculosis in the ICU: a retrospective cohort study Tuberculosis in the ICU: a retrospective cohort study R Duro, P Figueiredo, A Ferreira, S Xerinda, C Lima Alves, L Santos, A Sarmento Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 Introduction Changes in infection agents and their sensitivity to antibiotics are the main cause of severity of surgical infections. In spite of development and introduction of new drugs and methods of treatment, the number of patients with sepsis increases, so the problem in diagnosing and treatment is still far from resolution. Introduction Changes in infection agents and their sensitivity to antibiotics are the main cause of severity of surgical infections. In spite of development and introduction of new drugs and methods of treatment, the number of patients with sepsis increases, so the problem in diagnosing and treatment is still far from resolution. Methods A comparative retrospective analysis of 52 histories of patients with sepsis, which were treated in the Department of Surgical Infections in 1999 and 2013. g g Methods A comparative retrospective analysis of 52 histories of patients with sepsis, which were treated in the Department of Surgical Infections in 1999 and 2013. Results The number of patients with sepsis in 2013 was raised 2.7 times, in comparison with 1999. Mortality decreased from 79% in 1999 to 55% in 2013. In most cases sepsis was accompanied with immunosuppressive disorders, such as diabetes, oncology, alcohol and drug addiction, and HIV infection. We analyzed crops of discharge from the wound and blood cultures in 52 patients with sepsis. Crops of wound were taken during the initial surgical intervention, then every 3 to 7 days, as well as the surgical interventions being repeated. Blood cultures were performed in the presence or suspected diagnosis of sepsis, in accordance with the classifi cation Bone criteria. In comparison of spectrum of infection agents, Staphylococcus aureus is still leading (1999 – 36.6% of isolates, 2013 – 25%), and the percentage of MRSA was 0% in 1999 and 37.5% in 2013. The frequency of Gram-negative fl ora has increased: E. coli (8.5%/20%), P. aeruginosa (8.5%/12%), Klebsiella pneumoniae (0%/16%) and Acinetobacter spp. (0%/16%). Speaking about the resistance of microorganisms, there is still a high percentage of sensitivity to aminoglycoside antibiotics (79.4%/75%), glycopeptides (77.2%/71%), carbapenems (88.4%/78%) and also to the combination therapy (71.8%/62.4%), but also a reduction in sensitivity to the group of beta-lactam antibiotics (58.2%/32.5%) and fl uoroquinolones (64.6%/36.4%). Conclusion Cutaneous mucormycosis is less common than other clinical forms, most frequently seen in inmunocompetent patients but potentially lethal if treatment is not rapid. Patients at risk are those with disruption of the normal protective cutaneous barrier. Low-pathogenicity mycoplasma species induce immunoparesis and are highly prevalent amongst patients with ventilator-associated pneumonia TJ Nolan1, N Gadsby2, TP Hellyer3, K Templeton2, R McMullan4, J McKenna5, J Rennie1, CT Robb1, TS Walsh1, AG Rossi1, AJ Simpson3, A Conway Morris6 1University of Edinburgh, UK; 2NHS Lothian, Edinburgh, UK; 3Newcastle University, Newcastle, UK; 4Queen’s University, Belfast, UK; 5Belfast Health and Social Care Trust, Belfast, UK; 6University of Cambridge, UK Critical Care 2015, 19(Suppl 1):P89 (doi: 10.1186/cc14169) Introduction Ventilator-associated pneumonia (VAP) remains a signifi cant problem within ICUs. There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms of this immunoparesis remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent amongst patients with VAP than previously recognised, and that these organisms potentially impair immune cell function. Conclusion The number of patients with sepsis has increased; the mortality of sepsis has decreased. The frequency of S. aureus isolation is still high, MRSA is the same. The frequency of Gram- negative fl ora isolation has increased, especially K. pneumoniae and Acinetobacter spp. The resistance of microorganisms to beta-lactams and fl uoroquinolones is rising but the sensitivity to aminoglycosides, glycopeptides, and carbapenems is still maintained. y Methods Two cohorts [1,2], totalling 159 patients, were recruited from 12 UK ICUs; all patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage. VAP was defi ned as growth of organisms at >104 CFU/ml on conventional culture. Thirty- six healthy donors underwent lavage for comparison. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. Additionally, healthy donor monocytes and macrophages (MDM) were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed. P91 2. Hellyer T, et al. Thorax. 2014 [Epub ahead of print]. Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 In these patients, if signs are of sepsis it is very important to suspect the possibility of infection by Mucor and initiate empiric antifungal treatment with surgery to avoid high mortality. Surprisingly, in our series, determination of procalcitonin showed high levels in spite of not having value in fungal infection. 1. Conway Morris A, et al. Thorax. 2010;65:201-7. Infectious events and prescription of antimicrobials in the coronary ICU CE Bosso1, SV Ferreira2, GE Valerio2, JV Moraes2, V Raso2 1Instituto do Coração de Presidente Prudente, Brazil; 2Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P91 (doi: 10.1186/cc14171) Introduction The eff ectiveness of initially used antimicrobials represents an important factor in infectious events in coronary intensive care units (CICU) [1]. This study aimed to analyze the prevalence of infectious events and the prescribed antimicrobial in CICU. Introduction The eff ectiveness of initially used antimicrobials represents an important factor in infectious events in coronary intensive care units (CICU) [1]. This study aimed to analyze the prevalence of infectious events and the prescribed antimicrobial in CICU. Results Mycoplasmataceae were found in 48% of patients with VAP, compared with 14% of patients without VAP (P <0.0001). Patients with sterile lavage had a similar prevalence to healthy donor lavage (10 vs. 8%, P = 0.54). The commonest organism identifi ed was M. salivarium. Human blood monocytes and MDM incubated with M. salivarium displayed impaired cytokine responses to lipopolysaccharide and MDM demonstrated impaired phagocytosis. Methods We analyzed the data of 2,005 patients admitted to the CICU for 3 years. The infectious events were based on general characteristics, main sites and outbreaks of infectious events in addition to the main microorganisms and pathogens. The prescription of antimicrobials was analyzed based on the isolated or associated use of antimicrobials. We also analyzed the adequacy of initial empirical antimicrobial according to the microbiological evidence. The general characteristics of events – that is, time, evidence of infection, infections by multidrug-resistant pathogens – are also presented. y Conclusion This study demonstrates a high prevalence of Mycoplasmataceae amongst patients with VAP, with a markedly lower prevalence amongst patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The commonest organism found, M. salivarium, is able to profoundly impair the functions of key immune cells and thus suggests that Mycoplasmataceae may contribute to VAP pathogenesis. Results The prevalence of infection was 4% (n  = 81). Ventilator- associated pneumonia was 35% (n = 28), whereas urinary and primary bloodstream associated with catheters was 14% (n = 11) and 9% (n = 7), respectively. There was 82% (n = 66) evidence of microbiological infection. The main pathogens and microorganisms found were P88 Cutaneous mucormycosis in the ICU Cutaneous mucormycosis in the ICU EH Herrero, M Sánchez, A Agrifoglio, L Cachafeiro, MJ Asensio, B Galván, A García de Lorenzo Hospital La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P88 (doi: 10.1186/cc14168) Results Ninety-eight clinicians (anesthetics n  = 51, emergency medicine n = 21, intensive care medicine n = 26) completed surveys. The best knowledge (76% correct) was for SARS, with less knowledge for anthrax, plague, EVD, and smallpox (60%). We found limited knowledge for chemical warfare agents (20 to 30%). Sixty to 80% of all incorrect responses were over-rated. There was no diff erence in knowledge compared with previous published results [2]. Introduction Mucormycosis is a devastating disease most commonly seen in immunosuppressed individuals. It has the propensity to disseminate in humans and cause rhinocerebral, pulmonary, gastrointestinal, and cutaneous infections. This study focuses on cutaneous mucormycosis, incidence, epidemiologic characteristics and mortality in intensive care medicine. Conclusion Despite national and regional training since previous surveys [2], the results indicate that further training on PPE is required S31 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We present a descriptive study in an ICU between the years 2001 and 2013 on the incidence of patients with cutaneous mucormycosis. Sociodemographics, comorbidities and laboratory data were recorded. Clinical data were collected to calculate the APACHE score. The main outcome was to analyze the epidemiological characteristics of patients with cutaneous mucormycosis and mortality. Results Seven patients were identifi ed with cutaneous mucormycosis between the years 2001 and 2013. The mean age of patients was 52 ± 4, with an APACHE score of 19 ± 9, and 57% died. All patients were admitted for trauma-related injury suff ering blast, abrasive injuries or burns. Mortality among patients with signs of sepsis was 100%, and only in one of them was empirically antifungal therapy started; in the others antibiotic treatment was directed. Among patients without signs of sepsis, the survivor was treated with amputation where mucoral infection was isolated. Procalcitonin rose in all patients with signs of sepsis. P90 Comparative analysis of microfl ora and antibiotic resistance in patients with sepsis in 1999 and 2013 IV Avdoshin, LG Akinchits, ES Konstantinova, MA Shatil, ON Dobrydin, NA Bubnova Saint-Petersburg City Hospital of St. George, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P90 (doi: 10.1186/cc14170) Analysis of Gram-negative rod bacteremia in the surgical and medical ICU Analysis of Gram-negative rod bacteremia in the surgical and medical ICU D Adukauskiene, D Valanciene Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P92 (doi: 10.1186/cc14172) Introduction The aim was to analyze the Gram-negative bacteremia profi le and the predisposing factors for length of stay in the surgical and medical ICU and outcome. Methods Retrospective data analysis of patients during 4 years treated in a surgical and medical ICU with positive blood culture for Gram- negative rod. g Results Gram-negative rod monobacteremia (n = 160) cultures revealed: Escherichia coli (n  = 52, 32.5%), Acinetobacter spp. (n  = 47, 29.4%), Klebsiella spp. (n = 22, 13.7%), Enterobacter spp. (n = 20, 12.5%), Proteus spp. (n = 11, 6.9%), anaerobes (n = 3, 1.9%) and other Gram-negative rods, including Stenotrophomonas maltophilia, Haemophilus infl uenzae, Neisseria meningitidis, Achromobacter spp. and Actinobacillus limirensi (n = 5, 3.1%). Both E. coli and Acinetobacter spp. were responsible for the vast majority of Gram-negative rod monobacteremia (n  = 99, 61.8%, P = 0.0128). Also most often (n = 50, 72.5%, P = 0.049) primary bacteremia was caused by E. coli and Acinetobacter spp. Separate group’s multidrug resistance was found: E. coli in 12 (23.1%) cases, Acinetobacter spp. in 45 (95.7%, P = 0.02), Klebsiella spp. in nine (40.9%), Enterobacter spp. in 11 (55.0%), Proteus spp. in six (54.6%) cases. The vast majority of patients with multidrug-resistant bacteremia were aged over 65 years (n = 64, 77.1%, P = 0.042), stayed in the ICU less than 14 days (n = 70, 84.3%, P = 0.039), and had lethal outcome (n = 74, 89.2%, P = 0.03). Patients who stayed in the ICU less than 14 days presented with primary Gram-negative rod bacteremia (n = 67, 57.7%, P = 0.03), need for mechanical ventilation (n = 90, 77.6%, P = 0.043) and lethal outcome (n  = 112, 96.6%, P  =  0.01). Lethal outcome was confi rmed in patients with primary Gram-negative rod bacteremia (n = 55, 79.7%, P = 0.03), MDR strain (n = 74, 89.2%, P = 0.03), presence of shock (n = 120, 75.0%, P <0.001), mechanical ventilation (n = 133, 74.3%, P <0.001), cancer chemotherapy (n = 18, 90.0%, P = 0.03), and chronic obstructive pulmonary disease (n = 13, 100%, P = 0.03). Conclusion The carriage of MDRO in ICU-admitted patients is important, especially for ESBL-GN. Is carriage of multidrug-resistant organisms a risk factor for nosocomial infections in an infectious diseases ICU? Introduction The objective was to evaluate whether asymptomatic carriage of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and extended-spectrum beta- lactamase producing Gram-negative bacilli (ESBL-GN) on admittance to the ICU of the University Hospital of Infectious Diseases Cluj-Napoca, Romania is a risk factor for infection due to these multidrug-resistant organisms (MDRO) during hospitalization. fi Conclusion We conclude that infection is prevalent even in CICU, and that the microbiological profi le is quite diverse, as well as the antibiotics. This allows us to better understand the profi le of this kind of unit. Reference g g p Methods A prospective study during a 6-month period (June to November 2014), including all adult patients admitted to our ICU. All patients were screened on admittance for nasal MRSA, intestinal VRE and ESBL-GN carriage. Patients admitted with any localization of infections due to these organisms were excluded. Patients were monitored for developing nosocomial infections due to MDRO during hospitalization. We evaluated previous colonization as a risk factor for future infections. We used bioMerieux selective chromogenic media for MDRO for screening and Vitek2Compact for identifi cation. Statistical analysis was performed with chi-square test and univariate analysis. 1. Silva E, et al. Crit Care. 2004;8:R251-60. y p q y Results From 119 screened adult patients, 65 women (54.6%), average age 67  years, we had at screening on admittance into the ICU: 14 positive MRSA (11.8%), 63 positive ESBL-GN (52.9%  – 41 strains of Escherichia coli, 26 strains of Klebsiella sp., 11 strains of Proteus mirabilis and one strain of Enterobacter cloacae) and 35 positive VRE (29.4% – 33 strains of Enterococcus faecium and two strains of Enterococcus faecalis) without concomitant infection with these MDRO. The average duration of ICU stay was 7.32 days. During hospitalization, 14 patients (11.8%) developed nosocomial infections due to MDRO. Colonization with MDRO preceded nosocomial infections in: one of four patients with MRSA-positive blood cultures (P = 0.96), seven of eight patients with ESBL-GN infections (P = 0.10) and three of four patients with VRE urinary tract infections (P = 0.14). Although not statistically signifi cant, owing to the low number, most patients who developed infections with ESBL-GN had previous intestinal colonization. 1. Silva E, et al. Crit Care. 2004;8:R251-60. P93 Gram-positive bacteria (n = 24, 30%; Staphylococcus aureus (n = 16, 20%), Enterococcus faecalis (n = 4, 5%), Staphylococcus epidermidis (n = 3, 4%)), Gram-negative (n = 43, 53%; klebsiella sp. (n = 13, 16%), Pseudomonas aeruginosa (n = 7, 9%), Escherichia coli (n = 7, 9%)) and fungi (n = 5, 6%; candida sp. (n  = 2, 3%), Candida albicans (n  = 1, 1%), Candida dubliniensis (n  = 1, 1%)). The commonly prescribed antimicrobials were piperacillin/tazobactam (n = 32, 40%), vancomycin (n = 30, 37%), polymyxin B (n = 23, 28%), cefepime (n = 16, 20%), meropenem (n = 12, 15%), cefuroxime (n = 8, 10%), ciprofl oxacin (n = 6, 7%), tigecycline (n = 6, 7%), ampicillin (n = 5, 6%), clindamycin (n = 4, 5%), chloramphenicol (n = 4, 5%), oxacillin (n = 4, 5%) and others (n = 32, 28%). There was 75% (n = 46) infection during hospitalization in the unit. Approximately 32% of infections were caused by multidrug-resistant pathogens, although there was effi ciency of 81% in the proper use of initial antimicrobials. Conclusion We conclude that infection is prevalent even in CICU, and that the microbiological profi le is quite diverse, as well as the antibiotics. This allows us to better understand the profi le of this kind of unit. Reference Is carriage of multidrug-resistant organisms a risk factor for nosocomial infections in an infectious diseases ICU? M Lupse1, M Flonta2, L Herbel2, A Petrovan2, A Binder2, N Todor3, A Cioara1 1University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Teaching Hospital of Infectious Diseases, Cluj-Napoca, Romania; 3‘Ion Chiricuta’ Institute of Oncology, Cluj-Napoca, Romania Critical Care 2015, 19(Suppl 1):P93 (doi: 10.1186/cc14173) p g References S32 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P96 admission; 4.8% required the extrarenal depuration technique. In total, 497 patients (44.1%) were coronary, 49.5% male, mean age 66.18 (SD ±12.6), CI (64.88 to 67.48); mean APACHE II 9.74 (SD ± 6.1), CI (9.1 to 10.3); and a mean time stay of 3.62 days (SD ±4.7), CI (3.1 to 4.1). Mortality in this group was 3.7%. In 61.9% of cases the diagnosis of admission was AMI, 16% arrhythmia and 11.6% unstable angina. Of patients, 629 were polyvalent (55.8%), 53.85% male, mean age 58.05 (SD ±17.2), CI (56.7 to 59.4); mean APACHE II 14.6 (SD ±9.1), CI (13.8 to 15.3); and a mean time stay of 4.64 days (SD ±7.7), CI (4 to 5.25). Mortality was 11.6%. In 33.2% the cause of income was digestive, 23.2% acute or chronic exacerbated respiratory failure, 12.4% severe sepsis/septic shock and 10.1% postoperative cardiovascular surgery. The incidence density (ID) of catheter-related bacteremia was 5.5, 92.8% from the fourth day of ICU admission; ID of ventilator-associated pneumonia (VAP) was 5.94, 88.9% since the fourth day; and ID of urinary tract infections (UTI) related to urinary catheter was 2.88, 80% of them since the fourth day. From all patients who developed intra-ICU infections, mean APACHE II in admission was 21.3 (SD ±9.6) with a mean time stay of 23.4 days (SD ±12.9) and a mortality percentage of 19.6%. P95 Emergence of isolates that are intrinsically resistant to colistin in critically ill patients: are we selecting them out? MN Sivakumar, M Hisham, V Nandakumar, T Gopinathan Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P95 (doi: 10.1186/cc14175) Introduction Poor infection control practices along with irrational usage of antibiotics lead to emergence of multidrug-resistant (MDR) organisms. Increasing use of colistin for treating MDR infections leads to selection of organisms that are intrinsically resistant to colistin. There are limited Indian literatures which evaluated the incidence of intrinsically resistant isolates to colistin in critically ill patients. Our study aimed to investigate the incidence of true pathogen or colonizer with the prior antibiotic exposure and patient’s clinical outcome. Conclusion Being a broad-spectrum bactericidal agent usable both orally and parenterally with low toxicity profi les and lesser prevalence of cross-resistance with other antimicrobials, fosfomycin can be an alternative to other broad-spectrum agents to treat uncomplicated infections as well as in the case of infections with MDR organisms where treatment options are very few. This study possibly reveals a much- needed solution for the rising carbapenem resistance and also for the treatment of infections with such MDR pathogens, thereby bringing down the length of stay in hospital, cost of therapy and suff ering on the part of the patients. Methods The prospective, cross-sectional study was carried out from March 2013 to April 2014. Clinical samples included culture positivity for isolates intrinsically resistant to colistin from patients who were admitted to the ICU or had a prior ICU stay in the same admission. Methods The prospective, cross-sectional study was carried out from March 2013 to April 2014. Clinical samples included culture positivity for isolates intrinsically resistant to colistin from patients who were admitted to the ICU or had a prior ICU stay in the same admission. Results A total of 93 unusual Gram-negative rods were isolated from 76 patients. This included 19.4% (n = 18) Serratia marcescens, 12.9% (n = 12) Stenotrophomonas maltophilia, 14% (n = 13) Burkholderia cepacia, 24.7% (n = 23) Proteus mirabilis, 17.2% (n = 16) Morganella morganii, 9.7% (n = 9) Elizebethkingia meningoseptica and 2.1% (n = 2) Providencia species. A total of 68.4% (n = 52) patients had prior exposure to either colistin or carbapenems or both. In total, 71% (n  = 66) of the total isolates from patients had previous antibiotic exposure. 1. Rit K, et al. IJCP. 2013;24:451-5. 2. Garbati MA, et al. J Infect Dev Ctries. 2013;7:213-6. Analysis of Gram-negative rod bacteremia in the surgical and medical ICU The incidence of nosocomial infections with MDRO in the ICU is high. ESBL-GN intestinal colonization could be a risk factor for nosocomial infections but further studies are needed to confi rm this. Patient epidemiology in a level II hospital ICU and how main nosocomial infections aff ect morbidity and mortality M Muñoz, E Yuste, O Moreno, R Fernandez, R Ramirez Hospital Universitario San Cecilio, Granada, Spain Critical Care 2015, 19(Suppl 1):P94 (doi: 10.1186/cc14174) Introduction We describe the type of patient and the main nosocomial infections in a level II hospital ICU unit, 18 beds (12 polyvalent-general, six coronary). Introduction We describe the type of patient and the main nosocomial infections in a level II hospital ICU unit, 18 beds (12 polyvalent-general, six coronary). y Methods We used the ENVIN-HELICS database and made statistical calculations for all patients admitted to the ICU between 1 October 2012 and 30 September 2013 using SPSS v.15. g Results Patients admitted (1,126): 65.1% were male; mean age 61.72 (SD ±15.8), CI (60.7 to 62.7); mean APACHE II 12.6 (SD ±8.42), CI (12.12 to 13.11); and a mean time stay of 4.84 days (SD ±6.26). In total, 68.9% were provided from the community. A total of 44.1% were coronary, 2.84% trauma and 53.02% medical–surgical patients. A total of 29.8% had antibiotic therapy in the ICU, 20% had it before incoming. In total, 18.38% were treated with artifi cial airway (MV, tracheostomy). In total, 54.09% used a urinary catheter and 38.8% needed a central venous catheter. Fifteen percent of patients had some kind of surgery before Conclusion E. coli and Acinetobacter spp. – the most often pathogens of Gram-negative rod bacteremia – were mostly multidrug resistant. Multidrug-resistant bacteremia was related to age, length of stay less than 14  days, and lethal outcome. Predisposing factors for shorter length of stay: primary bacteremia, mechanical ventilation, lethal outcome, and for lethal outcome: primary bacteremia, multidrug resistance, presence of shock, mechanical ventilation, cancer chemotherapy, chronic obstructive pulmonary disease. S33 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P95 Among the total 93 intrinsically resistant isolates to colistin, 37.6% (n  = 35) of isolates from all clinical sources (endotracheal, pus, urine and blood samples) were true pathogens and the remaining 62.3% (n = 58) were colonizers. There was a statistically signifi cant increase in length of ICU stay and duration of hospitalization in the presence of true pathogen. Conclusion Selection pressure due to extensive use of higher antibiotics may lead to emergence of intrinsically resistant isolates, which narrows the therapeutic options in the ICU. Our study emphasizes the paramount importance of establishing clinical relevance of these organisms before treating them as true pathogens. This calls for judicious use of higher antibiotics, implementation of an antibiotic stewardship program and strict infection control practices. f Kolkata A Chakraborty, S Roy, S Chakraborty, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P96 (doi: 10.1186/cc14176) Introduction In the era of rising prevalence of serious infections caused by multidrug-resistant (MDR) organisms and the paucity of in-fl ow of newer antimicrobial agents, the relatively older antibiotics that had been left out of clinical practice for various reasons are now being increasingly considered as the potential agents to combat such infections. Fosfomycin, known for almost four decades, has a broad spectrum of activity against several Gram-negative and Gram-positive bacteria. Methods This study, conducted in the Microbiology Department of Medica Superspecialty Hospital between July and November 2014, was aimed at testing the in vitro sensitivity of fosfomycin against isolates identifi ed from various clinical specimens from diff erent parts of Kolkata. After confi rming the identity and antibiogram by Microscan Autoscan 4, the isolates were tested for fosfomycin sensitivity by the Epsilometer test. MIC values were interpreted in accordance with the currently recommended Clinical and Laboratory Standards Institute (CLSI) criteria for urinary tract isolates of Escherichia coli and Enterococcus faecalis and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria for Enterobacteriaceae and Staphylococcus aureus. y Conclusion In our ICU the main cause of admission was the polyvalent patient, who is younger and has more severity with not much diff erence in mean time of stay compared with the coronary patient. The intra-ICU infections provide an increase of morbi-mortality risk and consumption of resources. p y Results Out of the 1,895 isolates tested, fosfomycin displayed an overall in vitro susceptibility against 90%, but only 64% against MDR strains. Among the MDR organisms nearly 78% of E. coli and 70% of Klebsiella spp. and 40% of MRSA isolates showed provisional MICs in the sensitive range while among the sensitive strains fosfomycin showed around 92% susceptibility. Our study results were comparable with the results obtained from an Indian study published from CMC Vellore in 2013 showing a fosfomycin susceptibility of around 75% among MDR uropathogenic E. coli. P97 Results A total of 93 unusual Gram-negative rods were isolated from 76 patients. This included 19.4% (n = 18) Serratia marcescens, 12.9% (n = 12) Stenotrophomonas maltophilia, 14% (n = 13) Burkholderia cepacia, 24.7% (n = 23) Proteus mirabilis, 17.2% (n = 16) Morganella morganii, 9.7% (n = 9) Elizebethkingia meningoseptica and 2.1% (n = 2) Providencia species. A total of 68.4% (n = 52) patients had prior exposure to either colistin or carbapenems or both. In total, 71% (n  = 66) of the total isolates from patients had previous antibiotic exposure. Among the total 93 intrinsically resistant isolates to colistin, 37.6% (n  = 35) of isolates from all clinical sources (endotracheal, pus, urine and blood samples) were true pathogens and the remaining 62.3% (n = 58) were colonizers. There was a statistically signifi cant increase in length of ICU stay and duration of hospitalization in the presence of true pathogen. Antibiotic synergy testing for multidrug-resistant Gram-negative pathogens in a Greek ICU E Douka, E Perivoliot, E Kraniotaki, M Nepka, C Routsi, K Fountoulis, A Skoutelis, S Zakynthinos Evangelismos General Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P97 (doi: 10.1186/cc14177) Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma p A Chakraborty, S Roy, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P99 (doi: 10.1186/cc14179) A Chakraborty, S Roy, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P99 (doi: 10.1186/cc14179) Introduction Urinary tract infection (UTI) is one of the most common bacterial infections in humans. Gram-negative organisms being the most common causative agent, the rising prevalence of resistance to a number of antibiotics and more importantly the production of extended spectrum beta-lactamase (ESBL) by these organisms is a growing concern worldwide. As the scenario is no better in community isolates, the choice of empirical antimicrobials for such infections becomes a great challenge for the clinicians. fi Results Against 59 MDR A. baumannii strains, the synergy eff ect of CRB/ COL was 55.9%, RIF/COL 38.9%, CRB/GEN 22%, CRB/AMK 20.3% and TG/COL 16.9%, respectively. Against 41 K. pneumoniae strains, synergy rates were: CRB/COL 43.9%, CRB/GEN 31.7%, PIP/TAZ/GEN 29.2% and TG/COL 24.4% respectively. Against 64 P. aeruginosa strains, synergy rates were: AMK/PIP/TAZ 64.6%, AMK/AZT 64.6%, AMK/CEF 58.3%, CRB/ COL 52%, AMK/CRB 25%. Conclusion The most eff ective combination for both the A. baumannii and K. pneumoniae strains tested was CRB/COL. The next most eff ective combination was RIF/COL and CRB/GEN respectively. No competitive eff ect was observed for RIF/COL combination in all cases tested. The most eff ective combinations for P. aeruginosa strains were AMK plus PIP/TAZ or AZT or CEF. The next most eff ective combination was CRB/ COL. We recommend implementation of an antibiotic synergy test for MDR pathogens as a routine antimicrobial test in the hospitals’ microbiology laboratories, especially for critically ill patients, since some combinations seem to excel. Further studies are needed for the correlation of these combinations with clinical effi cacy. Methods In this retrospective observational study we aimed at knowing the prevalence of ESBL production by organisms causing UTI in the community and to study the antibiogram of such isolates. Urine samples from patients with suspected UTI in the community were cultured for uropathogen by routine microbiological methods and susceptibility testing was done on Microscan Autoscan 4 (Siemens). y Results Out of 527 isolates of Enterobactereaceae, 314 (59.58%) were ESBL producers from the community samples compared with 315 (67.30%) from hospital samples, with Escherichia coli being the most commonly isolated pathogen. Enterobacter spp. P100 Is it possible to predict multidrug-resistant organism colonization and/or infection at ICU admission? F Callejo-Torre1, JM Eiros2, S Ossa-Echeverri1, P Olaechea3, F Alvarez-Lerma4, M Palomar5, Envin-Helics Study Group1 1Hospital Universitario de Burgos, Spain; 2Hospital Clínico Universitario de Valladolid, Spain; 3Hospital de Galdakao-Usansolo, Galdakao, Spain; 4Hospital del Mar, Barcelona, Spain; 5Hospital Arnau de Villanova, Lleida, Spain Critical Care 2015, 19(Suppl 1):P100 (doi: 10.1186/cc14180) y Methods We retrospectively assessed two hypothetical empirical antibiotic treatment algorithms for VAP on an 18-bed ICU. Data on consecutive episodes of microbiologically confi rmed VAP were collected over a period of 22  months and divided into a derivation (1 February 2013 to 30 November 2013) and validation (1 December 2013 until 15 November 2014) cohort. We constructed two algorithms in the derivation cohort. One is a local ecology-based algorithm (LEBA), according to clinical risk factors for MDR and susceptibility results in our hospital. The other is a Gram stain-based algorithm (GSBA). The selection of antibiotics on GSBA was directed against pathogens predicted from the results of bedside Gram staining of tracheal aspirates collected just before antibiotic therapy. Subsequently, LEBA and GSBA were retrospectively reviewed and compared with actually prescribed antibiotics in the validation cohort.i p Critical Care 2015, 19(Suppl 1):P100 (doi: 10.1186/cc14180) Introduction We tried to develop a predictive model for patients colonized/infected by any multidrug-resistant organism (MDRO-C/I) at ICU admission based on risk factors easy to obtain (not depending on complex clinical records), being aware that foreseeing MDRO-C/I at ICU admission is key for appropriate empirical treatment and infection control. p Results The fi rst 50 VAP episodes made up the derivation cohort and the subsequent 50 VAP episodes the validation cohort. Antibiotic coverage rates by applying LEBA and GSBA were identical (96% vs. 96%). GSBA proposed more narrow spectrum therapy as compared with LEBA (P <0.001). GSBA recommended carbapenems in signifi cantly less episodes than LEBA (P <0.001) and the same episodes as actually prescribed initial therapy (P  =  1). However, there was signifi cant increase of antibiotic coverage rates in GSBA compared with the actually prescribed initial therapy (96% vs. 78%, P = 0.015). Conclusion Antibiotic coverage rates on GSBA were comparable with LEBA. The use of GSBA would result in a signifi cant reduction of the administration of broad-spectrum antibiotics. Bedside Gram staining may be useful to guide appropriate initial antibiotic therapy for VAP. Skewed antibiogram of community-acquired urinary isolates and the therapeutic dilemma showed highest prevalence (80%) of ESBL production from the community samples. Among the ESBL producing strains from the community, the sensitivity to ciprofl oxacin, levofl oxacin and nitrofurantoin was 18%, 21% and 44% respectively while in the non-ESBL producers the sensitivity rates were 52%, 51% and 73% respectively. P98 Development of antibiotic treatment algorithms based on Gram stain to restrict use of broad-spectrum antibiotics in the treatment of ventilator-associated pneumonia: a retrospective analysis J Yoshimura, T Kiguchi, A Matsushima, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P98 (doi: 10.1186/cc14178) P98 Development of antibiotic treatment algorithms based on Gram stain to restrict use of broad-spectrum antibiotics in the treatment of ventilator-associated pneumonia: a retrospective analysis J Yoshimura, T Kiguchi, A Matsushima, S Fujimi Osaka General Medical Center, Osaka, Japan Critical Care 2015, 19(Suppl 1):P98 (doi: 10.1186/cc14178) p y Conclusion Organisms producing the ESBL phenotype present with an added possibility of being resistant to other broad-spectrum antimicrobial agents which are commonly prescribed in the community to empirically treat such infections. This makes the choice of empirical antibiotic much more challenging in the community, drawing errors in judgment. A possibility of frequent overcorrection lies on the other side of the coin. This study also shows the possible need for empirical institution of class I carbapenems as one of the treatment options and outpatient parenteral antimicrobial therapy. Introduction Ventilator-associated pneumonia (VAP) is a common and serious problem in ICUs. Several studies have been conducted to determine the eff ectiveness of Gram stain of tracheal aspirates for diagnosing VAP. However, the eff ectiveness for predicting causative microorganisms and guiding appropriate initial antibiotic therapy has not been evaluated. The purpose of this study is to determine whether Gram stain of tracheal aspirates can guide appropriate initial antibiotic therapy for VAP. Antibiotic synergy testing for multidrug-resistant Gram-negative pathogens in a Greek ICU Introduction The emergence of multidrug-resistant (MDR) pathogens is a major cause of infection-related mortality among critically ill patients. The synergistic eff ect between commonly used antibiotics against diffi cult to treat nosocomial MDR Gram-negative strains, if present, could provide a viable option as an alternative therapy. The aim of this study was to investigate the potential of antibiotic synergy against MDR A. baumannii, K. pneumonia and P. aeruginosa strains, isolated from critically ill patients in a Greek ICU. y Methods We tested 59 A. baumannii, 41 K. pneumoniae and 64 P. aeruginosa strains, isolated during the period 2010 to 2013. All strains were resistant to carbapenems and showed reduced susceptibility or resistance to tigecycline or colistin (MIC >2), in accordance with CLSI guidelines. We evaluated double-drug combinations of carbapenem (CRB)/colistin (COL), tigecycline (TG)/COL, rifampicin (RIF)/COL, CRB/ gentamicin (GEN), CRB/amikacin (AMK) for A. baumannii, TG/COL, S34 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 CRB/COL, piperacillin–tazobactam (PIP/TAZ)/GEN, CRB/GEN for K. pneumoniae and AMK/(PIP/TAZ), AMK/aztreonam (AZT), AMK/cefepime (CEF), AMK/CRB and CRB/COL for P. aeruginosa strains. In order to perform synergy tests, the E-test methodology (BioMerieux, Marcy l’E’toile, France) was used. Synergy was defi ned as a fraction inhibitory concentration (FIC) index ≤0.5, additive eff ect 0.5 to 1, indiff erent or antagonistic eff ect >2 (Lorian defi nition). CRB/COL, piperacillin–tazobactam (PIP/TAZ)/GEN, CRB/GEN for K. pneumoniae and AMK/(PIP/TAZ), AMK/aztreonam (AZT), AMK/cefepime (CEF), AMK/CRB and CRB/COL for P. aeruginosa strains. In order to perform synergy tests, the E-test methodology (BioMerieux, Marcy l’E’toile, France) was used. Synergy was defi ned as a fraction inhibitory concentration (FIC) index ≤0.5, additive eff ect 0.5 to 1, indiff erent or antagonistic eff ect >2 (Lorian defi nition).f P103 Novel infl uenza A antibodies reduce severity of secondary pneumococcal pneumonia after infl uenza infection in mice KF Van der Sluijs, F Van Someren Greve, MD De Jong, MJ Schultz, NP Juff ermans Novel infl uenza A antibodies reduce severity of secondary pneumococcal pneumonia after infl uenza infection in mice KF Van der Sluijs, F Van Someren Greve, MD De Jong, MJ Schultz, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P103 (doi: 10.1186/cc14183) y Methods Data were collected prospectively from 69,894 patients admitted consecutively (stay >24  hours) to 147 Spanish ICUs participating in the National Surveillance Study of Nosocomial Infections in ICU registry (ENVIN) during April to June 2006 to 2010. Univariable and multivariable analysis was performed for both objectives but we used only easy-to-obtain variables for the predictive model exclusively from those admitted in 2010 (n  = 16,950, 2/3 for analysis and 1/3 for subsequent validation). Introduction Secondary bacterial pneumonia after infl uenza infection can cause severe disease with a high mortality. Recently, a new group 2 infl uenza A antibody (AT10_002) has been developed, which binds to multiple H3 and H7 subtypes. In a mouse model of primary infl uenza infection, treatment with AT10_002 as a fusion antibody protects against lethal infection, and reduces loss of bodyweight [1]. We hypothesized that treatment with AT10_002 reduces weight loss, lung injury and bacterial outgrowth, in a mouse model of viral infection followed by secondary pneumococcal infection. y q Results In the 2006 to 2010 period, 1,046 were C/I by MRSA (note that relative risks are not included due to space limitations). First objective: previous antibiotic, APACHE II score >18, skin-soft tissue or postsurgical superfi cial skin infections, trauma or medical patient, age >65 (especially >75), urinary catheter and admitted from a long- term care facility were independent risk factors for MRSA-C/I in ICU. Multicolonization increased signifi cantly the risk of MRSA-C/I, and immunodefi ciency and gender male emerged as protective factors. Second objective: independent risk factors on ICU admission were male gender, trauma critical patient, urgent surgery, admitted from other ICU, community or long-term facility, being immunosuppressed and skin-soft tissue infection. All confi gured the risk model for which, although showing good discrimination (AUC-ROC, 0.77; 95% CI, 0.72 to 0.82), sensitivity (67%) and specifi city (76.5%) were insuffi cient for the ICU setting. P101 P101 Methicillin-resistant Staphylococcus aureus in the ICU: risk factors and a predictive model to detect it at ICU admission F Callejo-Torre1, JM Eiros2, S Ossa-Echeverri1, P Olaechea3, M Palomar4, F Alvarez-Lerma5, Envin-Helics Study Group1 1Hospital Universitario de Burgos, Spain; 2Hospital Clínico Universitario de Valladolid, Spain; 3Hospital de Galdakao-Usansolo, Galdakao, Spain; 4Hospital Arnau de Villanova, Lleida, Spain; 5Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P101 (doi: 10.1186/cc14181) g y p y Results Among 41 patients, 15 (37%) underwent FMS. There was no diff erence in age, sex, underlying disease, target temperature management, and prophylactic antibiotic use between two groups. Mean duration of ECMO was 4 days in both groups. The incidence of bacteremia was none in the group with FMS and fi ve (19%) in the group without FMS. Within fi ve cases of bacteremia, three were caused by enterobacterium. Introduction Being capable of predicting MRSA on ICU admission is crucial to enhance infection control and to avoid inappropriate empirical treatment. Two objectives were studied: to describe risk factors for MRSA colonization/infection (MRSA-C/I) once admitted to the ICU; and to develop a predictive model at ICU admission, based on easy-to-obtain admission factors. Conclusion FMS may be protective against bacteremia for OHCA patients undergoing ECMO. Protective eff ect of a fecal incontinence management system against bacteremia for out-of-hospital cardiac arrest patients undergoing extracorporeal membrane oxygenation S Kikuta, R Miki, S Ishihara, S Nakayama Hyogo Emergency Medical Center, Chuo, Kobe, Hyogo, Japan Critical Care 2015, 19(Suppl 1):P102 (doi: 10.1186/cc14182) Introduction Recently, extracorporeal cardiopulmonary resuscitation (ECPR) has become a common measure against cardiopulmonary arrest. In cases with ECPR, we usually insert cannulae for extracorporeal membrane oxygenation (ECMO) via the femoral artery and vein. However, the cannulation site is often contaminated by feces due to incontinence. Moreover, patients tend to be compromised by hypothermia due to the target temperature management, so we often experience central line-associated bloodstream infection of patients undergoing ECMO. We investigated the protective eff ect of a fecal incontinence management system (FMS) against bacteremia in patients undergoing ECMO. Conclusion MDRO prediction at ICU admission could not be based merely on clinical–demographic risk factors. Taking into account local particularities and combining risk factors with a rapid laboratory test might be the most eff ective way forward. p g g Methods We studied 41 consecutive patients undergoing ECMO for out-of-hospital cardiac arrest (OHCA) between April 2010 and May 2014. Patients were divided into two groups according to the use or no use of FMS (Flexi-Seal™). Patients who died within 48 hours or from whom cannulae for ECMO were removed within 48  hours were excluded. Patients’ characteristics, underlying disease, target temperature management, prophylactic antibiotic use and incidence of bacteremia during admission were recorded and analyzed retrospectively. Results Among 41 patients, 15 (37%) underwent FMS. There was no diff erence in age, sex, underlying disease, target temperature management, and prophylactic antibiotic use between two groups. Mean duration of ECMO was 4 days in both groups. The incidence of bacteremia was none in the group with FMS and fi ve (19%) in the group without FMS. Within fi ve cases of bacteremia, three were caused by enterobacterium. g g Methods We studied 41 consecutive patients undergoing ECMO for out-of-hospital cardiac arrest (OHCA) between April 2010 and May 2014. Patients were divided into two groups according to the use or no use of FMS (Flexi-Seal™). Patients who died within 48 hours or from whom cannulae for ECMO were removed within 48  hours were excluded. Patients’ characteristics, underlying disease, target temperature management, prophylactic antibiotic use and incidence of bacteremia during admission were recorded and analyzed retrospectively. P102 (relative risk not shown due to space limitation): age 65 to 74, medical or surgical critical patient (especially urgent surgery), admitted from other ICU or long-term facility, immunosuppression and deep postsurgical skin or skin-soft tissue infections. Admitted from the community and female gender emerged as protective factors. Although the predictive model showed good discrimination (AUC-ROC = 0.775 (95% CI, 0.744 to 0.807)), sensitivity was only 67.4%. Validation with the remaining 4,952 patients (1/3) showed an AUC-ROC = 0.712 (95% CI, 0.665 to 0.759) and a P value on the Hosmer–Lemeshow goodness of fi t test of 0.855. Even creating a new model, including variables obtained after ICU admission (severity by APACHE score, mechanical ventilation, central venous, arterial or urinary catheter, immunodefi ciency, parenteral nutrition, ventricular derivation, extrarenal depuration, non-invasive ventilation, tracheotomy, enteral nutrition and nasogastric tube), prediction capability did not improve (AUC-ROC = 0.801 (95% CI, 0.774 to 0.828), sensitivity 71.4%). Protective eff ect of a fecal incontinence management system against bacteremia for out-of-hospital cardiac arrest patients undergoing extracorporeal membrane oxygenation S Kikuta, R Miki, S Ishihara, S Nakayama Hyogo Emergency Medical Center, Chuo, Kobe, Hyogo, Japan Critical Care 2015, 19(Suppl 1):P102 (doi: 10.1186/cc14182) P100 Results The fi rst 50 VAP episodes made up the derivation cohort and the subsequent 50 VAP episodes the validation cohort. Antibiotic coverage rates by applying LEBA and GSBA were identical (96% vs. 96%). GSBA proposed more narrow spectrum therapy as compared with LEBA (P <0.001). GSBA recommended carbapenems in signifi cantly less episodes than LEBA (P <0.001) and the same episodes as actually prescribed initial therapy (P  =  1). However, there was signifi cant increase of antibiotic coverage rates in GSBA compared with the actually prescribed initial therapy (96% vs. 78%, P = 0.015). Methods Data were collected prospectively from admission to discharge of 16,950 patients admitted consecutively (at least >24  hours) to 147 Spanish ICUs of the ENVIN (National Surveillance Study of Nosocomial Infections in ICUs) registry, from April to June 2010. To create the predictive model, 11,998 (2/3) patients were used for univariable and multivariable logistic regression model and 4,952 (1/3) for subsequent validation. Results With a MDRO prevalence of 2.12% (359 MDROs at ICU admission were detected in 314 patients), 87.58% patients had only one MDRO, meanwhile 12.42% were MDRO-C/I by two or more simultaneously. Risk factors used in the development of the predictive model and independently associated with MDRO-C/I at ICU admission were Conclusion Antibiotic coverage rates on GSBA were comparable with LEBA. The use of GSBA would result in a signifi cant reduction of the administration of broad-spectrum antibiotics. Bedside Gram staining may be useful to guide appropriate initial antibiotic therapy for VAP. S35 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Source of MDR infections in an ICU: busting the myth R Agrawal Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Results In total, 1,499 patients were included, of whom 265 patients (18%) had a viral respiratory tract infection with at least one virus. In 17 patients, two viruses were found; two patients had an infection with three viruses. The most prevalent was parainfl uenzavirus-3 (5.7%); 17 patients (1.1%) had an infection with infl uenza. The lowest prevalence of viral infections occurred in September (12%), the highest in October and February (both 26%). Of the patients tested positive in TA, only 46% also tested positive in NP. The median cp values were not signifi cantly diff erent between TA and NP swabs (31.1 vs. 31.6, P = 0.75). Introduction MDR infections in the ICU are not nosocomial all the time, as perceived commonly. We performed a 2-year retrospective study to analyze the source of culture positivity in a medical ICU and to identify which types of infections are more prevalent. Methods The data of a 35-bed medical ICU were analyzed from November 2012 to October 2014. The source of culture positivity was divided into three groups: patients admitted from the ER to the ICU who were referred from other hospitals or direct admissions, the second group was patients admitted within the hospital but outside the ICU for the fi rst 48 hours, and the third group was ICU-acquired infections. We also analyzed the data for type of infections, whether Gram-negative, Gram-positive or fungal. Conclusion The prevalence of viral respiratory tract infections is high in unselected ICU patients. Testing tracheal aspirate in combination with nasopharynx greatly increased detection of viruses, and yields similar cp values. Whether these viral infections are associated with prolonged mechanical ventilation and worse outcomes remains to be determined. g , p g Results There were 1,051 cultures positive in a 2-year period. In total, 46.8% (n = 492) of cultures were already positive on admission, which denotes community-acquired and referred patients from other hospitals. A total of 31.1% (n  = 327) of cultures were positive from patients admitted to general wards for more than 48 hours and then transferred to the ICU. Twenty-two percent (n = 232) of cultures were ICU-acquired infections. The data show community-acquired and hospital-acquired infections are the bulk of the culture load in an ICU. P103 Afterwards validation with the remaining 4,952 (1/3) showed AUC-ROC  = 0.72 (95% CI, 0.65 to 0.79) and P value on the Hosmer–Lemeshow goodness of fi t test = 0.539. The model did not improve even after including more complex variables (AUC-ROC = 0.82; 95% CI, 0.77 to 0.86, sensitivity 63.64%, specifi city 78.48%). y y p Methods Male C57Bl/6 mice were intranasally inoculated with 400 TCID50 Infl uenza A (H3N2). Two days after infection, mice were injected with either AT10_002 i.v. (n = 8) or a control antibody (n = 7). After 7 days, both groups were intranasally inoculated with 5 × 103 S. pneumoniae type 3 and were sacrifi ced 18  hours later. Outcome measures were weight loss, wet lung weight, cell count in bronchoalveolar lavage fl uid (BALF), and colony-forming units (CFUs) in lung homogenate. Data are represented as medians, and treatment groups are compared using nonparametric tests.i p Results Mice receiving AT10_002 showed signifi cantly lower weight loss at the time of sacrifi ce compared with the control group (+1% vs. –12% change in weight; P = 0.0003). Also wet lung weight was lower (68 vs. 96 mg; P = 0.0003), cell counts in BALF were lower (4.9 × 105 vs. 7.0 × 105 cells/ml; P = 0.0037) and CFUs in lung homogenate were lower (33 vs. 25 × 104 CFUs/mg; P = 0.0003) compared with controls. Conclusion Early treatment with infl uenza antibody AT10_002 signifi cantly reduces weight loss, lung injury and bacterial outgrowth, i Conclusion Independent risk factors for MRSA-C/I in the ICU and at ICU admission are described. To predict MRSA-C/I at ICU admission we should not rely on clinical–demographic risk factors alone. Its combination with a rapid laboratory test could be the way to proceed in future studies. Conclusion Early treatment with infl uenza antibody AT10_002 signifi cantly reduces weight loss, lung injury and bacterial outgrowth, S36 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 hospitals – 17 high-volume care units) in the Czech Republic from 1 January 2011 to 5 November 2013. Patients were divided into two groups: survivors (n = 274) and nonsurvivors (n = 261). in a mouse model of infl uenza infection followed by secondary pneumococcal pneumonia. Reference in a mouse model of infl uenza infection followed by secondary pneumococcal pneumonia. P103 Reference g Results Survivors versus nonsurvivors were similar in: age 65.8 (64.2; 67.5) versus 66.5 (64.7; 68.3) P = 0.583, men 159 (58.0%) versus 160 (62.0%) P = 0.376, APACHE II score 27 (15 to 40) versus 28 (15 to 40) P  =  0.737. Statistically signifi cant diff erences between survivors versus nonsurvivors were found in the parameter ‘Consensus initial antimicrobial therapy with microbial cultures’ 178 (79.5%) versus 128 (58.4%) P <0.001 and in the parameter ‘Administration antimicrobials within the fi rst hour’ 163 (59.9%) versus 171 (70.7%) P  =  0.001. Administration of 30  ml/kg crystalloid for hypotension or lactate 4 mmol/l (3 hours) and application of vasopressors (6 hours) were in both groups without statistically signifi cant diff erences. 1. Wagner K, et al. Proc Natl Acad Sci U S A. 2014;111:16820-5. Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock 1 l 1 Š 2 l 1 Š k1 Introduction The early identifi cation of severe sepsis and septic shock and early implementation of the SSC bundles were associated with reduced mortality [1]. The failure to initiate appropriate antimicrobial therapy increased mortality of septic shock patients [2]. We hypothesized that the parameter ‘Consensus initial antimicrobial therapy with microbial cultures’ correlates with outcome of septic shock patients. Conclusion Antibiotic stewardship and strict adherence to infection control protocols in hospitals and guidelines for general practitioners can signifi cantly reduce the load of resistant organisms in the ICU. This may eventually improve patient outcomes and help in preserving the antibiotics for future generations. Methods We analyzed 535 consecutive patients with septic shock (sepsis-induced hypotension persisting despite adequate fl uid resuscitation) from the EPOSS database (Data-based Evaluation and Prediction of Outcome in Severe Sepsis), which was developed to monitor and assess treatment effi cacy in patient with severe sepsis and septic shock. Patients were admitted to participating ICUs (12 P105 P105 Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock P Szturz1, P Folwarczny1, J Švancara2, R Kula1, P Ševèík1 1University Hospital and Faculty of Medicine Ostrava University, Ostrava, Czech Republic; 2Institute of Biostatistic and Analyses, Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P105 (doi: 10.1186/cc14185) P105 Adequate initial antimicrobial therapy as the factor assessing treatment effi cacy in human septic shock P Szturz1, P Folwarczny1, J Švancara2, R Kula1, P Ševèík1 1University Hospital and Faculty of Medicine Ostrava University, Ostrava, Czech Republic; 2Institute of Biostatistic and Analyses, Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P105 (doi: 10.1186/cc14185) Prevalence of viral respiratory tract infections in acutely admitted and ventilated ICU patients: a prospective multicenter observational study y F Van Someren Greve1, KF Van der Sluijs1, R Molenkamp1, AM Spoelstra-de Man2, OL Cremer3, RB De Wilde4, PE Spronk5, MD De Jong1, MJ Schultz1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU Medical Center, Amsterdam, the Netherlands; 3University Medical Center Utrecht, the Netherlands; 4Leiden University Medical Center, Leiden, the Netherlands; 5Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P104 (doi: 10.1186/cc14184) g p y gif Conclusion We found that correct choice of antibiotics improves outcome of septic shock patients. The choice of empirical antimicrobial therapy depends on complex factors related to the underlying disease, susceptibility of pathogens, patient’s history and clinical syndrome. Adequate initial antimicrobial therapy as an important factor of survival along with suitable initial fl uid resuscitation and application of vasopressors should be a priority for healthcare in human septic shock. References p p Critical Care 2015, 19(Suppl 1):P104 (doi: 10.1186/cc14184) Introduction The prevalence of viral respiratory tract infections in critically ill patients is uncertain, as well as the optimal diagnostic method to detect these. The aim of this study was to assess the prevalence of viral respiratory tract infections in mechanically ventilated patients, in both the upper and lower respiratory tract.i Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Source of MDR infections in an ICU: busting the myth R Agrawal FEHI, New Delhi, India Critical Care 2015, 19(Suppl 1):P106 (doi: 10.1186/cc14186) Source of MDR infections in an ICU: busting the myth R Agrawal This could be attributed to increased surveillance and adherence to infection control practices in the ICU which may not be followed stringently in other parts of the hospital. Overuse of broad-spectrum antibiotics in community and primary care hospitals has resulted in a spurt in growth of resistant infections. This has reached an alarming level in developing countries. Out of total cultures positive 78.3% (n = 822) were Gram-negative infections which included community-based and non-ICU infections. References 1. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 2. Kumar A, et al. Crit Care Med. 2006;34:1589-96. 1. Dellinger RP, et al. Crit Care Med. 2013;41:580-637. 2. Kumar A, et al. Crit Care Med. 2006;34:1589-96. Methods A prospective observational study was performed in fi ve ICUs in the Netherlands. From September 2013 to April 2014, consecutive acutely admitted, mechanically ventilated patients were included, regardless of diagnosis at admission. Nasopharyngeal (NP) swabs and tracheal aspirates (TA) were collected at intubation, and were tested via multiplex RT-PCR for the following viruses: infl uenza A and B, parainfl uenzaviruses, RSV, human metapneumoviruses, bocaviruses, coronaviruses, rhinoviruses, enteroviruses, parechoviruses and adenoviruses. Viral DNA/RNA copies were expressed by crossing-point (cp) values. Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Positive Agreement Sensitivity Specifi city culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/93.8 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/92.4 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/88.4 (BAL/ETA) Conclusion Sensitivity and specifi city of the new molecular approach for these main bacteria found in VAP could enable targeted fi rst-line antibiotic therapy. In the future, the development of this approach will aim at obtaining a bedside diagnostic in only a few hours. P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay for rapid (<1 hour) detection of bacteria carrying carbapenem-resistance genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study is to compare PCR with microbiological cultures in critically ill, abdominal Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Positive Agreement Sensitivity Specifi city culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/93.8 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/92.4 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/88.4 (BAL/ETA) Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ETA in VAP patients Introduction Antimicrobial resistance constitutes a growing global threat, driven in part by inappropriate antimicrobial prescribing [1]. Most hospitals implement antibiotic policies to promote antimicrobial stewardship. This audit examined the Royal Cornwall Hospital Trust (RCHT) Critical Care Department’s compliance with the current standard defi ned in our local antimicrobial policy. This states that all antimicrobial prescriptions are to have an indication and review date recorded [2]. Sequential strategies to improve compliance were introduced prior to re-auditing the eff ects. Methods The RCHT Critical Care Department utilizes the Phillips Care Vue electronic patient record. P107 C P107 the ICU stay. We obtained two rectal swab specimens and two drainage samples to perform PCR assay and classic culture tests. We used Cohen’s K to test concordance of results. We considered concordant those results of positive detection of carbapenemase-producing bacteria by both methods (even if a polymicrobial growth was observed by cultures) or negative results by both methods. Concordance was studied for rectal swab and drainage specimens. Antibiotic susceptibility testing was performed through a semiquantitative method. the ICU stay. We obtained two rectal swab specimens and two drainage samples to perform PCR assay and classic culture tests. We used Cohen’s K to test concordance of results. We considered concordant those results of positive detection of carbapenemase-producing bacteria by both methods (even if a polymicrobial growth was observed by cultures) or negative results by both methods. Concordance was studied for rectal swab and drainage specimens. Antibiotic susceptibility testing was performed through a semiquantitative method. P107 Concordance between a new molecular real-time approach and traditional culture in suspected VAP patients M Clavel1, O Barraud2, V Moucadel3, MC Ploy2, E Karam4, F Meynier3, B François for Valibi Study Group5 1Hopital Dupuytren, Limoges, France; 2UMRS-1092, Hopital Dupuytren, Limoges, France; 3bioMérieux SA, Grenoble, France; 4Service de Réanimation, Brive, France; 5Inserm, Limoges, France Critical Care 2015, 19(Suppl 1):P107 (doi: 10.1186/cc14187) Results Eight complete samples sets were collected from seven patients. Seven rectal swab specimens were negative for both PCR and cultures. In one patient a positive culture from carbapenem-resistant P. aeruginosa was detected from the rectal swab resulting negative to PCR. In one patient a positive culture from carbapenem-resistant A. baumanii was detected by drainage culture resulting negative to PCR. In two cases a positive result was observed from both PCR and cultures of rectal swab and drainage specimens. Vim and KPC genes were detected in one case and A. baumanii and K. pneumoniae with carbapenem resistance were isolated from cultures. A KPC gene was detected by PCR in the other case, and K. pneumoniae with carbapenem resistance was isolated from cultures. In all other cases a negative result was observed by both PCR and cultures. Cohen’s K of 0.71 (95% CI = 0.21 to 1) was observed for rectal swab and drainage specimens. Introduction Early microbiological documentation may reduce attributable mortality and excessive use of broad-spectrum antibiotics in ventilator-associated pneumonia (VAP). P107 C Using bronchoalveolar lavage (BAL) and endotracheal aspirates (ETA), we studied a new molecular biology-based approach to detect and quantify bacteria in less than 3 hours. This prospective multicenter trial aimed at comparing the microbiological results obtained using this molecular protocol (easyMAG® system) and semiquantitative culture in suspected VAP. y y q p Methods ETA and BAL samples were consecutively collected during 10 months in adult patients in four ICUs of France. The molecular method includes a preprocessing liquefaction for ETA before DNA extraction. DNAs were extracted using the easyMAG® system. Real- time PCR (qPCR) was run using the ABI7500FastDx PCR instrument. The results presented here concern: Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacteriaceae. Quantifi cation was performed using qPCR standard curves, by converting the cycle threshold to CFU/ ml. Conclusion We need more data to evaluate the performance of PCR for rapid detection of carbapenemase-producing bacteria from rectal swabs and drainage of critically ill surgical patients even though its concordance with cultures seems to be good. Results A total of 125 suspected VAP were included from 122 patients. In total, 125 BAL and 107 ETA were collected. Sex ratio (M/F) was 76%, and CPIS ≥6 was calculated in 74.6% of the suspected VAP patients. Mean ventilation duration before sampling was 6 days. Seventy-eight percent and 65% of the BAL and ETA culture were positive respectively. Correlations between molecular method and culture on BAL and ETA are reported in Table 1. Use of an electronic medical record system to improve antimicrobial stewardship P Allan, M Newman, J Collinson, L Bond, W English Royal Cornwall Hospital NHS Trust, UK Critical Care 2015, 19(Suppl 1):P109 (doi: 10.1186/cc14189) Data from this system were interrogated at three stages to assess our compliance with the trust’s antimicrobial policy. The fi rst data interrogation was performed prior to any intervention, and refl ected baseline antimicrobial prescribing habits. The second data interrogation was performed during a period of active antibiotic stewardship promotion. The third data interrogation was performed following the addition of a care bundle to the prescribing module of Care Vue. This daily tick-box prompt reminded clinicians to check that all antimicrobial prescriptions had an indication and review date recorded. The records of all of the patients admitted to the critical care department during the periods of data interrogations were assessed for antimicrobial indication and review date transcription.i References . Klevens, et al. Estimating healthcare associated infections. Public Health Rep. 2007;122:160-6. . Klevens, et al. Estimating healthcare associated infections. Public Health Rep. 2007;122:160-6. . Hecker, et al. Unnecessary use of antimicrobials. Arch Intern Med. 2003;163:972-8. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S37 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P107 Concordance between a new molecular real-time approach and traditional culture in suspected VAP patients M Clavel1, O Barraud2, V Moucadel3, MC Ploy2, E Karam4, F Meynier3, B François for Valibi Study Group5 1Hopital Dupuytren, Limoges, France; 2UMRS-1092, Hopital Dupuytren, Limoges, France; 3bioMérieux SA, Grenoble, France; 4Service de Réanimation Brive, France; 5Inserm, Limoges, France Critical Care 2015, 19(Suppl 1):P107 (doi: 10.1186/cc14187) Introduction Early microbiological documentation may red attributable mortality and excessive use of broad-spectrum antibio in ventilator-associated pneumonia (VAP). Using bronchoalveo lavage (BAL) and endotracheal aspirates (ETA), we studied a n molecular biology-based approach to detect and quantify bacteria less than 3 hours. This prospective multicenter trial aimed at compar the microbiological results obtained using this molecular proto (easyMAG® system) and semiquantitative culture in suspected VAP. Methods ETA and BAL samples were consecutively collected dur 10 months in adult patients in four ICUs of France. The molecu method includes a preprocessing liquefaction for ETA before D extraction. DNAs were extracted using the easyMAG® system. Re time PCR (qPCR) was run using the ABI7500FastDx PCR instrument. T results presented here concern: Staphylococcus aureus, Pseudomo aeruginosa and Enterobacteriaceae. Quantifi cation was perform using qPCR standard curves, by converting the cycle threshold to C ml. Results A total of 125 suspected VAP were included from 122 patie In total, 125 BAL and 107 ETA were collected. Sex ratio (M/F) was 76 and CPIS ≥6 was calculated in 74.6% of the suspected VAP patie Mean ventilation duration before sampling was 6 days. Seventy-ei percent and 65% of the BAL and ETA culture were positive respectiv Correlations between molecular method and culture on BAL and are reported in Table 1. Table 1 (abstract P107). Concordance between qPCR and culture on BAL/ in VAP patients Positive Agreement Sensitivity Specifi c culture qPCR (%) (%) (%) S. aureus 28/20 31/25 96.7/89.7 96.6/76.9 96.8/93 (BAL/ETA) P. aeruginosa 23/20 20/23 97.6/93.5 100/100 97.1/92 (BAL/ETA) Enterobacteriaceae 27/7 36/18 90.3/85.0 90.0/58.3 90.4/88 (BAL/ETA) Conclusion Sensitivity and specifi city of the new molecular appro for these main bacteria found in VAP could enable targeted fi rst- antibiotic therapy. References In the future, the development of this approach aim at obtaining a bedside diagnostic in only a few hours. P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay rapid (<1 hour) detection of bacteria carrying carbapenem-resista genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study is compare PCR with microbiological cultures in critically ill, abdom surgical patients. Methods We performed an observational study at University Hospita Giaccone’ Palermo. We enrolled abdominal surgical patients admit to the ICU with suspected abdominal sepsis or developing sepsis dur P108 P108 Use of Cepheid Xpert Carba-R® for rapid detection of carbapenemase-producing bacteria in critically ill, abdominal surgical patients: fi rst report of an observational study A Cortegiani, V Russotto, P Capuano, G Tricoli, DM Geraci, A Ghodousi, L Saporito, G Graziano, A Giarratano University of Palermo, Italy Critical Care 2015, 19(Suppl 1):P108 (doi: 10.1186/cc14188) Results From the fi rst data interrogation, antimicrobial prescriptions had an indication and review date transcription in 57% and 60% of cases respectively. Following the awareness campaign, the indication and review date transcription rate increased to 78% and 85% respectively. A daily electronic prompt was then added to our care bundle list. The fi nal data interrogation, performed after this intervention, demonstrated that the transcription rates for both the indication and the review date had increased to 96%. Introduction Xpert Carba-R® (Cepheid®, USA) is a PCR-based assay for rapid (<1 hour) detection of bacteria carrying carbapenem-resistance genes (KPC, NDM, VIM, OXA-48, IMP-1). The aim of the study is to compare PCR with microbiological cultures in critically ill, abdominal surgical patients. Conclusion We have demonstrated that the use of a daily prompt within an electronic patient record can greatly improve compliance in recording the indication and review date for all antimicrobials. These data support the widespread implementation of an electronic prescribing system where daily reminders are integrated in an eff ort to improve compliance with antimicrobial stewardship. Methods We performed an observational study at University Hospital ‘P. Giaccone’ Palermo. We enrolled abdominal surgical patients admitted to the ICU with suspected abdominal sepsis or developing sepsis during Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S38 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Eventually, the IC was confi rmed for 403/835 patients (peritonitis: 177; candidaemia: 141; deep candidiasis: 61; mixed infection sites: 24). Candida albicans was the main pathogen (67%), then C. glabrata (16%). At inclusion, CIC were treated with caspofungin (Cas): 55%, and fl uconazole (Flu): 34%, whereas these antifungals were administered to 46% and 45% of SIC, respectively. Patients with SIC were more severe than those with CIC. The two main criteria for initiating empirically an AFT were a central venous catheter (79%) and severe septic shock (70%). The rate of change of the initial AFT was higher in the CIC group (49%) than in the SIC group (33%, P <0.0001). P111 Epidemiological cohort study of systemic antifungal therapy for suspected or confi rmed invasive candidiasis in the ICU: the Amarcand2 study J Constantin1, JF Timsit2, JP Gangneux3, JP Mira4, P Montravers2, H Dupont5, P Perrigault6, O Lortholary7, E Azoulay8, O Leroy9 1CHU Estaing, Clermont-Ferrand, France; 2Paris Diderot University/Bichat Hospital, Paris, France; 3Rennes University Hospital, Rennes, France; 4Cochin University Hospital, Paris, France; 5Amiens University Hospital, Amiens, France; 6Montpellier University Hospital, Montpellier, France; 7Necker University Hospital, Paris, France; 8Saint-Louis University Hospital, Paris, France; 9Tourcoing University Hospital, Tourcoing, France Critical Care 2015, 19(Suppl 1):P111 (doi: 10.1186/cc14191) P108 In the CIC group, it was mostly for changing the antifungal agent (de-escalation Cas  Flu in half of the patients) based on mycological tests results. In the SIC group, the AFT was modifi ed almost as often for changing the drugs (including 22% de-escalation Cas  Flu) as for stopping the AFT. The 28-day mortality of candidaemia was 42% in cases of C. glabrata, 40% in cases of C. albicans, and 20% in cases of C. parapsilosis. Among survivors, the median duration of treatment was 17 to 21 days according to the infection site in cases of CIC, and 10 days in cases of SIC. Epidemiological cohort study of systemic antifungal therapy for suspected or confi rmed invasive candidiasis in the ICU: the Amarcand2 study J Constantin1, JF Timsit2, JP Gangneux3, JP Mira4, P Montravers2, H Dupont5, P Perrigault6, O Lortholary7, E Azoulay8, O Leroy9 1CHU Estaing, Clermont-Ferrand, France; 2Paris Diderot University/Bichat Hospital, Paris, France; 3Rennes University Hospital, Rennes, France; 4Cochin University Hospital, Paris, France; 5Amiens University Hospital, Amiens, France; 6Montpellier University Hospital, Montpellier, France; 7Necker University Hospital, Paris, France; 8Saint-Louis University Hospital, Paris, France; 9Tourcoing University Hospital, Tourcoing, France l l d y Conclusion This is the largest PK study of 100  mg daily of MCF in severely burned critically ill patients. The inverse correlation between MCF exposure and % burned TBSA suggests that patients with large burned TBSA may need higher doses of MCF. Nevertheless, MCF levels in plasma and burn eschar tissues after the fi rst and multiple doses were above the MIC90 against most clinically important Candida species. g y p g Critical Care 2015, 19(Suppl 1):P111 (doi: 10.1186/cc14191) Introduction Prescription of antifungal treatments (AFT) in ICUs in case of suspected or confi rmed invasive candidiasis (SIC or CIC) has been challenged by diff erent guidelines. The study aimed to describe the epidemiology of the invasive candidiasis (IC), analyze the criteria for the AFT initiation, the AFT type, and its changes during patient follow-up. Methods A prospective observational multicenter cohort study. Consecutive adult patients with SIC or CIC and treated with systemic AFT were included between October 2012 and September 2013 in 104 French ICUs. References 1. World Health Organisation. Antimicrobial resistance: global report on surveillance. 2014. http://www.who.int/drugresistance/documents/ surveillancereport/en/. 1. World Health Organisation. Antimicrobial resistance: global report on surveillance. 2014. http://www.who.int/drugresistance/documents/ surveillancereport/en/. 2. Royal Cornwall Hospital Trust. Automatic stop/review date policy for antimicrobials. 2012. Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries gi q Results A total of 102 mechanically ventilated patients, 75 men and 27 women, were included in the study. All patients showed VAP caused by MDR bacteria. They were stratifi ed by outcome into survivors and nonsurvivors. ICU mortality was 55%. Gender, cause of admission, the causative microbe, colonization of bronchial secretions and secondary bacteremia had no correlation with outcome. Age and APACHE II score were higher in nonsurvivors (P <0.01 and P <0.05 respectively). The time-onset of pneumonia after admission was longer in patients with VAP caused by Klebsiella or Pseudomonas than those with VAP caused by acinetobacter (P <0.01). Patients with Klebsiella or Pseudomonas pneumoniae needed more time on mechanical ventilation than those with pneumonia from acinetobacter (P <0.01). Introduction Micafungin (MCF) is an echinocandin agent with broad activity against Candida spp., which are frequently isolated in blood and eschar cultures of burned patients, who present diff erent pharmacokinetics (PK) characteristics. Due to the limited information about its PK, we investigate MCF levels in plasma and burn eschar tissues in this population. p p Methods A PK study of MCF at standard dosage (100 mg/day). Cmax (end of the infusion) and Cmin (before next dose) plasma levels of MCF were obtained after fi rst dose and at steady state (days 4 and 5 of therapy); and on day 5 in eschars (1 to 3 hours after infusion). They were measured by HPLC. Spearman’s rho test was used for bivariate correlations between MCF exposure and patient’s clinical factors. Conclusion VAP caused by MDR bacteria is a leading cause of ICU death. Age and APACHE II score are signifi cant risk factors of death. References 1. Golia S, et al. J Clin Diagn Res. 2013;7:2462-6. 1. Golia S, et al. J Clin Diagn Res. 2013;7:2462-6. 2. Charles MP, et al. Australas Med J. 2013;6:430-4. Results There were 10 patients (eight men; age: 18 to 77 years). Patients’ characteristics and PK are shown in Table  1. A high interindividual variability was observed in the concentrations of MCF. Peak plasma concentrations after the fi rst and repeated doses of MCF were inversely correlated with % burned TBSA (Spearman’s ρ  = –0.695 and –0.750 (P <0.05), respectively), but not with the time from burn injury. MCF concentrations in burn eschars were not correlated with % burned TBSA. MCF was well tolerated. One patient had candidemia. The crude mortality was 40%. P112 Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries A Agrifoglio1, MJ Asensio1, M Sánchez1, B Galván1, E Herrero1, L Cachafeiro1, E Perales1, S Luque2, A García de Lorenzo1 1La Paz/IdiPAZ University Hospital, Madrid, Spain; 2Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P112 (doi: 10.1186/cc14192) Micafungin concentrations 100 mg daily in plasma and burn eschars in patients with severe burn injuries A Agrifoglio1, MJ Asensio1, M Sánchez1, B Galván1, E Herrero1, L Cachafeiro1, E Perales1, S Luque2, A García de Lorenzo1 1La Paz/IdiPAZ University Hospital, Madrid, Spain; 2Hospital del Mar, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P112 (doi: 10.1186/cc14192) P113 P113 Tedizolid clearance by in vitro continuous renal replacement therapy model SJ Lewis, L Switaj, BA Mueller University of Michigan, Ann Arbor, MI, USA Critical Care 2015, 19(Suppl 1):P113 (doi: 10.1186/cc14193) Factors associated with survival of ICU patients with pneumonia caused by multidrug-resistant Gram-negative bacteria M Georgiadou, E Pappa, E Papandreou, H Pavlou, M Eforakopoulou KAT-EKA General Hospital Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P110 (doi: 10.1186/cc14190) Introduction Multidrug-resistant (MDR) bacterial pneumonia is associated with signifi cant morbidity and mortality in severely ill ICU patients. The assessment of factors associated with the onset and clinical course of MDR pneumonia may improve treatment eff ectiveness. The purpose of this study is to identify factors associated with outcome in mechanically ventilated patients with ventilator- associated pneumonia (VAP) caused by MDR bacteria. y Conclusion French ICU patients are treated with antifungal agents selected according to the candidiasis severity, contrary to ESCMID guidelines which recommend initiating with echinocandins regardless of severity. As recommended, the therapy was secondarily adapted to microbiological results. y Methods We studied retrospectively all mechanically ventilated patients treated in the A’ ICU of KAT General Hospital in Athens from 1 January 2011 to 31 December 2013 and showed ventilator-associated pneumonia from MDR Gram-negative bacteria. Standard demographic and clinical data, the causative organisms and outcome were recorded. For statistical signifi cance, chi-square and Student t tests were used. P112 P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration G Kennedy1, J Osborn1, S Flanagan2, N Alsayed3, S Bertolami1 1Cubist Pharmaceuticals, Lexington, MA, USA; 2Cubist Pharmaceuticals, San Diego, CA, USA; 3Cubist Pharmaceuticals, Zurich, Switzerland Critical Care 2015, 19(Suppl 1):P114 (doi: 10.1186/cc14194) Introduction Tedizolid phosphate, a novel oxazolidinone antibacterial prodrug recently approved by the US Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections, is available as oral (that is, tablets) and intravenous formulations. The clinical pharmacokinetics of tedizolid, the active moiety of tedizolid phosphate, are similar when orally administered tedizolid phosphate is given as powder in a capsule or as tablets. This suggests that crushing tablets prior to administration is unlikely to alter tedizolid pharmacokinetics, provided no drug is lost during administration. To determine whether the expected dose of tedizolid phosphate can be delivered via nasogastric (NG) tube in critically ill patients who have diffi culty swallowing, this study evaluated the stability and recovery of tedizolid phosphate 200 mg tablets after crushing, dispersion in water, and passage through an NG tube. Introduction Tedizolid phosphate, a novel oxazolidinone antibacterial prodrug recently approved by the US Food and Drug Administration for the treatment of acute bacterial skin and skin structure infections, is available as oral (that is, tablets) and intravenous formulations. The clinical pharmacokinetics of tedizolid, the active moiety of tedizolid phosphate, are similar when orally administered tedizolid phosphate is given as powder in a capsule or as tablets. This suggests that crushing tablets prior to administration is unlikely to alter tedizolid pharmacokinetics, provided no drug is lost during administration. To determine whether the expected dose of tedizolid phosphate can be delivered via nasogastric (NG) tube in critically ill patients who have diffi culty swallowing, this study evaluated the stability and recovery of tedizolid phosphate 200 mg tablets after crushing, dispersion in water, and passage through an NG tube. for treatment of nosocomial pneumonia, common in critically ill patients with acute kidney injury. There are limited data on tedizolid disposition in continuous renal replacement therapy (CRRT). This study’s purpose was to assess continuous hemofi ltration (CHF) and continuous hemodialysis (CHD) infl uence on tedizolid clearance. Methods Validated, bovine blood-based, in vitro CHF and CHD models were used with six new HF 1400 (polysulfone) and six new Multifl ow 150 (AN 69) hemodiafi lters. P113 Tedizolid clearance by in vitro continuous renal replacement therapy model P113 Tedizolid clearance by in vitro continuous renal replacement therapy model SJ Lewis, L Switaj, BA Mueller University of Michigan, Ann Arbor, MI, USA Critical Care 2015, 19(Suppl 1):P113 (doi: 10.1186/cc14193) Introduction Tedizolid is an oxazolidinone antibiotic approved to treat acute bacterial skin and soft tissue infection and is under investigation Results In total, 870 patients were included and 835 evaluable, the IC was confi rmed at study inclusion for 291 and suspected for 544 patients. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S39 Table 1 (abstract P112). Clinical and pharmacokinetic characteristics of patients Table 1 (abstract P112). Clinical and pharmacokinetic characteristics of patients Plasma Plasma Days from SOFA MCF dose Cmax/Cmin Cmax/Cmin Burn eschar admission at the % burned (mg/kg after fi rst dose at steady state tissue on day 5 to the start beginning LOS in BICU Patient TBSA % FT ABSI body weight) (μg/ml) (μg/ml) (μg/g) of MCF of MCF (days) 1 35 20 9 1.3 8.6/0.8 7.4/1.0 2.3 38 1 75 2 40 35 8 2.0 8.5/1.1 9.4/1.8 <LQ 15 6 23 3 23 16 8 1.3 6.4/0.8 10.3/1.2 <LQ 12 2 17 4 70 40 12 1.1 3.9/0.5 4.5/0.8 0.4 8 6 43 5 23 12 7 1.3 7.5/1.8 8.0/1.4 0.6 10 5 19 6 70 60 11 1.2 3.4/0.5 5.0/0.9 1.5 12 5 70 7 80 70 12 1.1 3.8/0.4 4.0/0.4 0.2 34 2 61 8 60 50 10 1.4 4.8/0.5 4.3/1.0 0.2 34 2 90 9 44 34 10 1.3 4.5/1.1 9.1/2.3 0.2 8 6 34 10 34 28 9 1.3 4.1/0.7 5.4/1.0 0.7 10 5 35 Median 42 34.5 9.5 1.3 4.7/0.7 6.4/1.0 0.5 12 5.0 39 IQR 31.3 to 70.0 19.0 to 52.5 8 to 11.3 1.1 to 1.4 3.9 to 7.5/0.5 to 1.1 4.5 to 9.1/0.9 to 1.4 0.3 to 1.1 9.5 to 19.8 3.5 to 5.6 22.7 to 71.3 ABSI, Abbreviated Burn Severity Index; BICU, burn intensive care unit; FT, full thickness; IQR, interquartile range; LOS, length of hospital stay; LQ, limit of quantifi cation (<0.1 μg/ml); SOFA, Sequential Organ Failure Assessment; TBSA, total body surface area. CLTM appears modest relative to total body clearance and is unlikely to require dose adjustments. CRRT adsorption in the clinical setting is likely less than what we observed in this in vitro, continuously recirculating blood model. CLTM appears modest relative to total body clearance and is unlikely to require dose adjustments. P113 Tedizolid clearance by in vitro continuous renal replacement therapy model CRRT adsorption in the clinical setting is likely less than what we observed in this in vitro, continuously recirculating blood model. Figure 1 (abstract P113). P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration G Kennedy1, J Osborn1, S Flanagan2, N Alsayed3, S Bertolami1 1Cubist Pharmaceuticals, Lexington, MA, USA; 2Cubist Pharmaceuticals, San Diego, CA, USA; 3Cubist Pharmaceuticals, Zurich, Switzerland Critical Care 2015, 19(Suppl 1):P114 (doi: 10.1186/cc14194) P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration P115 Introduction In daptomycin (DAP), 1,061  mg hour/l of the area under the concentration–time curve (AUC)/MIC was required to obtain clinical success [1], and a trough serum concentration (Cmin) cutoff point of 24.3 g/ml was most signifi cantly associated with CPK elevation [2]. Reportedly, DAP at a recommended dosage of 8 mg/kg is removed in patients undergoing high-fl ow continuous venovenous hemodiafi ltration (CVVHDF) (blood fl ow and fi ltration rates were 150 ± 48 and 2 l/hour). In Japan, CVVHDF is preferentially performed with lower fl ow rates. Investigating eff ects of fl ow rate on DAP removal during continuous renal replacement therapy is essential to adjust therapeutic dosages. We aimed to investigate the pharmacokinetics of DAP in CVVHDF patients in this setting. 5 Antiviral prophylaxis inhibits cytomegalovirus reactivation in critical illness NJ Cowley1, A Owen1, J Millar1, SC Shiels1, RL Woolley2, NJ Ives2, H Osman1, P Moss2, JF Bion1 1University Hospital Birmingham, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P115 (doi: 10.1186/cc14195) Introduction Reactivation of latent cytomegalovirus (CMV) can lead to viraemia or CMV disease and has been detected in up to 30% of critically ill patients without prior history of immune suppression. However, the clinical importance of this observation remains unclear. We report a proof-of-concept randomised controlled trial of two antiviral drugs in intensive care patients to determine their impact on CMV reactivation. Methods We conducted a single-centre randomised controlled study of high-dose valaciclovir or low-dose valganciclovir prophylaxis, as compared with standard care, in CMV seropositive patients in the ICU at Queen Elizabeth Hospital Birmingham, UK. Patients were excluded if CMV seronegative. Study participants randomised to a study drug received either 450  mg valganciclovir daily enterally (or ganciclovir intravenously) or 2 g valaciclovir four times daily enterally (or aciclovir intravenously) for a period of up to 28 days. Blood was collected for CMV viral load during the 28-day study period. The primary outcome measure was reactivation of CMV in blood above 20 copies.ml–1 (assay detection limit) by day 28. Methods DAP (6  mg/kg) was administered intravenously every 48 hours to CVVHDF patients in the ICU. Blood and fi ltrate samples were collected at 0, 1, 1.5, 2, 5, 12, 24, and 48 hours after infusion. All collected samples were analyzed using HPLC according to the method of Tobin and colleagues [3]. P115 Maximum concentration (Cmax), elimination half- life (t1/2), area AUC, Cmin, volume of distribution (Vd), clearance (CL), fraction unbound, and sieving coeffi cient (Sc) were evaluated. Patient characteristics and CVVHDF parameters including blood, dialysate, and fi ltration fl ow rates were recorded. il Results Three patients were included in the study. Mean blood, dialysate, and fi ltration fl ow rates were 86.7  ±  11.5  ml/minute, 417  ±  29  ml/hour, and 417  ±  29  ml/hour, respectively, confi rming that CVVHDF was performed under low-fl ow setting. Cmax was 50.1 ± 12.7 mg/l (31.9, 70.5, 49.7 mg/l); t1/2, 35.1 ± 34.8 hours (18.6, 11.5, 70.5 hours); AUC, 889 ± 399 mg hour/l (471, 967, 1,260 mg hour/l); Cmin, 16.0  ±  10.3  mg/l (2.3, 24.7, 14.0  mg/l); Vd, 26.0  ±  20.9 l (23.8, 6.34, 47.9 l); CL, 9.47  ±  4.56  ml/minute (14.7, 6.35, 7.37  ml/minute); and fraction unbound, 5.8% (5.7, 4.1, 7.6%). Sc and CL of dialyzer were 0.08  ±  0.03 (0.11, 0.04, 0.07) and 1.20  ±  0.39  ml/minute (1.70, 0.88, 0.96 ml/minute), respectively. Results A total of 124 patients were randomised; 44 control, 34 valaciclovir, and 46 valganciclovir. Recruitment to the valaciclovir arm was halted early because of an imbalance in mortality (44% mortality vs. 19% in other arms). Independent blinded review of all deaths did not reveal any deaths attributable to unexpected causes. Fourteen patients were excluded from the primary analysis because of baseline CMV reactivation. CMV reactivation occurred in 30% (12/40) of the control arm but only 3% (1/39) in the valganciclovir arm (RR: 0.09 (95% CI: 0.01, 0.6)). When the two treatment arms were considered together, reactivation was observed in only 4% (3/70) (RR: 0.1 (95% CI: 0.04, 0.5)). See Figure 1. Conclusion DAP (6  mg/kg daptomycin every 48  hours) in patients receiving low-fl ow CVVHDF resulted in showing variability of AUC and avoiding accumulation. Owing to small case numbers, it needs further study. g Conclusion This is the fi rst study in critical care to assess the feasibility of antiviral prophylaxis to prevent CMV reactivation in a mixed population of critically ill patients. Low-dose valganciclovir was shown to suppress CMV reactivation as eff ectively as higher-dose valaciclovir. P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration Tedizolid’s transmembrane clearances (CLTM) during CHF and CHD were assessed by measuring sieving (SC) and saturation (SA) coeffi cients at various ultrafi ltrate (Quf) (1, 2, 3 l/ hour) and dialysate fl ow rates (Qd) (1, 2, 3 and 6 l/hour), using a blood fl ow rate (Qb) of 200 ml/minute. Tedizolid adsorption was tested in a 1 l recirculating CHF model at Quf of 2 l/hour and Qb of 200 ml/minute over 4  hours. Adsorption (%) was calculated after correcting for the dilution by CHF priming volume. Urea was added as a control in all experiments. Methods For each assay, run in triplicate, one 200 mg tablet of tedizolid phosphate was crushed, dispersed in water, drained under gravity through one of two types of NG tubes (type 1, Kangaroo Nasogastric Feeding Tube, 10 Fr 43" (109 cm); type 2, Salem Sump Dual Lumen Stomach Tube, 18 Fr/CH (6.0 m) 48" (122 cm)), and collected for recovery analysis by high-performance liquid chromatography with UV detection. To analyze the chemical stability of the crushed tablet dispersed in water, the aqueous preparation was assayed initially after dispersion and again after 4 hours at room temperature, without NG tube passage. The prespecifi ed limit for tedizolid phosphate in recovery samples was 90 to 110% of the dose. Limits were also specifi ed for levels of certain impurities. Results Urea SC and SA were ~1 in all experiments. In CHF, mean tedizolid SC ranged from 0.52 to 0.57 for HF1400 and from 0.50 to 0.54 for M150. CLTM did not diff er between fi lter types for Quf of 1, 2, and 3 l/hour. In CHD, mean tedizolid SA ranged from 0.46 to 0.56 for HF1400 and from 0.38 to 0.44 for M 150. Tedizolid CLTM with the HF1400 was higher than M150 values at Qd of 6 l/hours (P <0.02). Tedizolid exhibited irreversible adsorption within 10 minutes. See Figure 1. Results The average and individual recovery values of tedizolid phosphate were within 90 to 110% of the 200 mg dose when crushed tablets, dispersed in water at room temperature, were transferred through the 2 NG tubes (type 1: 95.8%; type 2: 93.6%). There was Conclusion Tedizolid’s CLTM is dependent on hemodiafi lter type and Qd for CHD and Quf in CHF. P114 Stability of crushed tedizolid phosphate tablets for nasogastric tube administration At conventional CRRT rates, tedizolid S40 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Acknowledgements Research funded by the NIHR under the RfPB Programme (PG 1010 23225). Views expressed are of the authors and not necessarily the NHS, NIHR, or DOH. Acknowledgements Research funded by the NIHR under the RfPB Programme (PG 1010 23225). Views expressed are of the authors and not necessarily the NHS, NIHR, or DOH. no signifi cant change in recovery values after 4  hours of storage at room temperature (93.9% initially and 94.7% after 4  hours). Results for degradation products and impurities were also within specifi ed limits in NG recovery samples and in the 0-hour and 4-hour aqueous preparations. P116 p p Conclusion The stability and recovery of tedizolid phosphate were not infl uenced by crushing the tablets and passing through an NG tube. Therefore, administration of crushed tedizolid phosphate tablets to patients is unlikely to alter the pharmacokinetics of tedizolid compared with whole tablets. Pharmacokinetics of daptomycin in patients undergoing low-fl ow continuous venovenous hemodiafi ltration TI Ide1, Y Takesue1, K Ikawa2, S Nishi1 1Hyogo College of Medicine, Nishinomiya City, Japan; 2Hiroshima University, Hiroshima, Japan Critical Care 2015, 19(Suppl 1):P116 (doi: 10.1186/cc14196) PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? S De Winter1, J Wauters1, E Van Wijngaerden1, W Peetermans1, P Annaert2, J Verhaegen1, JB Gillet1, D Knockaert1, I Spriet1 1University Hospitals Leuven, Belgium; 2Catholic University Leuven, Belgium Critical Care 2015, 19(Suppl 1):P118 (doi: 10.1186/cc14198) Methods A total of four colistin-resistant (MIC ≥4) GNB were isolated from ICU patients with nosocomial MDR infections. All four isolates were Klebsiella pneumonia. Among these isolates three were from blood and one from endotracheal aspirate and all four isolates were sensitive to fosfomycin in vitro. All of these patients had multiple comorbidities with recent history of colistin exposure. Intravenous fosfomycin sodium (inj Fosmicin; Meiji, Japan) was started as a combination therapy with carbapenem. Introduction Studies in the ICU showed that a single amikacin dose of ≥25 mg/kg should be used in conditions of increased distribution volume (Vd) such as severe sepsis/shock [1]. However, no data are available for emergency department (ED) patients in the early phase of sepsis/septic shock. The purpose of this study was to determine whether a single amikacin dose of 25 versus 15 mg/kg results in PK/PD target attainment for ED patients. Results Among the three bacteremic patients, two recovered completely from sepsis as well as the patient with ventilator-associated pneumonia. There was clinical as well as microbiological cure with normalization of sepsis markers. The only one bacteremic patient who died during the course of therapy was later diagnosed to have azole- resistant fungemia as a superinfection. g p Methods ED patients with severe sepsis/shock were randomly treated with a single amikacin dose of 25 versus 15  mg/kg. Blood samples were collected at  +1 (peak),  +6  hours and  +24  hours (trough) after the start of infusion. Primary outcome was PK/PD target attainment defi ned as a peak/MIC >8, corresponding with both actual MIC values documented from isolated pathogens, as well as EUCAST susceptibility breakpoints for Enterobacteriaceae and P. aeruginosa; that is, 8 mg/l. Noncompartmental analysis was used to calculate PK parameters. g p Conclusion Based on the evidence of clinical experience and available studies, intravenous fosfomycin therapy may be considered as the last option for the treatment of MDR GNB infection where there is documented colistin resistance and where there is literally no other choice of antibiotic therapy. PK/PD of single-dose amikacin in emergency department patients with severe sepsis/shock: should we apply the ICU-based higher loading dose? Results During a study duration of 20 months, 50 patients were enrolled in each dosing regimen resulting in 100 peak concentrations, 92 and 88 +6 hours and +24 hours concentrations respectively. Target attainment using local MIC values (median 2  mg/l, documented in 56 isolated Gram-negative pathogens) was achieved in 95% in both groups (P = 0.98). Using EUCAST susceptibility breakpoints, the target Table 1 (abstract P118) Introduction In vitro studies suggest that there is signifi cant adsorption of amikacin, netilmicin, gentamicin and tobramycin to polyacrylonitrile haemofi lters. This occurs rapidly and has the potential to substantially reduce the peak aminoglycoside concentration, which will reduce effi cacy [1]. However, whether signifi cant adsorption occurs in vivo is unknown. We therefore carried out a controlled in vivo study of the eff ect of amikacin adsorption by polyacrylonitrile fi lters during haemofi ltration, using a porcine model of acute renal failure. l, clearance. aMann–Whitney U <0.05, 15 versus 25 mg/kg ED patients Conclusion The EUCAST-based PK/PD target was only attained in 76% of patients treated with 25 mg/kg. However, in contrast to ICU patients, the majority of ED patients are treated for community-acquired infections, so MIC values are signifi cantly lower than the EUCAST susceptibility breakpoints, warranting PK/PD target attainment in both 25 and 15 mg/kg dosing regimens when local epidemiology is taken into account. i g Methods A porcine model of acute renal failure was created by bilateral ligation of the renal arteries and veins. Eight pigs underwent haemofi ltration using a 0.6 m2 polyacrylonitrile fi lter, blood fl ow 200 ml/ minute, ultrafi ltration rate 1,000 ml/hour. All ultrafi ltrate was returned to the pigs via a separate venous catheter so that any elimination of amikacin by haemofi ltration could only be due to adsorption. Another eight pigs underwent sham haemofi ltration in which blood was pumped around a haemofi ltration circuit without a haemofi lter and without ultrafi ltration. Both groups of pigs were given intravenous amikacin, 15 mg/kg body weight over 30 minutes, and blood samples were taken from the arterial limb of the haemofi lter circuit at 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120, 150, and 180 minutes after the start of the amikacin administration to assay amikacin concentrations. Results Post-distribution peak concentration of amikacin was slightly, but signifi cantly, lower in the CRRT group than that in sham group (55.0 ± 4.5 vs. 61.1 ± 5.9 mg/l, P <0.05).f Reference 1. Taccone et al. Crit Care. 2013;14:R53. Intravenous fosfomycin therapy in critically ill patients infected with colistin-resistant enterobacteriacae Conclusion This study shows that the eff ect of adsorption by polyacrylonitrile haemofi lters on in vivo amikacin peak concentrations is small, and less than would be expected from in vitro data. Introduction Carbapenem-resistant enterobacteriacae emerged in recent years as one of the most challenging groups of antibiotic- resistant pathogens. Polymyxins are considered as the last resort for the treatment of infections with carbapenem-resistant Gram-negative bacilli (GNB). Inadequate or extensive use of colistin leads to emergence of colistin resistance in GNB, jeopardizing treatment options in ICUs, potentially increasing mortality and morbidity and necessitating prudent use of alternative antibiotics. Fosfomycin, a phosponic acid derivative which acts primarily by disrupting bacterial cell wall synthesis, is a broad-spectrum antibiotic. Fosfomycin tromethamine is an oral formulation approved for the treatment of uncomplicated urinary tract infection caused by multidrug-resistant (MDR) bacteria. Recently fosfomycin is also available as a sodium/disodium formulation for intravenous use, which is showing promising result against MDR/ potentially drug-resistant pathogens. Acknowledgement This work was supported by a grant from the Hong Kong Research Grants Committee, CUHK 4644/08M. Reference 1. Tian Q, Gomersall CD, Ip M, Tan PE, Joynt GM, Choi GY. Adsorption of amikacin, a signifi cant mechanism of elimination by hemofi ltration. Antimicrob Agents Chemother. 2008;52:1009-13. References 1. Safdar N, et al. Antimicrob Agents Chemother. 2004;48:63-8. 2. Bhavnani SM, et al. Clin Infect Dis. 2010;50:1568-74. . Safdar N, et al. Antimicrob Agents Chemother. 2004;48:63-8. 3. Tobin CM, et al. J Antimicrob Chemother. 2008;62:1462-3. Figure 1 (abstract P115). CMV reactivation over time. Each line represents a single patient. Figure 1 (abstract P115). CMV reactivation over time. Each line represents a single patient. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S41 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 was attained in 76% versus 40% in the 25 versus 15  mg/kg group, respectively (P < 0.0001). Single-dose PK parameters are displayed in Table 1 and compared with the ones reported in the ICU [1]. P117 P117 Adsorption of amikacin during continuous venovenous haemofi ltration in a swine model of acute renal failure CD Gomersall, Q Tian, D Reynolds, M Ip, G Choi, G Joynt The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P117 (doi: 10.1186/cc14197) Table 1 (abstract P118) PK parameter 15 mg/kg ED 25 mg/kg ED 25 mg/kg ICU Peak (mg/l) 58 (47 to 70)a 91 (72 to 105)a 73 (62 to 90) Trough (mg/l) 6 (3 to 12) 5 (3 to 15) 7 (2 to 15) Vd (l/kg) 0.3 (0.3 to 0.5) 0.4 (0.2 to 0.6) 0.4 (0.3 to 0.5) Cl (ml/minute/kg) 1.6 (1 to 2.3)a 2.2 (1.4 to 3)a 1.9 (1.3 to 3.5) Cl, clearance. aMann–Whitney U <0.05, 15 versus 25 mg/kg ED patients. 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation Introduction Amikacin Inhale is an integrated drug–device combi- nation in development by Bayer HealthCare through a collaboration with Nektar Therapeutics, to improve clinical outcome in intubated and mechanically ventilated patients with Gram-negative pneumonia. It is available in two confi gurations: on-vent for intubated patients and hand-held for extubated patients to complete aerosolized antibiotic therapy. Amikacin Inhale is a smart system that consists of the pulmonary drug delivery system with a vibrating mesh nebulizer and the specially formulated Amikacin Inhalation Solution (400 mg every 12  hours for 10  days). The objectives of this study were to evaluate the performance of the Amikacin Inhale hand-held confi guration with supplemental O2 concentration supplied at diff erent fl ow rates and in diff erent orientations. We hypothesize that the delivered dose of amikacin will not signifi cantly change with increased O2 fl ow rate or varying orientation.i q Results Preventive administration of IT as an adjunct to systemic antibiotics was associated with a lower incidence of NP in group 1 (group 1 33.3%, group 2 66.7%, χ2 = 6,000; P = 0.014) and a shorter duration of ICU stay (group 1 8.0  ±  4.6  days vs. 17.1  ±  18.4  days, P = 0.03). The mortality did not diff er between groups: 11.1% in group 1 and 22.2% in group 2 (P ≥0.99). On day 3 Acinetobacter spp. (30.5%), K. pneumoniae (22.0%), B. cepacia (13.2%) and P. aeruginosa (34.3%) were detected in BAL, there were no diff erences between groups. In group 1 CPIS remained stable and APACHE II decreased. CPIS and APACHE II were lower in group 1 on day 5 (P = 0.0004). y Conclusion Early administration of IT as an adjunct to systemic antibiotics is eff ective in prevention of NP in multiple trauma patients: it promotes decrease of NP incidence and decrease of ICU stay. Methods In the hand-held confi guration of Amikacin Inhale, amikacin is aerosolized into a holding chamber. Amikacin aerosol is inhaled with ambient air entering the bottom of the chamber through the inhalation valve. Supplemental O2 may be supplied through the O2 port and mixes with ambient air entering through the inhalation valve. O2 concentration and delivered dose at the mouthpiece exit were characterized in vitro at various O2 fl ow rates (2 to 10  l/minute). O2 concentrations were measured every minute until the end of dosing. Early preventive administration of inhaled tobramycin in severe polytrauma Early preventive administration of inhaled tobramycin in severe polytrauma A Kuzovlev1, A Shabanov2, T Chernenkaya2, V Moroz1, A Goloubev1 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.V. Sklifosofsky Research Institute, Moscow, Russia Critical Care 2015, 19(Suppl 1):P121 (doi: 10.1186/cc14201) y Results Overall, nine cases with CNS infection were recorded aged from 22 to 74, all males. LP was performed between the second and 17th day (average 8.3 days) and the CSF analysis showed 40 to 6,000 cells – mainly PMNs, protein 161 mg% to 287 mg% and glucose from 3 to 58 mg/dl. They were all colonized with Acinetobacter baumannii sensitive only to colistin. CSF cultures were negative for all patients besides one, who grew A. baumannii. Of those, seven (77%) were receiving i.v. colistin, eight (88%) carbapenems, and eight (88%) glycopeptides, all in combination with other antibiotics. Median i.t. administration time was 9.1 days. All patients responded to i.v. and i.t. antibiotics but there was one case in which fever relapsed and increased number of cells in subsequent LP was observed which was attributed to colistin, which was withdrawn. All these patients survived, and were discharged to the ward. p y A Kuzovlev1, A Shabanov2, T Chernenkaya2, V Moroz1, A Goloubev1 1V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2N.V. Sklifosofsky Research Institute, Moscow, Russia Critical Care 2015, 19(Suppl 1):P121 (doi: 10.1186/cc14201) Introduction Nosocomial pneumonia (NP) occurs in 30 to 50% of multiple trauma patients. It is mostly caused by multiresistant Gram- negative bacteria. Use of inhaled antibiotics as adjuncts to systemic antibiotics presents a great outlook for the prevention of NP in multiple trauma patients. The aim of the study was to evaluate the effi cacy of early administration of inhaled tobtamycin (IT) as an adjunct to systemic antibiotics for the prevention of NP in polytrauma. Methods Fifty-four ICU mechanically ventilated patients with multiple trauma (ISS >30; car accident 55.6%; fall 29.6%; train accident 11.1%; domestic 3.7%) were enrolled in the single-center randomized trial. Groups were comparable in ISS, age, sex, type of trauma, and blood loss. Patients were randomized into two groups: Group 1 (n  = 27), addition of IT to systemic antibiotics (ciprofl oxacin 800  mg/day; metronidazol 1,500 mg/day); Group 2 (n = 27), only systemic antibiotics (same regimen). Early preventive administration of inhaled tobramycin in severe polytrauma Inhaled tobramycin (300 mg twice daily via nebulizer) and systemic antibiotics were administered within the fi rst 24 hours Conclusion Patients treated with the abovementioned regime showed clinical and biochemical improvement. The above drug combination turned out to be successful in neurosurgical ICU patients with CNS infection. Intrathecal administration of colistin, vancomycin and amikacin for central nervous system infections in ICU neurosurgical patients P Alexandropoulos, S Georgiou, V Chantziara, A Tsimogianni, E Chinou, V Karagiannisa, G Michaloudis Introduction Central nervous system (CNS) infections in ICU patients after neurosurgery are a diffi cult and life-threatening complication demanding immediate action. In many cases intravenous (i.v.) administration of antibiotics is not suffi cient; thus, intrathecal (i.t.) administration is required. g pi pp 2 Results The mean O2 concentration ranged from 36 to 70% over 2 to 10 l/minute and was ≥40% at ≥3 l/minute. The delivered dose did not change substantially with increasing enriched O2 fl ow rate (72 to 82% of nominal dose). At 0° and 45° orientations, the delivered dose of amikacin was 74 to 80% and 73 to 76% of the nominal dose (400 mg), respectively. Methods From January 2013 to November 2014 all cases with CNS infections were recorded. Inclusion criteria were the presence of fever ≥38.5°C, increased infl ammatory markers, compatible lumbar puncture (LP) fi ndings (increased number of polymorphonuclear leukocytes, increased protein and low glucose compared with serum levels) and no evidence of other site of infection. All subjects were receiving appropriate i.v. antibiotic treatment based on cultures. Intrathecal administration of 300,000 iu colistin, 25 mg vancomycin and 25 mg amikacin was performed taking under consideration that neurosurgical patients in the ICU have CNS infection attributed to Gram-negative bacteria or/and to Staphylococcus species. Conclusion Amikacin Inhale was shown in vitro to be suitable for extubated patients who require supplemental O2. The delivered dose was independent of supplemental O2 and device orientation. Reference 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. 1. Antoniadou A, Kontopidou F, Poulakou G, Koratzanis E, Galani I, Papadomichelakis E, et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: fi rst report of a multiclonal cluster. J Antimicrob Chemother. 2007; 59:786-90. S42 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P120 after ICU admission. After obtaining the results of bronchoalveolar lavage microbiology, the antibiotic regimen was switched according to the sensitivity. The primary outcome measure was new onset of NP and duration of ICU stay. Microbiological, X-ray, CPIS, signs of sepsis and oxygenation index were used as objective indicators of the clinical progress. The secondary outcome measure was 30-day mortality. Diagnosis of NP was made according to the standard clinical and CPIS criteria. The data were statistically analyzed by SPSS 11.5 (M, σ, Newman–Keuls test; chi-square-test P <0.05). after ICU admission. After obtaining the results of bronchoalveolar lavage microbiology, the antibiotic regimen was switched according to the sensitivity. The primary outcome measure was new onset of NP and duration of ICU stay. Microbiological, X-ray, CPIS, signs of sepsis and oxygenation index were used as objective indicators of the clinical progress. The secondary outcome measure was 30-day mortality. Diagnosis of NP was made according to the standard clinical and CPIS criteria. The data were statistically analyzed by SPSS 11.5 (M, σ, Newman–Keuls test; chi-square-test P <0.05). P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation N Kadrichu1, K Corkery1, T Dang1, P Challoner2 1Novartis Pharmaceuticals, San Carlos, CA, USA; 2Nektar Therapeutics, San Francisco, CA, USA Critical Care 2015, 19(Suppl 1):P120 (doi: 10.1186/cc14200) P120 Performance of amikacin inhale: impact of supplemental oxygen and device orientation A ventilator was connected to the set up to simulate patient breathing. The delivered dose was measured at the exit of the mouthpiece. Drug distribution within the test setup compartments was analyzed using HPLC. The in vitro delivered amikacin dose was also measured at nebulizer orientations of 0° and 45° (n = 3 per orientation) using a simulated breathing profi le with no supplemental O2. P122 Intrathecal administration of colistin, vancomycin and amikacin for central nervous system infections in ICU neurosurgical patients P Alexandropoulos, S Georgiou, V Chantziara, A Tsimogianni, E Chinou, V Karagiannisa, G Michaloudis Saint Savvas Oncology Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P122 (doi: 10.1186/cc14202) Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Bl 1 N Ni 1 M B i l1 P S h 2 E Ull 3 I S Wid 1 Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Blum1, N Nigro1, M Briel1, P Schuetz2, E Ullmer3, I Suter-Widmer1, B Winzeler1, R Bingisser1, H Elsaesser3, D Drozdov2, B Arici2, SA Urwyler1, J Refardt1, P Tarr4, S Wirz4, R Thomann5, C Baumgartner6, H Duplain7, D Burki8, W Zimmerli3, N Rodondi6, B Mueller2, M Christ-Crain1 1University Hospital Basel, Switzerland; 2Medical University Clinic, Kantonsspital Aarau, Switzerland; 3Kantonsspital Baselland/Liestal, Liestal, Switzerland; 4Kantonsspital Baselland/Bruderholz, Bruderholz, Switzerland; 5Bürgerspital, Solothurn, Switzerland; 6Inselspital, Bern University Hospital, Bern, Switzerland; 7Hôpital du Jura, Site de Delémont, Delémont, Switzerland; 8Viollier SA, Basel, Switzerland Results We included nine trials enrolling 2,637 patients. Eight trials were of unclear risk of bias and one was classifi ed as having low risk of bias. In trials comparing heparin with placebo or usual care, the risk ratio for death associated with heparin was 0.88 (95% CI = 0.77 to 1.00, I2 = 0%, 2477 patients, six trials). In trials comparing heparin with other anticoagulants, the risk ratio for death was 1.30 (95% CI = 0.78 to 2.18, I2  = 0%, 160 patients, three trials). In trials comparing heparin with placebo or usual care, major hemorrhage was not statistically signifi cantly increased (risk ratio 0.79, 95% CI  = 0.53 to 1.17, I2 = 0%, 2,392 patients, three trials). In one small trial of heparin compared with other anticoagulants, the risk of major hemorrhage was signifi cantly increased (2.14, 95% CI = 1.07 to 4.30, 48 patients). Important secondary and safety outcomes, including minor bleeding, were sparsely reported. Critical Care 2015, 19(Suppl 1):P125 (doi: 10.1186/cc14205) Critical Care 2015, 19(Suppl 1):P125 (doi: 10.1186/cc14205) Introduction Clinical trials yielded confl icting data about the benefi t of adding systemic corticosteroids for community-acquired pneumonia (CAP). We evaluated whether short-term corticosteroid treatment reduces time to clinical stability in patients hospitalized for CAP. Methods This randomized, placebo-controlled multicenter trial compared prednisone 50  mg for 7  days with placebo in patients hospitalized with CAP. The primary endpoint was time to clinical stability. Results Overall, 802 patients were randomized in seven Swiss hospitals from December 2009 to May 2014. Time to clinical stability was shorter in the prednisone group compared with placebo (3.0 vs. 4.4  days, HR = 1.33, 95% CI = 1.15 to 1.50, P <0.001). The prednisone group as compared with the placebo group had a shorter time to hospital discharge (6 vs. i References 1. Alejandria MM, Lansang MD, Dans LF, Mantaring III JBlas. Intravenous immunoglobulin for treating sepsis, severe sepsis and septic shock. Cochrane Database Syst Rev. 2013;9:CD001090. doi:10.1002/14651858.CD001090.pub2. 2. Department of Health. Clinical guidelines for immunoglobulin use. London: Department of Health; 2011. y p 2. Department of Health. Clinical guidelines for immunoglobulin use. London: Department of Health; 2011. Methods We included randomized controlled trials from MEDLINE, EMBASE, CENTRAL, Global Health, Scopus, Web of Science, the International Clinical Trials Registry Platform (inception to April 2014), conference proceedings, and reference lists of relevant articles. Two reviewers independently identifi ed and extracted trial-level data from randomized trials investigating unfractionated or low molecular heparin administered to patients with sepsis, severe sepsis, septic shock or DIC associated with infection. Internal validity was assessed in duplicate using the Risk of Bias tool. Our primary outcome was mortality. Safety outcomes included hemorrhage, transfusion and thrombocytopenia. Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis g Conclusion The use of IVIg does not appear to aff ect mortality in sepsis. There was also no statistical benefi t or harm demonstrated by using IVIg. This also holds true whether IVIg is given either according to the guidelines or not; however, stricter adherence to the guidelines does have fi nancial implications. Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) Introduction Septic shock is characterized by systemic infl ammation coupled with upregulation of coagulation. Heparin is an inexpensive and widely available anticoagulant with anti-infl ammatory properties. The objectives our study were to evaluate the effi cacy and safety of heparin in patients with sepsis, septic shock or disseminated intravascular coagulation (DIC) associated with infection. Adjunct prednisone therapy for patients with community-acquired pneumonia: a randomized, placebo-controlled multicenter trial CA Bl 1 N Ni 1 M B i l1 P S h 2 E Ull 3 I S Wid 1 7 days (HR = 1.19, 1.04 to 1.38), P = 0.012) and a shorter duration of intravenous antibiotic treatment (4 vs. 5 days (diff erence, –0.89 days, –0.20 to –1.57 days), P = 0.011). All-cause mortality, ICU stay, recurrent pneumonia and rehospitalization rate were similar in both groups. Incidence of pneumonia-associated complications until day 30 tended to be lower in the prednisone group (2.8% vs. 5.6%, OR = 0.49, 0.23 to 1.02, P = 0.06). The prednisone group had a higher rate of in-hospital hyperglycemia needing insulin treatment (19.4% vs. 10.9%, OR = 1.96, 1.31 to 2.93, P = 0.001). Other adverse events compatible with corticosteroid use were rare and similar in both groups. Conclusion Heparin in patients with sepsis, septic shock, and DIC associated with infection may be associated with decreased mortality; however, the overall impact remains uncertain. Safety outcomes have been under-reported and require further study. Large randomized trials are needed to evaluate the effi cacy and safety of heparin in patients with sepsis, severe sepsis, and septic shock. P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis R Zarychanski1, AM Abou-Setta1, S Kanji2, AF Turgeon3, A Kumar1, DS Houston1, E Rimmer1, BL Houston4, L McIntyre2, AE Fox-Robichaud5, PC Hebert6, DJ Cook5, DA Fergusson2 1University of Manitoba, Winnipeg, MB, Canada; 2Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3Université Laval, Québec City, QC, Canada; 4University of Toronto, ON, Canada; 5McMaster University, Hamilton, ON, Canada; 6Centre hospitalier de l’Université de Montreal (CHUM) – Hopital Notre-Dame, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) Reference 1. Karaiskos I, Galani L, Baziaka F, Giamarellou H. Intraventricular and intrathecal colistin as the last therapeutic resort for the treatment of multidrug-resistant and extensively drug-resistant Acinetobacter baumannii ventriculitis and meningitis: a literature review. Int J Antimicrob Agents. 2013;41:499-508. S43 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P123 Effi cacy and safety of heparin in patients with sepsis: a systematic review and meta-analysis R Zarychanski1, AM Abou-Setta1, S Kanji2, AF Turgeon3, A Kumar1, DS Houston1, E Rimmer1, BL Houston4, L McIntyre2, AE Fox-Robichaud5, PC Hebert6, DJ Cook5, DA Fergusson2 1University of Manitoba, Winnipeg, MB, Canada; 2Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3Université Laval, Québec City, QC, Canada; 4University of Toronto, ON, Canada; 5McMaster University, Hamilton, ON, Canada; 6Centre hospitalier de l’Université de Montreal (CHUM) – Hopital Notre-Dame, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P123 (doi: 10.1186/cc14203) guidelines for prescription, with a mortality rate of 25%. Nine patients did not meet the national guidelines for prescription, with a mortality rate of 44%. The diff erence in mortality rates between the two groups did not reach statistical signifi cance (P = 0.6). There was no signifi cant diff erence in APACHE II scores between the two groups. There was also no diff erence in mortality between those receiving IVIg and those who did not. We also found no diff erence in those receiving single or double doses of IVIg. P126 Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) P124 P124 Use of intravenous immunoglobulin to treat sepsis in a general ICU Y Drakeford, J Kelly, P Morgan, J Melville, A Holland Surrey and Sussex Healthcare NHS Trust, Redhill, UK Critical Care 2015, 19(Suppl 1):P124 (doi: 10.1186/cc14204) Use of intravenous immunoglobulin to treat sepsis in a general ICU Y Drakeford, J Kelly, P Morgan, J Melville, A Holland Surrey and Sussex Healthcare NHS Trust, Redhill, UK Critical Care 2015, 19(Suppl 1):P124 (doi: 10.1186/cc14204) Introduction Sepsis is a major cause of admission to the ICU, and a leading cause of death for ICU patients. Intravenous immunoglobulin (IVIg) is indicated in the treatment of some patients with sepsis, although the evidence for this remains controversial. The use of IVIg is regulated due to its high cost, and prescription guidelines have been revised by the NHS, coordinated by the National Demand Management Programme for Immunoglobulin. Conclusion Prednisone treatment for 7 days in hospitalized patients with CAP shortens time to clinical stability, time to hospital discharge and duration of intravenous antibiotic treatment without an increase in complications. Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers Pharmacokinetics, safety and tolerability of human recombinant alkaline phosphatase in healthy volunteers E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model T Masuda1, C Mitaka2, M Khin Hnin Si2, K Kido2, Y Qi2, T Uchida2, S Abe2, T Miyasho3, M Tomita4 1Tokyo Medical and Dental University, Tokyo, Japan; 2Tokyo Medical and Dental University Graduate School, Tokyo, Japan; 3Rakuno Gakuen University, Hokkaido, Japan; 4Tokyo Medical and Dental University Hospital of Medicine, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P127 (doi: 10.1186/cc14207) g p gi Results The mortality rate of 28 days in the L-group was 32.8%, and was 18.8% in the H-group. Mean arterial pressure increased signifi cantly (P <0.01) in the H-group compared with the L-group. WBC counts in the L-group increased and in the H-group decreased (P <0.01) during PMX- DHP treatment. Platelet counts in both groups decreased signifi cantly (P <0.01).There was no signifi cant diff erence between before and after PMX-DHP in IL-6 levels. On the other hand, IL-1ra decreased signifi cantly before and after PMX-DHP. Also, IL-6 and IL-1ra in the L-group were signifi cantly higher than those in the H-group at the start of PMX- DHP. PCT values in the L-group were increased compared with the H-group at the start of PMX-DHP (P <0.01). PCT in the L-group increased signifi cantly (P <0.01), but no signifi cant changes in the H-group. PAI- 1 showed no signifi cant changes before and after PMX-DHP and no changes in both groups at the start of PMX-DHP. Introduction Direct hemoperfusion with a polymyxin B immobilized column (PMX-DHP) adsorbs endotoxin and has been used for the treatment of septic shock [1]. However, the mechanisms of action behind PMX-DHP are not fully understood. Therefore, the purpose of this study was to elucidate mechanisms of action behind PMX-DHP in a rat model of cecal ligation and puncture. Introduction Direct hemoperfusion with a polymyxin B immobilized column (PMX-DHP) adsorbs endotoxin and has been used for the treatment of septic shock [1]. However, the mechanisms of action behind PMX-DHP are not fully understood. Therefore, the purpose of this study was to elucidate mechanisms of action behind PMX-DHP in a rat model of cecal ligation and puncture. Methods Sprague–Dawley rats were anesthetized and were mechanical ventilated after tracheostomy. The right internal carotid artery was cannulated with a catheter for continuous measurement of the arterial pressure and heart rate. P128 y p Results RecAP administration resulted in a terminal elimination half- life and plasma clearance of 49 to 58 hours and 2.8 to 3.4 l/hour after single ascending doses, respectively, and 63 to 66 hours and 3.1 to 3.8 l/hour after multiple ascending doses. Peak recAP concentrations and AP activity levels were reached at the end of the 1-hour infusion and showed a rapid decline with about 10% of the maximum concentration remaining at 4 hours and less than 5% at 24 hours post start. Although the maximal concentration and total systemic exposure of recAP and AP activity increased slightly more than dose proportionally this is of no signifi cance in the estimated therapeutic dose range. RecAP treatment was generally well tolerated and anti-drug antibodies could not be detected in serum. P129 Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network White blood cell counts have an impact on septic patient outcome followed by polymyxin-B immobilized fi ber with direct hemoperfusion p H Tanaka, T Ikeda, S Ono, S Suda, T Ueno Tokyo Medical University, Hachioji Medical Center, Hachioji, Japan Critical Care 2015, 19(Suppl 1):P128 (doi: 10.1186/cc14208) Introduction The mortality rate of severe sepsis and septic shock is varied and high (25 to 70%). In our institute, the indication for polymyxin-B immobilized fi ber with direct hemoperfusion (PMX-DHP) has been that circulatory failure (systolic blood pressure <90 mmHg or required catecholamines and high lactacidemia) continued despite following early goal-directed therapy by the Surviving Sepsis Campaign guidelines 2012. Conclusion RecAP is characterized by a long serum terminal half-life, by stable serum AP levels and did not exert any safety concerns when administered to healthy volunteers. These results pave the way to investigate the potential of recAP as a new treatment option for sepsis- associated AKI in a phase II clinical trial, which will start at the end of 2014 [Clinical Trial Register:NCT02182440]. g Methods This study included 80 patients with severe sepsis or septic shock due to abdominal infection retrospectively. These subjects were divided into two groups: those with WBC counts <4,000 (L-group: 64 patients) and those with WBC counts >12,000 (H-group: 16 patients). Mean arterial pressure, WBC counts, platelet counts, interleukin-6 (IL-6), and plasminogen activator inhibitor-1 (PAI-1) were measured immediately before the initiation and after the completion of PMX- DHP. Statistical analysis was performed using the chi-squared test for background factors, with Wilcoxon’s rank-sum test for comparison within a group, and Mann–Whitney’s U test for comparison between groups. The signifi cance level was set at P <0.05. References 1. Pickkers P, et al. Crit Care. 2012;16:R14. , ; 2. Heemskerk S, et al. Crit Care Med. 2009;37:417-23.e1. Pharmacokinetics, safety and tolerability of h alkaline phosphatase in healthy volunteers 1 d2 3 l 2 Methods We conducted a retrospective audit of pharmacy records of IVIg prescriptions issued to ICU patients with severe sepsis and septic shock from 2009 to 2014 against national prescription guidelines. Microbiology results were examined to support prescriptions, and admission APACHE II scores and unit outcomes were examined. Results From 2009 to 2014, 644 patients were admitted to the ICU with severe sepsis and septic shock, with a mortality rate of 41%. Seventeen patients received IVIg. Of these, eight patients met the national Methods We conducted a retrospective audit of pharmacy records of IVIg prescriptions issued to ICU patients with severe sepsis and septic shock from 2009 to 2014 against national prescription guidelines. Microbiology results were examined to support prescriptions, and admission APACHE II scores and unit outcomes were examined. p p y E Peters1, J Arend2, R Tiessen3, A Van Elsas2, R Masereeuw1, P Pickkers1 1Radboudumc, Nijmegen, the Netherlands; 2AM-Pharma, Bunnik, the Netherlands; 3PRA Health Sciences, Zuidlaren, the Netherlands Critical Care 2015, 19(Suppl 1):P126 (doi: 10.1186/cc14206) Results From 2009 to 2014, 644 patients were admitted to the ICU with severe sepsis and septic shock, with a mortality rate of 41%. Seventeen patients received IVIg. Of these, eight patients met the national Introduction Clinical trials showed renal protective eff ects of bovine intestinal alkaline phosphatase in critically ill patients with S44 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion PMX-DHP improved hemodynamics, acid–base balance, and creatinine levels through reducing cytokines and renal tubular cell death in a rat model of cecal ligation and puncture. These fi ndings suggest the preventive role of PMX-DHP in the development of sepsis- related acute kidney injury. sepsis-associated acute kidney injury (AKI) [1,2]. Recently, human recombinant AP (recAP) was developed as a pharmacological attractive replacement. We conducted a phase I clinical trial to evaluate tolerability, safety and pharmacokinetics of recAP in healthy volunteers. Methods In a randomized, double-blind, placebo-controlled phase I trial, healthy volunteers received via a 1-hour i.v. infusion a single dose of recAP (200, 500, 1,000 or 2,000 U/kg; n = 33) or multiple doses of recAP (500 or 1,000 U/kg; n = 18) on three consecutive days (n = 18). Serum recAP concentrations, AP activity levels and anti-drug antibodies were measured, and safety parameters were monitored. Reference 1. Ronco C, et al. Polymyxin B hemoperfusion: a mechanistic perspective. Crit. Care. 2014;18:309. 1. Ronco C, et al. Polymyxin B hemoperfusion: a mechanistic perspective. Crit. Care. 2014;18:309. Rat polymyxin B hemoperfusion model: preventive eff ect on renal tubular cell death in a rat cecal ligation and puncture model The right femoral vein was cannulated with a catheter for infusion of saline (10 ml/kg/hour) during the study period. The rats were randomized into three experimental groups: cecal ligation and puncture (CLP) + dummy column (Dummy- DHP) group (n = 10), CLP + PMX-DHP group (n = 10), and sham group (n = 4). Four hours after CLP, Dummy-DHP or PMX-DHP was performed for 1  hour. Blood was drawn from the right internal carotid artery, perfused through PMX column or dummy column, and returned to the right femoral vein. The heart rate, mean arterial pressure, arterial blood gases, and plasma concentrations of creatinine, lactate, potassium, and cytokines (IL-6 and IL-10) were measured at baseline and at 4, 5, and 8 hours after CLP. At the completion of the experiment, the rats were killed overdose of pentobarbital. The kidney, liver, and lung were harvested, and histopathologic examinations of these organs were performed. g g p Conclusion The mortality rate of the L-group tended to be higher than that of the H-group. Infl ammatory and anti-infl ammatory cytokines in the L-group were higher than those of the H-group. These results indicate that leukopenia (WBC <4,000) in severe sepsis patients leads to more severe outcome and hypercytokinemia than leukocytosis (WBC >12,000) in severe sepsis patients. P129 Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network E Sevketoglu1, D Yildizdas2, O Horoz2, H Kihtir1, T Kendirli3, S Bayraktar4, J Carcillo5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Istanbul, Turkey; 2Cukurova University Medical Faculty, Adana, Turkey; 3Ankara University Medical Faculty, Ankara, Turkey; 4Haseki Research and Training Hospital, Istanbul, Turkey; 5University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Results Hypotension and metabolic acidosis occurred in the CLP  + Dummy-DHP group, whereas hemodynamics and acid–base balance were better maintained in the CLP  + PMX-DHP group. Plasma concentrations of lactate, creatinine, potassium, and cytokines were signifi cantly higher in the CLP + Dummy-DHP group than in the CLP + PMX-DHP group at 8 hours. Renal tubular cell death was observed in the CLP + Dummy-DHP group, but not in the CLP + PMX-DHP group. Critical Care 2015, 19(Suppl 1):P129 (doi: 10.1186/cc14209) Introduction Thrombocytopenia-associated multiple organ failure (TAMOF) can lead to high mortality in critically ill children, possibly related to consequences of thrombotic microangiopathy. Plasma Introduction Thrombocytopenia-associated multiple organ failure (TAMOF) can lead to high mortality in critically ill children, possibly related to consequences of thrombotic microangiopathy. Plasma S45 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 APACHE scores before and after ECAC therapy were available for eight patients who died; APACHE score decreased >5 points in fi ve patients after single application of ECAC. exchange therapy may improve thrombotic microangiopathy [1]. The purpose of this observational cohort study is to describe whether there is an association between use of plasma exchange therapy and outcome in the Turkish TAMOF network. Conclusion ECAC can be used as adjuvant therapy in treatment of severe sepsis/septic shock/MOF. Our patients had high PM and four could be saved with use of ECAC. We could expect a better outcome if ECAC was used early (<24 hours) during treatment. However, future well-designed studies are needed to clarify the role of ECAC in patients with MOF/septic shock. Methods We performed a retrospective cohort analysis in patients with TAMOF at three diff erent pediatric ICUs comparing those who received plasma exchange (+) plus standard therapies with those who did not receive plasma exchange (–) and only received standard therapies. Results Among the 42 TAMOF patients enrolled, all had a primary or secondary sepsis diagnosis. Use of therapeutic plasma exchange in children with thrombocytopenia-associated multiple organ failure in the Turkish TAMOF network Fifteen received plasma exchange therapy (PE(+) group) and 27 received standard medical treatment without plasma exchange (PE(–) group). The mean age was 17.69 months (8.24 to 54.22) in the PE(+) group, and 13.46 months (6.47 to 20.55) in the PE(–) group. Age (P = 0.232), gender (P = 0.206), thrombocyte count (P = 0.09), OFI score (P = 0.111) and Pelod score (P = 0.177) on admission were not statistically diff erent between groups. The overall 28-day mortality was higher in the PE(–) group 70.37% compared with 26.67% in the PE(+) group (univariate P =0.006; multivariate controlling for Pelod, OFI, PRISM scores and neurological failure P = 0.048). Length of stay was increased in the PE(+) group (P = 0.004). Clinical experience of using a novel extracorporeal cytokine adsorption column for treatment of septic shock with multiorgan failure Methods We retrospectively evaluated 387 patients who received a broad-spectrum antimicrobial treatment for septic shock due to GNB between January 2009 and December 2012 in the ICU of 10 Japanese tertiary hospitals. After alignment of the treatment time phase for each patient, we divided the patients into two groups according to whether PMX treatment was performed within 24  hours after ICU admission (PMX group: n  = 129 and non-PMX group: n  = 258). The primary endpoint was 28-day mortality. Ruby Hall Clinic, Pune, India Introduction Severe sepsis and multiorgan failure (MOF) are major causes of death in the ICU. The extracorporeal cytokine adsorption column (ECAC; Cytosorb®, CytoSorbents Corporation, USA), a critical care focused therapeutic device, results in rapid in vitro and in vivo elimination of several key cytokines and prevents organ failure. Use of ECAC in patients with sepsis is a new area of research with insuffi cient data to promote large prospective RCTs. Studies published to date have shown promising results. We report our clinical experience with ECAC for severe sepsis/septic shock/MOF patients. Results The mean (SD) age and SOFA scores on ICU admission were 72.5 (12.5) years and 10.0 (3.4), respectively. The infection site was intra-abdominal (47.0%), pulmonary (17.6%), and urinary tract (27.8%). Two-thirds of all patients had bacteremia due to GNB. No diff erence in 28-day mortality was observed between the two groups (PMX: 33.9% vs. non-PMX: 33.1%, P = 0.87). In the Cox regression analysis adjusted for age, sex and facilities, the PMX treatment (hazard ratio = 0.87; 95% confi dence interval, 0.53 to 1.43) did not improve the outcome.f Methods A retrospective evaluation of ECAC in patients admitted to a tertiary ICU from November 13 to October 14 to analyze: clinical safety; selection of a subgroup of patients where it could be used; selection of timing for initiation; number of device fi lters required per patient; and selective markers to identify above initiation. Patients were managed with standard of care (SOC; antibiotics, vasopressors, i.v. fl uids, sepsis dosed steroids) and ECAC as adjuvant therapy. Vitals, APACHE II and SOFA scores were measured. i Conclusion No diff erence in mortality rate was observed after adjustment for the endotoxin adsorption therapy with PMX in the patients with septic shock due to GNB. Reference Introduction Mortality from septic shock in the ICU remains high, ranging from 30 to 50%. In particular, Gram-negative bacilli (GNB) account for 40% of the causative bacteria of severe sepsis, which progresses to multiorgan failure due to signifi cant infl ammation. Hemoperfusion with polymyxin B-immobilized fi ber (PMX) adsorbs endotoxin and can reduce the infl ammatory cascade of sepsis due to GNB. However, the clinical effi cacy of this treatment has not been demonstrated. We aimed to verify the effi cacy of endotoxin adsorption therapy by using PMX. Reference 1. Nguyen TC, Carcillo JA. Bench-to-bedside review: Thrombocytopenia- associated multiple organ failure – a newly appreciated syndrome in the critically ill. Crit Care. 2006;10: 235. Reference 1. Nguyen TC, Carcillo JA. Bench-to-bedside review: Thrombocytopenia- associated multiple organ failure – a newly appreciated syndrome in the critically ill. Crit Care. 2006;10: 235. Eff ectiveness of polymyxin B immobilized fi ber hemoperfusion in patients with septic shock due to Gram-negative bacillus infection: the PMXHP study N Saito1, K Sugiyama2, T Ohnuma3, T Kanemura4, M Nasu5, Y Yoshidomi6, H Adachi7, H Koami8, Y Tsujimoto9, A Tochiki10, Y Wagatsuma11, T Myumi12 1Chiba Hokusou Hospital, Nippon Medical School, Chiba, Japan; 2Tokyo Metropolitan Bokutoh Hospital, Tokyo, Japan; 3Saitma Medical Center, Jichi Medical University, Saitama, Japan; 4National Hospital Organization Disaster Medical Center, Tokyo, Japan; 5Urasoe General Hospita, Okinawa, Japan; 6Saga-ken Medical Center, Koseikan, Saga, Japan; 7Iizuka Hospital, Fukuoka, Japan; 8Saga University Hospital, Saga, Japan; 9Yamagata Prefectural Central Hospital, Yamagata, Japan; 10Tsukuba Medical Center Hospital, Ibaraki, Japan; 11University of Tsukuba, Ibaraki, Japan; 12University of Occupational and Environment Health, Fukuoka, Japan Conclusion The positive association found between use of plasma exchange therapy and improved survival supports the potential of this therapy in Turkish children with TAMOF. The positive, although less so, associated treatment eff ect observed after controlling for illness severity provides further rationale for performing a randomized controlled trial in the pediatric Turkish TAMOF network. Sample size calculations call for a 100-patient trial with a pre hoc interim analysis after enrollment of 50 TAMOF patients. p Critical Care 2015, 19(Suppl 1):P131 (doi: 10.1186/cc14211) Introduction Mortality from septic shock in the ICU remains high, ranging from 30 to 50%. In particular, Gram-negative bacilli (GNB) account for 40% of the causative bacteria of severe sepsis, which progresses to multiorgan failure due to signifi cant infl ammation. Hemoperfusion with polymyxin B-immobilized fi ber (PMX) adsorbs endotoxin and can reduce the infl ammatory cascade of sepsis due to GNB. However, the clinical effi cacy of this treatment has not been demonstrated. We aimed to verify the effi cacy of endotoxin adsorption therapy by using PMX. Impact of evolving cardiac catheterisation services on admissions to a regional ICU Results Nineteen ICU patients (14 men, fi ve women; 24 to 72 years; average ICU stay 10 days; average ventilator days 9) with APACHE II >17 (except one with dengue shock syndrome), SOFA score ≥11 (n = 16) and the majority having infection largely in the lung (n = 8; alone or with UTI and blood infection) followed by the abdomen (n = 4), UTI (n = 3) and others (n = 4) were given ECAC (total ECAC = 31). Predicted mortality (PM) was >40% in 16, >30% in two and <30% in two (tropical infections) patients. Duration of therapy was 6 hours (no. of ECAC = 18) and 8 hours (n = 4; no. of ECAC = 5) for the majority of patients. Overall, four patients (two with tropical infections and two with PM >40%) survived; three of them had were ECAC early (<24 hours of admission). The majority of patients (n = 11) who died could be given ECAC only once. Of patients who died, seven were given ECAC late (>24 hours). g EA Gorman, D Trainor Royal Victoria Hospital, Belfast, UK Critical Care 2015, 19(Suppl 1):P132 (doi: 10.1186/cc14212) EA Gorman, D Trainor Introduction National UK audit data demonstrate cardiac catheterisation services, including percutaneous coronary intervention and noncoronary interventions, are increasing [1-3]. National mortality rates post cardiac catheterisation are also increasing, refl ecting an increasing proportion of sicker patients undergoing interventional procedures [3]. National audit procedures do not evaluate patients admitted to intensive care post cardiac catheterisation. We aimed to Introduction National UK audit data demonstrate cardiac catheterisation services, including percutaneous coronary intervention and noncoronary interventions, are increasing [1-3]. National mortality rates post cardiac catheterisation are also increasing, refl ecting an increasing proportion of sicker patients undergoing interventional procedures [3]. National audit procedures do not evaluate patients admitted to intensive care post cardiac catheterisation. We aimed to S46 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P133). evaluate the impact of an evolving regional cardiac catheterisation service on a regional intensive care unit (RICU) serving a population of 1.8 million. Methods A retrospective review was carried out. Patients admitted from the regional cardiac catheterisation laboratory to the regional ICU, between September 2009 and September 2014, were identifi ed using validated RICU admission records. Clinical data were extracted from computerised patient records. Impact of the introduction of e-learning prior to a basic transthoracic echo course P Madhivathanan1, S Jain2, D Walker3 Introduction Deep venous thrombosis (DVT) is an increasing major cause of mortality and morbidity. There is a need for quick, easy, cheap, convenient and reliable diagnostic tools. The objectives were to ascertain the diagnostic concordance of emergency doctor- performed ultrasound (EDUS) of the lower extremities with specialist doctor-performed (radiologist or vascular surgeon) echo-Doppler (SDED), in the diagnosis of DVT, and to identify possible causes of nonconcordance. P Madhivathanan1, S Jain2, D Walker3 P Madhivathanan1, S Jain2, D Walker3 1Barts Health NHS Trust, London, UK; 2Homerton University Hospitals NHS Foundation Trust, London, UK; 3University College London Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P133 (doi: 10.1186/cc14213) P Madhivathanan , S Jain , D Walker 1Barts Health NHS Trust, London, UK; 2Homerton University Hospitals NHS Foundation Trust, London, UK; 3University College London Hospitals NHS Foundation Trust, London, UK Critical Care 2015, 19(Suppl 1):P133 (doi: 10.1186/cc14213) Introduction Focused Intensive Care Echo accreditation is a nationally approved pathway for training and accreditation in basic transthoracic echocardiography (TTE) in the UK. Recently, an e-learning module, the Intensive Care Echo and Basic Lung Ultrasound (ICE-BLU), has been introduced to facilitate TTE learning [1]. Previous work from our group has shown that incorporating simulation-based teaching elements into a basic TTE course improves candidates’ satisfaction [2]. We assessed the impact of introducing the ICE-BLU e-learning programme prior to our simulation-based basic TTE course. Methods A prospective, multicentre study. Adult patients (>18 years old) with clinical suspicion of DVT, with high or moderate risk (on Wells scoring), or low risk with increased D-dimer levels, were eligible for the study. Emergency doctors performed two EDUS in femoral and popliteal areas (these results were blinded). After this, echo-Doppler was performed by specialist doctors. Both procedures were done within 24 hours of each other. The fi nal result was considered nonconcordant if one or both of the EDUS did not match with the SDED. These SDED were used as reference (as standard clinical practice). Methods Prior to the August 2014 course, all candidates were required to complete the ICE-BLU e-learning module. On the morning of the course, the candidates completed a questionnaire to assess the impact of the e-learning module. The survey included questions on the quality of content, user friendliness, whether the content was pitched at the right level and any problems faced whilst accessing the e-learning module. P133 p Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) Diagnostic concordance of emergency doctor-performed bedside ultrasonography versus specialist-performed echo-Doppler ultrasonography in the diagnosis of deep venous thrombosis of lower limbs 2. National Institute for Cardiovascular Outcomes. Myocardial ischaemia national audit project. Public report April 2012–March 2013. http://www.ucl.ac.uk/nicor/audits/minap/reports. 3. British Cardiovascular interventional society. National coronary interventional procedures. Public report. January 2012–December 2013. http://www.bcis.org.uk/resources/PCI_Audit_Report_2012_fi nal.pdf. DL Ly-Pen1, JP Penedo Alonso2, MS Sánchez Perez2, FR Roldán Moll2, MZ Zamorano Serrano2, LD Díaz Vidal2, SJ Justo3 1Southend University Hospital, Westcliff -on-Sea, UK; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) DL Ly-Pen1, JP Penedo Alonso2, MS Sánchez Perez2, FR Roldán Moll2, MZ Zamorano Serrano2, LD Díaz Vidal2, SJ Justo3 1Southend University Hospital, Westcliff -on-Sea, UK; 2Hospital Universitario Ramón y Cajal, Madrid, Spain; 3Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain Critical Care 2015, 19(Suppl 1):P134 (doi: 10.1186/cc14214) References 1. http://www.e-lfh.org.uk/programmes/icu-echoultrasound. Accessed 1 Dec 2014. 2. Madhivathanan PR, et al. Simulation-based transthoracic echo teaching: a tertiary centre experience. Intensive Care Med. 2014;40 Suppl 1:S158-9. 1. British Cardiovascular Interventional Society. Audit returns for adult interventional procedures. January 2013–December 2013. http://www.bcis. org.uk/documents/BCIS_Audit_2013_for_web_Version_23-11-2014.pdf. Impact of evolving cardiac catheterisation services on admissions to a regional ICU p p Results A total of 170 patients were identifi ed (representing 2.9% of critical care admissions during this time). Baseline characteristics: 71.7% male, median age 66 (IQR 55 to 74), median APACHE score 18 (IQR 15 to 23). Seventy-one patients (41.7%) had an APACHE score >20. Fifteen patients (8.8%) were aged >80  years. Admissions increased yearly – 20 in 2010, 26 in 2011, 35 in 2012, 47 in 2013, 37 at the end of the third quarter of 2014 (projected 59 admissions by year end 2014). Median length of stay was 3.5 days (IQR 1.8 to 7.2). Average length of stay reduced yearly (9.14 days in 2010 to 5.01 days in 2014). ICU bed- days per year remained static over the 5-year period. Critical care and hospital mortality rates were 33% and 39% respectively. There was a trend towards increasing mortality yearly, and with increasing age and APACHE score. Conclusion Our survey has shown that the e-learning initiative was welcome by the candidates. We conclude that introduction of e-learning prior to a simulation-based basic TTE course enhances candidates’ satisfaction and feedback. Conclusion An evolving cardiac catheterisation service is having a signifi cant impact on intensive care services within a regional centre. Increasing mortality trends in this critical care population refl ects post- cardiac catheterisation mortality trends nationally. We suggest intensive care admissions post cardiac catheterisation should be included in the national audit, to allow forward planning of intensive care services and to promote quality improvement within this population. References Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) y Results From January 2014 to December 2014, 32 patients with septic shock (study group) and 20 patients with sepsis but no septic shock (control group) were recruited. The baseline characteristics were similar. Conventional echocardiographic measurements, including LVEF (59.53% vs. 60.67% in the control group, P  =  0.450) and fractional shortening (31.98% vs. 32.98%, P = 0.323), did not diff er between the two groups. The study group had a greater degree of myocardial dysfunction measured by left ventricular global longitudinal strain (–14.6 vs. –17.6, P = 0.005, with a less negative value implying worse myocardial contractility). The hemodynamic profi les (cardiac index 3.49 l/minute/m2 vs. 3.41 l/minute/ m2 respectively, P = 0.764) were not statistically diff erent.i Introduction Myocardial depression is common among septic patients [1]. The aim of this study was to assess whether the values of cardiac index (CI) and velocity–time integral (VTI) calculated by echocardiography diff er between survivors and nonsurvivors of sepsis. Methods This was a prospective observational study. We included adult newly admitted septic patients, regardless of disease severity. Exclusion criteria were concomitant pregnancy or obstetric/gyne- co logical sepsis and co-existing or terminal diseases that may limit life expectancy. At the moment of recruitment, additional exclusion criteria included: concomitant pulmonary embolism, trauma or acute ischemic coronary disease; pericardial tamponade; aortic valve disease; tachyarrhythmias and absence of adequate echocardiographic windows. Echocardiographic evaluations were made within the fi rst 10 minutes of initiation of fl uid therapy in the emergency room. All measurements and images were obtained with a 1.5 to 3.5 MHz phased array transducer using a standard cardiac preset. CI is the quotient of the cardiac output (CO) divided by the body surface area. The CO is the product of the stroke volume by the heart rate. Stroke volume is calculated as the product between aortic VTI (measured using pulsed- wave Doppler) and aortic cross-sectional area. The latter is calculated in the long axis parasternal window using the left ventricular outfl ow tract diameter measurement. Impact of the introduction of e-learning prior to a basic transthoracic echo course We also analysed candidates’ feedback from our January and August 2014 courses (Figure 1). Results From September 2013 to September 2014, a total of 328 patients were enrolled. Fifty-one investigators from seven hospitals performed the EDUS. Each patient had the EDUS (femoral and popliteal areas) and SDED (also in femoral and popliteal areas). Of 328 pairs of US studies, 37 were nonconcordant between EDUS and SDED. Two EDUS were incomplete, so the concordance analysis was performed with 326 US studies, with 35 discordant. The percentage of agreement between EDUS and SDED was 89.26%. The kappa index was 0.76 (95% CI = 0.69 to 0.84), and this means a substantial agreement. Results The response rate of the survey was 100%. Eighty per cent of candidates completed the e-learning module. The e-learning module was rated high by most candidates (80%). However, nearly one-half of the candidates faced problems accessing the module, online. Analysis of candidates’ feedback (from the January and August 2014 courses) revealed that candidates’ overall impression was better with the introduction of e-learning prior to the course. g Conclusion There is substantial agreement between EDUS and SDED in the diagnosis of DVT, in routine clinical practice. This confi rms the results of previous papers. The largest nondiagnostic concordance in thrombus occurs in the early performances of emergency doctors, S47 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Comparing group I versus group II, the mortality rate was 45%, and there was a statistically signifi cant diff erence for temperature (P = 0.001), HR (P = 0.001) and WBC count (P = 0.01) on admission. Upon comparing survivors versus nonsurvivors in group I there was a statistical diff erence in HR on day 7 (P = 0.02), successful vasopressor withdrawal (P = 0.02), P/F ratio (P = 0.02) and ScVO2 on day 7 (P = 0.03). Regarding IL-1α, IL-1β, TNFα and troponin I there was no statistical signifi cant diff erence between groups I and II but IL-6, IL-10 and CRP showed statistically signifi cant diff erence on admission PV and CS. Pro- BNP shows statistically signifi cant diff erence in all CS samples between septic and nonseptic groups. Regarding echo upon comparing the survivors versus nonsurvivors, E’d/t on day 0 shows a statistically signifi cant diff erence between both groups. Reference 1. Angus DC. Epidemiology of severe sepsis in USA. Crit Care Med. 2001;29:1303-10. P135 P135 Sepsis survivors present with higher values of cardiac index and velocity time integral in the emergency department T Santos, M Schweller, C Gontijo-Coutinho, D Franci, P Nocera, T Guerra-Grangeia, J Matos-Souza, M Carvalho-Filho Unicamp, Campinas, Brazil Critical Care 2015, 19(Suppl 1):P137 (doi: 10.1186/cc14217) Conclusion This is a fi rst study in the adult population to show that the use of speckle tracking imaging can diagnose signifi cant sepsis- induced myocardial dysfunction, which was not otherwise detectable by conventional echocardiography. P135 Cardiac abnormalities in patients with septic shock detected by speckle tracking echocardiography PY Ng1, WC Sin1, AK Ng2, WM Chan1 1Queen Mary Hospital, Hong Kong; 2Grantham Hospital, Hong Kong Critical Care 2015, 19(Suppl 1):P135 (doi: 10.1186/cc14215) P135 Cardiac abnormalities in patients with septic shock detected by speckle tracking echocardiography PY Ng1, WC Sin1, AK Ng2, WM Chan1 1Queen Mary Hospital, Hong Kong; 2Grantham Hospital, Hong Kong Critical Care 2015, 19(Suppl 1):P135 (doi: 10.1186/cc14215) Introduction Sepsis-induced myocardial dysfunction is a well- recognized condition and confers worse outcomes in septic patients. However, the diagnostic criteria remain poorly described. Echocardiographic assessment by conventional parameters such as left ventricular ejection fraction (LVEF) is often aff ected by ongoing changes in preload and afterload conditions. Novel echocardiographic technologies such as speckle tracking imaging have evolved for direct assessment of the myocardial function. In this study, we investigate the measurement of myocardial strain by speckle tracking imaging for the diagnosis of sepsis-induced myocardial dysfunction. g p y p Conclusion Diastolic dysfunction was seen in 90% of patients. Fever, HR, and WBC counts are still good early indicators for diagnosis of sepsis. Vasopressor withdrawal on the seventh day was a good predictor for survival. Admission serum IL-6, IL-10 and CRP from PV were better indicators for sepsis than IL-1, pro-BNP and troponin I. Admission TNFα and seventh-day IL-6 levels were highly prognostic for mortality. CS samples proved that NT pro-BNP is a good indicator for sepsis diagnosis and a good predictor for survival. TNFα from CS samples was also a good predictor of mortality. SAPS II and a slower E’d/t on admission was a good predictor of mortality. R f Methods This is a prospective, case–control study at a university- affi liated tertiary care adult medical ICU. Consecutive patients admitted with a diagnosis of septic shock meeting the international consensus criteria were included. Patients with other causes of myocardial dysfunction were excluded. They are compared with age-matched, gender-matched, and cardiovascular risk factor-matched controls, who were admitted to hospital for sepsis but did not develop septic shock. Conventional echocardiographic parameters, as well as speckle tracking imaging of myocardial function, were obtained within 24  hours of diagnosis. Offl ine analyses of endocardial tracings were performed by two independent operators. Reference Impact of the introduction of e-learning prior to a basic transthoracic echo course SAPS II and seventh-day SOFA are good predictive scores for mortality in sepsis. especially until the fi fth performance. After the sixth one, the incidence of mismatches falls dramatically. It seems advisable to mentor the training programmes with at least fi ve shadowed performances in order to lower the incidence of mistakes. P136 Evaluating the eff ect of sepsis and septic shock on myocardial functions by echocardiography and serum biomarker level in peripheral veins and coronary sinus M Soliman1, A Alazab1, R El Hossainy1, M Nirmalan2, H Nagy1 1Cairo University, Cairo, Egypt; 2University of Manchester, UK Critical Care 2015, 19(Suppl 1):P136 (doi: 10.1186/cc14216) P138 Speckle tracking imaging for evaluation of eff ects of positive end-expiratory pressure level on right ventricular function M Türker, A Pirat, A Camkiran Firat, B Pirat, A Sezgin, G Arslan Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P138 (doi: 10.1186/cc14218) Figure 2 (abstract P139). Values of PEEP above which LS was not present. Introduction Positive end-expiratory pressure (PEEP) is commonly used to correct hypoxemia in the ICU. However, PEEP may impair right ventricular functions by increasing its afterload. Speckle tracking imaging (STI) is a new echocardiography strain analysis technique that provides direct assessment of myocardial contractility during systole and diastole. The aim of this study was to evaluate the eff ects of diff erent PEEP levels on right ventricular functions by using STI in patients undergoing coronary artery bypass grafting surgery. Methods After ethics committee approval and patients’ written consent, we prospectively analyzed 20 CABG surgery patients. After initiation of mechanical ventilation and before sternotomy, 5, 10, and 20 cmH2O PEEP were applied in 5-minute intervals consequently. After stabilization at each PEEP level, four-chamber and two-chamber images of the right ventricle were recorded using TEE. The right ventricle diameter, velocity, longitudinal strain, SR, and fractional area change (RVFAC) were calculated and evaluated from the recorded images. PEEP at which LS disappeared or reappeared were compared using the Wilcoxon signed-rank test to assess the infl uences of anatomical side and PEEP increase or decrease. The values of PEEP at disappearance of LS for the right lung were not statistically signifi cantly diff erent from the left lung (P  =  0.844 for increases, P  =  0.938 for decreases). The values of PEEP at which LS disappeared obtained during increases were not statistically signifi cantly diff erent from values obtained during decreases (P = 1.000 for left lung, P = 0.875 for right lung; Figure 1). From data pooled from both sides and protocols, the median value of PEEP at which LS disappeared as a false positive sign of pneumothorax was 25 cmH2O (interquartile range = 20 to 30 cmH2O). At PEEP = 10 cmH2O, no patient showed absence of LS, whereas at PEEP = 35 cmH2O, all patients showed absence of LS (Figure 2). g Results The mean age of study patients (85% male) was 59.7 ± 10.5 years. Intraoperative mean, systolic, and diastolic arterial blood pressures and heart rate were similar at the three PEEP levels. P139 Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP J Golub1, A Markota1, A Stožer2, G Prosen3, A Bergauer1, F Svenšek1, A Sinkovič1 1University Medical Centre Maribor, Slovenia; 2University of Maribor, Slovenia; 3Community Health Centre, Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P139 (doi: 10.1186/cc14219) P140 Lung ultrasound in quantifying lung water in septic shock patients L De Geer, A Oscarsson, M Gustafsson Linkoping University Hospital, Linkoping, Sweden Critical Care 2015, 19(Suppl 1):P140 (doi: 10.1186/cc14220) Evaluating the eff ect of sepsis and septic shock on myocardial functions by echocardiography and serum biomarker level in peripheral veins and coronary sinus M Soliman1, A Alazab1, R El Hossainy1, M Nirmalan2, H Nagy1 1Cairo University, Cairo, Egypt; 2University of Manchester, UK Critical Care 2015, 19(Suppl 1):P136 (doi: 10.1186/cc14216) Introduction Sepsis is a leading cause of death in critically ill patients despite the use of modern antibiotics and resuscitation therapies. Biomarkers and cardiovascular changes have an important place in this process. Myocardial depression occurs in 40% of septic patients. Methods Twenty patients (group I) with sepsis or septic shock were included and 10 patients (group II) served as the nonseptic group. Group I morbidity and mortality at day 28 in the ICU were targeted as the endpoint. Laboratory investigations, APACHE IV, SAPS II and SOFA scores were calculated. Biomarkers IL-1α, IL-1β, IL-6, IL-10, TNFα, CRP, NT-proBNP and troponin level were estimated on admission and day 7 in the peripheral vein (PV) and coronary sinus (CS). Transthoracic echocardiography and tissue Doppler imaging was done on admission and on day 7. Results In 3 months, 58 patients were included. The average age was 46.6 years, and 36 were male. Overall mortality was 14%. We included 16 patients with sepsis syndrome, 27 patients with severe sepsis and 15 patients with septic shock. Severe sepsis patients presented with higher values of CI, when compared with sepsis syndrome and septic shock patients (3.46, 3.08 and 2.92 l/minute/m2, respectively, P = NS). The same occurred with VTI (19.27, 18.81 and 16.74 cm for severe sepsis, sepsis syndrome and septic shock, respectively; P = NS). Mean values of CI were lower in nonsurvivors of sepsis (2.51 vs. 3.35 l/minute/ m2, P  =  0.018). Mean values of VTI were also lower in nonsurvivors (14.83 vs. 19.01 cm, P = 0.022). Conclusion In our study, nonsurvivors of sepsis presented with lower values of both CI and VTI in the emergency department. Therefore, CI S48 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P139). Absence of LS during increasing and decreasing PEEP. Figure 2 (abstract P139). Values of PEEP above which LS was not present. Figure 1 (abstract P139). Absence of LS during increasing and decreasing PEEP. and VTI may be good markers of sepsis severity and mortality in newly admitted patients. In addition, further studies are warranted to assess the role of CI and VTI as therapeutic targets. Reference 1. Silva E, Pedro Mde A, Sogayar AC, et al. Brazilian Sepsis Epidemiological Study (BASES study). Crit Care. 2004;8:R251-60. P138 Compared with 5 and 10 cmH2O PEEP, mean RVFAC signifi cantly decreased at 20 cmH2O PEEP (P  =  0.001). Right ventricle velocity reduced with incremental PEEP increases (P <0.05). Mean SR values decreased at 20 cmH2O PEEP when compared with 5 cmH2O PEEP (P = 0.03). Mean right ventricle diameter measurements decreased with incremental PEEP increases; however, this decrease was signifi cantly diff erent between 20 cmH2O PEEP and other two PEEP levels (P = 0.01). The mean right ventricle strain value signifi cantly decreased at 20 cmH2O PEEP when compared with other two PEEP levels (P <0.001 for both). p g Conclusion According to this study, higher PEEP levels correlate with disappearance of LS without pneumothorax. Absence of LS in patients with high PEEP should be interpreted with caution and other signs of pneumothorax should be sought before therapeutic interventions are attempted. Conclusion Compared with 5 and 10 cmH2O PEEP levels, right ventricle functions in terms of strain, SR, right ventricle diameter, and RVFAC were signifi cantly impaired at 20 cmH2O PEEP level. P140 Variations in near-infrared spectroscopy-derived oxygen downslope during a vascular occlusion test in critically ill patients: relationship with outcome p A Donati, E Damiani, A Carsetti, V Monaldi, E Montesi, P Pelaia A Donati, E Damiani, A Carsetti, V Monaldi, E Montesi, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P141 (doi: 10.1186/cc14221) Università Politecnica delle Marche, Ancona, Italy y Critical Care 2015, 19(Suppl 1):P141 (doi: 10.1186/cc14221) Results No changes in mean arterial pressure were observed. The perfused small vessel density tended to decrease at t1 and normalize at t2 (Figure 1) in the hyperoxia group. These variations appeared early after 2 minutes of FiO2 changes. A signifi cant increase in lactate levels over time (from 1.1 (0.9 to 1.7) at t0 to 1.4 (1.1 to 1.9) mmol/l at t2, P = 0.01) was seen in the hyperoxia group. Introduction Near-infrared spectroscopy (NIRS) with a vascular occlusion test (VOT) can be used to extrapolate information regarding the tissue oxygen extraction rate. We explored the meaning of variations in tissue oxygen saturation downslope (StO2down) during a VOT in critically ill patients. Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Figure 1 (abstract P142). y p Methods In this prospective observational study, NIRS (thenar eminence) was applied every day in 93 patients admitted to the ICU. A VOT was performed using a 40% StO2 target. The slope of the desaturation curve was assessed separately for the fi rst part (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between Down2 – Down1 was calculated.if Results No signifi cant diff erences were seen in StO2 Down1 or Down2 between ICU survivors (n  = 76) and ICU nonsurvivors (n  = 17) over Figure 1 (abstract P141). Figure 1 (abstract P142). Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Conclusion Hyperoxia induces an early decrease in microvascular perfusion, which appears to go back to normality at return to normoxia. Normobaric hyperoxia and the microcirculation in critically ill patients: a prospective observational study Normobaric hyperoxia and the microcirculation in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, E Montesi, S Ciucani, M Rogani, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P142 (doi: 10.1186/cc14222) Conclusion LUS non-invasively and user-independently quantifi es lung water in concordance with, but does not replace, invasive measurements. Further studies are needed establish the role of LUS as a monitoring and diagnostic tool in septic shock patients. References Introduction It is well known that oxygen acts as a vasoconstrictor. We evaluated the impact of normobaric hyperoxia on the sublingual microcirculation in critically ill patients. Introduction It is well known that oxygen acts as a vasoconstrictor. We evaluated the impact of normobaric hyperoxia on the sublingual microcirculation in critically ill patients. 1. Frassi F, et al. J Card Fail. 2007;13:830-5. 1. Frassi F, et al. J Card Fail. 2007;13:830-5. 2. Prosen G, et al. Crit Care. 2011;15:R114. Methods Forty mechanically ventilated (FiO2 ≤50%) patients with hemodynamic stability were enrolled in a prospective observational study. The fi rst 20 patients underwent a 2-hour period of hyperoxia (FiO2  = 100%), and 20 patients were studied as controls (no FiO2 variations). The sublingual microcirculation (three sites) was evaluated with sidestream dark-fi eld imaging at baseline (t0), after 2  hours of hyperoxia (t1), and 2  hours after return to baseline (t2). Continuous video recording was also performed during FiO2 variations on one and the same area (2-minute video). 3. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP The values of S49 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 guidelines [3]. The median number of B-lines was 15 (IQR 10 to 30) and the median (IQR) NT-proBNP, EVLWI and oxygenation index (OI) were 7,800 ng/l (3,690 to 15,050), 11 ml/kg (IQR 8 to 18) and 9.2 (5.7 to 15.7), respectively. None of the characteristics diff ered signifi cantly between survivors and nonsurvivors. The number of B-lines correlated to EVLWI (ρ = 0.45, P = 0.04; r2 = 0.20, P = 0.04), but not to NT-proBNP (ρ = –0.42, P = 0.06), OI (ρ = 0.25, P = 0.31) or ICU length of stay (ρ = 0.14, P = 0.57). On Bland–Altman analysis, mean diff erences and 95% limits of agreements between B-lines and EVLWI was 4.9 (–14.5 to 24.5), and 5.9 (–3.5 to 15.3) when assessing observer agreement. The ICC between methods was 0.52 (95% CI = –0.17 to 0.81) and 0.90 (95% CI = 0.73 to 0.92) between observers. the fi rst 10 days in the ICU, while Down2 – Down1 was higher in ICU nonsurvivors (Figure 1). Patients in the upper quartile of mean Down2 – Down1 showed the highest 90-day mortality (P = 0.014).l Conclusion ICU nonsurvivors tended to show a fl attening in the last part of the desaturation curve during a VOT, suggesting a reduced tissue oxygen extraction. This may depend on microvascular dysfunction and/or cellular hypometabolic status. P142 Absence of lung sliding is not a reliable indicator of pneumothorax in patients who require high PEEP Introduction Quantifi cation of lung ultrasound (LUS) artifacts (B-lines) is used to assess pulmonary congestion in emergency medicine and cardiology [1,2]. We investigated B-lines in relation to extravascular lung-water index (EVLWI) from invasive transpulmonary thermodilution in septic shock patients. Our aim was to evaluate the role of LUS in an intensive care setting. 1University Medical Centre Maribor, Slovenia; 2University of Maribor, Slovenia; 3Community Health Centre, Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P139 (doi: 10.1186/cc14219) Methods Twenty-one patients admitted with septic shock to a general ICU underwent LUS of eight zones, four per hemithorax, within 24  hours after ICU admission. EVLWI was calculated simultaneously by transpulmonary thermodilution using a pulse-contour continuous cardiac output system, and NT-proBNP and clinical data were collected. Two physicians blinded to other data independently quantifi ed the number of B-lines. Spearman’s rho was used to test the correlation of B-lines to EVLWI and clinical data, and linear regression and Bland– Altman analysis were used to assess the agreement between B-lines and EVLWI. Interobserver variability was tested using Bland–Altman analysis and intraclass correlation coeffi cient (ICC). Introduction The objective of our study was to estimate correlation between PEEP and disappearance of lung sliding (LS) due to lung overdistension in the absence of pneumothorax. Methods We performed a prospective study from September 2013 to May 2014 in adult patients with respiratory failure who required mechanical ventilation, lung CT and recruitment manoeuvre. Lung CT was used as the gold standard to exclude pneumothorax. A staircase recruitment manoeuvre was used with 5 cmH2O increases of PEEP from baseline to 35 cmH2O and decreases in reverse order. The duration of each step was 1 minute. Lung ultrasound was performed to evaluate LS at each step in one intercostal window in the highest point of left and right hemithoraces by physicians trained in lung ultrasound and blinded to changes in PEEP.i Results Fourteen patients (67%) were male, the median age was 62 years (IQR 55 to 68) and eight (38%) patients had cardiac comorbidities. In median, SAPS 3 was 64 (IQR 60 to 74), ICU length of stay was 3 days (IQR 2 to 8) and seven patients (33%) died within 30 days of ICU admission. All patients were mechanically ventilated and treated according to Results In all, eight patients were included; fi ve (62.5%) males, mean age 70.1 ± 7.4 years. Mean auto-PEEP was 0.7 ± 0.4 cmH2O. Prospective nonrandomized observational study of the use of an impedance threshold device in patients with spontaneous respiration and hemodynamic instability Conclusion The length of time to lactate normalization in the severe burn patient is a marker of survival and a simple parameter to guide the endpoint of resuscitation; however, the percentage of lactate clarifi ed in the fi rst 24 hours is not valid. Introduction The use of an impedance threshold device (ITD) in cardiac arrest victims has been shown to increase the systolic arterial pressure (SAP) by increasing venous return [1]. There are limited studies concerning the use of ITD in patients with spontaneous respiration and hemodynamic instability. The purpose of this study is to evaluate changes of hemodynamic parameters with the use of ITD in patients with spontaneous respiration and hemodynamic instability. Lactate in the burn patient This may refl ect a tissue hypometabolic status, which may be better elicited in the fi nal part of the ischemic challenge. Healthy volunteers (n = 27) were studied as controls. The slope of the desaturation curve was assessed separately for the fi rst (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between, Down2 – Down1, was calculated. Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P  <0.05); no diff erence was seen between ICU survivors (n  = 7) and nonsurvivors (n  = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P  <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P  <0.05); no diff erence was seen between ICU survivors (n  = 7) and nonsurvivors (n  = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P  <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Conclusion Tissue oxygen extraction was reduced in septic patients. Nonsurvivors showed a fl attening in the last part of the desaturation curve during a VOT, while the fi rst part of the StO2 downslope did not show any diff erence between survivors and nonsurvivors. This may refl ect a tissue hypometabolic status, which may be better elicited in the fi nal part of the ischemic challenge. y Results During a period of 2  years we studied 143 patients; their mean age was 46.98  ±  19.38 years, mean TBSA burn injury of the study population was 22.82 ± 20.25. The fl ame was the most frequent mechanism. A total of 83 patients were in mechanical ventilation and 13.6% of them developed ARDS. The mortality range in the study group was 17%. Serum lactate at admission ≥2 mmol/l was associated with 31.3% mortality versus 6% in patients with a serum lactate at admission <2 mmol/l (P <0.05). Length of time to lactate normalization variable is associated with mortality (P <0.02). The average lactate normalization time was 4.6 days in nonsurvivors while in survivors it was 2.02 days. A relation does not exist between the lactate clearance and mortality in all patients. P144 P144 Prospective nonrandomized observational study of the use of an impedance threshold device in patients with spontaneous respiration and hemodynamic instability C Pantazopoulos1, I Floros1, N Archontoulis1, D Xanthis1, D Barouxis2, N Iacovidou2, T Xanthos2 1Laiko General Hospital of Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P144 (doi: 10.1186/cc14224) P143 Near-infrared spectroscopy for assessing the tissue oxygen extraction rate during sepsis: relationship with outcome C S ll S d S C l Introduction Microcirculatory dysfunction impairs tissue oxygenation during sepsis. We applied near-infrared spectroscopy (NIRS) to evaluate the tissue oxygen extraction rate in sepsis and its relationship with outcome. Methods A prospective observational study; 14 septic patients underwent NIRS monitoring (thenar eminence) with a vascular occlusion test (using a 40% StO2 target) at admission to the ICU. Figure 1 (abstract P141). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S50 Figure 1 (abstract P143). 10 minutes after the intervention. Endpoint of the study was a change of patient’s SAP after application of ITD. Results The SAP of patients that were included in the study increased 15 to 22  mmHg (P  <0.05). Heart rate remained unchanged. Eighty percent of patients declared good to very good tolerance from ITD application. Conclusion In this observational study of patients with spontaneous respiration and hypotension, ITD increased the SAP, while it seems that it was well tolerated by patients. Additional and larger studies will be needed in the future in order to investigate the benefi ts of ITD when used to patients with spontaneous respiration and hemodynamic instability. Reference 1. Pirrallo RG, et al. Eff ect of an inspiratory impedance threshold device on hemodynamics during conventional manual cardiopulmonary resuscitation. Resuscitation. 2005;66:13-20. 1. Pirrallo RG, et al. Eff ect of an inspiratory impedance threshold device on hemodynamics during conventional manual cardiopulmonary resuscitation. Resuscitation. 2005;66:13-20. Lactate in the burn patient p EH Herrero, M Sánchez, L Cachafeiro, A Agrifoglio, B Galván, MJ Asensio, A García de Lorenzo Hospital La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P145 (doi: 10.1186/cc14225) Introduction Severe burns result in rapid loss of intravascular volume due to development of a severe capillary leak and hypovolemic shock. It is widely accepted that traditional markers, such as blood pressure and urinary output, are useful but do not suffi ciently refl ect global perfusion, regional microcirculation or reversal shock. Blood lactate concentration is widely used in ICUs as a reliable prognostic marker of global tissue hypoxia. Our aim is to determine whether the percentage of lactate clarifi ed in the fi rst 24 hours is valid as a guide for resuscitation. Methods We prospectively studied 143 consecutive burn patients admitted to our Burn Unit. Sociodemographics and comorbidities data were recorded. Clinical data were collected to calculate the Acute Burn Severity Index. Resuscitation according to the Parkland formula was guided by a urinary output of 0.5 to 1 ml/hour and the results of monitoring the blood pressure. Crystalloid solution (Ringer´s acetate) was given exclusively during the fi rst 24 hours. Early surgical excision of burn eschar and early coverage of excised burn wounds with autografts was performed. Initial and subsequent serum lactate levels were measured to calculate lactate clearance according to the formula: lactate basal – lactate at 24 hours / lactate basal × 100. The primary outcome was mortality. Figure 1 (abstract P143). Healthy volunteers (n = 27) were studied as controls. The slope of the desaturation curve was assessed separately for the fi rst (StO2 Down1) and the last part (StO2 Down2) of the curve and the diff erence between, Down2 – Down1, was calculated. Results StO2 Down1 was lower in healthy volunteers as compared with septic patients (P  <0.05); no diff erence was seen between ICU survivors (n  = 7) and nonsurvivors (n  = 7). StO2 Down2 was similar between healthy volunteers and ICU survivors, while it was higher in nonsurvivors (P  <0.01 vs. healthy). ICU nonsurvivors showed higher Down2 – Down1 as compared with ICU survivors (P <0.01, Figure 1). Conclusion Tissue oxygen extraction was reduced in septic patients. Nonsurvivors showed a fl attening in the last part of the desaturation curve during a VOT, while the fi rst part of the StO2 downslope did not show any diff erence between survivors and nonsurvivors. Is the shock index a universal predictor in the emergency department? A cohort study Results Twenty-three patients underwent VA ECMO for refractory CS in the study period. Etiologies of CS were: 11 acute myocarditis, fi ve acute myocardial infarction and seven acute decompensation of chronic cardiomyopathy. The median time of ECMO was 10 days (4 to 15). Thirteen patients died during hospital stay and 10 survived. Three patients were bridged to LVAD and two to heart transplant; eight were bridged to recovery. The main cause of ICU death was multiple organ dysfunction (12/13). Nonsurvivors showed signifi cantly higher LAC0 (5 (2 to 6) vs. 8 (5 to 11), P = 0.021). Lactate clearance at 48 hours was not signifi cantly diff erent between survivors and nonsurvivors (79%, 95% CI  = 67 to 86 vs. 60%, 95% CI  = 32 to 72, P  =  0.08). However, LAC48 was predictive for ICU mortality (AUC 0.82; 95% CI = 0.64 to 1.0; P = 0.011). ROC curve analysis identifi ed the accuracy was highest by setting the lactate <2 mmol/l. Patients that did not normalize lactate (LAC <2 mmol/l) after 48 hours despite hemodynamic restoration had poorer outcome at 30 days, as is shown in the Kaplan–Meier curve (log- rank P = 0.006) (Figure 1). Introduction The shock index (SI; heart rate/systolic blood pressure) is a widely reported tool to identify acutely ill patients at risk for circulatory collapse in the emergency department (ED). Because old age, diabetes, essential hypertension, and β-/Ca2+ channel-blockers might reduce the compensatory increase in heart rate and mask blood pressure reductions in shock or pre-shock states, we hypothesized that these factors weaken the association between SI and mortality, reducing the utility of SI to identify patients at risk. Methods This was a cohort study from Odense University Hospital of all fi rst-time visits to the ED between 1995 and 2011 (n = 111,019). The outcome was 30-day mortality. We examined whether age ≥65 years, diabetes, essential hypertension, and use of β-/Ca2+ channel-blockers modifi ed the association between SI and mortality. The prognostic value of SI ≥1 was evaluated with diagnostic likelihood ratios. Conclusion Failing to normalize patient’s LAC in the fi rst 48 hours of VA ECMO assistance for CS is a predictor of ICU mortality. Targeting LAC level <2  mmol/l at 48  hours post ECMO institution might be a reasonable goal for these patients. Results We observed a 30-day mortality of 3%. Blood lactate normalization following venoarterial ECMO institution for refractory cardiogenic shockfi Methods A 5-month prospective nonrandomized observational study that included 50 adult patients with spontaneous respiration and hypotension in the emergency room and the wards of multiple causes except trauma. After measurement of the SAP and verifi cation of hypotension (SAP ≤90  mmHg), a mask-style ITD was added. Hemodynamic parameters were evaluated every 1  minute and for M Bottiroli, D Decaria, T Maraffi , S Nonini, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P146 (doi: 10.1186/cc14226) Introduction Venoarterial (VA) ECMO is used to support patients with refractory cardiogenic shock (CS). Elevated lactate level (>2  mmol/l) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S51 Figure 1 (abstract P146). an inotropic score as a predictor of mortality and morbidity among children who diagnosed septic shock. Figure 1 (abstract P146) Methods A multicenter retrospective chart review was performed in two pediatric ICUs. A total of 93 children with septic shock were recruited. Hourly doses of following inotropes were recorded for the fi rst 48 hours after admission: dopamine, dobutamine, adrenaline and noradrenaline. The inotrope score for every hour, minimum, maximum and mean values for the fi rst 24 hours, and subsequent 24 hours were calculated. In our analysis, the inotrope score was calculated as described by Wernovsky. We expanded this formula to include norephinephrine as follows: Wernovsky Inotrope Score = dopamine dose (μg/kg/minute) + dobutamine dose (μg/kg/minute) + 100 × epinephrine dose (μg/kg/ minute). Our adjusted inotrope score = Wernovsky Inotrope Score + 100 × norepinephrine dose (μg/kg/minute).f p p μg g Results Forty-two of 93 patients died. Age and sex were not diff erent between survivors and nonsurvivors. Signifi cantly higher mean and maximum inotropic score for the fi rst 24 hours and fi rst 48 hours were found in nonsurvivors than those of survivors (P <0.05). Using 15 as a cutoff point for predicting mortality, the sensitivity and specifi city were 69.76% and 50.98% respectively. The association between Prism scores and minimum, mean and maximum inotrope scores were statistically signifi cant for 0 to 24 hours, 25 to 48 hours and 0 to 48 hours. Mean 0 to 24 hours and maximum 0 to 48 hours inotrope scores were weakly associated with prolonged ICU stay (P = 0.047, P = 0.042 respectively). There were no signifi cant relationships between inotrope scores and receiving mechanical ventilation.i Figure 1 (abstract P146). Is the shock index a universal predictor in the emergency department? A cohort study With SI <0.7 as reference, a SI of 0.7 to 1 was associated with an adjusted OR of 2.9 (CI 2.7 to 3.2) for 30-day mortality while the adjusted OR for SI ≥1 was 10.3 (CI 9.2 to 11.5). ORs for SI ≥1 were reduced (but still signifi cant) in patients who were older, hypertensive, or on β-/Ca2+ channel-blockers, whereas diabetes had no eff ect. The OR for SI ≥1 in patients ≥65 years was 8.2 (CI 7.2 to 9.4) compared with 18.9 (CI 15.6 to 23.0) in younger patients. β-/Ca2+ channel-blocked patients had an OR of 6.4 (CI 4.9 to 8.3) versus 12.3 (CI 11.0 to 13.8) in nonusers, and the OR for hypertensive patients was 8.0 (CI 6.6 to 9.4) versus 12.9 (CI 11.1 to 14.9) in nonhypertensive patients. The OR for SI ≥1 of 9.3 (CI 6.7 to 12.9) in diabetics did not diff er from the OR of 10.8 (CI 9.6 to 12.0) in nondiabetic patients. A SI of 0.7 to 1 was associated with ORs signifi cantly greater than 1 (range: 2.2 to 3.1) with no evident diff erences within the subgroups. A SI measurement ≥1 was associated with lower positive likelihood ratios in patients ≥65 years, with hypertension, diabetes or using β-/Ca2+ channel-blockers (range 4.9 to 6.5) compared with patients not exposed to these factors (range 7.6 to 11.6). Blood lactate normalization following venoarterial ECMO institution for refractory cardiogenic shockfi is an indicator of end-organ hypoperfusion. We hypothesize that the lactate (LAC) normalization had prognostic value in this cohort of patients. Methods We performed a retrospective observational study on patients admitted to the ICU for refractory CS from January 2010 to November 2014. Patients with postcardiotomy and/or post-transplant CS were excluded. Demographics, clinical, hemodynamic and biochemical values were collected. LAC was measured on arterial blood before ECMO institution (LAC0) and after 48 hours (LAC48). Lactate clearance was calculated as follows: (LAC0  – LAC48) / LAC0  ×  100. Data were analyzed by comparative statistic; sensibility and specifi city were tested with ROC. Conclusion The mean and maximum inotropic scores in the fi rst 24 hours and 0 to 48 hours are an independent predictor of mortality in critically ill children with septic shock. P148 Is the shock index a universal predictor in the emergency department? A cohort study AK Kristensen1, JG Holler1, J Hallas2, A Lassen1, N Shapiro3 1Odense University Hospital, Odense, Denmark; 2University of Southern Denmark, Odense, Denmark; 3Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P148 (doi: 10.1186/cc14228) P149 Risk factors for severe vasodilatory shock after cardiac surgery J Almeida, F Galas, J Fukushima, E Almeida, A Gerent, E Osawa, C Park, R Nakamura, A Leme, M Sundin, R Kalil Filho, F Jatene, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P149 (doi: 10.1186/cc14229) Introduction Vasodilatory shock is a well-known complication in patients who undergo cardiac surgery with cardiopulmonary bypass (CPB) and its occurrence is associated with higher morbidity and mortality. Despite that, clinical characteristics of vasoplegic shock and its spectrum of severity are poorly described. The aim of this study was to compare patients who developed mild to moderate vasoplegic shock with patients who developed a severe form and to identify predictive factors for the severe form of vasoplegic shock. Results Heterogeneity of LS eff ects was registered in a number of values. The most signifi cant positive eff ect which allowed one to evaluate myocardial reserve was demonstrated by decrease of PPA (93.5% of patients) and increase of EF (77.6% of patients). The most signifi cant changes were also noted in decrease of TrI, PH and MI (in 53.2%, 36.6% and 36% of patients, respectively). In 69.2% of patients with noncoronarogenic dilated cardiomyopathy the eff ect of LS exposure was marked. In the majority of patients with ischemic cardiomyopathy the eff ect was moderate. In case of the absence of LS-positive eff ect, perioperative use of mechanical circulatory support was considered. Conclusion Preoperative use of LS allows one to evaluate myocardial reserves and prepare high-risk patients with CHF for surgery. Our fi ndings may serve as one of the additional criteria to choose the type of surgical treatment: reconstructive surgery (with or without perioperative mechanical circulatory support) or heart transplantation. Methods We performed an observational study in 300 patients who underwent cardiac surgery with CPB and presented within the fi rst 24  hours after surgery with refractory hypotension and used a vasopressor agent. Severe vasoplegic shock was defi ned as a requirement of norepinephrine higher than 1  μg/kg/minute or the use of two or more vasopressors. Baseline characteristics, laboratorial, clinical and intraoperative data, such as amount of fl uids, bleeding, blood transfusion, inotropes and length of CPB were collected at ICU admission. Logistic regression was performed using severe vasodilatory shock as the outcome. Results There were 46 (15%) patients who develop the severe form of vasodilatory shock within 24  hours after cardiac surgery. Reference Reference 1. Jochberger S, et al. Intensive Care Med. 2009;35:489-97. 1. Jochberger S, et al. Intensive Care Med. 2009;35:489-97. P149 In a univariate analysis, patients with the severe form were more likely to be older, to receive more blood transfusion and inotropic agents, to have higher levels of serum lactate, lower hemoglobin concentration and lower SvO2 at the end of the procedure, lower cardiac output index, higher heart rate and higher levels of reactive C protein at ICU admission. These patients also experienced more postoperative organ dysfunction, had a longer length of ICU stay and higher mortality. There were no diff erences between patients regarding amount of fl uids and length of CPB. In a multivariate analysis we identify age (OR = 1.04, 95% CI = 1.01 to 1.08, P = 0.016), intraoperative use of epinephrine (OR = 5.49, 95% CI = 2.42 to 12.43, P <0.001), higher serum lactate at the end of the procedure (OR = 1.04, 95% CI = 1.01 to 1.06, P = 0.001) and intraoperative blood transfusion (OR = 5.06, 95% CI = 2.19 to 11.69, P <0.001). P151 Levosimendan versus dobutamine in cardiac surgery MD Delgado-Amaya, EC Curiel-Balsera, CJ Joya-Montosa Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P151 (doi: 10.1186/cc14231) Introduction Early studies suggested a signifi cant increase in survival in patients treated with levosimendan compared with dobutamine or placebo (LIDO, RUSSLAN and CASINO trials). However, two subsequent studies (SURVIVE and REVIVE II) have not confi rmed these fi ndings. Methods A prospective observational study of all patients undergoing cardiac surgery at Malaga’s Regional Hospital from March 2009 to March 2013. We analyzed patients who used levosimendan compared with those that used dobutamine in the fi rst  hours after cardiac surgery, discarding patients in which neither of these two drugs were used or surgical cases that arrived at the ICU with both inotropics. We analyzed demographic variables as well as clinical complications in the ICU and overall perioperative mortality of patients. We performed a second analysis using the propensity score, obtaining the probability of patients being treated with either drug, pairing each patient who received levosimendan with its nearest neighbor receiving dobutamine. Results We collected 875 patients: 331 received one of the two drugs, 50 received both drugs and 494 did not receive any drug. ICU mortality was 7.2% (levosimendan group) and 12.5% (dobutamine group), P = 0.1. After adjustment for severity and type of surgery, the use of levosimendan in the postoperative period was not a protective factor for ICU mortality (P = 0.18, OR = 0.5, 95% CI = 0.18 to 1.3). In the matched sample, mortality was 7.4% (levosimendan group) and 5.9% (dobutamine), P = 0.73. After logistic regression adjusted for severity, measured with EuroSCORE and type of surgery, levosimendan was not a protective factor for ICU mortality (P = 0.8, OR = 1.2, 95% CI = 0.26 to 5.45).f Conclusion This study demonstrated that older patients, intraoperative blood transfusion and utilization of epinephrine were independently associated with a more severe form of vasodilatory shock after cardiac surgery with CPB. Also, we identifi ed that a higher lactate at the end of the procedure was an independent predictive factor for this severe form of shock. Is an inotrope score a predictor of mortality and morbidity in children with septic shock? E Sevketoglu1, A Anil2, S Kazanci1, O Yesilbas1, M Akyol1, S Bayraktar3, N Aksu4, S Hatipoglu1, M Karabocuoglu5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Bakýrkoy, Turkey; 2Izmir Katip Celebi University Medicine Faculty, Izmir, Turkey; 3Haseki Research and Training Hospital, Istanbul, Turkey; 4Tepecik Research and Training Hospital, Izmir, Turkey; 5Sisli Memorial Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P147 (doi: 10.1186/cc14227) E Sevketoglu1, A Anil2, S Kazanci1, O Yesilbas1, M Akyol1, S Bayraktar3, N Aksu4, S Hatipoglu1, M Karabocuoglu5 1Bakirkoy Dr. Sadi Konuk Research and Training Hospital, Bakýrkoy, Turkey; 2Izmir Katip Celebi University Medicine Faculty, Izmir, Turkey; 3Haseki Research and Training Hospital, Istanbul, Turkey; 4Tepecik Research and Training Hospital, Izmir, Turkey; 5Sisli Memorial Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P147 (doi: 10.1186/cc14227) Introduction Inotropes and vasoactive drugs in septic shock are commonly used to maintain cardiac output, tissue perfusion and oxygenation. We undertook this study with the purpose of evaluating S52 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion SI is independently associated with 30-day mortality in a broad population of ED patients. Old age, hypertension and β-/Ca2+ channel-blockers weaken this association, but the association remains prognostic. SI ≥1 suggests substantial risk of 30-day mortality in all ED patients. for the surgical intervention (2 to 4  days before surgery). In total, 44.9% of the patients had CHF caused by noncoronarogenic dilated cardiomyopathy and 55.1% by ischemic cardiomyopathy. Indication for LS therapy was left ventricular ejection fraction (EF) <35% (28 ± 6%). Seventy percent of patients had mitral insuffi ciency (MI), grades II to IV, 63% tricuspid insuffi ciency (TrI), grades II to III, 84% pulmonary hypertension (PH), 47% arterial hypertension, grades II to III, and 27% of the patients had left ventricular aneurysm. Mean level of BNP was 1,803 ± 124 pg/ml. LS was administered as i.v. infusion in doses of 0.025 to 0.1 μg/kg/minute without previous bolus injection. Mean duration of infusion was 27.5 ± 5.3 hours. After infusion all patients underwent control assessment of values. All patients were operated: 25 (23.3%) underwent reverse cardiac remodeling, 63 (58.9%) myocardium revascularization (MR) with mitral or aortic valve replacement, 17 (15.9%) MR and/or resection of left ventricular aneurism and two (1.9%) heart transplantation. Levosimendan in the emergency department: a useful tool to improve cellular perfusion Introduction Levosimendan was originally developed for the treat ment of decompensated heart failure in situations for which conventional therapy is not suffi cient. It is an eff ective calcium- sensitising drug with vasodilatory and inotropic eff ects and improves cardiac contractility. Trials have shown positive outcome benefi t with the use of levosimendan [1]. We reviewed the usage levosimendan at our institution and outcome of these patients. Introduction Levosimendan is an inotropic seldom used in emergency departments (EDs). It has shown improved mortality in patients with shock [1] with few adverse eff ects [2]. This work denotes the experience of levosimendan use in the ED of Hospital Universitario Mayor between 2012 and 2014. p Methods We reviewed the use of levosimendan at Harefi eld from January 2013 through December 2013. Patient demographics, logistic EuroSCORE (Figure  1), diagnosis, surgical or intervention details, inotropic support, dosage and duration of levosimendan use, length of stay in the ICU, cost (Table 1) and patient outcome were collected. Results Levosimendan was used in 30 patients, 23 (77%) male and seven (23%) female. Median age was 69 (59 to 72.8). Levosimendan was used post cardiac surgery, post angioplasty and in patients with ventricular assist devices (VAD) and extracorporeal membrane oxygenator (ECMO). Most of the patients received a standard regimen of 12.5 mg administered at a dose of 0.1 μg/kg/minute for 24 hours. Concurrent noradrenaline was used in most of the patients ranging from 0.02 to 0.2 μg/kg/minute. The median length of stay in the ICU was nine (6 to 14.5) days for survivors and 23.5 (7.5 to 36) days in nonsurvivors. Sixteen patients (55%) survived and were discharged from the hospital. Methods We reviewed the use of levosimendan at Harefi eld from January 2013 through December 2013. Patient demographics, logistic EuroSCORE (Figure  1), diagnosis, surgical or intervention details, inotropic support, dosage and duration of levosimendan use, length of stay in the ICU, cost (Table 1) and patient outcome were collected. Methods We present a retrospective study which analyzes the eff ect, after 24  hours, of levosimendan administration on perfusion parameters of 166 ED patients. Patients had to have shock diagnosis of any cause. Diff erences between the initial and fi nal mean value of the following parameters were evaluated: lactate, central venous oxygen saturation (ScvO2) and venoarterial diff erence of CO2 (DvaCO2). Data were stratifi ed according to levosimendan categories (initial or rescue). Reference Reference Preoperative treatment with levosimendan helps to evaluate myocardial reserves in cardiosurgical patients with chronic heart failure Conclusion In our environment, we have observed diff erences in the use of levosimendan compared with dobutamine (higher rate of men undergoing CABG, diabetes and worse EF). After homogenizing the sample of patients by propensity score, an eff ect on mortality is discarded and we observed a signifi cant need for use of norepinephrine and a nonsignifi cant trend for prolonged mechanical ventilation and renal failure requiring renal replacement therapy, both probably related with the greatest need for vasopressors observed. Introduction The aim of the study was to assess the possibility of preoperative levosimendan (LS) administration in myocardial reserve evaluation and choice of the method of surgical treatment in patients with chronic heart failure (CHF). Introduction The aim of the study was to assess the possibility of preoperative levosimendan (LS) administration in myocardial reserve evaluation and choice of the method of surgical treatment in patients with chronic heart failure (CHF). Methods LS was used in 107 (female and male) patients (mean age 53  ±  3  years) as a component of CHF therapy to prepare them S53 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P152 Reference 1. Follath F, et al. Lancet. 2002;360:196-202. Levosimendan in the emergency department: a useful tool to improve cellular perfusion In addition, association between diff erent variables with mortality was sought. Diff erences were considered statistically signifi cant at probability levels below 0.05.f y , ( ) p Results Levosimendan was used in 30 patients, 23 (77%) male and seven (23%) female. Median age was 69 (59 to 72.8). Levosimendan was used post cardiac surgery, post angioplasty and in patients with ventricular assist devices (VAD) and extracorporeal membrane oxygenator (ECMO). Most of the patients received a standard regimen of 12.5 mg administered at a dose of 0.1 μg/kg/minute for 24 hours. Concurrent noradrenaline was used in most of the patients ranging from 0.02 to 0.2 μg/kg/minute. The median length of stay in the ICU was nine (6 to 14.5) days for survivors and 23.5 (7.5 to 36) days in nonsurvivors. Sixteen patients (55%) survived and were discharged from the hospital. p y Results There were no diff erences in APACHE II values between patients who received levosimendan as initial therapy from those who received it as a rescue measure. A total of 41 patients fulfi lled lactate normalization requirements (lactate <2.0 or clearance >50%) (Table 1). Forty-four patients reached normal values of SvcO2 and 37 patients of DavCO2 after levosimendan initiation. There were no associations between the normalization of lactate, SvO2 and DvaCO2 and diff erent types of shock. Twenty-nine patients who received initial therapy with levosimendan normalized their lactate values and 12 who received it as a rescue therapy (P <0.05). Sixty-three patients developed hypotension, and none had adverse eff ects requiring discontinuation of the drug. Hospital mortality was 47.7%. Variables associated with mortality in the study group were lactate value at admission (OR = 1.3, 95% CI = 1.0 to 1.7), the use of vasopressin after start levosimendan (OR = 7.5, 95% CI = 1.9 to 28.6) and the use of norepinephrine before starting (OR = 10.8, 95% CI = 1.9 to 60.7). Figure 1 (abstract P152). Logistic EuroSCORE of all patients. Table 1 (abstract P153). Perfusion variables before and after levosimendan Variable Initial Final Lactate (mmol/l) 3.3 (0.5 to 18) 2.38 (0.4 to 17)* SvcO2 (%) 56.3 (23 to 85) 65.2 (37 to 91)* DvaCO2 (mmHg) 8.2 (–16 to 26) 7.3 (–2 to 21) *P <0.01. McNemar test. Table 1 (abstract P153). Perfusion variables before and after levosimendan Variable Initial Final Figure 1 (abstract P152). Logistic EuroSCORE of all patients. Figure 1 (abstract P152). Levosimendan in the emergency department: a useful tool to improve cellular perfusion Logistic EuroSCORE of all patients. Conclusion Levosimendan use in the ED, as initial or rescue therapy, normalizes lactate values and improves the SvcO2 after 24  hours, without an increase in adverse eff ects. f Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Levosimendan Adrenaline Milrinone Maximum dose (μg/kg/minute) 0.2 1 0.7 Cost per vial (£) 894 2.30 16 Cost per 24 hours (£) 894 7 112 Conclusion We have successfully used this drug in high-risk patients during the perioperative period with good results without major complications. Levosimendan seems to reduce catecholamine requirement, the need for mechanical circulatory support, and the duration of critical care, which can justify the cost of this drug. It can be also useful in weaning patients from short-term VAD and ECMO. Larger studies are required in this area. Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Table 1 (abstract P152). Cost of levosimendan based on a 70 kg patient Levosimendan Adrenaline Milrinone P153 Levosimendan in the emergency department: a useful tool to improve cellular perfusion P153 Levosimendan in the emergency department: a useful tool to improve cellular perfusion G Devia, J Torres, S Lopez Hospital Universitario Mayor, Bogotá, Colombia Critical Care 2015, 19(Suppl 1):P153 (doi: 10.1186/cc14233) Levosimendan: use, cost-eff ectiveness and outcome in a tertiary cardiothoracic centre Levosimendan: use, cost-eff ectiveness and outcome in a tertiary cardiothoracic centre A Ranjan, N Bhudia, I McGovern, C Walker, L Kuppurao Royal Brompton & Harefi eld NHS Foundation Trust, Harefi eld, London, UK Critical Care 2015, 19(Suppl 1):P152 (doi: 10.1186/cc14232) A Ranjan, N Bhudia, I McGovern, C Walker, L Kuppurao Royal Brompton & Harefi eld NHS Foundation Trust, Harefi eld, London, UK Critical Care 2015, 19(Suppl 1):P152 (doi: 10.1186/cc14232) P153 References 1. Landoni G, et al. Minerva Anestesiol. 2010.76:276-86. 2. Mebazaa A, et al. JAMA. 2007.297:1883–91. Conclusion We have successfully used this drug in high-risk patients during the perioperative period with good results without major complications. Levosimendan seems to reduce catecholamine requirement, the need for mechanical circulatory support, and the duration of critical care, which can justify the cost of this drug. It can be also useful in weaning patients from short-term VAD and ECMO. Larger studies are required in this area. Introduction The inotropic agents used in the ICU are dobutamine and milrinone; unfortunately, they have shown signifi cant side eff ects when used for myocardial depression during septic shock. Our objective is to describe Mn behavior in septic shock. S54 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 adaptive threshold for low amplitude QRS detection; QRS fi lter with an extended frequency range; management of VT alarm priorities. Conclusion A majority of the FPs was linked to user settings and patient conditions. The algorithm changes resulted in a clear reduction of ECG algorithm-related FP alarms, while the magnitude of the reduction depends strongly on the settings at the bedside. References 1. ECRI Institute. 2013;42:354-9. 2 Drew BJ et al PLoS ONE 2014 9: e110274 Methods We reviewed 72 clinical records of patients with diagnosis of septic and mixed shock who received Mn through January to December 2013. Demographic, hemodynamic, metabolic and gasometric data were recorded before and after Mn infusion. The PiCCO monitoring system was used. Data were expressed as mean and standard deviation. The statistical analysis used Student’s t test. P <0.05 was considered signifi cant. adaptive threshold for low amplitude QRS detection; QRS fi lter with an extended frequency range; management of VT alarm priorities. Conclusion A majority of the FPs was linked to user settings and patient conditions. The algorithm changes resulted in a clear reduction of ECG algorithm-related FP alarms, while the magnitude of the reduction depends strongly on the settings at the bedside. References 1. ECRI Institute. 2013;42:354-9. 2. Drew BJ, et al. PLoS ONE. 2014.9: e110274. 1. ECRI Institute. 2013;42:354-9. P155 y Admission to the ICU with cerebrovascular event (CVE) and trauma was related to arrhythmia (P = 0.021, P = 0.032, respectively). There was a signifi cant relationship between VIP and sepsis presence (P  <0.001, P <0.001). Atrial fi brillation was the most frequent type of arrhythmia (53%), and the most frequently used medication was diltiazem (28.5%). The patients who had arrhythmias had a longer ICU stay (P = 0.021). The mortality rate for all patients was 48.1%. There was no statistically signifi cant relationship between arrhythmia and mortality (P >0.05). False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) False arrhythmia alarms can be reduced by algorithm improvements while the magnitude of the reduction is aff ected by alarm settings M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 M Kaski1, J Vanhatalo1, S Treacy2, H Viertio-Oja1 y j 1GE Healthcare, Helsinki, Finland; 2GE, Milwaukee, WI, USA , , ; , , , Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) Critical Care 2015, 19(Suppl 1):P155 (doi: 10.1186/cc14235) Introduction The ECRI Institute has identifi ed alarm fatigue as the number one health technology hazard [1]. A recent study on 461 ICU patients investigated 2,558,760 alarms [2]. In total, 88.8% of the annotated 12,671 arrhythmia alarms were false positives (FPs). It was concluded that the excessive number of alarms is ‘a complex interplay of inappropriate user settings, patient conditions, and algorithm defi ciencies’. Nine conditions causing alarms, four of which were ECG algorithm related, were reported [2]. In this study, we investigated a new algorithm in which improvements targeting three of the reported four ECG-related conditions were implemented: low amplitude QRS; wide QRS; nonactionable ventricular tachycardia (VT). gi p y y Conclusion Older age, higher APACHE II scores, trauma, CVE, VIP and sepsis increases arrhythmia risk in critically ill patients. Atrial fi brillation is most common and the most preferred treatment for all arrhythmias is diltiazem. References Results Seventy-two patients were studied: 36.5% were women, mean and SD of age, APACHE II, mechanical ventilation days and long ICU stay were: 67 ± 16 years, 18.5 ± 5.9 points, 14.9 ± 12.9 and 24.5 ± 21.9 days, respectively. A total 20.3% of the patients received dobutamine. Thirty- nine percent presented mixed shock. Global mortality was 23%. After Mn infusion: cardiac index (CI) increased: 3.1 ± 1 to 3.3 ± 1.1, cardiac rate increased: 82.4 ± 14.4 to 88.3 ± 18 and ScvO2 increased: 71.1 ± 10.3 to 76.1 ± 7.3 (P <0.05). PaCO2 arteriovenous diff erence and lactate were reduced: 7.36 ± 3.3 to 6.04 ± 3.6 and 18.7 ± 14.9 to 13.1 ± 9.1 (P <0.05). CVP, MAP, RVSI, VSTI, EVLWI and base excess showed no signifi cant diff erence. Mn initial average dose was 0.35  ±  0.13. NE before and after Mn infusion showed no signifi cant diff erence: 0.11 ± 0.20 versus 0.12 ± 0.22. P156 Arrhythmia incidence and risk factors in critically ill patients G Tasdemir, N Kelebek Girgin, A Aydin Kaderli, E Cizmeci, R Iscimen, F Kahveci, A Aydinlar Uludag University Medical Faculty, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P156 (doi: 10.1186/cc14236) Introduction Cardiac arrhythmias may be observed at any time during the ICU stay. The prognosis may suff er due to these arrhythmias. In our study, we aimed to evaluate incidence and risk factors of arrhythmias occurring in patients in the ICU. Introduction Cardiac arrhythmias may be observed at any time during the ICU stay. The prognosis may suff er due to these arrhythmias. In our study, we aimed to evaluate incidence and risk factors of arrhythmias occurring in patients in the ICU. Conclusion Mn optimizes cardiovascular performance in septic shock and mixed shock, without aff ecting hemodynamic variables and global tissue perfusion. In addition, we observed that the IC, ScvO2, PaCO2 arteriovenous diff erence and lactate are related variables. References Methods Patients treated in the ICU were included in the study if they fulfi lled the following: age >18, no cardiac valvular disease, no cardiac surgery in the recent 6-month period, no history of myocardial infarction (MI), need for mechanical ventilation, and one or more organ failure. Demographic, hemodynamic and laboratory parameters, APACHE II score, presence of sepsis, acute renal failure, MI, and VIP during the ICU stay were recorded. Therapies used for arrhythmia and response to therapies were also recorded. 1. Holmes CL, et al. Vasoactive drugs for vasodilatatory shcok in ICU. Curr Opin Crit Care. 2009;15:398-402. 2. Jentzer JC, et al. Pharmacotherapy update on the use of vasopressors and inotropes in the intensive care unit. J Cardiovasc Pharmacol Ther [Epub ahead of print].l p p Results Two hundred and fourteen patients were included in the study. Twenty-six percent (n = 56) of patients had arrhythmias. Incidence was higher in females (P = 0.045). Average age of arrhythmic patients was 69 (19 to 86), and they were older than nonarrhythmic patients (P <0.001). APACHE II scores were higher in arrhythmic patients (P = 0.001). 3. Deep A, et al. Evolution of haemodynamics and outcome of fl uid-refractory septic shock in children. Intensive Care Med. 2013;39:1602-9. 3. Deep A, et al. Evolution of haemodynamics and outcome of fl uid-refractory septic shock in children. Intensive Care Med. 2013;39:1602-9. References 1. Kanji S, et al. Crit Care Med. 2008;36:1620-4 1. Kanji S, et al. Crit Care Med. 2008;36:1620-4. j , ; 2. Annane D, et al. Am J Respir Crit Care Med. 2008;178:20-5. 2. Annane D, et al. Am J Respir Crit Care Med. 2008;178:20-5. High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery N De Mey, I Brandt, C Van Mieghem, K De Decker, G Cammu, L Foubert OLV AALST, Aalst, Belgium Critical Care 2015, 19(Suppl 1):P158 (doi: 10.1186/cc14238) N De Mey, I Brandt, C Van Mieghem, K De Decker, G Cammu, L Foubert OLV AALST, Aalst, Belgium Introduction Hs-cTn is the new standard cardiac biomarker for the diagnosis of myocardial necrosis. We conducted a prospective study to compare the course and values of Hs-cTn and CK-MB after CABG and OPCAB. We also evaluated the relationship between values >10 × 99th percentile URL of CK-MB and Hs-cTn as a possible marker for perioperative myocardial infarction. g g Results The cTnI concentrations measured in the control group ranged from 0.4 to 17.2, mean 2.1 (95% CI: 1.6 to 2.6) ng/l, without age dependency. Men revealed higher levels than women, means (IQR) were 2.8 (1.2 to 2.6) and 1.1 (0.7 to 1.3) ng/l. The CLSI nonparametric method revealed a 99th percentile value of 16 ng/l. The quantifi cation of cTnI above the LoD (1.0 ng/l) and below the 99th percentile was possible in 79 of 120 individuals. The imprecision profi le according to NCCLS revealed 20%, 10% and 5% CVs at concentrations of 2, 3 and at 20 ng/l, respectively. The discharge diagnosis was NSTEMI in 71 patients. The cTnI median values at 0, 3 and 6 hours were 46, 166 and 399 ng/l, respectively. AUC values at 0, 3 and 6  hours were 0.923, 0.964 and 0.969 for hs-cTnT and 0.919, 0.962 and 0.958 for cTnI, respectively. cTnI revealed AUC values of absolute changes from admission to 3 hours and from admission to 6 hours were 0.920 and 0.931, respectively.i Methods All adult patients undergoing cardiac surgery between February and August 2014 were included. Exclusion criteria were urgent surgery, GFR <60  ml/minute/1.73 m2, CK-MB >4  μg/l and/or Hs-cTn >14 ng/l at baseline (BL). Hs-cTn and CK-MB were measured before induction (BL), upon arrival in the ICU and at fi xed times after arrival. Patients with perioperative AMI as defi ned by the third universal defi nition of AMI were excluded for post hoc analysis [1]. Results Of the 93 patients admissible for inclusion, 40 in the CABG and 14 in the OPCAB group met all inclusion criteria in this preliminary dataset. High-sensitive cardiac troponins and CK-MB concentrations in patients undergoing cardiac surgery CK-MB values are higher from ICU arrival up to T24 versus baseline in both CABG and OPCAB (P <0.0001) with a peak at T3. For Hs-cTn, ICU up to T48 values are higher (P <0.01) in CABG with a peak Conclusion The PATHFAST cTnI demonstrated complete fulfi llment of the analytical criteria for high-sensitive cTn assays: The imprecision (CV) at the manufacturer-recommended 99th percentile value was 5%. The quantifi cation of cTnI in was possible in 65.8% of healthy individuals. The examination of the diagnostic characteristics revealed complete concordance with the hscTnT assay for detection of NSTEMI and for assessment of absolute changes of cTn values (rise and/or fall) in NSTEMI patients. PATHFAST cTnI showed highly sensitive detection of NSTEMI with increasing sensitivity at admission and after 3 to 6 hours, not going along with decreased specifi city. The PATHFAST cTnI might be useful at the point-of-care setting for early rule-in and rule-out diagnosis of NSTEMI. Figure 1 (abstract P158). Figure 1 (abstract P158). P158 yi g y Methods cTnI was determined using PATHFAST in 120 healthy individuals (60 men and 59 women, 21 to 69  years old, median 42  years). Cardiac disorders were excluded by cardiac magnetic resonance imaging. The diagnostic characteristics were investigated by determination of cTnI and cTnT (Roche hs-cTnT) in 181 patients admitted to the chest pain unit at presentation, and 3 and 6 hours later. The results were related to the discharge diagnoses. Pulmonary hypertension, right ventricular dysfunction and acute heart failure: a portentous consortium y Methods The false alarm rate of the new algorithm (GE Carescape, 2012) was compared with that of the algorithm evaluated in the study (GE Solar, 2003) [2] on the collected ECG waveform data. User settings such as QRS detection sensitivity (high/normal) were not available. Therefore, normal sensitivity was assumed for both versions. With the old algorithm, 10 patients with low QRS amplitudes gave a signifi cantly higher number of FPs than were reported [2]. For those patients, both sensitivity modes were tested with the old algorithm. Sixty-six percent of patients with a pacemaker did not have the pacemaker mode selected [2]. Outlier patients in which false alarms were due to user settings (20 patients with a pacemaker) or patient condition (four patients with a bundle branch block) rather than algorithm defi ciency were excluded. HD Michalopoulou, HM Michalopoulou, P Stamatis, E Kostetsos, D Stamatis Metaxa Hospital, Athens, Greece Critical Care 2015, 19(Suppl 1):P157 (doi: 10.1186/cc14237) Introduction Pulmonary hypertension and right ventricular dysfunc- tion (RVD) are frequently encountered in patients with acute heart failure. We sought a better understanding of the coupling between RVD and pulmonary hypertension in the setting of acute decompensated heart failure (ADHF) as it might improve the prognostic stratifi cation and infl uence the survival rates. l Methods Echocardiography was performed in 329 patients with ADHF and right ventricular function was assessed by measuring the right ventricular fractional area, and a right ventricular ejection fraction (RVEF) <35% was taken as the cutoff value for RV systolic dysfunction. The systolic pulmonary pressure (PASP) was calculated from the tricuspid regurgitation signal applying the modifi ed Bernoulli equation, Results Improved algorithm resulted in 66% reduction of FP alarms. When using the high-sensitivity mode for the 10 patients with low QRS, FP reduction was 18%. No compromises regarding detection of true events were found. The 24 outlier patients contributed to 81.3% of FP alarms. The algorithm changes responsible for the reduced FPs were: S55 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 at T6, and from T3 to T48 in OPCAB (P <0.05) versus baseline (Figure 1). In CABG patients CK-MB levels are higher versus OPCAB from ICU up to T12 (P <0.03), and from ICU to T48 for Hs-cTn levels (P <0.02). Pulmonary hypertension, right ventricular dysfunction and acute heart failure: a portentous consortium In 39 CABG patients (97.5%) and 10 OPCAB patients (71.4%) all individual Hs-cTn values are above 140 ng/l (= 10 × 99th percentile of URL). at T6, and from T3 to T48 in OPCAB (P <0.05) versus baseline (Figure 1). In CABG patients CK-MB levels are higher versus OPCAB from ICU up to T12 (P <0.03), and from ICU to T48 for Hs-cTn levels (P <0.02). In 39 CABG patients (97.5%) and 10 OPCAB patients (71.4%) all individual Hs-cTn values are above 140 ng/l (= 10 × 99th percentile of URL).i and pulmonary hypertension was considered as PASP >35  mmHg. Based on the values of PASP and RVEF the study group was classifi ed into four subgroups: group 1, normal PASP/preserved RVEF; group 2, high PASP/preserved RVEF; group 3, normal PASP/ low RVEF; group 4, high PASP/low RVEF. The primary endpoint was all-cause mortality. The median follow-up was 18 months. Survival analysis was performed according to the Cox regression method, adjusted for age, gender, LV function, estimated glomerular fi ltration rate, troponin I, hemoglobin, serum sodium and BNP levels. Conclusion Both CK-MB and Hs-cTn levels increase signifi cantly after cardiac surgery. Postoperative Hs-cTn levels exceed the 10  ×  99th percentile of URL in nearly all CABG patients. Our data show an important discrepancy between the 10  ×  99th percentile for both biomarkers, and suggest that a diff erent defi nition for postoperative AMI may be needed when Hs-cTn is used. Results Pulmonary hypertension was found in 78% of the patients (median PASP: 53 mmHg). As compared with the patients with normal PASP the patients with pulmonary hypertension were more likely to be in New York Heart Association functional class (NYHA) III or IV (86% vs. 49%, P <0.001), had a lower RVEF (23 ± 9% vs. 32 ± 8%, P <0.001), and had signifi cantly higher BNP levels (280 ± 107 pg/ml vs. 540 ± 320 pg/ ml, P <0.001). In a Cox model, compared with patients with normal right ventricular function and without pulmonary hypertension (group 1), the adjusted hazard ratio for mortality was 3.1 (95% CI: 1.6 to 4.2, P <0.01) in group 2, 0.3 (95% CI: 0.2 to 1.9, P = 0.3) in group 3 and 4.2 (95% CI: 1.9 to 6.1, P <0.001) in group 4. y Reference y Reference 1. Thygesen et al. J Am Coll Cardiol. 2012;60:1581-98. Analytical and diagnostic characteristics of the high-sensitivity PATHFAST troponin I assay p y E Spanuth1, R Thomae2, E Giannitsis3 E Spanuth1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P159 (doi: 10.1186/cc14239) E Spanuth1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P159 (doi: 10.1186/cc14239) Conclusion Among ADHF patients, the coupling of pulmonary hypertension and RVD carries an incremental risk, having a portentous impact on the survival rate. Introduction The POC PATHFAST cTnI assay (Mitsubishi Medience, Japan) has shown promising analytical validity. We thought to evaluate whether the assay could be classifi ed ‘highly-sensitive’. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Of the 3,835 nontraumatic patients treated in the ED, 3,196 were adults. In this adult population, 500 ECGs were performed in patients whose symptoms suggest ACS. The median age was 62.3 years and the sex ratio was 1.16. Clinical presentation was chest pain (31%), dyspnea (14%), palpitations (5%), disturbance of consciousness (3%) or others (47%). Fifty-six (11.2%) were diagnosed as ACS, including 20 ST-elevation myocardial infarction (STEMI), 28 non-STEMI and eight unstable angina. Of the 20 STEMI patients, eight (40%) and fi ve (25%) were diagnosed as STEMI complicated by right ventricular and posterior wall ischemia respectively, which means that these complications could have been missed by standard 12-lead ECG. Methods A total of 112 patients undergoing PPCI and MIH were compared with 32 comparable consecutive patients who underwent PPCI but no MIH. We hypothesized that combining both methods lead to better survival rate. MIH was induced (propofol, fentanyl, saline 4 ml/kg BW, 2°C) and maintained for 24 hours, targeting 32 to 34°C. Spontaneous rewarming was allowed (0.5°C).if g Results There were no signifi cant diff erences between the MIH and Control group in general characteristics, cardiac arrest circumstances and angiographic features. Except for decreases in heart rate during MIH, there was no diff erence between MIH and no MIH groups in arterial pressure, peak lactate (7.7 vs. 6.2 mmol/l; P = 0.36), need for vasopressors (57% vs. 41%; P = 0.09), aortic balloon counterpulsation (13% vs. 22%; P  =  0.19), repeat cardioversion/defi brillation (17% vs. 25%; P = 0.30). There was lower incidence of inotropic use (36% vs. 59%; P = 0.01) and use of antiarrhythmics (11% vs. 53%; P = 0.002). There was no diff erence in FiO2 during mechanical ventilation and in renal function. See Table 1. y Conclusion Eighteen-lead ECG with synthesized right-sided and posterior precordial leads was an effi cient method to diagnose ACS in a Caucasian population within 10 minutes of ED arrival. It is particularly performant to detect right ventricular ischemia early, which can modify acute therapeutic strategy. References 1. Thygesen K, et al. Circulation. 2007;116:2634-53. 2. Katoh T, et al. J Nippon Med Sch. 2011;78:22-9. 3. Tamura A, et al. Am J Cardiol. 2014;114:1187-91. References 1. Thygesen K, et al. Circulation. 2007;116:2634-53. 2. Katoh T, et al. J Nippon Med Sch. 2011;78:22-9. 3. Tamura A, et al. Am J Cardiol. 2014;114:1187-91. Table 1 (abstract P160). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Survival after 12 months MIH Control P CPC 1/2 50 (45%) 5 (15%) 0.002 CPC 3/4 0 0 NS Cardio 20 (18%) 6 (19%) 0.95 CNS 18 (16%) 17 (53%) 0.00001 Table 1 (abstract P160). Survival after 12 months Conclusion Hospital survival with CPC 1/2 was signifi cantly better in the MIH group (45% vs. 15%; P = 0.01). Our study clearly demonstrates that PPCI and MIH are feasible and may be combined safely in comatose survivors of ventricular fi brillation in STEMI setting. Such strategy improves survival with good neurological recovery. References Introduction After an acute myocardial infarction with ST-segment elevation (STEMI) treated with percutaneous coronary intervention (PCI), the left ventricle (LV) can undergo negative remodeling (R–). We aimed to investigate whether global longitudinal strain (SGL) of the left ventricle (LV) predicts remodeling. e e e ces 1. HACA Study Group. Mild therapeutic hypothermia to improve the neurological outcome after cardiac arrest. N Engl J Med. 2002;346:549-56. 2. Knafelj R, Radsel P, Ploj T, Noc M. Primary percutaneous coronary intervention and mild induced hypothermia in comatose survivors of ventricular fi brillation with ST-elevation acute myocardial infarction. Resuscitation. 2007;74:227-34. 1. HACA Study Group. Mild therapeutic hypothermia to improve the neurological outcome after cardiac arrest. N Engl J Med. 2002;346:549-56. g g ; 2. Knafelj R, Radsel P, Ploj T, Noc M. Primary percutaneous coronary intervention and mild induced hypothermia in comatose survivors of ventricular fi brillation with ST-elevation acute myocardial infarction. Resuscitation. 2007;74:227-34. Methods Transthoracic echocardiography with speckle tracking imaging (TTE-STI) was performed 2 to 3 days after primary PCI and 6 months later in patients with diagnosis of STEMI. LV R– criteria were: LVEF increase ≤5% and end-diastolic volume increase ≥15%. Logistic regression and ROC curve analysis was used for the statistical analysis. Results Eighty-three patients (56  ±  11  years) with STEMI at any LV localization and subjected to primary PCI were studied during 2012: LV P160 P160 Combining therapeutic hypothermia and primary coronary intervention in comatose survivors of ventricular fi brillation due to ST-elevation myocardial infarction R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) Combining therapeutic hypothermia and primary coronary intervention in comatose survivors of ventricular fi brillation due to ST-elevation myocardial infarction R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) y R Knafelj, M Noc Universty Clincal Center, Ljubljana, Slovenia Critical Care 2015, 19(Suppl 1):P160 (doi: 10.1186/cc14240) Introduction Primary percutaneous coronary intervention (PPCI) is the preferred reperfusion strategy for ST-elevation acute myocardial infarction (STEMI). In comatose survivors of cardiac arrest, mild induced hypothermia (MIH) improves neurological recovery. Figure 1 (abstract P158). S56 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) y y Methods A consecutive registry of patients seen in 2013 (January to October) in the ICU of a hospital without a HU. The total transfer time is considered from the call to the Emergency Coordination Center until arrival at the HU. In turn, this time is divided into activation time, arrival time of the relocation team, patient preparation time and transfer time. Methods A consecutive registry of patients seen in 2013 (January to October) in the ICU of a hospital without a HU. The total transfer time is considered from the call to the Emergency Coordination Center until arrival at the HU. In turn, this time is divided into activation time, arrival time of the relocation team, patient preparation time and transfer time. In the case of primary PCI, the door-to-balloon time was estimated by adding to the total transfer time the initial assessment and completion time of catheterization and balloon infl ation. The times are expressed in minutes, as the median and interquartile range. In the case of primary PCI, the door-to-balloon time was estimated by adding to the total transfer time the initial assessment and completion time of catheterization and balloon infl ation. The times are expressed in minutes, as the median and interquartile range. Introduction Diastolic dysfunction as evaluated by tissue Doppler imaging (TDI), particularly by E/E’ ratio (peak early diastolic transmitral velocity/peak early diastolic mitral annular velocity) and mitral annular E’-wave, is common and crucial in critical illness. Our prospective observational study assessed the prognostic signifi cance of TDI variables versus cardiac biomarkers, B-type natriuretic peptide (BNP), troponin-T (TnT) and investigated determinants of plasma BNP rise, in septic shock. g Results During 10 months of 2013, we treated 162 STEMI. Of these, 104 had evidence of reperfusion (64%). Primary PCI was performed in 24 patients (23%), of which 10 were transferred from the hospital to the HU. Fibrinolytic therapy was used in 62 patients (59%), of these 20 (32.2%) required rescue PCI. The transfer time for primary PCI was 0:39:44 (0:31:41 to 0:44:32) minutes. The transfer time for rescue PCI was 0:38:56 (0:37:25 to 0:51:29) minutes. Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) p y gi Results Mean  ±  SD APACHE II score was 21.22  ±  7.28, mean  ±  SD admission SOFA score was 10.25 ± 2.76. Hospital mortality was 55%. Nonsurvivors had increased E/E’ (15.56  ±  1.48; P  <<0.00001) and reduced E’ 6.32  ±  0.68 cm/second (P  <<0.00001) compared with survivors, who exhibited inverse correlations with an E/E’ signifi cantly lower (9.30 ± 2.88) and higher E’ (9.01 ± 0.85 cm/second). In contrast, BNP and TnT levels displayed remarkably lower statistical signifi cance in nonsurvivors (P  =  0.005, P  =  0.007 respectively). The ROC curves had an area under the curve of 0.98 for the E’, and 0.92 for the E/E’. Vasopressor management (noradrenaline dose) (P  =  0.0001), fl uid balance (P <0.001) and E/E’ (P = 0.00004) were independent predictors of plasma BNP concentration. Introduction The goal of the study was to analyse cerebral blood fl ow in pregnancy complicated by preeclampsia. Introduction The goal of the study was to analyse cerebral blood fl ow in pregnancy complicated by preeclampsia. Methods This was a prospective study. I group: 45 patients, 17 to 38 years (mean age 27.5 ± 5.3 years) with verifi ed diagnosis of severe preeclampsia; control group: 72 healthy women with normal pregnancy, third trimester, 19 to 34 years (mean age 24.5± 4.3 years). Exclusion criteria: potentially haemodynamically signifi cant stenosis; congestive heart disease; arrhythmia; large changes in haemorheology; diabetes mellitus; and craniospinal trauma and syncope. Study of cerebral fl ow was improved by the method of transcranial dopplerography (TCD). All patients underwent duplex scan of extracranial portions of brachiocephalic arteries and transcranial duplex scan in the area of middle cerebral artery (MCA) (segment M1). During duplex scan of brachiocephalic arteries lumen, the presence of extravasal causes for basic blood fl ow disturbances was estimated. We determined lumen of large basilar arteries and quantitative features of blood fl ow in MCA. Synthesized 18-lead electrocardiogram as routine myocardial ischemia detection in an emergency department: a preliminary evaluation in Europe Figure 1 (abstract P162). Clinique Notre-Dame de Grâce, Gosselies, Belgium q g Critical Care 2015, 19(Suppl 1):P161 (doi: 10.1186/cc14241) Introduction Standard 12-lead electrocardiogram (ECG) is, with biomarkers, the most accurate method in the diagnosis of acute coronary syndrome (ACS). However, posterior (V7-V8-V9) and right (V3R-V4R-V5R) derivations are not systematically performed due to the time-consuming procedure involved, despite major therapeutic implications (fl uid loading instead of nitrates use in right ventricular involvement) and published guidelines [1]. Recently, an 18-lead ECG system, standard 12-lead ECG and six additional synthesized leads (assessing posterior and right ventricular areas) in only one recording procedure has been developed. The reliability of this material (ECG 2550; Nihon Kohden Co. Ltd, Japan) was already validated in this indication in an Asian population [2,3]. Methods We conducted a prospective, observational study with patients admitted to our emergency department (ED), during a 6-month period. Requirement for ECG was guided by physician’s discretion according to patient’s history. All patients with chest pain, dyspnea, palpitations, disturbance of consciousness, malaise or abdominal complaint underwent synthesized 18-lead ECG within 10 minutes of ED arrival. The aim of the study was to evaluate the eff ectiveness of the synthesized 18-lead ECG as an ischemia triage tool in the ED, and particularly the ability to early detect a right ventricular involvement. Figure 1 (abstract P162). S57 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 R– patients (n = 35, 42%) and no LV R– patients (n = 48, 58%). Diabetes mellitus (41% vs. 19%; P <0.001) and TnI levels (1.2 ± 2.1 μg/l vs. 0.4 ±0.3 μg/l; P = 0.005) showed higher incidence in LV R– patients. SGL was –12.5 ± 5.6% in no LV R– patients and –6.5 ± 3.4 in LV R– patients. In the regression analysis just LV SGL and SL in left anterior descending territory remained signifi cant, OR: 1.85 (1.24 to 2.76) (P <0.001) and OR: 1.63 (1.15 to 2.31) (P <0.001), respectively. P165 Prognostic comparison of tissue Doppler indices of diastolic dysfunction and cardiac biomarkers in septic shock V Karali, V Voutsas, N Loridas, M Konoglou, M Papaioannou, A Alexiou, V Hatsiou, C Fitili, M Bitzani Papanikolaou Hospital, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P165 (doi: 10.1186/cc14245) The door-to-balloon time estimated for primary PCI was 80 minutes. Methods Twenty-seven mechanically ventilated adult patients admitted to our ICU were evaluated within 72 hours of evolving septic shock. Patients underwent two transthoracic echocardiographies within 72 hours of the onset of septic shock: shortly after diagnosis and 24 hours later (confi rmatory), alongside relevant measurements of cardiac biomarkers. Peak mitral infl ow E and A velocity waves were recorded using pulsed-wave Doppler at the mitral valve tips from the apical four-chamber view, peak early (E’) and late (A’) diastolic myocardial velocities were obtained by TDI at the septal mitral annulus in the apical four-chamber view. E/E’ was calculated. P ≤0.01 was reported as statistically signifi cant. Conclusion Times for interhospital transfer of patients with STEMI who had undergone urgent catheterization are within the range considered optimal. In the case of primary PCI, times are lower than the 90 to 120 minutes recommended practice guidelines. Synthesized 18-lead electrocardiogram as routine myocardial ischemia detection in an emergency department: a preliminary evaluation in Europe The analysis of ROC curves revealed that at the cutoff level of –12.46%, SGL identifi es LV R– with a sensibility of 81% and a specifi city of 86% (AUC = 0.88: 95% CI: 0.79 to 0.96; P <0.001) (Figure 1).i fl ow velocity (Vps), maximal end-diastolic velocity (Ved), time-adjusted maximal velocity (TAMX), resistance index (RI), pulsative index (PI), and systolic/diastolic ratio (S/D) were determined. Signifi cance of mean value diff erences were calculated using the STATISTICA 6.0 program with determination of Student’s t criteria with normal spread in the group. g p Results All haemodynamic values in the M1 segment of MCA in preeclamptic patients were decreased in comparison with the same values in healthy pregnant women with diff erent signifi cance: PI (mean 0.77 vs. 0.84, P <0.01); RI (mean 0.52 vs. 0.54, P <0.05); Vps (mean 90.22 vs. 104.74 cm/second, P <0.001); Ved (mean 43.25 vs. 48.53 cm/second, P <0.001); TAMX (mean 61.48 vs. 67.30 cm/second, P <0.01); and S/D (mean 2.02 vs. 2.06, P  <0.05). Found pathophysiological changes of cerebral haemodynamics were consistent with a dopplerographic pattern of diminished perfusion and are typical for vascular segments, which are located proximally to the zone of abnormally high haemodynamic resistance: prestenotic arterial segments, episodes of arterial hypertension and distal vasoconstriction. Conclusion SGL assessment in the fi rst days after primary PCI is useful in the prediction of LV R– independently of the myocardial infarction localization. P164 Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka The State Budgetary Healthcare Institution of Moscow Area ‘Moscow’s Regional Research Clinical Institute n.a. M.F. Vladimirsky’, Moscow, Russia Critical Care 2015, 19(Suppl 1):P164 (doi: 10.1186/cc14244) Analysis of the interhospital transfer times in patients with ST-elevation acute coronary syndrome for undergoing urgent coronariography Analysis of the interhospital transfer times in patients with ST-elevation acute coronary syndrome for undergoing urgent coronariography FM Acosta Diaz, O Moreno, M Muñoz, R Fernandez, J Soto, M Colmenero San Cecilio Universitary Hospital, Granada, Spain Critical Care 2015, 19(Suppl 1):P163 (doi: 10.1186/cc14243) y Conclusion With TCD we obtained a possibility to determine and estimate changes in cerebrovascular fl ow in pregnant patients with severe preeclampsia. This enhances diagnostic possibilities of some serious pregnancy complications, and gives us deep understanding of some components of pathogenesis and increased treatment effi cacy. y p p Critical Care 2015, 19(Suppl 1):P163 (doi: 10.1186/cc14243) Introduction The aim was to analyze the related assistance times to transfer patients with ST-elevation acute coronary syndrome (STEMI) referred to another hospital with a hemodynamics unit (HU) for performing emergency catheterization (primary or rescue PCI). P165 P168 Results A total of 207 male and 144 female patients, APACHE II score 21  ±  7, 62  ±  14  years old, one to 126 TPTDs per patient. Diagnosis: cirrhosis/liver failure n = 112 patients (31.9%), sepsis 55 (15.7%), ARDS 46 (13.1%), GI aff ection 21 (6.0%), cardiogenic shock 19 (5.4%), various 98 (27.9%). Patients with liver failure were slightly younger than the other patients (58 ± 11 vs. 64 ± 15 years; P <0.001). All other baseline characteristics were comparable including APACHE II (20 ± 7 vs. 21 ± 8; NS), SAPS (39 ± 12 vs. 41 ± 14; NS), height (172 ± 9 vs. 170 ± 9 cm; NS) and weight (76 ± 20 vs. 73 ± 17 kg; NS). Among haemodynamic parameters, preload markers GEDVI (753  ±  168 vs. 790  ±  226  ml/m2; P  =  0.182) and CVP (14.4 ± 8.8 vs. 14.9 ± 7.1 mmHg; P = 0.250) were comparable. Despite comparable preload parameters, the following parameters were signifi cantly diff erent: patients with acute or chronic liver failure had signifi cantly higher cardiac index (4.3 ± 1.3 vs. 3.3 ± 1.3 l/minute/ m2; P <0.001), stroke volume index (50 ± 15 vs. 37 ± 15; P <0.001), pulse pressure (75 ± 19 vs. 65 ± 21 mmHg; P = 0.021) and cardiac power index (0.7 ± 0.24 vs. 0.60 ± 0.28 W/m2; P <0.001). By contrast, MAP (77 ± 15 vs. 80 ± 15 mmHg; P = 0.045), SVRI (1,305 ± 638 vs. 1,877 ± 898 dyn*s/ cm5*m2; P <0.001) and heart rate (84 ± 19 vs. 92 ± 22/minute; P <0.001) were signifi cantly lower in patients with liver failure. P168 Room temperature transpulmonary thermodilution (TPTD) with increased indicator 20 ml TPTD bolus compared with standard TPTD with 15 ml iced saline: a prospective observational study W Huber1, E Maendl1, A Beitz1, M Messer1, T Lahmer1, B Henschel1, S Rasch1, C Schnappauf1, RM Schmid1, ML Malbrain2 1Klinikum rechts der Isar, Technical University of Munich, Germany; 2Ziekenhuis Netwerk, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P168 (doi: 10.1186/cc14248) Introduction Use of ice-cold saline is assumed to provide best accuracy of TPTD to obtain the cardiac index (CI), global end-diastolic volume (GEDVI) and extravascular lung-water (EVLWI). However, room- temperature injectate might facilitate TPTD outside the ICU. Accidental intra-arterial injection: an under-reported preventable never event femoral TPTD (Pulsion Medical Systems, Germany). Results Fifteen female and 16 male patients, APACHE II score 21 ± 7. Mean values of CI (4.02 ± 0.98 vs. 3.96 ± 0.91 l/minute*m2; P <0.001), GEDVI (800 ± 166 vs. 796 ± 163 ml/m2; P = 0.011) and EVLWI (10.3 ± 3.7 vs. 9.7 ± 3.6 ml/kg; P <0.001) were slightly higher when measured at room temperature compared with cold saline. Mean bias and PE values were 0.06  ±  0.37 l/minute*m2 and 18.6% for CI, 4  ±  81  ml/m2 and 20.2% for GEDVI and 0.5 ± 1.1 ml/kg and 22.7% for EVLWI. Bias values in case of femoral compared with jugular indicator injection were not signifi cantly diff erent for CI (0.04 ± 0.41 vs. 0.11 ± 0.30 l/minute*m2; P = 0.161) and EVLWI (0.56 ± 1.19 vs. 0.42 ± 1.07 ml/kg; P = 0.492). Bias for GEDVI-room was signifi cantly lower for femoral CVC compared with jugular indicator injection (–6.0 ± 81.1 vs. 18.9 ± 78.3 ml/m2; P = 0.008). Conclusion Compared with previous data using 15  ml room- temperature injectate, our data with 20 ml room-temperature injectate in general provide acceptable bias and percentage error when compared with standard TPTD with 15 ml iced saline. This also applies for femoral CVC room-temperature TPTD which might also be related to a new PiCCO-2 algorithm correcting for femoral CVC site. Reference Queen Elizabeth Hospital, Kings Lynn, UK Critical Care 2015, 19(Suppl 1):P166 (doi: 10.1186/cc14246) Introduction Depending upon the medication administered, accidental administration of medication into the arterial line can cause devastating complications. This wrong-route injection is a never event in the UK but may be under-reported especially when occurring in the unconscious patient who may not notice associated pain temporally. Under-reporting may occur because resultant complications may be delayed a number of hours and the accountable healthcare worker may not recognise or choose not to report the error. In 2008 the UK National Patient Safety Agency (NPSA) reported only 76 incidents related to poor sampling technique but few wrong route arterial injections. Of these 21% suff ered moderate to severe harm [1]. The NPSA suggests that training and the use of clear labelling alongside red arterial tubing and standard red lock caps be used to prevent arterial sampling errors. Cerebrovascular haemodynamics in preeclamptic patients E Shifman, S Floka By the transtemporal approach in the MCA M1 segment, peak systolic Conclusion Diastolic dysfunction as evaluated by E/E’ and E’ constitutes a major independent predictor of outcome in septic shock, compared with cardiac biomarkers, suggesting that echocardiographic techniques assessing diastolic dysfunction in sepsis may replace cardiac biomarkers for mortality prediction. Fluid balance, vasopressor management and diastolic dysfunction are independent predictors of BNP elevation in septic shock. Our fi ndings should be confi rmed by an extended prospective study. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S58 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P166 the PiCCO-2 device with the latest algorithm correcting GEDVI for femoral TPTD (Pulsion Medical Systems, Germany). Accidental intra-arterial injection: an under-reported preventable never event Methods In 2014, we conducted a national postal survey of ICUs in the UK to attempt to determine the rate of accidental intra-arterial injections. The survey was sent to the clinical director of every ICU and they were asked whether they were aware of any unintentional arterial line injection having occurred in their hospital in the last 5 years. 1. Huber W, et al. J Crit Care. 2014;29:1133. 1. Huber W, et al. J Crit Care. 2014;29:1133. y Results Of the 56 ICUs that responded, 16 (28.5%) reported that they had personally seen an accidental injection into the arterial line. References Methods We analyzed a prospectively maintained database including 6,016 TPTD measurements in 351 patients. To account for diff erent numbers of TPTDs in diff erent patients, comparison of fi rst measurements of patients with and without liver failure was the primary endpoint. Statistics: Wilcoxon test for unpaired samples; IBM SPSS Statistics 22. 1. Rapid response alert 06. NPSA; 2008. 1. Rapid response alert 06. NPSA; 2008. 2. Mariyaselvam et al. Anaesthesia. 2014;70:51-5. 3. Mukhopadhyay et al. Crit Care. 2010;14:R7. P169 Transpulmonary thermodilution-derived haemodynamics in patients with liver failure: a prospective study in 351 patients W Huber, A Breitling, B Henschel, S Mair, S Goetz, J Tschirdewahn, J Frank, A Beitz, RM Schmid Klinikum rechts der Isar, Technical University of Munich, Germany Critical Care 2015, 19(Suppl 1):P169 (doi: 10.1186/cc14249) Conclusion Despite the arterial line safety recommendations made by the NPSA in 2008, we demonstrate that intra-arterial injection is still a problem and that it remains under-reported. Our incidence is likely to be an underestimate as it relies on the recollections of a single individual in each institution. Medical errors can be mitigated by consideration of human factors and system engineering to improve patient safety. A focus on clinical awareness, colour coding and training may lead to improvements; however, institutions and clinical directors also bear a responsibility to prevent never events and a number of engineered solutions are now available such as needle-free non-injectable arterial sampling devices to protect the healthcare environment and make this error impossible [2,3]. Introduction Patients with acute or chronic liver failure are considered to have an altered pattern of haemodynamics. Nevertheless, there is a lack of studies systematically investigating haemodynamics in patients with liver failure. Therefore, it was the aim of this study to compare transpulmonary thermodilution (TPTD)-derived haemodynamics of 112 patients with acute or chronic liver failure with 239 patients without liver failure. Acknowledgement Funding from Eastern Academic Health Science Network, UK. References Acknowledgement Funding from Eastern Academic Health Science Network, UK. Reference 1. Romano M, et al. Care Med. 2002;30:1834-41. Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report Measures were performed by a single trained operator, who was blind to CVP values. Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. Measures were performed by a single trained operator, who was blind to CVP values. Results Forty-eight patients were included. A poor correlation was found between CVP and IJV and EJV circumference and area in mechanically ventilated patients. A strong correlation was found between CVP and EJV circumference (r: 0.74; P = 0.0004; 95% CI: 0.421 to 0.897) and area (r: 0.702; P = 0.0012; 95% CI: 0.35 to 0.88) in spontaneously breathing patients. Conventional receiver-operating characteristic curves were generated to assess the utility of EJV circumference and area to predict low (≤8  mmHg) versus high (>8  mmHg) CVP values. AUC for EJV circumference and area was 0.935 (P <0.0001; 95% CI: 0.714 to 0.997) and 0.87 (P <0.0001; 95% CI: 0.63 to 0.98) respectively (Figure 1). Conclusion These results highlight a potentially evolving role of Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report Introduction There are few methods of cardiac output (CO) estimation validated in children. The aim of this study is to investigate the reliability of an uncalibrated pulse contour method of CO estimation, the pressure recording analytical method (PRAM), in pediatric patients scheduled for diagnostic right and left heart catheterization, compared with the oxygen-direct Fick method. yg Methods Cardiac index (CI) was simultaneously estimated by Fick, and PRAM applied to pressure signals recorded invasively from a femoral catheter. All measurements were performed in steady-state condition. PRAM CI measurements were obtained for 10 consecutive beats simultaneously during the Fick CI estimation. Agreement between Fick and PRAM was assessed using the Bland–Altman method. Correlation coeffi cient, bias, and percentage of error were calculated. fi Results Forty-three CI measurements were performed in 43 patients. The data showed good agreement between CIFick and CIPRAM: r2 = 0.98; bias –0.0074 l/minute/m2; limits of agreement from –0.22 to 0.22  l/minute/m2. The percentage error was 8%. Figure  1 shows the Bland–Altman plot. Figure 1 (abstract P171). Figure 1 (abstract P170). Bland–Altman plot of the cardiac index measured with Fick versus PRAM. Figure 1 (abstract P170). Bland–Altman plot of the cardiac index measured with Fick versus PRAM. Figure 1 (abstract P171). Nonetheless, the external jugular vein (EJV) circumference and area have not been evaluated. Considering the role of EJV visual assessment in the clinical estimation of CVP, we hypothesized that EJV ultrasound evaluation could be used to reliably estimate CVP. Conclusion PRAM provides reliable estimates of cardiac output in hemodynamically stable pediatric cardiac patients compared with the Fick method. Reference Conclusion PRAM provides reliable estimates of cardiac output in hemodynamically stable pediatric cardiac patients compared with the Fick method. f Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. Measures were performed by a single trained operator, who was blind to CVP values. Methods Patients with a CVC placed as part of clinical management were evaluated. EJV and internal jugular vein (IJV) measurements were performed at the left cricoid level. IJV and EJV were visualized in short axis view; diameters, circumferences and areas were obtained at end expiration with simultaneous CVP measurement. P170 P170 Measurement of cardiac output in children: comparison between direct Fick method and pressure recording analytical method: preliminary report J Alonso Iñigo1, F Escribá1, J Carrasco1, J Encarnación1, M Fas2, M Barberá1 1Hospital Universitario y Politécnico La Fe, Valencia, Spain; 2Hospital Universitario de la Ribera, Alzira, Spain Critical Care 2015, 19(Suppl 1):P170 (doi: 10.1186/cc14250) P168 A recent study [1] showed acceptable bias and percentage error (PE) for CI-room derived from TPTD with 15  ml room temperature saline compared with CI-cold using 15 ml iced saline for TPTD. However, GEDVI-room and EVLWI-room had borderline PE values close to 30%, and the bias of GEDVI-room markedly increased with higher values of GEDVI and in case of femoral CVC. Since imprecision of TPTD-room might be reduced by a larger volume of injectate, it was the aim of our study to compare CI, GEDVI and EVLWI derived from TPDT using 20 ml room temperature injectate with standard TPTD with 15 ml iced saline. Conclusion Our data derived from a large TPTD database demonstrate markedly diff erent haemodynamics in patients with cirrhosis or acute liver failure with the only exception of static preload markers GEDVI and CVP. These fi ndings should be considered in instable patients with liver failure. Methods In 31 patients 236 sets with two 20 ml TPTDs with 21°C and subsequently two standard TPTDs with 4°C saline were obtained using S59 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P171). P170 P172 Do intravascular hypovolaemia and hypervolaemia result in changes in pulmonary blood volume? g p y JJ Vos1, TW Scheeren1, SA Loer2, A Hoeft3, JK Wietasch1 1University of Groningen, University Medical Center Groningen, the Netherlands; 2Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, the Netherlands; 3University of Bonn, Germany Critical Care 2015, 19(Suppl 1):P172 (doi: 10.1186/cc14252) Introduction Hypovolaemia is generally believed to induce centrali sa- tion of blood volume. Therefore, we evaluated whether hypovolaemia and hypervolaemia result in a change in central blood volume (that is, pulmonary blood volume (PBV)) and we explored the eff ects on the distribution between PBV and circulating blood volume (Vd circ). Introduction Hypovolaemia is generally believed to induce centrali sa- tion of blood volume. Therefore, we evaluated whether hypovolaemia and hypervolaemia result in a change in central blood volume (that is, pulmonary blood volume (PBV)) and we explored the eff ects on the distribution between PBV and circulating blood volume (Vd circ). Methods Twenty anesthetized Landrace/Large-White pigs (19 ± 2 kg, 10 to 15 weeks) were subjected to a fi xed hemorrhage (50% over 30 minutes). The pigs were randomly allocated into two groups (n = 10 per group). In group A, ITD was the only treatment for hypotension, while in group B, an intravenous administration of 1 l Ringer lactate was applied for treatment of hypotension. Hemodynamic parameters were continuously assessed for the fi rst 30 minutes after blood loss. Methods After local District Governmental Animal Investigation Committee approval, blood volume was altered in both directions randomly in steps of 150  ml (mild) to 450  ml (moderate) either by haemorrhage, retransfusion of blood, or infusion of colloids in six Foxhound dogs. The anaesthetised dogs were allowed to breathe spontaneously. Blood volumes were measured using the dye dilution technique: PBV was measured as the volume of blood between the pulmonary and aortic valve, and Vd circ by two-compartmental curve fi tting [1,2]. The PBV/Vd circ ratio was used as a measure of blood volume distribution. A linear mixed model was used for analysing the infl uence of blood volume alterations on the measured haemodynamic variables and blood volumes. yi Results Mean systolic arterial pressures (SAPs) 30  minutes after the intervention in each group were as follows: group A 80 ± 5 mmHg and group B 90 ± 4 mmHg. Maximum SAPs during the assessment period were: group A 89 ± 2 mmHg and group B 128 ± 5 mmHg. p P Balsorano, S Romagnoli, A De Gaudio P Balsorano, S Romagnoli, A De Gaudio AOUC Careggi, Florence, Italy AOUC Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P171 (doi: 10.1186/cc14251) AOUC Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P171 (doi: 10.1186/cc14251) Introduction Although recognized as a questionable indicator of the intravascular volume, central venous pressure (CVP) is integrated in many therapeutic algorithms for hemodynamic resuscitation of critically ill patients [1]. In an attempt to simplify CVP estimation, several clinical and ultrasonographic approaches have been suggested [2-5]. p y g Conclusion These results highlight a potentially evolving role of EJV circumference and area in the hemodynamic management S60 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of spontaneously breathing patients. An important aspect of the suggested approach is its simplicity, requiring basic technical skills and making it suitable in any scenario where an ultrasound machine is available. P175 Relation between global end-diastolic volume and left ventricular end-diastolic volume A Pironet1, P Morimont1, S Kamoi2, N Janssen1, PC Dauby1, JG Chase2, B Lambermont1, T Desaive1 1University of Liège, Belgium; 2University of Canterbury, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P175 (doi: 10.1186/cc14255) (Figure 1). Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. References 1. Anesthesiology. 1994;81:76-86. 2. Intensive Care Med. 2001;27:767-74. Figure 1 (abstract P172). Figure 1 (abstract P172). 1University of Liège, Belgium; 2University of Canterbury, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P175 (doi: 10.1186/cc14255) Introduction Measurement of global end-diastolic volume (GEDV) is provided by cardiovascular monitoring devices using thermodilution procedures. The aim of this study was to assess the relation between this clinically available index and left ventricular end-diastolic volume (LVEDV), which is typically not available at the patient bedside. Methods Measurements were performed on six anaesthetised and mechanically ventilated pigs. Volume loading via successive infusions of saline solution was fi rst performed and was followed by dobutamine infusion. These two procedures provided a wide range of LVEDV values. During these experiments, GEDV was intermittently measured using the PiCCO monitor (Pulsion AG, Germany) during thermodilutions and LVEDV was continuously measured using an admittance catheter (Transonic, NY, USA) inserted in the left ventricle. Results Table  1 presents the linear correlations obtained between LVEDV and GEDV. These correlations are good to excellent, with r2 values from 0.59 to 0.85. However, the coeffi cients of the linear regressions present a large intersubject variability, which prevents the precise estimation of LVEDV using GEDV. Nevertheless, variations in LVEDV are well reproduced by the GEDV index. The variations in LVEDV actually equal 21 to 48% of those in GEDV. The coeffi cient b is always nonzero, indicating that some proportion of the GEDV index is actually not linked to LVEDV. Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. R f Conclusion Mild to moderate alterations of blood volume result in changes of PBV and Vd circ. Comparative study between fl uidless resuscitation with permissive hypotension using the impedance threshold device versus aggressive fl uid resuscitation with Ringer lactate in a swine model of hemorrhagic shock C Pantazopoulos1, I Floros1, N Archontoulis1, D Xanthis1, D Barouxis2, N Iacovidou2, T Xanthos2 1Laiko General Hospital of Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P174 (doi: 10.1186/cc14254) References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. References 1. Dellinger RP, et al. Intensive Care Med. 2013;39:165-228. 2. Cook DJ. Am J Med Sci. 1990;299:175-8. 3. Donahue SP, et al. Am J Emerg Med. 2009;27:851-5. 4. Beigel R, et al. J Am Soc Echocardiogr. 2013;26:1033-42. 5. Rudski LG, et al. J Am Soc Echocardiogr. 2010;23:685-713. Introduction Permissive hypotension, which results in avoidance of intravascular overpressure and thereby avoidance of platelet plug dislodgement early in the clotting mechanism, improves the results after trauma and hemorrhage. The research hypothesis is that augmentation of negative intrathoracic pressure with the use of an impedance threshold device (ITD) will improve hemodynamic parameters, without aff ecting permissive hypotension or causing hemodilution. On the other hand, aggressive resuscitation with Ringer lactate will cause hemodilution and intravascular pressures that are very high for permissive hypotension, capable of platelet plug dislodgement. 2. Intensive Care Med. 2001;27:767-74. P172 Do intravascular hypovolaemia and hypervolaemia result in changes in pulmonary blood volume? Mean pulse pressure was higher in the ITD group versus the fl uid resuscitation group (P  <0.05). After the assessment period, mean hematocrit in group A was 24 ± 2%, while in group B it was 18 ± 1% (P <0.001). Conclusion In our study, the ITD increased SAP and pulse pressure without overcompensation. On the other hand, aggressive fl uid resuscitation led to a signifi cant increase of SAP >100 mmHg capable of clot dislodgement and in addition led to hemodilution. Results A total of 68 alterations in blood volume resulted in changes in Vd circ ranging from –33 to +31% (Figure 1). PBV decreased during mild and moderate haemorrhage, while during retransfusion PBV increased during moderate hypervolaemia only. The PBV/Vd circ ratio remained constant during all stages of hypovolaemia and hypervolaemia (Figure 1). P175 R l i 1. Anesthesiology. 1994;81:76-86. P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) p S Hutchings1, P Hopkins1, A Campanile2 pp Methods This prospective, single-center study included 80 patients with sepsis from the Department of Critical Care Medicine of Zhejiang Hospital. Patients were randomly assigned to either Group A or Group B, with patients of in Group A fi rst taking the passive leg raising test and then taking the fl uid infusion test, while patients in Group B followed the opposite protocol. NICOM was used to continuously record hemodynamic parameters such as cardiac output (CO), heart rate (HR) and central venous pressure (CVP), at baseline1, PLR, baseline2, and volume expansion (VE). Fluid responsiveness was defi ned as the change in CO (ΔCO) ≥10% after VE. Introduction We performed an evaluation of three devices used for assessment of volume status in critically ill patients in our institution: transthoracic echocardiography (TTE) (CX50; Philips Ultrasound), bio reactance (NICOM; Cheetah Medical) and pulse contour-based thermodilution (PiCCO; Pulsion Medical). Methods Ten mechanically ventilated critically ill patients with PiCCO monitoring in situ and a good quality of images on transthoracic view were included. All study measurements were made in triplicate. A single trained cardiologist, blinded to the results from the other monitors, performed the TTE study. Diff erences among the three methods were assessed for signifi cance using one-way ANOVA, Spearman’s coeffi cient and Bland–Altman analysis. All statistical analyses were performed using Graph-pad Prism 5 and P <0.05 was taken as signifi cant. g ( ) Results CO increased during PLR (from 5.21 ± 2.34 to 6.03 ± 2.73 l/ minute, P <0.05); and after VE (from 5.09 ± 1.99 to 5.60 ± 2.11 l/minute, P <0.05). The PLR-induced change in CO (ΔCOPLR) and the VE-induced change in CO (ΔCOVE) were highly correlated (r = 0.80 (0.64 to 0.90)), while the CVP and ΔCOVE were uncorrelated (r = 0.12 (–0.16 to 0.32)). The areas under the ROC curves of ΔCOPLR and ΔCVP for predicting fl uid responsiveness were 0.868 and 0.514 respectively. ΔCOPLR ≥10% was found to predict fl uid responsiveness with a sensitivity of 86% and a specifi city of 79%.l g p p gi Results Ninety measurements were obtained. NICOM and TTE-derived stroke volume appeared well matched but PICCO-derived values showed signifi cant variation (F = 2.4, P = 0.09). P175 However, against the traditional belief of centralisation we could show that the cardiovascular system preserves the distribution of blood between central and circulating blood volume in anaesthetised dogs. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S61 Table 1 (abstract P175). Linear regressions between LVEDV and GEDV Subject a b (ml) r2 1 0.26 7.64 0.82 2 0.43 –47.10 0.66 3 0.21 –12.99 0.75 4 0.25 –11.42 0.59 5 0.41 –65.42 0.85 6 0.48 –65.75 0.68 LVEDV = a × GEDV + b. regards fl uid administration between PiCCO and echocardiography. NICOM appeared unreliable in this setting. regards fl uid administration between PiCCO and echocardiography. NICOM appeared unreliable in this setting. Conclusion The results show that GEDV and LVEDV are generally well correlated, but the correlation coeffi cients are subject specifi c. A preliminary calibration step (for instance using echocardiography) is thus necessary to infer LVEDV from GEDV. Introduction Fluid administration is always important and diffi cult during the therapy of patients with sepsis. Accurately predicting fl uid responsiveness and thus estimating whether the patient will benefi t from fl uid therapy seems particularly important. The present study intended to predict fl uid responsiveness in patients with sepsis using a bioreactance-based passive leg raising test, and to compare this approach with the commonly used central venous pressure (CVP) approach. P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) There was no correlation between TTE velocity time integer (VTI) and NICOM stroke volume variation (SVV) (r = 0.24, P = 0.20; Figure 1A) but a good correlation and small bias between TTE-VTI and PiCCO-SVV (r = 0.76, P <0.0001; Figure 1B). Applying the following indications for volume expansion (PiCCO and NICOM SVV >15% and TTE VTI variability >15%) we found an agreement in 71% of cases between TTE and PiCCO and in 42% of cases between echocardiography and NICOM. i Conclusion Bioreactance-based PLR could predict fl uid responsiveness in patients with sepsis, while CVP could not. P176 P176 Volume assessment in critically ill patients: echocardiography, bioreactance and pulse contour thermodilution S Hutchings1, P Hopkins1, A Campanile2 1King’s College Hospital, London, UK; 2Papworth Hospital, Cambridge, UK Critical Care 2015, 19(Suppl 1):P176 (doi: 10.1186/cc14256) Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment H Barrasa, J Maynar, S Castaño, Y Poveda, P Garcia Domelo, A Tejero, G Baziskueta, A Quintano, B Fernández Miret, M Iturbe, S Cabañes, F Fonseca Alava University Hospital-Santiago, Vitoria, Spain Critical Care 2015, 19(Suppl 1):P180 (doi: 10.1186/cc14260) Introduction Dynamic parameters are becoming standard for fl uid responsiveness assessment. Cutoff values are diff erent in the literature. The aim was to assess the accuracy of diff erent preload parameters to predict fl uid responsiveness using pulse power analysis and to compare diff erent levels of hemodynamic response due to passive leg raising (PLR) against the eff ect of a fl uid challenge (FC). y Results We evaluated 27 patients, age 68 (95% CI: 61 to 74) and APACHE II score 22 (95% CI: 18 to 26). Seven patients were high responders, eight patients were moderate responders and 12 were nonresponders. DO2 was signifi cantly increased in high responders (37  ±  35%, P <0.01) as compared with moderate responders or nonresponders. Furthermore, nonresponders had a decrease in their DO2 (–10 ± 7%, P <0.01), while moderate responders showed no change in their DO2 (1.6% ± 10, P = 0.73) after fl uid challenge. We found no diff erences in changes in lactate levels and central venous oxygen saturation (ScvO2) between high responders, moderate responders and nonresponders. No diff erences in the changes of VCO2 or VO2/VCO2 ratio were found between high responders, mild responders and nonresponders too. Changes in DO2/VCO2 ratio were found to be signifi cantly increased only in high responders (47 ± 73% vs. –14 ± 31%, P = 0.02) and not in mild responders (15 ± 54% vs. –14 ± 31%, P = 0.15) as compared with nonresponders. g gfl g Methods A prospective study in a 17-bed mixed ICU. Patients were fully ventilated and CO monitored with LiDCOplus® and underwent a FC due to hypotension and/or hypoperfusion and preload dependence (SVV and/or PPV >10%). PLR was performed before FC. Hemodynamic data were recorded prePLR, postPLR and postFC with 0.5 l crystalloids. We compared diff erent cutoff values of increase in CO and SV (10 to 15%) to assess the ability of PLR, SVV, PPV and CVP to predict the response to FC. Statistical analysis: continuous variables expressed as mean ± SD. P180 80 Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment H Barrasa, J Maynar, S Castaño, Y Poveda, P Garcia Domelo, A Tejero, G Baziskueta, A Quintano, B Fernández Miret, M Iturbe, S Cabañes, F Fonseca Alava University Hospital-Santiago, Vitoria, Spain Critical Care 2015, 19(Suppl 1):P180 (doi: 10.1186/cc14260) Global end-diastolic volume: a better indicator of cardiac preload in patients with septic shock L Mirea, R Ungureanu, D Pavelescu, I Grintescu g Clinical Emergency Hospital of Bucharest, Romania g y p , Critical Care 2015, 19(Suppl 1):P179 (doi: 10.1186/cc14259) g y p , Critical Care 2015, 19(Suppl 1):P179 (doi: 10.1186/cc14259) Introduction The aim of the study was to assess the value of the global end-diastolic volume (GEDV) evaluated by transpulmonary thermodilution as an indicator of cardiac preload comparing with stroke volume variation (SVV) in patients with septic shock. g p p Conclusion Our results support the idea that a reversible FC (PLR; CO cutoff 12.6%) is best at identifying responder patients to a FC. Dynamic parameters (SVV/PPV) are also eff ective when appropriate. Beat-to- beat SV and CO using pulse power analysis is a valid tool for these tests. Methods A prospective, observational study performed in an interdisciplinary ICU including 91 patients with septic shock. Hemodynamic monitoring was performed with a new calibrated pulse wave analysis method (VolumeView/EV1000; Edwards Lifesciences) in 37 patients (group EV1000) or with an uncalibrated method (FloTrac/ Vigileo; Edwards Lifesciences) in 54 patients (group Vigileo) during the fi rst 72 hours. All patients were receiving mechanical ventilation and vasopressors. Measurements were performed before and immediately after volume loading using 500 ml Ringer solution over a short period (<30 minutes).l P181 P181 Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) p References p p Referencesl 1. De Backer D. Can passive leg raising be used to guide fl uid administration? Crit Care. 2006;10:170. 2. Pinsky MR. Functional haemodynamic monitoring. Curr Opin Crit Care. 2014;20:288-93. 3. Teboul JL, Monnet X. Pulse pressure variation and ARDS. Minerva Anestesiol. 2013;79:398-407. Conclusion Stroke volume produced by bioreactance appeared to be comparable with that measured by echocardiography but not with PiCCO. There was a good agreement between decision-making as 4. Monnet X, Teboul JL. Volume responsiveness. Curr Opin Crit Care. 2007;13:549-53. Figure 1 (abstract P176). Figure 1 (abstract P176). t P176) S62 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P178 Conclusion The transpulmonary thermodilution GEDV is a better indicator of cardiac preload than SVV in patients with septic shock. Acknowledgements This paper was cofi nanced from the European Social Fund, through the Sectorial Operational Programme Human Resources Development 2007–2013, project number POSDRU/159/1.5/S/138907 ‘Excellence in scientifi c interdisciplinary research, doctoral and postdoctoral, in the economic, social and medical fi elds – EXCELIS’; coordinator, The Bucharest University of Economic Studies. References Acute changes of metabolic parameters after fl uid challenge T Nguyen, D De Bels, M Pustetto, P Cottignies, J Devriendt, C Pierrakos Brugmann Hospital, Brussels, Belgium Critical Care 2015, 19(Suppl 1):P178 (doi: 10.1186/cc14258) Introduction The detection of heart response to fl uid administration is still a challenge in clinical practice. Changes in metabolic parameters may be useful to detect changes in cardiac output (CO) after fl uid expansion. 1. Michard F, et al. Chest. 2003;124:1900-8. 2. Bendjelid K, et al. Br J Anaesth. 2013;111:573-9. Methods This is a prospective observational study in adult critically ill patients. CO was measured either by echocardiography or by a thermodilution method (PiCCO, Swan–Ganz catheter). Hemodynamic measurements and blood gas analysis were obtained before and after a fl uid challenge with either 1,000 ml crystalloid or 500 ml colloid. Arterial and central venous blood gas samples were taken simultaneously. Oxygen delivery (DO2), oxygen consumption (VO2) and carbon dioxide production (VCO2) were calculated according to well-known formulas. Patients were divided into three groups (high responders, mild responders and nonresponders) according to their change in CO (>20%, 10 to 20%, <10%, respectively). Predicting fl uid responsiveness in ICU patients: comparison of diff erent parameters and cutoff limits using pulse power analysis assessment Comparison before and after was done using a paired Student’s t test, and receiver operating characteristic (ROC) curves were generated by varying the discriminating threshold of each variable. p Conclusion Only signifi cant increases of CO (>20%), after fl uid administration, lead to improved oxygen delivery; DO2 may be decreased in nonresponders. The changes of ScvO2 and lactate levels do not track the changes of CO after fl uid challenge. The DO2/VCO2 ratio may be a useful index to identify signifi cant increases of CO after fl uid challenge in cases where CO measurement is not feasible. Results Thirty-one patients were included. Baseline parameters: MAP 70.5 mmHg (SD 13.3) 87% under catecholamine, SV 55.32 ml (SD 20.2), CO 5.2 l (SD 2), SVV 16.8% (SD 12), PPV 19.1% (SD 14), HR 96 bpm (SD 18) and CVP 9.2 mmHg (SD 2.5). In total, 41.9% of patients increased 15% CO after FC (selected as responders), 38.7% after the PLR. Diff erences in responders versus nonresponder patients were: baseline SVV (23.9 vs. 11.6; P = 0.02) and PPV (28.4 vs. 12.4; P = 0.01). Diff erences in SV and CO were not statistically signifi cant. The best parameter to predict positive response to FC was PLR with cutoff 12.6% for CO increase: sensitivity 84.6% (95% CI = 65 to 104), specifi city 94.4% (95% CI = 84 to 105) and AU ROC 0.94 (95% CI = 0.86 to 1.0). ROC was also good for SVV 0.835 (95% CI = 0.66 to 1.0; P = 0.002) and PPV 0.833 (95% CI = 0.681 to 0.985; P = 0.002) in this cutoff value. In SV increase, PLR, SVV and PPV had P <0.05, but with worse ROC. In addition, SVV <13% identifi ed patients who will not increase MAP with FC: sensitivity 91.7% (95% CI = 76 to 107.3%), negative predictive value 93.5 (95% CI  = 80.7 to 106). CVP failed to distinguish responders from nonresponders. Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU Respiratory variations in aortic blood fl ow velocity and inferior vena cava diameter as predictors of fl uid responsiveness in mechanically ventilated children using transthoracic echocardiography in a pediatric PICU References volume status. In this way, dynamic echocardiographic parameters have been proposed in mechanically ventilated children [1,2], using the heart–lung interactions. This study aimed to investigate whether respiratory variations of aortic blood fl ow velocity (DELTA Vpeak ao) and inferior vena cava diameter (DELTA IVC) by transthoracic echocardiography (TTE) could accurately predict fl uid responsiveness in ventilated children. volume status. In this way, dynamic echocardiographic parameters have been proposed in mechanically ventilated children [1,2], using the heart–lung interactions. This study aimed to investigate whether respiratory variations of aortic blood fl ow velocity (DELTA Vpeak ao) and inferior vena cava diameter (DELTA IVC) by transthoracic echocardiography (TTE) could accurately predict fl uid responsiveness in ventilated children. 1. Monnet X, et al. Esophageal Doppler monitoring predicts fl uid responsiveness in critically ill ventilated patients. Intensive Care Med. 2005;31:1195-201. 2. Tibby SM, et al. Are transoesophageal Doppler parameters a reliable guide to paediatric haemodynamic status and fl uid management? Intensive Care Med. 2001;27:201-5. 2. Tibby SM, et al. Are transoesophageal Doppler parameters a reliable guide to paediatric haemodynamic status and fl uid management? Intensive Care Med. 2001;27:201-5. Methods A prospective observational and interventional study conducted in a pediatric ICU investigated 40 mechanically ventilated children with preserved left ventricular (LV) function using TTE. Each patient had tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs. Intervention: standardized volume expansion (VE). P183 Fluid management in mechanically ventilated children with acute circulatory failure based on the pleth variability index in a pediatric ICU H Bouguetof, M Negadi, K El Halimi, L Zemour, D Boumendil, Z Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P183 (doi: 10.1186/cc14263) Results The VE-induced increase in LV stroke volume was ≥10% in 28 patients (responders) and <10% in 12 patients (nonresponders). Before VE, the DELTA Vpeak ao and DELTA IVC in responders was respectively higher than that in nonresponders (18.75% (12 to 32) vs. 13.5% (6 to 16) and 31% (18 to 57) vs. 17.5% (14 to 25)). The prediction of fl uid responsiveness was higher with DELTA Vpeak ao (ROC curve area 0.894 (95% CI = 0.756 to 0.969), P = 0.0001) and DELTA IVC (ROC curve area 0.869 (95% CI = 0.717 to 0.957), P = 0.0001). P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Introduction Prediction of fl uid responsiveness is defi ned by an increase in stroke volume (SV) of at least 10% after volume expansion. Dynamic [1] and static [2] esophageal Doppler (OD) parameters have been proposed in mechanically ventilated children to guide fl uid therapy. This study aimed to compare dynamic parameters using the respiratory variation in aortic blood fl ow with static parameters using Doppler corrected fl ow times (FTc) obtained by OD. Introduction The accuracy of predicting fl uid responsiveness (FR) using IVC collapsibility is high in patients on controlled mechanical ventilation, but remains unknown in spontaneously breathing patients with mechanical ventilation. Also, adequate ultrasound images of IVC are diffi cult to obtain in a substantial number of patients. The aim of the current study is to evaluate utility of collapsibility of jugular veins (IJV) and subclavian veins (SCV) in comparison with collapsibility of IVC in patients on pressure support ventilation. ppl y Methods A prospective, observational and interventional study was conducted in our pediatric ICU from March 2012 to September 2014. We investigated 18 mechanically ventilated children with acute circulatory failure (ACF)  – tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs – using OD for each patient. Intervention: standardized volume expansion (VE). Results The VE-induced increase in stroke volume was ≥10% in 14 patients (responders) and <10% in four patients (nonresponders). Before VE, the DELTA Vpeak ao in responders was higher than in nonresponders (19.5% (12 to 29) vs. 11.5% (7 to 13)), whereas FTc was lower in responders than in nonresponders (262.5 milliseconds (180 to 340) vs. 285 milliseconds (205 to 300)). The prediction of fl uid responsiveness was higher with DELTA Vpeak ao (ROC curve area 0.964 (95% CI = 0.756 to 1.000); P = 0.0001) than with FTc (ROC curve area 0.562 (95% CI = 0.314 to 0.790); P = 0.7203). pediatric PICU pediatric PICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P181 (doi: 10.1186/cc14261) Results A total of 211 fl uid challenges were studied in 91 patients. We observed a signifi cant relationship between the GEDV index before volume loading and the percentage increase in GEDV index in the EV1000 group and changes in GEDV index were signifi cantly correlated with changes in stroke volume index (r = 0.75, P <0.001), but an insignifi cant relationship between SVV variation and cardiac index variation (P >0.05) in the Vigileo group. Introduction Volume expansion remains the fi rst treatment step for most children with acute circulatory failure in order to assess blood Introduction Volume expansion remains the fi rst treatment step for most children with acute circulatory failure in order to assess blood S63 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Prediction of fl uid responsiveness in mechanically ventilated children using dynamic and static parameters by esophageal Doppler in a pediatric ICU Prediction of fl uid responsiveness in mechanically ventilated children using dynamic and static parameters by esophageal Doppler in a pediatric ICU K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P182 (doi: 10.1186/cc14262) Conclusion In this study, PVI seems to predict fl uid responsiveness in ventilated children with ACF. pp p K El Halimi, M Negadi, H Bouguetof, L Zemour, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P182 (doi: 10.1186/cc14262) References The best cutoff value for DELTA Vpeak ao was 16% with sensitivity and specifi city predictive values of 71.6% and 83.3%, respectively, and DELTA IVC was 20% with sensitivity and specifi city predictive values of 88.5% and 90.9%, respectively. Introduction The pleth variability index (PVI) is a new dynamic index obtained by automatic estimation of respiratory variations in the pulse oximeter waveform amplitude. This noninvasive and continuous hemodynamic monitoring has been recently proposed in mechanically ventilated patients to guide fl uid therapy. We recently acquired a PVI monitor in 2014. PVI is calculated by measuring changes in perfusion index (PI) during the respiratory cycle as follows: PVI = ((PImax – Pimin) / PImax) × 100. This study aimed to investigate whether PVI at baseline can predict fl uid responsiveness. pl p Methods In our pediatric ICU we started a prospective and observational study. Between January and November 2014, nine mechanically ventilated children were investigated using PVI and transthoracic echocardiography for each patient with acute circulatory failure (ACF): tachycardia, hypotension, oliguria, delayed capillary refi lling or hemodynamic instability despite vasopressor drugs. Intervention: standardized volume expansion. Conclusion In this study, DELTA Vpeak and DELTA IVC were appropriate variables to predict fl uid responsiveness by TTE in ventilated children. References 1. Durand P, et al. Respiratory variations in aortic blood fl ow predict fl uid responsiveness in ventilated children. Intensive Care Med. 2008;34:888-94. 1. Durand P, et al. Respiratory variations in aortic blood fl ow predict fl uid responsiveness in ventilated children. Intensive Care Med. 2008;34:888-94.l 2. Choi DY, et al. Respiratory variation in aortic blood fl ow velocity as a predictor of fl uid responsiveness in children after repair of ventricular septal defect. Pediatr Cardiol. 2010;31:1166-70. 2. Choi DY, et al. Respiratory variation in aortic blood fl ow velocity as a predictor of fl uid responsiveness in children after repair of ventricular septal defect. Pediatr Cardiol. 2010;31:1166-70. Results Signifi cant changes in stroke volume were observed after volume loading (VL) ≥10% in eight patients (responders (R)) and <10% in one patient (nonresponder (NR)). Before VL, PVI was signifi cantly higher in R than NR at baseline ((19.75  ±  3.15%) vs. (9%  ±  0.00%), P <0.0001), and decreased signifi cantly in R from baseline to after VL ((19.75% ± 3.15) vs. (12.5% ± 2.828), P <0.0001). P187 Assessing fl uid status with the vascular pedicle width: relationship to IVC diameter, IVC variability and lung comets N Salahuddin, I Hussain, Q Shaikh, M Joseph, H Alsaidi, K Maghrabi King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P187 (doi: 10.1186/cc14267) Assessing fl uid status with the vascular pedicle width: relationship to IVC diameter, IVC variability and lung comets Methods Mechanically ventilated patients having cardiac arrhythmia who have been considered for volume expansion were recruited in this prospective study. Each patient was sedated, paralyzed and monitored with a central venous catheter and a thermistor-tipped femoral arterial VolumeView catheter connected to the EV1000 monitor. We assessed hemodynamic changes after PLRT via a pulse wave contour analysis method. Then we compared it with hemodynamic changes after volume expansion (NSS 500 ml in 15 minutes) via the transpulmonary thermodilution (TPTD) method. N Salahuddin, I Hussain, Q Shaikh, M Joseph, H Alsaidi, K Maghrabi King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P187 (doi: 10.1186/cc14267) Introduction This study attempts to determine a vascular pedicle width (VPW) cutoff value that identifi es a fl uid replete state defi ned as an IVC diameter ≥2 cm and ≤15% respiratory variation. p y Methods In a cross-sectional design, consecutive, critically ill patients underwent simultaneous chest radiographs and ultrasounds. The Research Ethics Committee approved the study. Results A total of 17 patients were included in this study. Six patients were volume responders (TPTD cardiac index change ≥15%). A PLRT change cardiac index ≥10% from the pulse wave contour analysis method had predicted VR with a sensitivity of 50%, a specifi city of 72.7% and an area under the ROC curve of 0.591 (P = 0.546). Results Eighty-four data points on 43 patients were collected. VPW correlated with IVC diameter (r  = 0.64, P ≤0.001) and IVC variation (r = –0.55, P ≤0.001). No correlation was observed between VPW and number of lung comets (r = 0.12, P = 0.26) or positive fl uid balance (r  = 0.3, P  =  0.058). On multivariate linear regression, standardized coeffi cients demonstrated that a 1 mm increase in IVC diameter corresponded to a 0.28 mm (Beta) increase in VPW. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) Results A total of 204,680 patients met the search criteria, and 76,807 patients developed one or more postsurgical complications (morbidity rate 37.5%). In patients with and without complications, hospital costs (including 30  days readmission costs) were $27,607  ±  32.788 and $15.783  ±  12,282 (P  <0.0001), median (interquartile range) hospital lengths of stay (fi rst stay) were 7 (4 to 10) days and 4 (3 to 5) days (P  <0.0001), and 30-day readmission rates were 17.2% and 11.9% (P <0.0001), respectively. With PGDT, the morbidity rate was anticipated to decrease from 26.6 to 31.1%, yielding gross cost savings of $153 million to 263 million for the study period, $61 million to 105 million per year, or $754 to 1,286 per patient. used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean difference in total costs. volume was 9.8 ml/predicted body weight. The area under the ROC curve of IVC collapsibility was 0.576 (95% confi dence interval (CI): 0.38 to 0.77), while the area under the ROC curves of right IJV, left IJV, right SCV and left SCV collapsibility were 0.870 (95% CI: 074 to 1.0), 0.54 (95% CI: 0.34 to 0.74), 0.62 (95% CI: 0.43 to 0.81) and 0.54 (95% CI: 0.34 to 0.74), respectively. Greater than 11% of right jugular vein collapsibility predicted fl uid responsiveness with a sensitivity of 79% and a specifi city of 94%. volume was 9.8 ml/predicted body weight. The area under the ROC curve of IVC collapsibility was 0.576 (95% confi dence interval (CI): 0.38 to 0.77), while the area under the ROC curves of right IJV, left IJV, right SCV and left SCV collapsibility were 0.870 (95% CI: 074 to 1.0), 0.54 (95% CI: 0.34 to 0.74), 0.62 (95% CI: 0.43 to 0.81) and 0.54 (95% CI: 0.34 to 0.74), respectively. Greater than 11% of right jugular vein collapsibility predicted fl uid responsiveness with a sensitivity of 79% and a specifi city of 94%. pi y Conclusion Our results suggest collapsibility of the right jugular vein can be a useful predictor of fl uid responsiveness in patients on pressure support ventilation, compared with other central large veins. Collapsibility of IVC does not predict FR in those patients. P185 Passive leg raising cannot predict volume responsiveness in septic shock patients having cardiac arrhythmia P Ratanawatkul1, A Wattanathum2 1Srinagarind Hospital, Khon Kaen, Thailand; 2Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P185 (doi: 10.1186/cc14265) P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) p y p p Conclusion Postsurgical complications occurred in more than one- third of our study population and had a dramatic impact on hospital costs, length of stay, and readmission rates. Potential cost savings with PGDT were $754 to 1,286 per patient. These projections should help hospitals estimate the return on investment for implementation of PGDT. Reference 1. Pearse et al. Eff ect of a perioperative, cardiac output-guided hemodynamic therapy algorithm on outcomes following major gastrointestinal surgery: a randomized clinical trial and systematic review. JAMA. 2014;311:2181-90. Introduction The passive leg raising test (PLRT) is a self-volume challenge used in order to predict volume responsiveness (VR) in both spontaneous and mechanically ventilated critically ill patients. However, there were small numbers of arrhythmic patients included in previous studies. Therefore, the accuracy of the PLRT for prediction of VR in arrhythmic patient is still inconclusive. We hypothesized that the PLRT can predict VR in mechanically ventilated patients having cardiac arrhythmia. P187 P187 ROC curve analysis yielded an AUC of 0.843 (95% CI = 0.75 to 0.93), P ≤0.001 and provided the best accuracy with a cutoff VPW value of 64 mm (sensitivity 81%, specifi city 78%, PPV = 88.5%, NPV = 66%, correct classifi cation rate = 79.6%). See Figure 1. Conclusion The PLRT may not be used for prediction of VR in mechanically ventilated patients having cardiac arrhythmia; however, further and larger studies are needed to confi rm this fi nding. References 1. Monnet X, et al. Crit Care Med. 2006;34:1402-7. 1. Monnet X, et al. Crit Care Med. 2006;34:1402-7. 2. Cavallaro F, et al. Intensive Care Med. 2010;36:1475-83. 2. Cavallaro F, et al. Intensive Care Med. 2010;36:1475-83. Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. P184 Collapsibility of jugular veins, subclavian veins and inferior vena cava as predictors of fl uid responsiveness in patients on pressure support ventilation: a prospective cohort study Y Iizuka1, M Sanui1, T Nomura2 1Jichi Medical University Saitama Medical Center, Saitama, Japan; 2Shonan Kamakura General Hospital, Kamakura, Japan Critical Care 2015, 19(Suppl 1):P184 (doi: 10.1186/cc14264) The best cutoff value for DELTA Vpeak ao was 13% with sensitivity and specifi city predictive values of 85.7% and 100%, respectively; and the best cutoff value for FTc was 265 milliseconds with sensitivity and specifi city predictive values of 57.1% and 75%, respectively. Methods Patients on pressure support ventilation were prospectively included when fl uid challenges were clinically indicated. Bilateral IJV were examined at the level of cricoid cartilage. Bilateral SCV were measured where the veins crossed the clavicle. Anteroposterior diameter, cross-sectional area (CSA) of IJV and SCV were measured using frame by frame analysis. IVC was measured 2 cm from the right atrial border in a long axis view. Fluid responsiveness was defi ned as 8% increase of stroke volume calculated by the Vigileo monitor (Vigileo, FloTrac; Edwards Lifesciences) after passive leg raising (started from supine position). Receiver operating characteristic (ROC) curves were generated using EZR. Results Twenty-nine patients (35 measurements) were included. Nineteen measurements had fl uid responsiveness. The mean tidal Conclusion In our study, DELTA Vpeak was the most appropriate variable to predict fl uid responsiveness by OD in ventilated children with ACF. S64 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean diff erence in total costs. Results A total of 204,680 patients met the search criteria, and 76,807 patients developed one or more postsurgical complications (morbidity rate 37.5%). In patients with and without complications, hospital costs (including 30  days readmission costs) were $27,607  ±  32.788 and $15.783  ±  12,282 (P  <0.0001), median (interquartile range) hospital lengths of stay (fi rst stay) were 7 (4 to 10) days and 4 (3 to 5) days (P  <0.0001), and 30-day readmission rates were 17.2% and 11.9% (P <0.0001), respectively. With PGDT, the morbidity rate was anticipated to decrease from 26.6 to 31.1%, yielding gross cost savings of $153 million to 263 million for the study period, $61 million to 105 million per year, or $754 to 1,286 per patient. used to estimate the expected reduction in postsurgical morbidity with PGDT. Potential cost-savings were calculated from the actual and anticipated morbidity rates using the mean diff erence in total costs. Bioimpedance as a measure of fl uid overload in patients recently admitted to intensive care Bioimpedance as a measure of fl uid overload in patients recentl admitted to intensive care M O’Connor, E Galtrey, CJ Kirwan, JR Prowle Barts Health NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) y Barts Health NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) Critical Care 2015, 19(Suppl 1):P188 (doi: 10.1186/cc14268) Introduction Fluid overload is associated with adverse outcomes in critical illness; however, better methodology is required for its quantifi cation. Bioelectrical impedance analysis (BIA) represents a non- invasive method for quantifi cation of fl uid overload [1], but has not been widely taken up in the ICU. Results Fifty patients were included, 40.8% of them were responders. The proportion of responders increases with the increase of dose of fl uids (Table 1). The regression equation was: change of Pmsf (%) = 4.4 (dose of fl uids ml/kg, 95% CI 2.3 to 6.5) – 1.6 (95% CI 7.4 to 4.3, R2 = 0.28, F(1.47) = 17.8, P <0.001). The predicted dose of fl uids required to achieve a change in Pmsf of 15% is 3.7 ml/kg crystalloids. y p Methods We assessed changes in fl uid balance and performed daily BIA (using a Maltron BioScan 920-II; Maltron International Ltd, UK) over 3 days in consecutive ICU admissions with LOS >72 hours. Table 1 (abstract P189). Change of Pmsf-arm and CO Table 1 (abstract P189). Change of Pmsf-arm and CO 1 ml/kg 2 ml/kg 3 ml/kg 4 ml/kg (n = 12) (n = 12) (n = 13) (n = 13) P ΔPmsf-arm (%) 0.0 6.5 9.0 18 0.05 (–4 to 9) (3 to 21) (8 to 16) (9 to 21) ΔCO (%) 3.9 6 9.9 12.9 0.1 (0.4 to 10) (2.1 to 9.1) (–1.6 to 14.3) (2.6 to 23.5) Responders (%) 25.0 18.2 46.2 69.2 0.04 Values are median (interquartile range). Results Of 24 patients 71% were male, median age was 65 years and APACHE II score was 15. Eleven patients had a medical diagnosis and 13 a surgical or trauma reason for admission. Seventy-one percent were mechanically ventilated and 67% were on vasopressors or inotropes. Median BIA-estimated extracellular water was 25.2 l (IQR 22 to 28) on day 1, equating to excess fl uid of 7.2 l (IQR 5 to 13.9). Median right body resistance normalized to height at 50 kHz (R50/h) on day 1 was 214 Ω/m (IQR 187 to 256). Return on investment for implementation of perioperative goal-directed therapy in major surgery: a nationwide database study Introduction Preventable postsurgical complications are increasingly recognized as a major healthcare burden. A recent meta-analysis showed a 17 to 29% decrease in complications after major surgery with perioperative goal-directed therapy (PGDT) [1]. We assessed the fi nancial consequences of postsurgical complications in a large population from 541 US hospitals in order to predict potential savings with PGDT. Introduction Preventable postsurgical complications are increasingly recognized as a major healthcare burden. A recent meta-analysis showed a 17 to 29% decrease in complications after major surgery with perioperative goal-directed therapy (PGDT) [1]. We assessed the fi nancial consequences of postsurgical complications in a large population from 541 US hospitals in order to predict potential savings with PGDT. Methods Data from adults who had any one of 10 major noncardiac surgical procedures between January 2011 and June 2013 were selected from the Premier research database. Twenty-six postsurgical complications were tabulated. Hospital costs, length of stay, and readmission rates were compared in patients with and without complications. Risk ratios reported by Pearse’s meta-analysis were Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. Figure 1 (abstract P187). ROC curve for VPW discriminating fl uid repletion by IVC ultrasound. S65 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 The objective of this study was to determine the minimum volume of intravenous fl uid required to signifi cantly increase the Pmsf. Conclusion A VPW value of 64 mm accurately identifi es a fl uid replete state. Increased extravascular lung water, however, was not relatable to the VPW measurements. The VPW can be confi dently used to discriminate fl uid repletion from fl uid responsiveness. li Methods Patients following cardiac surgery were randomly allocated to receive 1, 2, 3 or 4 ml/kg (body weight) of crystalloid over 5 minutes using a 60 ml syringe. Pmsf was measured using the arterial pressure after stopping blood fl ow in the arm with a pneumatic tourniquet infl ated for 1 minute. Cardiac output (CO) was also recorded at baseline and immediately after the fl uid infusion. CO was measured with LiDCO or pulmonary artery catheter, and a positive response was considered an increase of 10% from baseline. From previous data, the least signifi cant change for Pmsf was 15%. Return on investment for implementation of perioperative goal-directed therapy in major surgery: a nationwide database study Medians were compared using the independent samples media test, and proportions were compared using a chi-square test. Statistical signifi cance was considered when P <0.05. P188 Bioimpedance as a measure of fl uid overload in patients recently admitted to intensive care Daily change in ECW and R50/h correlated with daily fl uid balance between BIA measurements (R2 = 0.48 and 0.37 respectively) (Figure 1).i y g Conclusion BIA suggests many patients already have signifi cant fl uid overload on the fi rst day of ICU admission. Overall, changes in device-specifi c algorithms for ECW estimation and measured resistances correlated with recorded fl uid balance; however, there were inconsistencies in the number of individual patients. Prospective assessment is required to establish whether BIA measurements can be used to assist fl uid management in the ICU. Conclusion The minimum volume required to perform an eff ective fl uid challenge is 4 ml/kg infused in 5 minutes. However, only 30% of the variation of change in Pmsf can be explained by the dose of i.v. fl uid given. The proportion of responders increases with the volume of fl uids. 1. Earthman C, et al. Bioimpedance spectroscopy for clinical assessment of fl uid distribution and body cell mass. Nutr Clin Pract. 2007;22:389. P189 P189 Minimal volume for a fl uid challenge in postoperative patients H Aya, A Rhodes, RM Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P189 (doi: 10.1186/cc14269) Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery 1ICESP, São Paulo, Brazil; 2Heart Institute, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P191 (doi: 10.1186/cc14271) Introduction After cardiac surgery, about 15% of patients develop vasoplegic shock, characterized by systemic vasodilation, increased capillary permeability and edema. We hypothesized that large-volume resuscitation, resulting in positive fl uid balances in the fi rst 24 hours of ICU admission, would be associated with mortality and would not be protective against AKI in this subset of patients. Conclusion Postsurgical complications have a signifi cant impact on hospital margins. Enhanced Recovery Programs have the potential not only to improve quality of care but also to improve hospital margins. References g Methods This is a retrospective analysis of 362 patients submitted to cardiac surgery at the Heart Institute of University of São Paulo in a period of 2 years. Of a total of 2,383 patients, we enrolled 362 patients. Vasoplegic shock was diagnosed if in the 24 hours of ICU admission patients had hypotension, need of vasoactive drugs after fl uid replacement and cardiac index ≥2.2 l/minute/m2. Data were analyzed in logistic regression models for 30-day mortality and acute kidney injury through Acute Kidney Injury Network (AKIN) score as outcomes. Results The mean age of patients was 57 years. Of 362 patients, 53 died at 30 days (14.6%). Nonsurvivors as compared with survivors were slightly older (59 ± 12 vs. 55 ± 13, P = 0.063), had a higher prevalence of AKI through AKIN score ((0) 6.9%, (1) 11.1%, (2) 28.9%, (3) 31.9%, P  <0.001), a higher 24-hour fl uid balance (421  ml (–55 to 695) vs. 2,686 ml (1,321 to 2,856), P <0.001), and higher lactate levels at the intraoperative and at 48 hours (5 mmol/l (4.0 to 7.6) vs. 4.4 (3.33 to 6.55), P <0.001; and 8.11 (5.49 to 12.3) vs. 1.5 (1.33 to 1.88), P <0.001). In the multivariate analysis, positive fl uid balance in the fi rst 24 hours (OR = 1.006, 95% CI = 1.003 to 1.008, P <0.001) and higher lactate after 48 hours (OR = 1.204, 95% CI = 1.072 to 1.353, P = 0.002) were predictors of 30-day mortality. Forty-three percent of patients developed AKI during 30 days. P192 Impact of postsurgical complications on hospital costs and margins R Lavender1, M Mythen2, J Bao3, RH Chapman3, F Michard1 1Edwards Lifesciences, Irvine, CA, USA; 2UCLH National Institute of Health Research, London, UK; 3Avalere Health, Washington, DC, USA Critical Care 2015, 19(Suppl 1):P192 (doi: 10.1186/cc14272) y Results Three hundred and thirty-nine patients were included; mean age was 51 ± 20.4 years, 167 (49%) patients were male. Mean APACHE II score was 22 ± 12.8 and SAPS II score was 35.4 ± 18.9. Severe sepsis/ septic shock was the admitting diagnosis in 129 (38%) patients, 108 (32%) patients were postoperative. AKI developed in 148 (44%) patients; Risk 29 (9%); Injury 26 (8%); Failure 89 (26%) by the RIFLE criteria. On univariate regression analysis; positive fl uid balance >2 l on the fi rst ICU admission day, OR 2 (95% CI = 1.3, 3.3, P = 0.002); age, OR 2.7 (95% CI = 1.7, 4.2, P = 0.000); CHF, OR 3.1 (95% CI = 1.2, 7.9, P = 0.013); APACHE II score, OR 1.02 (95% CI = 1.0, 1.04, P = 0.006); SAPS II score, OR 1.04 (95% CI = 1.02, 1.05, P = 0.000); mean MAP on admission, OR 0.98 (95% CI = 0.96, 0.99, P = 0.033); need for vasopressors on admission, OR 2.7 (95% CI = 1.7, 4.2, P <0.001) and for >24 hours, OR 2.7 (95% CI = 1.7, 2.5, P <0.001); and vancomycin use, OR 1.5 (95% CI = 1.02, 2.53, P = 0.04) signifi cantly predicted the development of AKI. On multivariate regression, CHF, OR 3.8 (95% CI = 1.4, 10, P = 0.007); age, OR 1.02 (95% CI = 1.01, 1.03, P = 0.001); vasopressors for >24 hours, OR 2.6 (95% CI = 1.6, 4.2, P <0.001) and a >2 l positive fl uid balance on the fi rst ICU day, OR 1.6 (95% CI = 1.02, 2.7, P = 0.04) remained signifi cant predictors. Introduction The impact of postsurgical complications (PSC) on hospital cost has been studied but the impact on margins remains controversial [1]. We assessed economic consequences of PSC in US Medicare patients, and benefi ts expected from reducing PSC by 14% to 40% with Enhanced Recovery Programs [2]. y Methods Data from patients with ≥1 comorbidity and major cardiac, vascular, gastrointestinal and orthopedic surgeries in 2011 were extracted from Medicare Standard Analytic Files. Hospital margin was calculated as payment minus cost. P192 Patients with and without PSC were compared, and the economic impact of a 14 to 40% relative reduction in PSC was calculated. Results Of 303,432 patients, 37% had ≥1 PSC. Median length of stay was 10 days for patients with ≥1 PSC and 6 days without (P <0.0001 with vs. without PSC), with readmissions for 21% and 16%, respectively (P <0.0001 with vs. without PSC). Average margins for cases with PSC converted into without PSC would be $1,870 higher. A 14 to 40% reduction in patients with PSC (from 37% to 32% to 22%) would result in saving $153 million to $438 million, and increase hospital margins overall by $28 million to $79 million. See Table 1. i Conclusion Fluid overload, defi ned as a >2 l positive fl uid balance on the fi rst day of ICU admission, is an independent risk factor for the development of AKI in the general ICU population. Table 1 (abstract P192) With PSC Without PSC Mean 2011 US$/patient Cost ($) Margin ($) Cost ($) Margin ($) Cardiac 46,535 –2,286 32,887* –778* Gastrointestinal 33,280 –3,088 18,942* –752* Orthopedic 20,798 –3,567 15,194* –1,872* Vascular 31,042 –4,782 17,667* –2,267* *P <0.0001 with versus without PSC. Table 1 (abstract P192) Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery In the multivariate analysis, positive fl uid balance in the fi rst 24 hours (OR = 1.001, 95% CI = 1.000 to1.001, P <0.001) and higher lactate at 48 hours (OR = 1.011, 95% CI = 1.000 to 1.021, P = 0.0043) were predictors of 30-day AKI. 1. Eappen S, et al. JAMA. 2013;309:1599-606. P191 Positive fl uid balance as a risk factor for mortality and acute kidney injury in vasoplegic shock after cardiac surgery A Rezende1, L Camara2, A Leme2, J Ribeiro2, I Bispo2, S Zeferino2, J Jardim2, C Park1, E Osawa2, J Almeida2, A Gerent1, F Galas2, D Fonseca2, J Fukushima1, L Hajjar2 1ICESP, São Paulo, Brazil; 2Heart Institute, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P191 (doi: 10.1186/cc14271) Minimal volume for a fl uid challenge in postoperative patients H A A Rh d RM G d M C i H Aya, A Rhodes, RM Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK g Critical Care 2015, 19(Suppl 1):P189 (doi: 10.1186/cc14269) Introduction In critical illness, fl uid overload may predispose to acute renal dysfunction by a number of mechanisms. Once acute kidney Introduction An eff ective fl uid challenge should increase the mean systemic fi lling pressure (Pmsf) in order to increase the venous return. Figure 1 (abstract P188). Change in BIA-measured ECW (a) or R/h at 50 kHz (b) versus daily fl uid balance. Hz (b) versus daily fl uid balance. Figure 1 (abstract P188). Change in BIA-measured ECW (a) or R/h at 50 kHz (b) versus daily fl uid balance. S66 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Positive fl uid balance after cardiac surgery is an independent risk factor for mortality and for acute kidney injury in patients presenting vasoplegic shock. injury (AKI) develops, positive fl uid balance has been described as a risk factor for overall mortality and delayed renal recovery. We hypothesized that fl uid overload may be an independent risk factor for AKI in the critically ill. injury (AKI) develops, positive fl uid balance has been described as a risk factor for overall mortality and delayed renal recovery. We hypothesized that fl uid overload may be an independent risk factor for AKI in the critically ill. Methods In a cross-sectional design, we collected data on consecutive, critically ill, adult patients admitted over a 5-month period to the medical and surgical ICUs of a single center. AKI was defi ned according to the RIFLE Classifi cation. Logistic regression analysis was performed to determine the predictive ability of variables for AKI. The institutional Research Ethics Committee approved the study. P195 Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Results We included a total of 1,267 patients. The median age was 68 (quartiles: 59, 76), 32% were female, 68% underwent coronary artery bypass grafting and 59% underwent valve surgery. Median length of hospital stay was 6 days (quartiles: 5, 9). Median length of stay in the normal, elevated and high lactate groups were 5 days (quartiles: 4, 7), 6 days (quartiles: 5, 9) and 9 days (quartiles: 6, 17), P <0.001 for comparison. In multivariable analysis, patients with an elevated lactate had a 1.12 times (95% CI: 1.02 to 1.23, P = 0.02) longer length of stay compared with those with normal lactate. Patients with a high lactate had a 1.30 times (95% CI: 1.10 to 1.53, P = 0.002) longer length of stay compared with those with normal lactate. Introduction National University Hospital, Singapore, recently formed a Division of Critical Care – Respiratory Therapy. This service rapidly expanded to provide 24/7 Respiratory Therapy Services in the cardiothoracic intensive care unit (CTICU). One goal of service expansion was a reduction in duration of mechanical ventilation after cardiac surgery. We hypothesized that introduction of a team- based extubation protocol would reduce the duration of mechanical ventilation and ultimately aff ect ICU length of stay. Conclusion Postoperative lactate levels are associated with increased length of hospital stay in patients undergoing major cardiac surgery. Interventions aimed at decreasing postoperative lactate levels may decrease hospital length of stay. f Methods A multidisciplinary group created a team-based extubation protocol. The protocol was applied to all elective postoperative cardiac surgery patients. To assess the protocol’s impact, data were collected in a registry 3 months before and 3 months after protocol initiation. Data collection included cardiopulmonary bypass time, McCormack airway assessment, ICU admission time, initial pH, lactate, inotropes upon arrival at the CTICU, blood gas analysis prior to extubation, time of extubation and length of stay. Patients were excluded from data analysis if they experienced events which contraindicated application of the protocol, such as signifi cant intraoperative or postoperative complications. These events were explicitly stated in the extubation protocol. Lactate levels after major cardiac surgery are associated with hospital length of stay Introduction The objective of the study was to evaluate whether postoperative lactate values are associated with hospital length of stay in patients undergoing major cardiac surgery. Previous studies have shown an association between postoperative lactate levels and increased morbidity and mortality after major cardiac surgery. However, the association between lactate and hospital length of stay has not been adequately characterized. Methods We performed a retrospective analysis of all patients presenting for coronary artery bypass grafting and/or valve surgery between 2002 and 2014 at a tertiary care center in Boston, who had a lactate level measured within 3 hours of skin closure. Lactate values S67 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 were categorized into clinical meaningful categories: 0 to 2  mmol/l (normal), 2 to 4 mmol/l (elevated) and ≥4 mmol/l (high) to allow for nonlinear eff ects. The unadjusted association between lactate group and length of stay was assessed with the Kruskal–Wallis test and post hoc Wilcoxon rank-sum tests. To assess the association between postoperative lactate levels and hospital length of stay we performed multivariable Poisson regression with robust variance estimates. We adjusted for more than 30 variables including patient demographics, comorbidities, cardiac characteristics (for example, New York Heart Association class and ejection fraction), and surgical characteristics (for example, year, status (elective, urgent, emergent), type of procedure, perfusion time, and cross clamp time). severe hypotension (MAP <65 mmHg) and the study was not stopped in any case. The length of the hospital stay was shorter among patients in the intensive group (10.9 (9.9 to 11.9) vs. 12.4 days (11.3 to 13.6); P = 0.045). Conclusion An intensive alveolar recruitment protocol did not result in hemodynamic instability in hypoxemic patients after cardiac surgery (NCT01502332). P195 P195 Team-based extubation protocol in cardiac surgical patients reduces ventilation time and reduces length of stay in the ICU JM Taculod, MJ Dajac, A Del Rosario, J Gammad, S Mahaju, O Siow Eng, P Oh, R Kollengode, G Maclaren, ME Cove National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P195 (doi: 10.1186/cc14275) Singapore’s Domain-specifi c review board granted waiver of patient consent to analyze and present these data. Hemodynamic behavior in a randomized trial of intensive alveolar recruitment after cardiac surgery recruitment after cardiac surgery A Leme, M Amato, E Osawa, J Fukushima, M Feltrim, E Nozawa, J Almeida L Hajjar, F Galas Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P194 (doi: 10.1186/cc14274) A Leme, M Amato, E Osawa, J Fukushima, M Feltrim, E Nozawa, J Almeida, L Hajjar, F Galas jj Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P194 (doi: 10.1186/cc14274) Introduction The potential benefi ts of a protocol of intensive alveolar recruitment may be outweighed by its detrimental eff ects in hemodynamic stability after cardiac surgery. The aim of this study was to analyze the hemodynamic behavior of patients included in a trial of intensive alveolar recruitment after cardiac surgery. p y p Results A total of 201 patients undergoing elective open cardiac surgery were included; 99 patients before protocol implementation (pre-protocol) and 102 patients after implementation (post-protocol). There was no signifi cant diff erence in mean age (60 vs. 61 P = 0.823), gender (79.8% vs. 79.4% P  =  1.00), EuroSCORE (26 vs. 32 P  =  0.576) and proportion receiving bypass surgery (72% vs. 80% P = 0.206) or valve surgery (21% vs. 19% P = 0.722) between the two groups. Median extubation time was reduced by 3.5 hours (620 minutes vs. 408 minutes P <0.001). ICU length of stay was also reduced following introduction of the pre-protocol 48 hours versus 24 hours post protocol (P <0.05).i Methods In this randomized trial, we assigned adult patients with PaO2/ FIO2 <250 at a PEEP of 5 cmH2O to either intensive alveolar recruitment or a standard protocol, both using low-tidal volume ventilation (6 ml/ kg/ibw) after adequate volemia status. Our hypothesis was that an intensive alveolar recruitment protocol with controlled pressure of 15 cmH2O and PEEP of 30 cmH2O during 1  minute, repeated three times at 1-minute intervals between each maneuver, would not cause hemodynamic instability. Conclusion A team-based extubation protocol signifi cantly reduced the duration of mechanical ventilation and this translated to reduced ICU length of stay in patients undergoing elective open-heart surgery. Results In total, 163 patients were included in the standard and 157 in the intensive group. Patients of the intensive group had a signifi cant reduction of the MAP at T1, T2 and T3 (1 hour, 2 hours and 3 hours of the protocol), returning to baseline after T4 (Figure 1). No patients had P196 Impact of patient frailty on outcome in cardiothoracic surgery J Brohan, P Delaney, B O’Brien Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 JL Iribarren, J Jiménez, N Perez, M Brouard, R Perez, E Hurtado, S Diosdado, M Buitrago, A Arbesu, R Martinez, M Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P199 (doi: 10.1186/cc14279) Introduction Cardiopulmonary bypass can lead to postoperative hemodynamic disorders. Several genetic polymorphisms have been studied in this setting. We investigated the possible existence of a synergism between polymorphisms of plasminogen activator inhibitor type 1 (PAI-1) and tumoral necrosis factor beta (TNF-B) on hemodynamic response after cardiac surgery. Introduction Cardiopulmonary bypass can lead to postoperative hemodynamic disorders. Several genetic polymorphisms have been studied in this setting. We investigated the possible existence of a synergism between polymorphisms of plasminogen activator inhibitor type 1 (PAI-1) and tumoral necrosis factor beta (TNF-B) on hemodynamic response after cardiac surgery. g Results A total of 8,026 were recorded, in 77 of them an IABP was inserted before the surgery. We performed a propensity score analysis by pairing 72 patients with and without BCIAO based on epidemiological factors and type of surgery. In the analysis of all-cause 30-day mortality, 27% of patients in whom IABP was inserted prior surgery died versus 13.1% of patients without IABP preoperative implantation (P = 0.043). A combined endpoint that included need for prolonged mechanical ventilation over 24 hours or reoperation or mediastinitis or stroke after surgery or 30-day mortality was performed and occurred in 58.3% of patients with preoperative IABP versus 41.7% without it (P = 0.046). When stratifi ed by preoperative risk (analyzed with EuroSCORE), no diff erence between groups was observed (P = 0.62, OR 0.75 (0.23 to 2.35)) for mortality rate and (P = 0.11, OR 0.47 (0.19 to 1.18)) for the combined endpoint. The patients with preoperative IABP implantation had a higher ICU length of stay (10.6  ±  7.7 vs. 4.6  ±  6.7, P  =  0.046) with no diff erences in terms of overall hospital stay (21.8  ±  18.7 vs. 18.9 ± 22.08, NS). y y Methods We prospectively studied the association between hemodynamic response and polymorphisms of TNF-B and PAI-1 in 563 patients undergoing elective cardiac surgery during the years 2008 to 2011. We tested the Hardy–Weinberg equilibrium in the sample. V18 SPSS was used.if Results We studied 563 patients. We found signifi cant diff erences in TNF-B polymorphisms regarding norepinephrine requirements at 4 hours (F: 15.9; P <0.001), post hoc Scheff é (GG vs. P198 Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery J Muñoz-Bono, MD Delgado-Amaya, E Curiel-Balsera, C Joya-Montosa, G Quesada-García Hospital Regional de Málaga, Spain Critical Care 2015, 19(Suppl 1):P198 (doi: 10.1186/cc14278) Results A total of 120 patients were included in this study, including 100 patients who underwent cardiac surgery and 20 patients who underwent thoracic surgery. Eighty-fi ve patients (70.8%) were male. The mean age was 65.4 years (range 25 to 89 years). The mean baseline frailty score also varied widely within our cohort. Four patients died in the ICU following their surgery (3% ICU mortality rate). Mean length of ICU stay was 2.7 days (range 0 to 20 days), with a mean duration of ventilation of 20 hours (range 0 to 264 hours). Follow-up of these patients at 6 months following their surgery is currently underway. Introduction The aim of the study is to analyze IABP use in patients undergoing cardiac surgery included in the ARIAM Registry of Cardiac Surgery. Introduction The aim of the study is to analyze IABP use in patients undergoing cardiac surgery included in the ARIAM Registry of Cardiac Surgery. g y Methods An observational, retrospective, multicenter study of all patients undergoing cardiac surgery included in the ARIAM- ANDALUCIA database of Cardiac Surgery from March 2008 to July 2012. We used the chi-square test and Student t test as needed, establishing the level of statistical signifi cance at 95%. y y y Conclusion Owing to advances in life expectancy, health and perioperative medicine, it has become more diffi cult to determine fi tness for major surgery. Our data suggest that frailty may be a useful prognostic measure to help inform such decisions. R f gi Results Of the 8,026 patients who underwent cardiac surgery during the study period, BCIAO was implemented in 358 (4.5%) of them. In total, 65.4% were male. Surgical times in those patients where IABP was implanted were 146  ±  81  minutes and 90  ±  66  minutes (cardiopulmonary and aortic clamping times, respectively). The in- surgery room mortality was 4.7%, 30-day mortality in these patients was 40.2%. Patients in whom IABP was implanted had a mortality rate eight times higher than those who did not require it during surgery or postoperatively (40.2% vs. 8.4%, P = 0.0001. OR 8.1, 95% CI (6.4 to 10.3)). Impact of patient frailty on outcome in cardiothoracic surgery J Brohan, P Delaney, B O’Brien Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) Figure 1 (abstract P194). J Brohan, P Delaney, B O’Brien , y, Cork University Hospital, Cork, Ireland Cork University Hospital, Cork, Ireland Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc14276) y p , , Critical Care 2015, 19(Suppl 1):P196 (doi: 10.1186/cc142 Introduction Frailty is defi ned as a multidimensional syndrome involving loss of physical and cognitive reserve leading to greater vulnerability to adverse events [1]. Such events include susceptibility to unplanned hospital admissions, and death [1-3]. Frailty is associated with increased ICU and 6-month mortality, and reduced quality of life [4]. The aim of this study is to investigate the impact of baseline frailty on postoperative quality of life indicators and postoperative frailty following cardiothoracic surgery. Methods Adult patients undergoing cardiac surgery or thoracic surgery (involving thoracotomy) were included in this study. Baseline measures of frailty [4] and performance status were prospectively recorded using validated tools. Informed consent was obtained prior to inclusion. Outcome measures of APACHE II scores, duration of ventilation, length S68 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P198 Intraortic balloon pump use in cardiac surgery: analysis of data from the ARIAM Registry of Cardiac Surgery Besides mortality was higher, the later IABP was implanted the higher the mortality rate was (29.6% of the preoperative, 44.2% of surgical and 54.4% of those starting in ICU, P = 0.015). The ICU length of stay was 9 ± 22 days while the hospital length of stay was 21 ± 28 days. In patients who needed IABP, the ICU stay was higher than for those who did not need it (9 ± 22 vs. 5 ± 10 days, P = 0.002) whereas there was no diff erence in hospital stay (21 ± 28 vs. 20 ± 24 days, P = 0.054). 1. Rockwood K, et al. A global clinical measure of fi tness and frailty in elderly people. CMAJ. 2005;173:489-95. 2. Rockwood K, et al. Changes in relative fi tness and frailty across the adult lifespan: evidence from the Canadian National Population Health Survey. CMAJ. 2011;183:E487-94. 3. Makary MA, et al. Frailty as a predictor of surgical outcomes in older patients. J Am Coll Surg. 2010;210:901-8. 4. Le Maguet P, et al. Prevalence and impact of frailty on mortality in elderly ICU patients: a prospective, multicentre observational study. Intensive Care Med. 2014;40:674-82. P198 of ICU stay and mortality were recorded. Follow-up at 6 months was conducted by telephone to assess recovery patterns. of ICU stay and mortality were recorded. Follow-up at 6 months was conducted by telephone to assess recovery patterns. Registry of Cardiac Surgery MD Delgado-Amaya, C Joya-Montosa, J Muñoz-Bono, E Curiel-Balsera Hospital Regional de Málaga, Spain MD Delgado-Amaya, C Joya-Montosa, J Muñoz-Bono, E Curiel-Balsera Hospital Regional de Málaga, Spain f p y y Conclusion The intra-aortic balloon pump was used by 4.5% of surgeries performed during the study period and in patients with an increased risk of perioperative complications, estimated by EuroSCORE. ICU length of stay was higher in patients requiring IABP, with no diff erences in overall hospital stay. Mortality rate was 40% higher, and increases with the delay in the implantation. p g g p Critical Care 2015, 19(Suppl 1):P197 (doi: 10.1186/cc14277) g g Critical Care 2015, 19(Suppl 1):P197 (doi: 10.1186/cc14277) Introduction The aim of our study was to assess whether the preoperative use of IABP is benefi cial in patients undergoing cardiac surgery of any kind. Introduction The aim of our study was to assess whether the preoperative use of IABP is benefi cial in patients undergoing cardiac surgery of any kind. Methods An observational, retrospective, multicenter study of all patients undergoing cardiac surgery and included in the ARIAM- ANDALUCIA Registry of Cardiac Surgery from March 2008 to July 2012. The probability of placing IABP in the preoperative period has been calculated, making a propensity analysis to obtain two homogeneous groups treated with or without the IABP, based on personal history, functional status and type of surgery. Seventy-seven patients with preoperative IABP were matched with 77 patients without BCIAO with the nearest propensity score. We used the chi-square test or Student t test as needed and binary logistic regression for multivariate analysis so we can rule out possible confounding variables. We used the statistical package R v2.12 for MAC. P199 Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 JL Iribarren, J Jiménez, N Perez, M Brouard, R Perez, E Hurtado, S Diosdado, M Buitrago, A Arbesu, R Martinez, M Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P199 (doi: 10.1186/cc14279) P200 Pharyngeal oxygenation during apnoea following conventional pre-oxygenation and high-fl ow nasal oxygenation D Stolady, M Mariyaselvam, H Young, E Fawzy, M Blunt, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P200 (doi: 10.1186/cc14280) y Methods We performed a prospective randomized cross-over study on hypoxemic non-intubated patients (PaO2/FiO2 ≤300 mmHg) admitted to the ICU of the San Gerardo Hospital and prescribed to receive oxygen by facial mask. We delivered the same air/oxygen mix by HFNC (Optifl ow; Fisher & Paykel Healthcare, Auckland, New Zealand) and facial mask (20 minutes per step). Continuous recordings of regional lung volumes by EIT (Pulmovista 500; Drager Medical GmbH, Lubeck, Germany) and of inspiratory eff ort by esophageal pressure (Pes) were obtained and analyzed offl ine by dedicated software. Introduction We hypothesised that pharyngeal oxygen concentrations would be maintained higher and for longer with transnasal humidifi ed rapid insuffl ation ventilatory exchange (THRIVE) than conventional bag-mask pre-oxygenation (CPO). CPO requires the mask to be removed during laryngoscopy; this means that air may enter the mouth so subsequent apnoeic oxygenation will be less eff ective. Oral suctioning could exacerbate this process. However, if high pharyngeal oxygen concentrations and an open airway are maintained, apnoeic oxygenation could be substantially improved. Methods used have included NO-DESAT [1] and recently THRIVE [2], which has been shown to extend apnoea times for up to 1 hour. yfl y Results We enrolled 15 patients (10 male), age 57 ± 16 years. Compared with standard facial mask, HFNC signifi cantly improved PaO2/FiO2 (199 ± 60 vs. 150 ± 46, P <0.001) and end-expiratory lung impedance (corresponding to aeration) (866  ± 568 au vs. baseline, P  <0.001). Moreover, HFNC decreased the respiratory rate (22 ± 5 bpm vs. 20 ± 5 bpm, P <0.001), as well as negative Pes swings (ΔPes 8.3 ± 5 mmHg vs. 6.6 ± 1 mmHg, P <0.01) and corrected minute ventilation (that is, actual MV × actual PaCO2 / 40 mmHg) (49,887 ± 16,176 au vs. 41,811 ± 14,042 au, P <0.001). Finally, central venous pressure increased (6 ± 5 mmHg vs. 4 ± 5 mmHg, P <0.01), possibly indicating positive end-expiratory pressure eff ect. Methods A volunteer with a nasopharyngeal sampling catheter underwent simulated emergency airway management (EAM), using both CPO and THRIVE, with and without suction. Reference 1. Sotello D, Rivas M, Mulkey Z, Nugent K. High-fl ow nasal cannula oxygen in adult patients: a narrative review. Am J Med Sci. 2015;349:179-85. Results Pharyngeal oxygen concentrations (mean and SEM) are shown in Figure 1 (all points are signifi cant P <0.05). Vasoplegic syndrome in cardiac surgery: role of synergism between polymorphism of tumor necrosis factor beta and plasminogen activator inhibitor type 1 AA, 0.32 (0.11 to 0.65) vs. 0.06 (0.04 to 0.09) μg/kg/minute, P <0.001; GG vs. AG, 0.32 (0.11 to 0.65) vs. 0.06 (0.03 to 0.08), P <0.001)) and at 24 hours (F: 8; P = 0.005), post hoc Scheff é (GG vs. AA, 0.27 (0.01 to 0.52) vs. 0.10 (0.06 to 0.14), P = 0.019; GG vs. AG, 0.27 (0.01 to 0.52) vs. 0.07 (0.04 to 0.09), P = 0.003)). Unfavorable TNF-B (G homozygous vs. allele A) and PAI-1 unfavorable (4G homozygous vs. allele 5G) were grouped, after adjusting for perioperative signifi cant variables. The homozygous GG and 4G alleles were signifi cant for NA 4 hours (F: 5.5; P = 0.02 and F: 4.1; P = 0.04, respectively) and GG–4G allele interaction (F: 6; P = 0.01) (Figure 1), Conclusion The use of IABP prior to cardiac surgery in patients at high risk does not reduce the mortality rate nor the combined endpoint described above. ICU length of stay was greater in those patients in whom IABP was implanted prior to surgery; there were no diff erences in overall hospital stay. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S69 THRIVE. Assessment of NO-DESAT (15 l/minute) was abandoned due to discomfort. R f Figure 1 (abstract P199). THRIVE. Assessment of NO-DESAT (15 l/minute) was abandoned due to discomfort. Figure 1 (abstract P199). P200 Following 3 minutes of pre-oxygenation with CPO (FiO2 = 1, FEO2 >0.8) or THRIVE (60 l/minute; Optifl ow, Fisher and Paykel), EAM was simulated by voluntary apnoea and pharyngoscopy with the laryngoscope blade tip placed 2 cm from the posterior pharyngeal wall. Capnography at the laryngoscope tip confi rmed apnoea. Pharyngeal gas samples (20  ml) were collected during apnoea, and after 5 seconds of oropharyngeal suctioning. Pre- oxygenation was repeated between sampling. Samples (n = 100) were analysed using calibrated fuel cells. f Conclusion In non-intubated hypoxemic critically ill patients, HFNC improves oxygenation and end-expiratory aeration; moreover, HFNC reduces the inspiratory eff ort and the minute ventilation needed to maintain normal arterial CO2 tension. 0 Eff ects of high-fl ow nasal cannula therapy on oxygenation, lung volumes and CO2 removal in critically ill hypoxemic patients: preliminary results p y T Mauri1, N Eronia2, G Bellani2, G Grasselli2, R Marcolin2, S Marocco Arrigoni2, A Pesenti2 1IRCC Ca’ Granda Maggiore Policlinico Hospital, Milan, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P201 (doi: 10.1186/cc14281) Figure 1 (abstract P199). Figure 1 (abstract P199). while for NA at 24 hours statistics showed GG (F: 3.2; P = 0.07), 4G allele (F: 2; P = 0.15) and interaction (F: 3.6, P = 0.05). Introduction High-fl ow nasal cannula (HFNC) is increasingly proposed as respiratory support for hypoxemic non-intubated acute respiratory failure patients. Clinically, HFNC therapy decreases dyspnea, improves patient’s comfort, improves oxygenation and enhances clearance of upper airway secretions [1]. We present preliminary results from a clinical study aimed at measuring the eff ects of HFNC on gas exchange, lung volumes and inspiratory eff ort in hypoxemic non-intubated critically ill patients. Conclusion GG homozygous polymorphism TNF-B is associated with an increased dependence on norepinephrine after cardiopulmonary bypass, showing a synergistic action with the 4G allele of PAI-1. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness A similar pattern was seen was seen in the vast majority of ICUs: a single institution reported no capnography available. However, in 141 (66.8%) of the hospitals surveyed, no facility to measure ETCO2 was present on the general wards. Where available, 86.7% used capnography to confi rm ETT placement. Less than 50% used ETCO2 to determine CPR eff ectiveness and 8% to prognosticate.i is located in the same position as the light source on the standard Macintosh blade thus providing a view angle of up to 290° and the USB camera is connected to a laptop. A total of the fi rst 50 patients who presented to the emergency department over a period of 6 months in need of intubation were included in the study and every alternate patient participated in the evaluation of the assembled video laryngoscope (VAL). Information about patient demographics and airway characteristics, Cormack-Lehane (C/L) views and the ease of intubation using the VAL was collected. Failure was defi ned as more than one attempt at intubation. examining practice regarding intubation for cardiac arrest and the availability and utilisation of capnography within the ED, ICU and general wards. Questions were directed at the anaesthetist or intensive care doctor ‘responding to cardiac arrest calls’. The respondent was given the option to decline participation. All data were anonymised. Results A total of 211 hospitals met the inclusion criteria. The response rate was 100%. Arrest calls were mainly attended by anaesthesia (47.8%) and ICU doctors (38.3%) with around 2% physicians only. Most were a registrar grade (56.3%). The ability to measure ETCO2 was available in all but four EDs; most used waveform capnography. A similar pattern was seen was seen in the vast majority of ICUs: a single institution reported no capnography available. However, in 141 (66.8%) of the hospitals surveyed, no facility to measure ETCO2 was present on the general wards. Where available, 86.7% used capnography to confi rm ETT placement. Less than 50% used ETCO2 to determine CPR eff ectiveness and 8% to prognosticate.i Results Excellent (C/L1) or good (C/L2) laryngeal exposure was obtained in 92% and 8% of patients respectively. In 25 patients in whom VAL was performed, there was a comparable or superior view. Intubation with direct laryngoscopy was successful in 95.2% of patients and VAL was successful in 95.4% of patients. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness Three patients from the VAL group and four patients from the direct laryngoscopy group were excluded. See Figure 1. Conclusion We believe this is the fi rst study of its kind to follow NAP4 and investigate the availability of capnography throughout for use during cardiac arrest. Whilst equipment levels appear adequate (albeit not perfect) in resuscitation areas, there appears a lack of availability of suitable devices on general wards. Figure 1 (abstract P202). New video laryngoscope connected to laptop. New assembled video laryngoscope: a study on effi cacy and cost-eff ectiveness i Conclusion Pharyngeal oxygen concentration rapidly falls following CPO. This may be detrimental for apnoeic oxygenation during con ven- tional laryngoscopy. Conversely, THRIVE maintains high pharyngeal oxygen concentrations over time. Suction has an immediate negative eff ect on pharyngeal oxygen concentration that is attenuated by f SM Ayyan, Z Ali Figure 1 (abstract P200). Pharyngeal oxygen concentration. Introduction Video laryngoscopes have been introduced in recent years as an alternative choice to facilitate tracheal intubation. Diffi culties with tracheal intubation are mostly caused by diffi cult direct laryngoscopy with impaired view to the vocal cords. Many endoscopic intubation laryngoscopes have been designed to visualize the vocal cords around the corner looking through a proximal viewfi nder. Although they are useful devices, they have limitations for doing direct laryngoscopy and are very expensive, hence they are not used for routine tracheal intubation.i Methods A Macintosh intubating laryngoscope has been modifi ed by attaching a waterproof USB camera with a inbuilt light source, which S70 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 is located in the same position as the light source on the standard Macintosh blade thus providing a view angle of up to 290° and the USB camera is connected to a laptop. A total of the fi rst 50 patients who presented to the emergency department over a period of 6 months in need of intubation were included in the study and every alternate patient participated in the evaluation of the assembled video laryngoscope (VAL). Information about patient demographics and airway characteristics, Cormack-Lehane (C/L) views and the ease of intubation using the VAL was collected. Failure was defi ned as more than one attempt at intubation. examining practice regarding intubation for cardiac arrest and the availability and utilisation of capnography within the ED, ICU and general wards. Questions were directed at the anaesthetist or intensive care doctor ‘responding to cardiac arrest calls’. The respondent was given the option to decline participation. All data were anonymised. Results A total of 211 hospitals met the inclusion criteria. The response rate was 100%. Arrest calls were mainly attended by anaesthesia (47.8%) and ICU doctors (38.3%) with around 2% physicians only. Most were a registrar grade (56.3%). The ability to measure ETCO2 was available in all but four EDs; most used waveform capnography. P203 P203 Availability of appropriate airway monitoring at UK in-hospital cardiac arrest S Turle1, PB Sherren1, T Callaghan1, S Nicholson2, SJ Shepherd1 1Pan-Thames Intensive Care Training Scheme, London, UK; 2KSS Intensive Care Training Scheme, London, UK Critical Care 2015, 19(Suppl 1):P203 (doi: 10.1186/cc14283) Using a laryngoscope and endotracheal tube succeeds in a diffi cult case of nasogastric tube insertion J P k Y L Using a laryngoscope and endotracheal tube succeeds in a diffi cult case of nasogastric tube insertion J Park, Y Lee Ewha Womans Unversity Hospital, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P204 (doi: 10.1186/cc14284) , Ewha Womans Unversity Hospital, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P204 (doi: 10.1186/cc14284) Introduction Nasogastric (NG) tube insertion is necessary in a variety of critically ill patients for intra-abdominal decompression, prevention of aspiration, route of medication administration and nutrition. However, it often fails in patients who showed sedated or comatose mentality with poor cooperation during the procedure. Although there are many reports inserting a NG tube in diffi cult cases, most methods need a special guide wire, tube or nasoendoscope. We report a case of NG tube insertion in a comatose patient using a laryngoscope and endotracheal tube which are easily available. Figure 1 (abstract P202). New video laryngoscope connected to laptop. Conclusion This new assembled VAL is the cheapest video-assisted laryngoscope available costing around $60, which can even be introduced into primary healthcare setup in developing countries. VAL consistently yielded a comparable or superior glottic view compared with direct laryngoscopy despite the limited or lack of prior experience with the device. Because the device can be used for both routine as well diffi cult tracheal intubation, it may be a helpful tool to intubate trauma cases where C-spine immobilization is unavoidable. The presented video-assisted laryngoscope is a useful tool for documentation, teaching and monitoring tracheal intubation. Figure 1 (abstract P204). Insertion of a nasogastric tube through an endotracheal tube in the esophagus. References 1. Grmec S. Intensive Care Med. 2002;28:701-4. 2. Deakin CD, et al. Resuscitation. 2010;81:1305-52. 3. Cook TM, et al. Br J Anaesth. 2011;106:617-31. 1. Grmec S. Intensive Care Med. 2002;28:701-4. Admissions with airway emergencies in the ICU at a tertiary referral centre Results Forty-fi ve patients (31 male) were included with a mean age of 67  ± 15  years. The commonest admission diagnoses were sepsis (10), cardiogenic shock (10), primary respiratory failure (nine) and intracranial haemorrhage (eight). The median transfer delay time was 47 minutes. Only 27 (60%) patients were actually transferred and they were signifi cantly younger than nontransferred patients (62 vs. 73 years, P = 0.02). In-transit mortality was zero. Mean length of stay in the critical care centre was 14.8 ± 16.8 days. Survival to discharge was signifi cantly higher in transferred (14/27) compared with nontransferred (3/18) patients (52% vs. 17%, P = 0.017). Overall mortality rates were 62% and 69% at 1 and 6 months respectively and were signifi cantly lower in the transferred group (P = 0.02). M Gresoiu, M Singer M Gresoiu, M Singer University College London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P205 (doi: 10.1186/cc14285) Introduction As a tertiary referral centre for ENT and maxillo-facial surgery, our ICU receives complex elective and emergency cases. The frequency, aetiology and outcomes of airway emergencies are poorly described. Understanding these factors is key to improving management. g Methods We conducted a retrospective review of the ICU electronic patient database examining unplanned admissions with airway emergencies between December 13 and November 14. Data on demographics, aetiology of airway obstruction (including postprocedural), APACHE II score, therapeutic intervention(s) administered, and outcomes were collected. Conclusion Overall mortality rates of medical patients intubated urgently at BGH were high. Forty per cent of intubated patients were not transferred, indicating signifi cant modifi cation of the protocol over time. Patients transferred to the critical care centre were younger and had signifi cantly better outcomes than patients remaining in BGH, probably due to decisions not to transfer patients with poor prognoses. Most patients who survived to discharge were still alive 6 months later. Reference Results Of 1,516 unplanned admissions, airway emergencies represented 6.3% (96 patients) of whom 40 (41.7%) had malignancy (26 maxillo-facial/trachea, three pulmonary, four haematological, seven other) and 24 infection (abscesses, epiglottitis, Ludwig’s angina). Referring specialties were maxillo-facial surgery (n  = 34), internal medicine (n = 25), ENT (n = 21) and other surgical specialties (n = 16). Thirteen patients had complications post bronchoscopy (vocal cord palsy, need for NIV or intubation), one post microlaryngoscopy, and 20 were admitted after diffi cult intubation. P206 Methods A NG tube was inserted using a laryngoscope and endotracheal tube. Outcomes of medical patients requiring emergency intubation in a rural Irish hospital Outcomes of medical patients requiring emergency intubation in a rural Irish hospital Results A 15-year-old male patient admitted due to abrupt mental change and brain imaging showed severe subdural hemorrhage. NG tube insertion was done for enteral feeding but failed several times though changing position. As we had no guide wire and no nasoendoscope, an endotracheal tube was used as guidance for the NG tube. After making a longitudinal midline cut on the endotracheal tube, it was inserted into the esophagus under a laryngoscope. The NG tube was pushed into the endotracheal tube, and then the endotracheal tube was removed through the cut, reserving the NG tube. We checked the position of the NG tube by air sound and X-ray, and started enteral feeding without complication, such as nasal bleeding, emphysema, and gastric perforation. See Figure 1. p A O’ Connor, MP D’Alton, B Carey Bantry General Hospital, Co. Cork, Ireland Critical Care 2015, 19(Suppl 1):P206 (doi: 10.1186/cc14286) Introduction Bantry General Hospital (BGH) is a small rural hospital serving a large, geographically isolated part of southwest Ireland. Following an infl uential national review of adult critical care services [1], a protocol was introduced in late 2010 mandating the immediate transfer of all medical patients intubated on an emergency basis to a large critical care centre 100 km away. Similar mandatory transfer protocols were introduced at the same time throughout the island of Ireland but few data are available regarding patient outcomes. We designed a study to look at the outcomes of all patients encompassed by the protocol at BGH. Conclusion We report a new method of NG tube insertion using a laryngoscope and endotracheal tube. y Methods We retrospectively reviewed the charts and electronic data of medical patients requiring emergency intubation at BGH from November 2010 to December 2013. We recorded the following data: age, sex, admission diagnosis, comorbidities, time delay to transfer, in- transit mortality, length of stay, survival to discharge and 1-month and 6-month mortality.i p Reference 1. Towards Excellence in Critical Care. Review of adult critical care services in the Republic of Ireland fi nal report. Submitted to the Health Service Executive; September 2009. Admissions with airway emergencies in the ICU at a tertiary referral centre Eighteen were admitted post drainage of abscess (dental, retropharyngeal) and seven for observation for epiglottitis. Thirteen patients had stridor (three tracheal stenosis, one vocal cord cyst, one post CVA, four post vocal cord palsy, one post oesophagoscopy, one post thyroidectomy, two post decannulation). Seven were admitted after emergency tracheostomy, one after blocked tracheostomy, one after emergency laryngectomy, six post bleeding (epistaxis, haemoptysis, bleed form laryngectomy site), and four post evacuation haematoma. Three were admitted following anaphylaxis/ angioedema and one after laryngospasm. Twenty-nine patients required medical management only (for example, steroids, nebulisers, and so forth), 25 were extubated post diffi cult intubation and six needed haemostasis control. Ten (nine surgical) tracheostomies were performed during their ICU stay. Sixteen patients died in hospital, of whom fi ve were in the ICU at the time; 14 of these had an underlying malignancy. Twenty-three patients deteriorated during their ICU stay including HAP (n = 3), bleeding from airway (n = 3), PEA arrest (n = 1), airway swelling (n = 2), blocked laryngectomy (n = 1), and tracheostomy dislodgement (n = 1). 03 Availability of appropriate airway monitoring at UK in-hospital cardiac arrest S Turle1, PB Sherren1, T Callaghan1, S Nicholson2, SJ Shepherd1 1Pan-Thames Intensive Care Training Scheme, London, UK; 2KSS Intensive Care Training Scheme, London, UK Critical Care 2015, 19(Suppl 1):P203 (doi: 10.1186/cc14283) Introduction Airway complications are more common outside the operating theatre and in emergency situations. Capnography remains the gold standard of confi rming correct endotracheal tube (ETT) placement, retaining high sensitivity and specifi city in cardiac arrest [1]. The 2010 European Resuscitation Council guidelines for adult advanced life support recommended waveform capnography in this setting [2]. Failure to use capnography was also identifi ed as a major contributor to airway-related morbidity and mortality in a national UK audit [3]. We sought to investigate current practice relating to the availability and use of capnography equipment cardiac arrest within UK hospitals. Methods Between June and November 2014, a telephone survey was conducted of all UK acute hospitals with adult level 3 ICUs and an emergency department (ED). Hospitals were identifi ed using nationally available data. A standardised telephone questionnaire was developed Figure 1 (abstract P204). Insertion of a nasogastric tube through an endotracheal tube in the esophagus. S71 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes Centro Hospitalar Tâmega e Sousa, Penafi el, Portugal Critical Care 2015, 19(Suppl 1):P209 (doi: 10.1186/cc14289) Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes g Centro Hospitalar Tâmega e Sousa, Penafi el, Portugal Critical Care 2015, 19(Suppl 1):P209 (doi: 10.1186/cc14289) g g j Results A total of 348 patients were included. VL attained better glottic visualization than DL (92.3% vs. 82.6%, respectively: P <0.001). In total, 299 patients with good glottic visualization were included in the analysis. Of these patients, 185 (61.9%) were male, median age and body mass index were 69 (interquartile range (IQR), 51 to 77) and 22 (IQR, 20 to 24) respectively. In univariate analysis, VL group had less respiratory failure (18.3% vs. 46.8%: P <0.001) and included more trauma patients (21.1% vs. 7.9%: P <0.001). The fi rst-attempt success rates were similar between two groups (82.6% vs. 77.4%: P = 0.286). Multivariable logistic regression analysis adjusted for potential confounders showed that the success rate of VL was similar to that of DL (odds ratio, 1.17; 95% confi dence interval, 0.57 to 2.39).i Introduction β-Adrenergic agonists increase the ciliary beat frequency in experimental models, raising the possibility that they may be useful for airway hygiene [1]. Salbutamol increases large airway mucociliary clearance [2], although this may not be true for smaller airways [3]. There are no data from ICU patients, so we decided to test the eff ectiveness of transtracheal instillation of a mixture of ipratropium and salbutamol, assessing the reduction of the number of aspirations and hours of ventilation. Methods An open randomized prospective study was held during 2014. All admitted patients were alternately selected as the study or control group and included if submitted to invasive ventilation for at least for 24 hours. Four patients who had secondary cardiac rhythm eff ects attributable to β-adrenergic agonists were excluded. In the study group, 3 ml of a dilution of 2.5 ml ipratropium (0.52 mg) plus salbutamol (2.5 mg) with 2.5 ml distilled water was instilled after the aspiration of secretions. During the ventilation period we noted for both groups, in addition to the demographic data, the number of tracheal aspirations by day and hours of ventilation. The statistical analysis was done with XLSTAT 2014 and we used the Kolmogorov– Smirnov test to compare the normal distribution of the groups. P210 Simultaneous use of a heat and moisture exchanger and a heated humidifi er causes critical airway occlusion in less than 24 hours M Mariyaselvam, A Doyle, G Wijewardena, N English, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P210 (doi: 10.1186/cc14290) p , g y , Critical Care 2015, 19(Suppl 1):P210 (doi: 10.1186/cc14290) q Results There were 62 RSI intubations using THRIVE (ICU and ED = 30; OR = 33). Diffi cult airway equipment used in 36 cases (videolaryngoscopy in 23). Mean apnoea time was 118 seconds (30 to 480 seconds), with a median SpO2 fall of 1% (0 to 33%). There was no correlation between arterial desaturation and apnoeic time. OR cases had a mean apnoea of 113 seconds with a median SpO2 fall of 0% (0 to 13%). ICU and ED cases had a mean apnoea time of 119 seconds and median SpO2 fall of 1% (0 to 33%). THRIVE was universally readily accepted. Reasons cited included: simplifi cation of pre-oxygenation (hands free) and increased confi dence. Six outlying arterial desaturation events suggested poor airway maintenance at induction or use in particularly high-risk patients. Many anaesthetists reinstituted THRIVE following extubation in selected patients (for example, obesity). No complications occurred during implementation. Introduction We hypothesised that the simultaneous use of a heat and moisture exchanger (HME) and a heated humidifi er (HH) would increase the incidence of airway occlusion over a 24-hour period in comparison with each device in isolation. This bench study compares the incidence of airway occlusion when using (group 1) no airway humidifi cation, (group 2) a HME alone, (group 3) a HH alone and (group 4) both a HME and a HH in combination. Tracheal intubation requires the use of artifi cial humidifi cation systems. HMEs are less effi cient but convenient especially for a short period of intubation and HHs are commonly more expensive. Both devices are often used in close proximity on the ICU depending on the particular clinical scenario and/ or clinical practitioner. Following a critical incident of HME obstruction due to waterlogging on our ICU we realised that HH and HME may be used together inadvertently. This airway obstruction was only resolved by the removal of the HME from the patient’s breathing circuit. Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction Transnasal humidifi ed rapid insuffl ation ventilatory exchange for pre-oxygenation and apnoeic oxygenation during rapid sequence induction M Mariyaselvam, D Stolady, G Wijewardena, M Blunt, P Young Queen Elizabeth Hospital, King’s Lynn, UK Critical Care 2015, 19(Suppl 1):P208 (doi: 10.1186/cc14288) Results These preliminary results included 107 patients. The only normal distribution, according to the Kolmogorov–Smirnov test, was for the number of hours of ventilation: 76.3 ± 100 (24; 478) in the study group versus 111 ± 167 (24; 780) in the control group.i Conclusion The preliminary analysis, referring to the fi rst 6 months of study, showed a tendency in the reduction of both ventilation hours and length of stay in the study group. No signifi cant diff erence was found in the number of aspirations, which may be explained by ICU nursing routines. Further studies will try to fi nd whether signifi cant diff erences in the incidence of VAP exist, allowing this procedure to be implemented in ICU routines. Introduction Rapid sequence induction (RSI) in the ICU, emergency department (ED) and operating room (OR) carries the risk of hypoxemia if laryngoscopy is prolonged especially in high-risk patients. Bag and mask pre-oxygenation is normally used to extend the apnoea time; however, arterial desaturation may still rapidly occur. Transnasal humidifi ed rapid insuffl ation ventilatory exchange (THRIVE) is a new technique that provides modest CPAP during pre-oxygenation and crucially also continuous oxygenation of the pharyngeal space throughout the apnoeic period. In elective surgery, THRIVE provides apnoea times as long as 60 minutes due to apnoeic oxygenation [1]. We report the fi rst implementation of THRIVE with emergency patients into the ICU, ED and OR. References 1. Frohock JI, Wijkstrom-Frei C, Salathe M. Eff ects of albuterolenantiomers on ciliary beat frequency in ovine trachealepithelial cells. J Appl Physiol. 2002;92:2396-402. 1. Frohock JI, Wijkstrom-Frei C, Salathe M. Eff ects of albuterolenantiomers on ciliary beat frequency in ovine trachealepithelial cells. J Appl Physiol. 2002;92:2396-402. 2. Lafortuna CL, Fazio F. Acute eff ect of inhaled salbutamol onmucociliary clearance in health and chronic bronchitis. Respiration. 1984;45:111-23. 2. Lafortuna CL, Fazio F. Acute eff ect of inhaled salbutamol onmucociliary clearance in health and chronic bronchitis. Respiration. 1984;45:111-23. 3. Svartengren K, Philipson K, Svartengren M, Camner P. Eff ect ofadrenergic stimulation on clearance from small ciliatedairways in healthy subjects. Exp Lung Res. 1998;24:149-58. Methods Following training a THRIVE system was installed in each location either as a fi xed system on the anaesthetic machine (OR) or a mobile solution on a wheeled stand (ICU, ER). This was a simplifi ed Optifl ow system (Fisher and Paykel, New Zealand) consisting of a high-fl ow rotameter, a reusable humidifi er, a reusable circuit and a disposable nasal interface. Anaesthetists of all grades were encouraged to use THRIVE (60 l/minute) prior to and during all high-risk intubations. Prospective data of pre and post intubation SpO2 and time to intubate were collected. Anaesthetists were interviewed on acceptability of the technique. 1. Patel A, et al. Anaesthesia. 2014 [Epub ahead of print]. Airway hygiene in the ICU: can ipratropium plus salbutamol help? N Magalhaes i Conclusion Despite the possible poor alignment of airway, the fi rst- attempt success rate of VL is similar to that of DL among patients with good glottic visualization. P210 M th d A l i l t d t t ll d til ti Conclusion We conclude that THRIVE provides a convenient, safe and easy to implement technique for pre-oxygenation and apnoeic oxygenation during laryngoscopy. P209 The primary exposure was use of VL. Potential confounders of success rate examined were age, sex, primary indication of intubation, methods of intubation, and operator level of training and specialty. Among patients with good glottic visualization, we conducted a multivariable logistic regression adjusted for potential confounders. Comparison of video laryngoscopy with direct laryngoscopy in patients with good glottic visualization: an observational study of 348 emergency intubations g y Y Kato, H Okamoto, H Uchino, T Fukuoka Kurashiki Central Hospital, Kurashiki Okayama, Japan Critical Care 2015, 19(Suppl 1):P207 (doi: 10.1186/cc14287) g y Y Kato, H Okamoto, H Uchino, T Fukuoka Introduction Video laryngoscopy (VL) is known to improve glottic visualization and the fi rst-attempt success rate compared with direct laryngoscopy (DL) in emergency tracheal intubations (ETIs). Since VL does not align the oral, pharyngeal, and laryngeal axes of the upper airway, it sometimes leads to failed intubation despite good glottic visualization. We tested the hypothesis that VL has a lower fi rst- attempt success rate of ETI than DL among patients with good glottic visualization. Methods We performed a prospective observational study examining ETIs at our tertiary care institution from July 2012 to June 2014. All consecutive patients who underwent ETIs in the emergency department and ICU were included. Patients under 18  years of age, intubated with VL not using C-MAC, were excluded. After each ETI eff ort, the operator completed a standardized data collection form. We classifi ed glottic visualization as good (C-L grade 1 or 2), and poor (C-L grade 3 or 4). The primary outcome was the fi rst-attempt success rate. Conclusion Marked variation was seen in the type and aetiology of airway emergencies admitted to the ICU. A broad training programme is thus required to off er wide-ranging awareness of potential problems, communication (including an emergency airway plan), and acute management. S72 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P211 P211 Endobronchial streptokinase for airway thrombus: a case series D Lloyd, J Bomford, M Barry, W Berry, N Barrett, L Camporota, N Ioannou, B Lams, C Langrish, C Meadows, A Retter, D Wyncoll, G Glover Guys and St Thomas’ NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P211 (doi: 10.1186/cc14291) Introduction Pulmonary haemorrhage (PH) is common in patients receiving mechanical ventilation and especially during ECMO, due to severe lung pathology and systemic anticoagulation. Whilst PH manifests as worsening ventilation and gas exchange, in ECMO patients who already have low tidal volume and who do not rely on pulmonary gas exchange, deterioration may not be evident until extensive airway thrombus (AT) has developed. Management of AT is challenging, with lavage, suctioning, mechanical disruption and extraction of limited effi cacy in severe cases. Limited reports suggest that topical thrombolytics may have a role in the management of AT [1]. We report the safety and effi cacy of endobronchial streptokinase (EBSK) in patients with extensive AT. Results Control group: 100 patients who received standard intubation and treatment in 2009 to 2010. Of these, four dropped out due screening failure of pneumonia on the day of enrollment. Study group: in 2011 to 2014, 192 patients were screened. Of these, 49 were found eligible and were enrolled. Of these, 14 dropped out (four screening failures with pneumonia on day of enrollment and 10 withdrawals with MV of less than 24 hours). Mean age was 51 (control) and 49 (study). Males were 75% of both groups and mean weight was 81 kg in both. VAP was diagnosed in 26 (27%) of controls and only three (8.5%) of the study group (P = 0.03). Mean time from admission to VAP diagnosis was 4.7 days in controls versus 5.12 in the study group (NS). No serious adverse events occurred. Methods A retrospective case series in a UK ECMO centre. Patients who received EBSK between 2010 and 2014 were identifi ed from pharmacy records. Conclusion Patients connected to the AnapnoGuard system demonstrated a statistically signifi cant lower VAP rate compared with the control group (8.5% vs. 27% respectively, P = 0.03). The estimated relative risk of VAP occurring in the control group was more than three times higher than the study group. Rinsing and aspiration of subglottic secretions combined with cuff pressure and seal management may be an eff ective method to prevent VAP. P211 Results Five patients were identifi ed, 80% were male. Median age was 40 years, APACHE II score 36.5 and Murray score 3.75. Four were on ECMO with systemic heparin. All had ARDS secondary to lung infections (community-acquired pneumonia (two), lung abscess (one), TB (one) and PJP (one)). All had extensive AT, diagnosed on bronchoscopy, causing occlusion of the trachea or major bronchi, refractory to physiotherapy, lavage, suctioning ± rigid bronchoscopy. Patients received up to three administrations of EBSK, 1,000 u/ml in saline 0.9% under bronchoscopic guidance. Dose per administration was 30,000 to 80,000 u and total dose was 30,000 to 150,000 u (375 to 1,500  u/kg), with interval bronchoscopy after several hours for lavage and suctioning of lysed clot. In all cases EBSK was well tolerated with no immediate complications and no clinically signifi cant change in systemic laboratory coagulation parameters at 12 or 24  hours compared with pretreatment baseline. In all cases, signifi cant clearance of airway thrombus was achieved. Median tidal volume increased from 60 ml pre treatment to 170 ml at 24 hours. Median PaO2 during the ‘FiO2 1.0 test’ improved from 9.0 to 17.6 kPa at 24 hours. No major bleeding, intracerebral haemorrhage or ECMO cannulae bleeding was seen up to 7 days post treatment.i 3 Risk factors for bleeding complications after percutaneous dilatational tracheostomy: a 10-year institutional analysis K Pilarczyk1, G Marggraf1, M Dudasova1, E Demircioglu1, D Wendt1, B Huschens2, H Jakob1, F Dusse1 1West German Heart Center Essen, University Hospital Essen, Germany; 2University Hospital Essen, Germany Critical Care 2015, 19(Suppl 1):P213 (doi: 10.1186/cc14293) Introduction Percutaneous dilatational tracheotomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. Bleeding complications after PDT are infrequently observed but have a tremendous impact on further clinical course CFA risk stratifi cation for patients scheduled for PDT. Introduction Percutaneous dilatational tracheotomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. Bleeding complications after PDT are infrequently observed but have a tremendous impact on further clinical course CFA risk stratifi cation for patients scheduled for PDT. Conclusion In this series, the largest reported to date, and the fi rst on ECMO, EBSK was highly eff ective in achieving clearance of AT with subsequent improvements in pulmonary mechanics and gas exchange. No major disturbance of systemic coagulation parameters or major haemorrhagic complications occurred. P212 test periods for each group (n = 24). A HME (Filta-Therm; Intersurgical, Berkshire, UK) was placed between the breathing circuit and catheter mount or the HH (MR850; Fisher & Paykel, Auckland, New Zealand) was used, or both in combination. Circuit manipulation was performed 4-hourly to simulate patient movement. Hourly Vt was recorded to determine airway occlusion. Critical airway occlusion (defi ned as a drop on the TV to <50 ml) was assessed using a Fisher’s exact test. 1. Keane et al. Chest. 1999;115:293-300. P211 The use of EBSK may be considered for refractory AT. Methods We retrospectively reviewed the records of all patients who underwent PDT (using the Ciaglia technique with bronchoscopic guidance) on our cardiothoracic ICU between 2003 and 2013. Patients were stratifi ed into two groups: patients suff ering from acute moderate, severe or major bleeding (Group A) and patients who presented none or only mild bleeding (Group B). Reduction of ventilator-associated pneumonia using the AnapnoGuard system Reduction of ventilator-associated pneumonia using the AnapnoGuard system p y Y Bar-Lavie Rambam Medical Center, Haifa, Israel Critical Care 2015, 19(Suppl 1):P212 (doi: 10.1186/cc14292) Introduction Ventilator-associated pneumonia (VAP) is a common complication in mechanically ventilated patients. Frequently the pathogens responsible derive from aspirated secretions of the upper respiratory tract or the stomach. In order to prevent aspiration, two missions should be attained: a good tracheal cuff seal with a well- tolerated pressure, together with continuous evacuation of secretions from the subglottic space. These two goals can be achieved using the AnapnoGuard system and its related endotracheal tube (ETT). Results In all seven of the breathing circuits in group 4 (both a HME and a HH in combination), critical airway occlusion occurred suddenly between 19 and23  hours. No episodes occurred in the other three groups (P <0.0001). g p Conclusion The combination of the use of HME and HH within a single ICU risks inadvertent dual use in a single patient and if uncorrected this is likely to result in a ventilator circuit obstruction. Medical errors can be mitigated by consideration of human factors and system engineering to improve patient safety. A focus on clinical awareness and training may lead to improvements; however, the numbers, experience and turnover of critical care staffi ng would indicate that a systems approach is appropriate and either HME or HH should be used exclusively in an ICU. y Methods A single-center, open-label study in a general ICU. Control group: (retrospective data) mechanically ventilated patients on standard of care regular ETT, manual suction of the trachea and oral– pharyngeal space by nursing staff . Study group: (prospective data) connected at all times to the AnapnoGuard system: an ETT with two above-the-cuff suction ports and a third port and lumen for rinsing and CO2 measurement. A triple lumen harness is connected to a control system designed to measure CO2 levels above the cuff (to identify leaks), infl ate the cuff accordingly, rinse and suction secretions above the cuff . To be included in the study patients had to have no pneumonia on admission and at least 3 days of mechanical ventilation. VAP was diagnosed for a new chest X-ray infi ltrate accompanied by fever, leucocytosis and positive sputum culture. The study was approved by the hospital IRB. Reference Methods A lung simulator underwent pressure-controlled ventilation (Pinsp = 25 cmH2O; PEEP = 5 cmH2O; Vt = 500 ml) for 24 hours for seven S73 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Reference Study groups showed signifi cant diff erences in Simplifi ed Acute Physiology Score (SAPS) on the day of PDT (Group A: 47.0 ± 13.1, Group B: 32.9 ± 11.2, P = 0.042), renal replacement therapy on the day of PDT (Group A: 53 (60.2%), Group B: 439 (48.1%), P  = 0.026), presence of coagulopathy (Group A: 48 (54.5%), Group B: 393 (43.0%), P = 0.043), platelet count (Group A: 91.6 ± 59.2, Group B: 111.5 ± 79.8 × 1,000/μl, P = 0.037), fi brinogen levels (Group A: 373.6 ± 159.1, Group B: 450.6 ± 259.0 mg/dl, P = 0.012), proportion of PDTs performed by residents (Group A: 72 (81.8%), Group B: 632 (69.2%), P = 0.034) and moderately to very diffi cult PDT (Group A: 31 (35.2%), Group B: 141 (15.4%), P = 0.001). Using logistic regression analyses, diffi cult PDT, low-experienced operator, SAPS >40 and low fi brinogen were independent predictors of relevant bleedings after PDT. Methods The study was conducted in a Danish eight-bed, non- university ICU. Since 2007, all patients admitted to the ICU have been registered on an electronic patient record system, in which daily vital values, diagnoses, procedures and healthcare providers’ notes are entered. When searching for ‘percutaneous dilatation tracheostomy’ in the electronic system, we found all patients who had undergone this specifi c procedure. Afterwards we analyzed each of these patients’ hospital records, looking for any periprocedure or postprocedure complications noted within 7 days. In addition we registered patients’ age, sex, BMI, SOFA score, methods used in procedures and experience of operators. p Results A total of 136 patients admitted to the UCI had undergone a PDT between 2007 and 2014. Of these, two were excluded due to the PDT being performed in another hospital before admission to our ICU. All 134 PDTs were performed with the Ciaglia Blue Rhino Method. No PDTs were performed with bronchoscopic guidance. In 12 cases some kind of complication due to the PDT was registered: six cases with need of surgical hemostasis, three cases of bleeding with need of transfusion of blood products, one case of PDT displacement, one case of ventilation-related problems during procedure and, fi nally, one case of tracheal cartilage fracture. There were no incidents of pneumothorax. No PDTs had a lethal outcome due to the procedure itself. The total complication rate was 9.0%. Reference Results A total of 1,001 patients (46% male, mean age 68.1  years) that underwent PDT were analyzed. In the majority of patients, no or S74 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of PDT are described in the literature, each with disadvantages and benefi ts. The aim of this study was to analyze complications due to PDTs performed without the use of bronchoscopic assistance. only mild bleeding during PDT occurred (none: 425 (42.5%), mild: 488 (48.8%)). In 84 patients (8.4%), bleeding was classifi ed as moderate. Three patients suff ered from severe bleeding, only one major bleeding with need for emergency surgery was observed. Study groups showed signifi cant diff erences in Simplifi ed Acute Physiology Score (SAPS) on the day of PDT (Group A: 47.0 ± 13.1, Group B: 32.9 ± 11.2, P = 0.042), renal replacement therapy on the day of PDT (Group A: 53 (60.2%), Group B: 439 (48.1%), P  = 0.026), presence of coagulopathy (Group A: 48 (54.5%), Group B: 393 (43.0%), P = 0.043), platelet count (Group A: 91.6 ± 59.2, Group B: 111.5 ± 79.8 × 1,000/μl, P = 0.037), fi brinogen levels (Group A: 373.6 ± 159.1, Group B: 450.6 ± 259.0 mg/dl, P = 0.012), proportion of PDTs performed by residents (Group A: 72 (81.8%), Group B: 632 (69.2%), P = 0.034) and moderately to very diffi cult PDT (Group A: 31 (35.2%), Group B: 141 (15.4%), P = 0.001). Using logistic regression analyses, diffi cult PDT, low-experienced operator, SAPS >40 and low fi brinogen were independent predictors of relevant bleedings after PDT. only mild bleeding during PDT occurred (none: 425 (42.5%), mild: 488 (48.8%)). In 84 patients (8.4%), bleeding was classifi ed as moderate. Three patients suff ered from severe bleeding, only one major bleeding with need for emergency surgery was observed. Percutaneous dilatational tracheostomy: complications and safety without the use of bronchoscopic guidance Sygehus Lillebælt, Vejle, Denmark Sygehus Lillebælt, Vejle, Denmark yg j Critical Care 2015, 19(Suppl 1):P214 (doi: 10.1186/cc14294) Reference Of the 12 cases, four (33%) complications occurred during the procedure, the rest (66%) occurred after the procedure. The overall periprocedure complication rate was 3%. Conclusion In this study, PDTs without the use of bronchoscopic guidance were performed safely with a low rate of complications. Conclusion Periprocedural bleeding complications during PDT are rare. However, low fi brinogen levels as well as diffi cult PDT, low-experienced operator and SAPS >40 are associated with an increased risk for bleeding complications. Therefore, preprocedural risk evaluation for bleeding complications should include these factors and further studies are necessary to prove whether modifi cation of risk factors – for example, substitution of fi brinogen prior to PDT – is able to reduce incidence of bleeding complications. P215 Introduction Since the introduction and development of percu- taneous dilatational tracheostomy (PDT), this procedure is accepted and incorporated in ICUs worldwide. In spite of obvious benefi ts for the patients, who obtain more comfort and mobility and less use of sedatives, the procedure also implies the risk of several complications, some of which may be lethal. Severe complications include hemorrhage, displacement and pneumothorax. Diff erent methods Impact of antibiotic therapy during a bedside percutaneous tracheotomy procedure in an ICU Impact of antibiotic therapy during a bedside percutaneous tracheotomy procedure in an ICU E Brotfain1, A Borer1, L Koyfman1, A Frenkel1, S Gruenbaum2, A Smolikov1, A Zlotnik1, M Klein1 1Ben Gurion University of The Negev, Soroka Medical Center, Beer Sheva, Israel; 2Yale University School of Medicine, New Haven, CT, USA Critical Care 2015, 19(Suppl 1):P216 (doi: 10.1186/cc14296) Results In total 12,839 records of patients discharged from the ICU were analyzed. Tracheostomy was present in 133 patients. Two groups were defi ned: (1) TCU (n = 56) and (2) GW (n = 77). Patients of the TCU group were older (60.1 ± 13.1 vs. 54.9 ± 15.8 years; P <0.05) with higher APACHE II score (23 (CI: 21.5 to 25.6) vs. 18.5 (CI: 17.1 to 19.9); P <0.001), and had longer stay in the ICU (45.8 (CI: 38.2 to 53.3) vs. 28.4 (CI: 24.2 to 32.6) days; P <0.001) and on the ward (71.1 (CI: 57.4 to 84.8) vs. 46.1 (CI: 33.7 to 58.2) days; P <0.001) than those of the GW group. The GW group had category 1 of Sabadell score more frequently than the TCU group (25.9% vs. 8.9%; P = 0.019). Rates of nosocomial infections were similar in both groups. No signifi cant diff erences on vasoactive use (50% vs. 40.2%), renal failure (23.2% vs. 20.7%), blood transfusions (25% vs. 23.2%), parenteral nutrition (10.7% vs. 12.9%), in-hospital deaths (14.3% vs. 24.6%), decannulation (55% vs. 50%), or discharge to home (53.6% vs. 37.7%) were found between groups 1 and 2, respectively. Introduction Percutaneous bedside tracheostomy (PBT) is a frequently done procedure in the ICU. PBT is a clean-contaminated procedure, and the duration of the procedure is 15 to 20 minutes depending on the physician’s procedural skills. The rate of infectious complications and effi cacy of perioperative therapy in reducing infections after PBT is currently unknown. Currently there have been no defi nitive recommendations for prophylactic antibiotic therapy before PBT in the ICU. Methods All clinical and microbiological data were retrospectively collected and analyzed during the ICU stay before PBT performance and 72 hours after the PBT procedure from 110 patients in our ICU. Controls were defi ned as patients in whom the PBT procedure was performed in the ICU, with antibiotics administered 72 hours prior to and during the procedure (Group 1, n  = 82). Impact of antibiotic therapy during a bedside percutaneous tracheotomy procedure in an ICU Cases were defi ned as patients in whom the PBT procedure was performed in the ICU without antibiotics administered 72 hours prior to and during the procedure (Group 2, n = 28). Secondary bacteremia, line sepsis and VAP during the 72 hours after PBT were considered infectious complications. Two- tailed P <0.05 was considered to be signifi cant.f Conclusion In our setting the TCU helps to care more safely for severe tracheostomized patients after ICU discharge, and furthermore facilitates discharge home. References 1. Martinez GH, et al: Respir Care. 2009;54:1644-52. 1. Martinez GH, et al: Respir Care. 2009;54:1644-52. 2. Fernandez R, et al: Crit Care Med. 2011;39:2240-5. 2. Fernandez R, et al: Crit Care Med. 2011;39:2240-5. i Results No diff erences were found in age, gender, admission diagnoses, length of ICU stay and in-hospital mortality rate between the two study groups. Overall Gram-negative, Gram-positive and fungal fl ora were Reference Methods We retrospectively reviewed medical records of ICU adults patients who had TQ when discharged to a new TCU and to a GW. The study was carried out in two tertiary care university hospitals from January 2007 to November 2014. Study variables were age, sex, APACHE II score, principal diagnosis, associated major procedures, length of stay in ICU and out in hospital, TCU and GW, Sabadell score, in-hospital mortality, types of tracheotomy procedure, decision to decannulate and discharge to home or long-care facilities. 1. Kollig E, et al. Injury. 2000;31:663-8. 1. Kollig E, et al. Injury. 2000;31:663-8. 1. Kollig E, et al. Injury. 2000;31:663-8. Decision-making algorithm for TS in the ICU Second University of Naples, Italy Critical Care 2015, 19(Suppl 1):P215 (doi: 10.1186/cc14295) Introduction Nowadays more percutaneous dilatational tracheostomy (PDT) methods are in use, but there is no ideal risk-free technique. We Figure 1 (abstract P215). Figure 1 (abstract P215). Figure 1 (abstract P215). S75 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 similar in both groups before and after PBT. Patients who received antibiotic therapy had a lower incidence of new ventilator-associated pneumonia (VAP) episodes (15/82 (18.2%) in Group 1 vs. 14/28 (50%) in Group 2, P  <0.001 (0.23, 0.87 to 0.13)) (Table  1). There were no diff erences in the incidence of bacteremia or line sepsis (Table 1). Conclusion Our fi ndings highlight the importance of conducting a prospective randomized control trial to better understand the role of antibiotic prophylaxis in PBT. have outlined a decisional algorithm to choose the most appropriate technique in each case to reduce the incidence of complications. have outlined a decisional algorithm to choose the most appropriate technique in each case to reduce the incidence of complications. Methods A retrospective review was performed using data from the last 14 years. Two hundred patients were selected. Patients were divided into two groups: one including the fi rst 100 PDTs treated without the algorithm (nA-group) and the other including the last 100 patients treated with the algorithm (A-group). Valuation of clinical and anatomical features of the patients, neck ultrasound and fi brobronchoscopy came before the procedure [1]. The algorithm was formulated by our experience with PDT techniques, comparing the specifi c characteristics of each one with the physiopathological characteristics of each patient. Conclusion Our fi ndings highlight the importance of conducting a prospective randomized control trial to better understand the role of antibiotic prophylaxis in PBT. P217 P217 Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards J Rubio1, JA Rubio2, E Palma2, R Sierra1, F Carmona1, F Fuentes2 1Hospital Universitario Puerta del Mar, Cadiz, Spain; 2Hospital Infanta Cristina, Badajoz, Spain Critical Care 2015, 19(Suppl 1):P217 (doi: 10.1186/cc14297) Outcomes of patients with tracheostomy discharged from ICU to Transitional Care Unit and general wards Results We recorded complications (bleeding, tracheoesophageal fi stula, subglottic stenosis, tracheal rings’ fracture, diffi culty of placement, change of procedure) related to PDTs performed with and without applying the algorithm. We considered complications that occurred in our experience and we changed our modality in technique choice (Figure 1). Compared with the complications reported in the nA-group, use of the algorithm as a guide to choose the kind of PDT technique seems to reduce the incidence of complications (37% vs. 19%; P = 0.001 chi-square test). Introduction Patients with a tracheostomy (TQ) tube in place discharged from the ICU to a general ward (GW) are a fragile group and the TQ may be a risk factor for morbidity and mortality [1,2]. For this reason they need closer monitoring and more airway care. The Transitional Care Unit (TCU) assists patients with serious medical conditions and bridges the gap between the ICU and home or long-term care facilities providing the necessary medical, nursing, psychological and rehabilitative care. The purpose of this study was to evaluate the impact of the TCU on outcomes of tracheostomized patients discharged from the ICU. Conclusion In our experience the application of the proposed algorithm may reduce the incidence of complications related to PDT in the ICU. However, a randomized controlled multicenter study would be necessary in order to confi rm the effi ciency and validity of the proposed algorithm. P218 Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT procedure Infection Group 1 Group 2 P value OR Bacteremia 21/82(25.6%) 11/28(39.3%) 0.149 Colonization 67/82(82.7%) 23/28(82.1%) 0.718 Line sepsis 10/82 (12%) 4/28 (14.3%) 0.39 New VAP (%) 15/82(18.2%) 14/28 (50%) 0.001 Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT procedure Table 1 (abstract P216). Clinical data of new infections 72 hours after the PBT d Introduction Incidence of obesity in developed countries is rising. Currently, Europe has a prevalence of 9 to 30% with signifi cant impact on public health systems. Obese patients in the ICU require special management and treatment. Altered anatomy in obese patients complicates procedures such as mechanical ventilation. Obesity aff ects cardiopulmonary physiology and requires elevated ventilation pressures. In our retrospective study, we determined the eff ect of early S76 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion There is a clear reduction in the need for unplanned IPPV in both patient groups. An audit in 2013 showed incomplete protocol adherence in the ITU, therefore benefi ts may be underestimated. Conclusion There is a clear reduction in the need for unplanned IPPV in both patient groups. An audit in 2013 showed incomplete protocol adherence in the ITU, therefore benefi ts may be underestimated. percutaneous dilatational tracheotomy (PDT) and cessation of sedation on respiratory parameters in severely obese patients. percutaneous dilatational tracheotomy (PDT) and cessation of sedation on respiratory parameters in severely obese patients. p y p y p Methods From June 2010 to July 2014, we included all patients with a body weight of >130  kg, respiratory failure and PDT who were admitted to the ICU of the University Hospital of Muenster. We compared respirator parameters and blood gas analysis before and after PDT. Parameters were recorded on days –1, 0, 1, 3, 5, and 10, with day 0 describing the day of PDT. P220 P220 Is the gastric tube a burden for noninvasive ventilation? I Minev, C Stefanov University Hospital ‘St. Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H K d J N i J S id l Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H Kennedy, J Navein, J Seidel Doncaster Royal Infi rmary, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P219 (doi: 10.1186/cc14299) H Kennedy, J Navein, J Seidelifi y Doncaster Royal Infi rmary, Sheffi eld, UK Methods In this study, six of the COPD patients admitted to our ICU, who required NGT placement, were ventilated with the Draeger Evita 2 dura through a modifi ed reusable silicone face mask (UMDNS code: 12-453 with 22 mm ID connection; sizes 4 and 5) with silicone headgear and a hook ring. All of them had a NGT during their stay in the ICU. We evaluated the effi cacy of our modifi cation comparing the achieved Vt with modifi ed and unmodifi ed face mask, during two periods of 10 minutes. The mode and parameters of ventilation were not changed. We assessed patient comfort with a visual analogue scale. Introduction Respiratory failure is a well-known complication of aortic aneurysm surgery. We describe the impact of a protocol, using CPAP after elective surgery to reduce the need for unplanned invasive ventilation. Methods In 2012 we introduced a CPAP protocol for patients undergoing elective aortic aneurysm surgery, either open (AAA) or as an endovascular repair (EVAR). According to pre-existing risk factors (see Table 1) and arterial blood gas analysis in the anaesthetic room, they were assigned to two alternative options on the ITU: prophylactic CPAP for 9 hours in each of the fi rst two postoperative nights or oxygen via face mask. CPAP was applied at any time in the patients stay, if their P/F ratio dropped below 40. Criteria to stop CPAP were also predefi ned. Previously, CPAP was initiated at the discretion of nursing staff , P/F ratios were not utilised. p g Results The average duration of NIV was 3.5  days (SD  = 1.6). We examined two sets of 10 consequent breathing cycles for every patient. The mean Vt was 472 ml (SD = 76 ml) with standard face mask and 460 ml (SD = 86 ml) with the modifi ed one. There was statistically signifi cant correlation between the two datasets (P  <0.05). No additional leaks were detected. According to the VAS evaluation, fi ve of the patients (83%) had comfort improvement with the modifi ed mask. Table 1 (abstract P219). Criteria for the use of prophylactic CPAP FEV1: <1.5 l/minute Poor exercise tolerance (<100 yards) due to chest problems Poor exercise tolerance (<100 yards) due to chest problems SpO2 <92% on air SpO2 <92% on FiO2 >0.4 in theatre SpO2 <92% on FiO2 >0.4 in theatre Early postoperative use of CPAP reduces need for unplanned IPPV in elective vascular patients H K d J N i J S id l Conclusion With this modifi cation of the face mask we achieved adequate drainage of the stomach and/or the enteral nutrition of the patients and improvement in their comfort during NIV, compared with the ventilation with a standard mask, without additional air leaks and at a low cost. Table 1 (abstract P219). Criteria for the use of prophylactic CPAP P218 Rapid amelioration of respiratory parameters in severely obese patients after percutaneous dilatational tracheotomy S Kaese, M Zander, J Waltenberger, P Lebiedz University Hospital of Muenster, Münster, Germany Critical Care 2015, 19(Suppl 1):P218 (doi: 10.1186/cc14298) George’, Plovdiv, Bulgaria Critical Care 2015, 19(Suppl 1):P220 (doi: 10.1186/cc14300) y g y Results Twenty-one patients were included in the study. Mean age was 56 ± 10.3 years and 14 (66.7%) of the patients were male. Body weight was 164.5 ± 39.4 kg, body height accounted for 176.8 ± 8.7 cm (n = 20) and body mass index was 49.7 ± 16.9 kg/m2. Patients stayed in the ICU for 18.4 ± 13.8 days. Mean time of mechanical ventilation by endotracheal tube was 2.4 ± 1.5 days (n = 20) and via tracheostomy 9.8  ± 7.0  days. After PDT, peak inspiratory pressure (P  <0.0001), positive end-expiratory pressure (P <0.0001) and insuffl ated oxygen concentration (P <0.0001) could signifi cantly and rapidly be reduced. Respiratory minute volume increased signifi cantly (P = 0.004). PDT was not associated with relevant complications. Introduction The application of noninvasive ventilation (NIV) in ICUs has spread widely during the years. It is used in the treatment of diff erent forms of acute respiratory failure and COPD exacerbations. Although NIV is thought to be more comfortable for patients than invasive mechanical ventilation, its failure rates in the ICUs range between 10 and 40%. Except for the interface-related problems, there are some specifi c considerations for the patient–ventilator interaction and the applied mechanical forces. During NIV there is a predisposition for the stomach to be infl ated with gas, which could cause severe respiratory complications, especially in COPD patients, and thus prolong the mechanical ventilation and the weaning process. This remains one of the major causes for NIV failure. Although a lot of face masks with diff erent interfaces are available on the market, just a few have additional ports for a NGT. They are characterized by higher price and a complex setup. In order to perform NIV in patients, requiring NGT placement, without additional air leaks and to be able to ensure their enteral nutrition and/or stomach drainage, we installed a port for a NGT on a standard face mask. Conclusion Early PDT rapidly improves respiratory distress in severely obese patients due enabling of spontaneous breathing and reduction of dead space ventilation. Lung ultrasound aeration assessment: comparison of two techniques S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 S Mongodi1, F Mojoli1, A Stella1, I Godi1, G Via1, G Tavazzi1, A Orlando1, B Bouhemad2 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France 1Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 2Centre Hospitalier Universitaire Dijon, France Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) Critical Care 2015, 19(Suppl 1):P222 (doi: 10.1186/cc14302) p p Methods The authors prospectively enrolled 17 patients with septic shock who were admitted to the medical ICU, Phramongkutklao Hospital between September 2013 and June 2014. EVLWI was measured by TPTD (VolumeView Set, EV1000; Edwards Lifesciences) method. According to international evidence-based recommendations for point-of-care lung ultrasound 2012, three methods of LUS (LOGIQ e ultrasound; GE Healthcare) were compared to assess EVLW daily in each patient until no indication for invasive blood pressure monitoring [1]. Firstly, B-lines were measured in 28 lung zones. The total numbers of B-lines seen in each patient were counted as total B-line scores (TBS). Secondly, upper and lower BLUE points were anterior two-region scans each side marked by physician hands. Pulmonary edema was diagnosed if three or more B-lines were presented in all regions. Lastly, scanning eight regions, two anterior and two laterals per side, was considered abnormal if more than one scan per side had three or more B-lines.i Introduction Lung ultrasound (LUS) allows semiquantifi cation of lung aeration in PEEP trials [1], pneumonia [2] and weaning [3]. LUS score is based on number/coalescence of vertical artifacts (B-lines) in longitudinal scan (LONG) [4]: the pleura is identifi ed between two ribs and its visualization limited by intercostal space (ICS) width. We hypothesized that a transversal scan (TRANSV) aligned with ICS would visualize longer pleura and a higher number of artifacts, with better assessment of loss of aeration (LoA). Methods LONG and TRANSV were performed in six areas per lung (anterior, lateral and posterior, each divided into superior and inferior). Once LONG was performed, TRANSV was obtained by a probe rotation until the ribs disappeared. We considered pleural length, B-line number/coalescence, and subpleural/lobar consolidations. P221 P221 Lung ultrasonography as a marker of pulmonary edema in cardiac surgery patients: visual versus quantitative evaluation F Corradi1, C Brusasco2, T Manca3, F Nicolini3, F Nicosia1, T Gherli3, V Brusasco2, A Vezzani3 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria di Parma, Italy Critical Care 2015, 19(Suppl 1):P221 (doi: 10.1186/cc14301) Results We compare patient cohorts in the years 2010 and 2011 (pre protocol) with 2013 and 2014 (post protocol). Results are reported as the split between open surgery and endovascular repair. Table  2 presents requirements for invasive ventilation (IPPV) and length of stay (LOS) for both patient groups. Table 2 (abstract P219). IPPV requirements and length of stay data Table 2 (abstract P219). IPPV requirements and length of stay data 2010 to 2011 2013 to 2014 LOS LOS LOS LOS ITU hospital ITU hospital IPPV (days) (days) IPPV (days) (days) EVAR 2/67 (3%) 2 6 1/77 (1.3%) 2 5 AAA 10/46 (21.7%) 5 9 6/37 (16.2%) 5 12 Introduction Lung ultrasonography (LUS) has been used for non- invasive detection of pulmonary edema. LUS visual scores (V-LUS) based on B-lines are poorly correlated with either pulmonary capillary wedge pressure (PCWP) or extravascular lung water (EVLW). A new quantitative LUS analysis (Q-LUS) has been recently proposed [1,2]. The aim of the study was to investigate whether Q-LUS is better correlated with PCWP and EVLW than V-LUS, and to what extent positive end- expiratory pressure (PEEP) aff ects the assessment of pulmonary edema by Q-LUS and V-LUS. S77 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Thirty-nine patients mechanically ventilated with PEEP of 5 cmH2O (n = 47) or 10 cmH2O (n = 30) and PCWP (n = 77) or EVLW (n = 38) monitored were studied.i Methods Thirty-nine patients mechanically ventilated with PEEP of 5 cmH2O (n = 47) or 10 cmH2O (n = 30) and PCWP (n = 77) or EVLW (n = 38) monitored were studied.i versus 3.9 ± 0.1 cm (range 3.0 to 4.3; variance 0.1) (P <0.0001), B-lines per scan were 1.1 ± 1.6 versus 1.8 ± 2.5 (P <0.0001), coalescent B-lines were detected in 24 versus 30% (P <0.05) and subpleural consolidations in 16 versus 22% (P <0.05), respectively. LUS scores’ prevalence signifi cantly diff ered in LONG versus TRANSV (Figure 1). P221 Results PCWP was signifi cantly and strongly correlated with Q-LUS Grey Unit value (r2 = 0.64) but weakly with V-LUS B-line score (r2 = 0.19). EVLW was signifi cantly and strongly correlated with QLUS Grey Unit mean value (r2 = 0.65) more than with V-LUS B-line score (r2 = 0.42). Q-LUS showed a better diagnostic accuracy than V-LUS for the detection of PCWP >15 mmHg or EVLW >10 ml/kg. With a PEEP of 5 cmH2O, the correlations with PCWP or EVLW were stronger with Q-LUS than V-LUS. With a PEEP of 10 cmH2O, the correlations with PCWP or EVLW were still signifi cant for Q-LUS but insignifi cant for V-LUS. Intraobserver repeatability and interobserver reproducibility were much better for Q-LUS than V-LUS. f Conclusion TRANSV visualizes signifi cantly longer pleura and greater number of artifacts useful for lung disease assessment. References f Conclusion TRANSV visualizes signifi cantly longer pleura and greater number of artifacts useful for lung disease assessment. References 1. Bouhemad B, et al. Am J Resp Crit Care Med. 2011;183:341-7. 2. Bouhemad B, et al. Crit Care Med. 2010;38:84-92. 3. Soummer A. et al. Crit Care Med. 2012;40:264-72. 4. Volpicelli G. et al. Int Care Med. 2012;38:577-91. Ultrasound assessment for extravascular lung water in patients with septic shock Conclusion Both V-LUS and Q-LUS are acceptable indicators of pulmonary edema in patients mechanically ventilated with low PEEP but at high PEEP only Q-LUS provides data that are signifi cantly correlated. Computer-aided Q-LUS has the advantages of being not only independent of operator perception but also of PEEP. References P Pirompanich1, A Wattanathum2 1Thammasat University, Pathumthani, Thailand; 2Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P223 (doi: 10.1186/cc14303) 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. Introduction Extravascular lung water (EVLW) refers to fl uid within the lung but outside the vascular compartment. Increment of EVLW was associated with mortality in critically ill patients. Extravascular lung water index (EVLWI) >10 ml/kg was found in patients with cardiogenic pulmonary edema and correlated with pulmonary capillary wedge pressure >20 mmHg. Measurement of EVLW needs sophisticated tools and an invasive method by transpulmonary thermodilution (TPTD) technique. In contrast, multiple B-lines by lung ultrasound (LUS) have been recently proposed to correlate with increased EVLW in patients with pulmonary edema. This study aims to compare three methods of LUS and EVLWI measured by TPTD to assess pulmonary edema in patients with septic shock. bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. Lung ultrasound aeration assessment: comparison of two techniques LUS score was assigned: 0 normal lung, 1 moderate LoA (≥3 well-spaced B-lines), 2 severe LoA (coalescent B-lines), 3 complete LoA (tissue-like pattern). Results We enrolled 38 patients (21 males, age 60 ± 16 years, BMI 24.7 ± 4.7 kg/m2) corresponding to 456 ICSs. In 63 ICSs, a tissue-like pattern was visualized in both techniques. In the other 393, LONG versus TRANSV pleural length was 2.0 ± 0.6 cm (range 0.8 to 3.8; variance 0.31) Results A total of 40 comparisons were obtained. Signifi cant positive linear correlations were found between TBS and EVLWI determined by TPTD (r  = 0.637, P <0.001). The TBS ≥39 has sensitivity of 91.7% and specifi city of 75.0% to defi ne EVLWI >10  ml/kg. There was low sensitivity (33.3% and 50.0% respectively) but high specifi city (100% and 96.0% respectively) of the positive BLUE points and eight regions to defi ne EVLWI >10 ml/kg. Figure 1 (abstract P222). LUS scores. *P <0.01 TRANSV versus LONG. i Conclusion TBS is the best method for assessing EVLW compared with BLUE points and eight regions. These data support the benefi t of LUS with summation of B-line scores of 28 rib interspaces for assessment of the increment of EVLW in septic shock patients. f Reference Reference 1. Volpicelli G, et al. Intensive Care Med. 2012;38:577-91. 1. Volpicelli G, et al. Intensive Care Med. 2012;38:577-91. Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) Introduction One of the aims of lung recruitment is to improve oxygenation [1], but it has not yet been investigated in spontaneously breathing patients. Our objective was to evaluate the eff ects of recruitment maneuvers on oxygenation in patients ventilated in CPAP/ pressure support (CPAP/PS) mode. Methods In a prospective, observational study, 30 patients with a Lung Injury Score ≥2 were recruited. Following baseline measurements (t0) PEEP was increased by 5 cmH2O (t1). Recruitment maneuver was applied for 40 seconds with 40 cmH2O PS. Measurements were taken immediately after recruitment (t2) then 15 minutes (t3) and 30 minutes later (t4).f 2 3 4 Results According to the diff erence of PaO2/FiO2 between t2 and t0, three groups were defi ned: nonresponders (NR: diff erence of PaO2/FiO2 ≤0%, n = 8), low responders (LR: diff erence of PaO2/FiO2 = 0 to 50%, n = 11) and high responders (HR: diff erence of PaO2/FiO2 >50%, n = 11). In the NR-group, PaO2/FiO2 decreased signifi cantly: median (interquartile), PaO2/FiO2 = 178 (159 to 240) versus 165 (118 to 210) mmHg; in the LR- group and in the HR-group there was signifi cant improvement: 119 (98 to 164) versus 161 (123 to 182) mmHg and 141 (130 to 183) versus 239 (224 to 369) mmHg, P <0.05, respectively. Dynamic compliance (Cdyn) signifi cantly dropped at t2 as compared with t0 in the NR-group, Cdyn = 62 (48 to 87) versus 53 (43 to 78) ml/cmH2O, while there was no signifi cant change in the LR- and HR-groups, P <0.05. At the same time points the dead space to tidal volume ratio (Vds/Vte) signifi cantly increased in the NR-group, Vds/Vte = 30 (23 to 37) versus 37 (26 to 42)%, but not in the LR- and HR-groups, P <0.05. Figure 1 (abstract P224). Sample ultrasound recording. Lines 1 and 2 are diaphragm thickness measurements. Results We were successfully able to record the diaphragm thickness in all included patients. Median time on the ventilator was 9 days (IQR 4 to 15 days). Mean baseline thickness was 1.9 mm (SD ±0.4 mm), and mean nadir was 1.3 mm (SD ±0.4 mm), corresponding with a mean change in thickness of 32% (SD ±18%). As early as after only 72 hours of MV, we already noted an average drop of diaphragm thickness of 20%, illustrating the rapid progression of the atrophy in VIDD. P226f Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) References 1. Grosu H, et al. Chest. 2012;142:1455-60. Conclusion Recruitment maneuvers improved PaO2/FiO2 in the majority of patients (73%) without aff ecting Cdyn or Vds/Vte; therefore it may be a safe approach to improve oxygenation in patients ventilated in CPAP/PS mode. 1. Grosu H, et al. Chest. 2012;142:1455-60. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. 2. McCool F, et al. Am J Resp Crit Care Med. 1997;155:1323-28. Eff ect of lung recruitment on oxygenation in patients with acute lung injury ventilated in CPAP/pressure support mode A Lovas, D Trasy, M Nemeth, I Laszlo, Z Molnar University of Szeged, Hungary Critical Care 2015, 19(Suppl 1):P226 (doi: 10.1186/cc14306) Conclusion On average, diaphragm thickness decreased 32% in our cohort. The decrease occurred rapidly, with two-thirds of the maximal thinning already present after 72 hours of MV. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Thirty-two spontaneously breathing patients with suspected community-acquired pneumonia undergoing computed tomography examination were consecutively enrolled. Each hemithorax was evaluated for the presence or absence of abnormalities by chest X-ray and quantitative or visual ultrasonography. Methods Thirty-two spontaneously breathing patients with suspected community-acquired pneumonia undergoing computed tomography examination were consecutively enrolled. Each hemithorax was evaluated for the presence or absence of abnormalities by chest X-ray and quantitative or visual ultrasonography. diaphragmatic atrophy can be assessed using ultrasound, the biggest trial in humans published so far included seven patients and only measuring the thickness at two moments during the disease process [1]. We aimed to assess the time course of diaphragm atrophy in a larger cohort of MV patients using ultrasound. y Results Quantitative ultrasonography showed higher sensitivity (93%), specifi city (95%), and diagnostic accuracy (94%) than chest X-ray (64%, 80%, and 69%, respectively), or visual ultrasonography (68%, 95%, and 77%, respectively), or their combination (77%, 75%, and 77%, respectively). Methods A total of 54 patients from an adult ICU were included in this prospective single-centre cohort trial. Patients who needed <72 hours of MV or had been recently admitted to an ICU were excluded. Patients were ventilated in a controlled, assisted, and/or hybrid ventilation mode. The thickness of the diaphragm was assessed daily; the fi rst recording was within 24 hours after the start of mechanical ventilation and we continued the measurements until the patients were extubated or tracheotomised. We measured the diaphragm at the zone of apposition, as described by McCool and colleagues [2] using a linear 13 MHz ultrasound probe. Figure 1 shows a sample measurement. y Conclusion Quantitative lung ultrasonography was considerably more accurate than either chest X-ray or visual ultrasonography in the diagnosis of community-acquired pneumonia and it may represent a useful fi rst-line approach for confi rmation of clinical diagnosis in emergency settings. Quantitative ultrasonography for pneumonia F C di1 C B 2 T M 3 F Ni i 1 A V 1. Lachman B. Intensive Care Med. 1992;18:319-21. g p y p F Corradi1, C Brusasco2, T Manca3, F Nicosia1, A Vezzani3, V Brusasco2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria, Parma, Italy Critical Care 2015, 19(Suppl 1):P225 (doi: 10.1186/cc14305) y F Corradi1, C Brusasco2, T Manca3, F Nicosia1, A Vezzani3, V Brusasco2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Università degli Studi di Genova, Italy; 3Azienda Ospedaliero-Universitaria, Parma, Italy Critical Care 2015, 19(Suppl 1):P225 (doi: 10.1186/cc14305) References Figure 1 (abstract P224). Sample ultrasound recording. Lines 1 and 2 are diaphragm thickness measurements. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 2. Quanta Imaging Technology. www.quanta.camelotbio.com. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. 1. Corradi F, Ball L, Brusasco C, Riccio AM, Baroffi o M, Bovio G, et al. Assessment of extravascular lung water by quantitative ultrasound and CT in isolated bovine lung. Respir Physiol Neurobiol. 2013;187:244-9. g p y 2. Quanta Imaging Technology. www.quanta.camelotbio.com. A better way to determine sample size to detect changes in length of mechanical ventilation? A better way to determine sample size to detect changes in length of mechanical ventilation? YS Chiew1, C Pretty1, D Redmond1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P227 (doi: 10.1186/cc14307) Atrophy of diaphragm muscle visualized with ultrasound in mechanically ventilated patients T Schepens, M Mergeay, W Verbrugghe, P Parizel, M Vercauteren, PG Jorens Antwerp University Hospital, Edegem, Belgium p y p , g , g Critical Care 2015, 19(Suppl 1):P224 (doi: 10.1186/cc14304) Introduction Mechanical ventilation (MV) induces diaphragmatic muscle atrophy and contractile fi bre dysfunction, the so-called ventilator-induced diaphragm dysfunction (VIDD). Although Figure 1 (abstract P222). LUS scores. *P <0.01 TRANSV versus LONG. S78 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 A better way to determine s of mechanical ventilation? Introduction Chest-X-ray is recommended for routine use in patients with suspected pneumonia, but its use in emergency settings is limited. In this study, the diagnostic performance of a new method for quantitative analysis of lung ultrasonography was compared with bedside chest X-ray and visual lung ultrasonography for detection of community-acquired pneumonia, using thoracic computed tomography as a gold standard. YS Chiew1, C Pretty1, D Redmond1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P227 (doi: 10.1186/cc14307) Introduction Estimation of eff ective sample size (N/arm) is important to ensure power to detect signifi cant treatment eff ects. However, S79 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 monitoring of the eff ect of benzodiazepines on respiratory status in non-intubated patients has been diffi cult, putting patient safety at risk. A non-invasive respiratory volume monitor (RVM) that provides continuous measurement of minute ventilation (MV), tidal volume (TV) and respiratory rate (RR) was used to quantify the eff ects of midazolam on respiratory status in spontaneously breathing patients. traditional parametric sample size estimations depend upon restrictive assumptions that often do not hold in real data. This study estimates N to detect changes in length of mechanical ventilation (LoMV) using Monte-Carlo simulation (MCS) and mechanical ventilation (MV) data to better simulate the cohort. Methods Data from 2,534 MV patients admitted to Christchurch Hospital ICU from 2011 to 2013 were used. N was estimated using MCS to determine a sample size with power of 80%, and compared with the Altman’s nomogram for two patients groups, (1)  all patients and (2)  targeted patients with 1 <LoMV ≤15 days. MCS allows any range of intervention eff ect to be simulated, where this study tested a 10 and 25% diff erence in LoMV (0.5 to 1.25 days for mean LoMV of 5 days). The simulated LoMV for the intervention group is compared with the LoMV in a control group using the one-sided Wilcoxon rank-sum test, Student t test, and Kolmogorov– Smirnov test to assess central tendency and variation. Methods An impedance-based RVM (ExSpiron; Respiratory Motion Inc., Waltham, MA, USA) was used in 30 patients who received 2 mg midazolam prior to induction of anesthesia and were sedated but spontaneously breathing. A better way to determine s of mechanical ventilation? Further studies are ongoing to quantify hypoventilation after administration of other anesthetic medications. Conclusion Continuous monitoring with RVM provides a valuable depiction of hypoventilation from benzodiazepines, not demonstrated by other methodologies such as pulse oximetry and RR alone. RVM monitoring can help uncover potentially life-threatening hypoventilation in older patients. Further studies are ongoing to quantify hypoventilation after administration of other anesthetic medications. Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. A better way to determine s of mechanical ventilation? Eleven of these patients (58  ± 19  years, average BMI 27.7) received midazolam at least 20  minutes prior to induction. Digital RVM data were collected and MV, TV and RR calculated and evaluated from 30-second segments 10 minutes before and after the fi rst dose of midazolam. Ten patients were analyzed as a group and one patient was analyzed separately (due to idiosyncratic reaction). y Results The distribution of LoMV is heavily skewed. Altman’s nomogram assumes a normal distribution and found N >1,000 to detect a 25% LoMV change. Figure 1 panels (1) and (2) show N for 80% power if all patients were included, and panels (3) and (4) for the targeted patient group. Panels (1) and (3) show that it is impossible to achieve 80% power for a 10% intervention eff ect. For 25% eff ect, MSC found N = 400/arm (all patients) and N = 150/arm (targeted cohort). Results Following administration of midazolam, the group MV and TV decreased an average of 19 ± 7% and 16 ± 5%, respectively (mean ± SEM, P <0.01, both) while RR remained essentially unchanged (decrease of 3 ± 8%, P >0.3). In the younger half of the cohort (45 ± 16 years), the decreases in MV and TV were not signifi cant, only 6 ± 3% and 8 ± 5%, respectively. The older half of the cohort (72 ± 8 years) displayed fourfold greater MV and TV decreases (32 ± 11%, P <0.05 and 25 ± 6%, P <0.05), when compared with the younger cohort, P <0.01, Figure 1). Figure 1 (abstract P227). Conclusion Traditional parametric sample size estimation may overestimate the required patients. MCS can estimate eff ective N/arm and evaluate specifi c patient groups objectively, capturing local clinical practice and its impact on LoMV. It is important to consider targeting specifi c patient groups by applying patient selection criteria that can be easily translated into trial design. P228 Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1 D Ladd2 Figure 1 (abstract P227). Figure 1 (abstract P228). Figure 1 (abstract P228). Figure 1 (abstract P227). Conclusion Continuous monitoring with RVM provides a valuable depiction of hypoventilation from benzodiazepines, not demonstrated by other methodologies such as pulse oximetry and RR alone. RVM monitoring can help uncover potentially life-threatening hypoventilation in older patients. Nebulized heparin for patients under mechanical ventilation: a conventional data meta-analysis y GJ Glas1, A Serpa Neto2, J Horn1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Israelita Albert Einstein, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P229 (doi: 10.1186/cc14309) P229 P229 Nebulized heparin for patients under mechanical ventilation: a conventional data meta-analysis GJ Glas1, A Serpa Neto2, J Horn1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Israelita Albert Einstein, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P229 (doi: 10.1186/cc14309) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 1Vidant Medical Center, Greenville, NC, USA; 2Respiratory Motion, Inc., Waltham, MA, USA Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 1Vidant Medical Center, Greenville, NC, USA; 2Respiratory Motion, Inc., Waltham, MA, USA Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 Introduction Mechanical ventilation has the potential to induce pulmonary coagulopathy. Local treatment by nebulization of heparin could be benefi cial in ventilated patients. The aim of this data meta- analysis is to determine the association between nebulization of heparin and outcome of mechanically ventilated critically ill patients. Methods PubMed, Scopus, EMBASE, and Web of Science were searched for relevant articles. Articles were selected if they compared Critical Care 2015, 19(Suppl 1):P228 (doi: 10.1186/cc14308) Introduction Benzodiazepines are used in many of settings to induce sedation, but can cause a reduction in respiratory drive. Objective Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S80 Figure 1 (abstract P229). Eff ect of heparin nebulization on mortality. Figure 1 (abstract P229). Eff ect of heparin nebulization on mortality. nebulization of heparin with standard care. The primary endpoint was overall mortality. Secondary endpoints included occurrence of pneumonia and number of ventilator-free days and alive at day 28.i pulse oximetry with derived heart rate; snoring and nasal airfl ow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic diffi culties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI). pulse oximetry with derived heart rate; snoring and nasal airfl ow with nasal pressure transducer and nasal thermistor; rib cage, abdominal motion and body position with abdominal and thoracic belts. American Academy of Sleep Medicine 2014 recommendations were used for the diagnosis of OSA and OHS. Because of the diagnostic diffi culties of hypopnea in hypoxemic patients, we evaluated only the obstructive apnea index (OAI) instead of the apnea hypopnea index (AHI). Results Thirty-one patients with the mean age of 67 ± 9 years were included in the study. Their mean APACHE II score was 16 ± 5 and BMI was 33 ± 9 kg/m2. Admission arterial blood gases were as follows (mean ± SD); pH: 7.33 ± 0.07, PaO2: 74 ± 12 mmHg, PaCO2: 69 ± 11 mmHg, HCO3 –: 31 ± 5, O2Sat%: 92 ± 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 ± 6 in patients with OAI >5. Diagnosis of obstructive sleep apnea with respiratory polygraph in hypercapnic ICU patients P231 G Gursel, A Zerman, M Aydogdu, B Basarik Aydogan, K Gonderen, S Memmedova, N Sevimli, I Koroglu, Z Isikdogan, M Badoglu, O Ozdedeoglu Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P230 (doi: 10.1186/cc14310) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 Diagnosis of obstructive sleep apnea with respiratory polygraph in hypercapnic ICU patients G Gursel, A Zerman, M Aydogdu, B Basarik Aydogan, K Gonderen, S Memmedova, N Sevimli, I Koroglu, Z Isikdogan, M Badoglu, O Ozdedeoglu Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P230 (doi: 10.1186/cc14310) Non-invasive respiratory volume monitoring for quantifi cation of respiratory depression after benzodiazepine administration G Mullen1, D Ladd2 Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identifi ed to have OSA. Conclusion Although it underestimates AHI, RPLG is important and technically feasible in ICU patients in suggesting the presence of OSA and in providing information for appropriate NIV management. Results Six articles were found: fi ve retrospective cohorts with historical controls, one randomized controlled trial, covering 423 patients. Dosages of nebulized heparin varied from 30,000 to 150,000 IU/day. Fifty out of 222 patients (22.5%) receiving nebulized heparin and 48 out of 201 patients (23.9%) receiving standard care died (risk ratio (RR) 0.79 (95% CI 0.47 to 1.35)) (see Figure 1). Occurrence of pneumonia (RR 1.36 (95% CI 0.54 to 3.45); I2 = 59%), and number of ventilator-free days and alive at day 28 (standardized mean diff erence 0.11 (95% CI –0.14 to 35); I2 = 0%), were not diff erent between the two groups. Results Thirty-one patients with the mean age of 67 ± 9 years were included in the study. Their mean APACHE II score was 16 ± 5 and BMI was 33 ± 9 kg/m2. Admission arterial blood gases were as follows (mean ± SD); pH: 7.33 ± 0.07, PaO2: 74 ± 12 mmHg, PaCO2: 69 ± 11 mmHg, HCO3 –: 31 ± 5, O2Sat%: 92 ± 4. Admission diagnoses of the patients were OHS (36%) and COPD (68%). Mean OAI was 13 ± 6 in patients with OAI >5. Eighty-one percent (n = 25) of the recordings were interpretable and clinical and RPLG data supported a new diagnosis of OSA in 14 (56%) patients, and EPAP levels were increased. Laboratory sleep study was recommended to 19% of the patients. At the end of the study 56% of the COPD and 72% of the OHS patients were identifi ed to have OSA. Conclusion Nebulization of heparin is not associated with improved outcome in mechanically ventilated critically ill patients. This meta- analysis is limited by methodological problems in most included studies. Only one randomized controlled trial could be included. Also, most patients in the meta-analyzed studies suff ered from inhalation trauma, and heparin dosages diff ered widely. Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU G Besch1, J Revelly2, P Jolliet2, L Piquilloud-Imboden2 1CHRU Besançon, France; 2CHUV, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P232 (doi: 10.1186/cc14312) G Besch1, J Revelly2, P Jolliet2, L Piquilloud-Imboden2 1CHRU Besançon, France; 2CHUV, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P232 (doi: 10.1186/cc14312) y 2 2 Results The study group was comprised of four men and three women. Median age was 69 (IQR: 61.0 to 72.5) years old. The reason for admission was ARDS (n  = 5) and acute exacerbation of idiopathic pulmonary fi brosis (n = 2). The reasons for ARDS are bacterial pneumonia (n = 3), necrotizing fasciitis (n = 1) and extensive burn (n = 1). We identifi ed NETs in the bronchial aspirates of all the patients. NET formation had persisted in four cases during the study period, their P/F ratio did not improve and all patients were dead due to respiratory failure. On the other hand, NETs decreased and vanished in three cases, their P/F ratio improved and all patients recovered from ARDS. Introduction As the objective clinical criteria [1] are imperfect to assess patients before extubation, simple physiological parameters are used to try to improve extubation failure (EF) prediction. The rapid shallow breathing index (RSBI) (respiratory rate (RR) over tidal volume (VT) ratio) recorded during a T-piece spontaneous breathing trial (SBT) is known as the most reliable physiologic predictor. However, RSBI is nowadays usually computed during a pressure support (PS) SBT using the values displayed on the ventilator screen and not based on spirometry measurements without any assist as initially published. The aim of the present study was to prospectively assess the ability of currently measured RSBI to predict EF. Conclusion NET formation was observed in bronchial aspirates of all the patients diagnosed with ARDS or acute exacerbation of idiopathic pulmonary fi brosis. It may be one of the prognostic factors of ARDS or acute exacerbation of idiopathic pulmonary fi brosis. Methods Retrospective analysis of prospectively collected data from patients intubated for more than 48 hours admitted in the medico- surgical ICU of Lausanne, Switzerland, from January 2007 to December 2008. P233 A variety of IMT techniques were employed including inspiratory threshold loading (eight studies), biofeedback to increase inspiratory eff ort (one study), chair-sitting (one study) and diaphragmatic breathing pattern training (one study). Threshold loading was achieved by application of an external device (six studies) or increases in the inspiratory pressure trigger setting (two studies). Most studies implemented IMT in the weaning phase (n = 5) or after diffi cult weaning (n = 5); one study implemented IMT within 24 hours of intubation. IMT was associated with greater increases in maximal inspiratory pressure compared with control (six studies, mean diff erence 7.6 cmH2O (95% CI 5.8, 9.3), I2 = 0%). There were no signifi cant diff erences in the duration of MV (six studies, mean diff erence –1.1 days (95% CI –2.5, 0.3), I2 = 71%) or the rate of successful weaning (Figure 1; fi ve studies, risk ratio 1.13 (95% CI 0.92, 1.40), I2 = 58%). The GRADE quality of evidence was low for all these outcomes; risk of bias was high for most studies and summary eff ects were imprecise and inconsistent. No serious adverse events related to IMT were reported. Presence of neutrophil extracellular traps in bronchial aspirate of patients diagnosed with acute respiratory distress syndrome or acute exacerbation of idiopathic pulmonary fi brosis M Ojima1, N Yamamoto1, T Hirose1, S Hamaguchi1, N Matsumoto1, R Takegawa1, M Seki1, O Tasaki2, K Tomono1, T Shimazu1 1Osaka University Graduate School of Medicine, Suita, Japan; 2Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Critical Care 2015, 19(Suppl 1):P233 (doi: 10.1186/cc14313) Introduction Neutrophil extracellular traps (NETs) are fi brous structures that are produced extracellularly from activated neutrophil. They can trap and kill various pathogens, and their release is one of the fi rst lines of immune system. Meanwhile, recently it was reported that NETs also exert adverse eff ects in infl ammatory diseases. Acute respiratory distress syndrome (ARDS) and acute exacerbation of idiopathic pulmonary fi brosis is acute-onset respiratory failure. It is characterized by excessive neutrophil infi ltration into the alveolus, and great amounts of neutrophil elastase are released. The purpose of this study is to evaluate whether NETs are produced in bronchial aspirate of patients with ARDS or acute exacerbation of idiopathic pulmonary fi brosis, and to identify correlations with the respiratory function. p Conclusion IMT in mechanically ventilated patients appears safe and well tolerated and improves respiratory muscle function. University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) Introduction Acute lung injury (ALI) and its hypoxemic form, acute respiratory distress syndrome (ARDS), has no approved pharma co- logical treatment and is a condition with high mortality. Vascular leakage is one of the early events of ALI/ARDS. CD73 activity (ecto- 5’-nucleotidase) maintains the endothelial barrier function of lung capillaries via its enzymatic endproduct adenosine. Interferon-beta (IFNβ) increases synthesis of CD73 and has been eff ective in a mouse model of ALI [1]. y Results A total of 478 extubated patients were included, 25 of whom (5.2%) were reintubated. ICU mortality (ICU-m) and in-hospital mortality (H-m) were higher in reintubated patients: ICU-m = 6 (24) versus 22 (5), P = 0.002 and H-m = 9 (36) versus 63 (15), P = 0.009. The reasons for EF were: acute lung failure (n = 15), congestive heart failure (n = 4) and aspiration/bronchial congestion (n = 6). Demographic data were similar between patients successfully and nonsuccessfully extubated: age: 58 ± 17 versus 58 ± 19 years, P = 0.85; male gender: 15 (60) versus 263 (61), P = 0.99. SAPS II score was higher in the EF group: 30 ± 22 versus 42 ± 27, P = 0.04. RSBI were signifi cantly higher in patients who experienced EF: RSBI=59 ± 44 versus 43 ± 26, P = 0.04. The area under the ROC curve for currently measured RSBI was: 0.617 (95% CI = 0.571 to 0.662), P = 0.035. Methods Therefore we conducted a phase I/II trial [2], in which intravenously administered human recombinant IFNβ1a (FP-1201) was used in the study, which consisted of dose escalation (phase I) and expansion (phase II) parts to test the FP-1201 safety, tolerability and effi cacy in ALI/ARDS patients. CD73, MxA (a marker for IFN β response) and other biomarkers were measured to follow pharmacokinetics/ dynamics of the intravenously administered FP-1201 and therapeutic effi cacy. Conclusion In a cohort of 458 medico-surgical ICU patients, RSBI measured during a pressure support SBT was higher in patients experiencing EF but very imperfect to predict EF. Reference fi y Results The optimal tolerated dose of FP-1201 (10 μg/day for 6 days) resulted in maximal MxA stimulation. Also soluble CD73 values increased, while IL-6 decreased in sera of the FP-1201-treated ALI/ARDS patients. Prospective assessment of the ability of rapid shallow breathing index computed during a pressure support spontaneous breathing trial to predict extubation failure in ICU EF was defi ned as the need for reintubation within 48 hours after extubation. Reintubations for a procedure requiring general anesthesia were not considered as EFs. RR and VT during the PS SBT were recorded from the ventilator and RSBI was computed accordingly. Baseline characteristics and currently measured RSBI were compared between patients who experienced EF versus success (t test or chi-square test as appropriate). The ability of currently measured RSBI to predict EF was assessed using ROC curve analysis. P234 1. MacIntyre NR, et al. Chest. 2001;120:375S. P233 IMT was not associated with accelerated liberation from mechanical ventilation. However, because the included studies had important methodological limitations and employed varying methods of IMT, we cannot draw fi rm conclusions about the eff ect of IMT on clinical outcomes. i y y Methods This was a prospective observational study of seven patients admitted to the ICU of a large urban tertiary referral hospital. All patients were mechanically ventilated at the time of admission. The bronchial aspirates were collected serially from the patients by suction through a tracheal tube. To identify NETs, extracellular components including DNA, histone H3, and citrullinated histone H3 were simultaneously detected using immunohistochemistry. The respiratory function was evaluated by PaO2/FiO2 ratio (P/F ratio). Respiratory muscle training during mechanical ventilation: a systematic reviewfi a systematic review D Brace, M Parrotto, C Urrea, A Goffi , A Murray, E Fan, L Brochard, N Ferguson, E Goligher UHN, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P231 (doi: 10.1186/cc14311) Introduction The most frequent reasons for hypercapnic respiratory failure (HRF) in ICUs are COPD and in recent years obesity hypoventilation syndrome (OHS) and obstructive sleep apnea (OSA). Even 15 to 30% of COPD patients also have accompanying OSA. Due to increased upper airway resistance, those patients require higher expiratory pressures (EPAP) during noninvasive ventilation (NIV). In order to prescribe optimal mode and pressures during the ICU stay and at discharge, the intensivist should diagnose the underlying OSA. Portable recording devices have been developed and they were approved at least for the diagnosis in high pretest probability patients with results equal to in-laboratory polysomnography. The aim of this study is to assess whether respiratory polygraph (RPLG) can be used for obtaining diagnostic information of OSA in hypercapnic ICU patients. Methods Patients, with HRF requiring NIV, were included in the study. RPLG studies were conducted under nasal oxygen before NIV, using the Philips Respironics Alice PDx® device, which provides the records of Introduction Respiratory muscle weakness is common in mechanically ventilated patients and impairs liberation from ventilation. Inspiratory muscle training (IMT) might accelerate liberation from mechanical ventilation. We undertook to summarize previously published IMT protocols and the impact of IMT on respiratory muscle function and clinical outcomes. Methods We searched multiple databases using a sensitive search strategy combining MeSH headings and keywords for studies of IMT during MV. Studies were adjudicated for inclusion and data were abstracted independently and in duplicate. Methodological quality was assessed using the GRADE system. Results Eleven studies met the inclusion criteria; of these, six were randomized controlled trials and fi ve were observational studies. Figure 1 (abstract P231). Eff ect of IMT on the rate of successful weaning from mechanical ventilation. S81 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 New molecules controlling endothelial barrier S Jalkanen New molecules controlling endothelial barrier S Jalkanen New molecules controlling endothelial barrier S Jalkanen University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314 References 1. Crit Care Med. 2006;34:532-7. 2. J Crit Care. 2012;27:522.e11-7. 3. Crit Care. 2013;17:R132. Methods Forty-one CD-1 mice were divided into two groups: an HCl group underwent orotracheal instillation of hydrochloric acid on day 0, and a group control. Mice were evaluated on days 0, 1, 2 and 4 after a 30-minute period of mechanical ventilation. Blood and lung edema fl uid (EF) were sampled. Before initiation of MV, all mice received a tracheal instillation of bovine serum albumin (5%) to detect changes in alveolar protein levels over 30 minutes. Plasma levels of sRAGE and total protein levels were measured. AFC rate values were corrected after measurement of mouse serum albumin in EF. P235 P235 Endocan can be a predictive marker of severity of sepsis but cannot be a marker of acute respiratory distress syndrome in ICU patients M Mizunuma, H Ishikura, Y Nakamura, K Muranishi, S Morimoto, H Kaneyama, Y Izutani, T Nishida, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P235 (doi: 10.1186/cc14315) f References 1. Kiss et al. Eur J Immunol. 2007;37:3334-8. 2. Bellingan G, et al. Lancet Respir Med. 2014; 2:98-107. 1. Kiss et al. Eur J Immunol. 2007;37:3334-8. 2. Bellingan G, et al. Lancet Respir Med. 2014; 2:98-107. 1. Kiss et al. Eur J Immunol. 2007;37:3334-8. 2. Bellingan G, et al. Lancet Respir Med. 2014; 2:98-107. P237 Elevated levels of soluble RAGE predict impaired alveolar fl uid clearance in a translational mouse model of acute respiratory distress syndrome R Blondonnet1, M Jabaudon1, G Clairefond2, J Audard1, D Bouvier2, G Marceau1, P Blanc2, P Dechelotte1, V Sapin2, JM Constantin1 1CHU Clermont-Ferrand, France; 2Laroratoire R2D2-EA7281, Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P237 (doi: 10.1186/cc14317) Results We enrolled 70 ARDS patients during the investigation periods. We met six patients with nonsepsis, 27 with severe sepsis and 37 with septic shock. The serum Endocan levels were signifi cantly higher in patients with septic shock (3.7 to 3.9 ng/ml) than in patients with severe sepsis (1.7 to 2.3 ng/ml, P <0.05), nonsepsis (0.6 to 0.3 ng/ml, P <0.05) and healthy donors (0.4 to 0.1 ng/ml, P <0.05). However, there was no signifi cant correlation between the Endocan level and the severity of ARDS. In addition, signifi cant correlation between the Endocan level and EVLWi and PVPI was not observed. Introduction Receptor for advanced glycation endproducts (RAGE) is a transmembrane receptor expressed in the lung and primarily located on alveolar type I cells. RAGE is implicated in acute respiratory distress syndrome to alveolar infl ammation and, when its soluble form sRAGE is assayed in plasma or pulmonary edema fl uid, as a marker of AT I cell injury. Functional activity of AT 1 cells can be assessed by the measurement of the alveolar fl uid clearance (AFC) rate [1], but the relationship between sRAGE plasma levels of sRAGE and AFC rates has never been investigated. Our objectives were to report plasma levels of sRAGE in a mouse model of direct acid-induced epithelial injury, and to test their correlation with AFC rates. Conclusion These results suggested that there was good relationship between the Endocan level and the severity of sepsis. But unlike the trauma patients, correlation between the severity of ARDS and Endocan value was not recognized. P236 Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Q Chen, A Liu, H Qiu, Y Yang Southeast University, Nanjing, China Critical Care 2015, 19(Suppl 1):P236 (doi: 10.1186/cc14316) Endocan can be a predictive marker of severity of sepsis but cannot be a marker of acute respiratory distress syndrome in ICU patients M Mizunuma, H Ishikura, Y Nakamura, K Muranishi, S Morimoto, H Kaneyama, Y Izutani, T Nishida, A Murai Fukuoka University Hospital, Fukuoka, Japan Critical Care 2015, 19(Suppl 1):P235 (doi: 10.1186/cc14315) l Results The permeability signifi cantly increased after LPS stimulation in a dose-dependent and time-dependent manner (P <0.01). Mean- while, MSC-EC interaction more signifi cantly decreased endothelial permeability induced by LPS (P  <0.05 or P  <0.01). Moreover, HGF levels in the MSC-EC interaction group were much higher than those of the MSC group (P  <0.01). However, neutralizing HGF with anti- HGF antibody inhibited the role of MSC-EC interaction in improving endothelial permeability (P  <0.05). Compared with the MSC group, MSC-EC interaction increased VE-cadherin protein expression (P <0.01), and restored remodeling of F-actin and junctional localization of VE- cadherin. However, the MSC eff ect was signifi cantly blocked by anti- HGF antibody (P <0.05 or P <0.01). Introduction Endocan (endothelial cell specifi c molecule-1), a 50 kDa dermatan sulfate proteoglycan, is expressed by endothelial cells in the lung and kidney. It was reported that the serum Endocan level is related to the severity of sepsis and positive correlation with the mortality rate. On the other hand, it was also reported that lower levels of serum Endocan are associated with subsequent development of chronic kidney disease, and chronic liver disease acute lung injury (ALI) in trauma patients. The aim of this study is confi rmation of the relationship between serum Endocan level and the severity of sepsis, and also the severity of acute respiratory distress syndrome (ARDS) in septic patients. y Conclusion These data suggest that MSC-EC interaction decreased endothelial permeability induced by LPS, which was mainly attributed to HGF secreted by hMSCs. The main mechanisms of HGF restoring the integrity of EC monolayers are remodeling of endothelial intercellular AJs and decreasing caveolin-1 protein expression. p p Methods This study was conducted as a single-center, retrospective, observational study in the emergency department of Fukuoka University Hospital from April 2010 to August 2013. Blood samples were collected within 2 hours when the patients were diagnosed with ARDS. In this time we adopted the Berlin defi nition as the categorized of ARDS severity. Furthermore, we evaluated the extravascular lung water index (EVLWi) and pulmonary vascular permeability index (PVPI) as a condition of ARDS using the transpulmonary thermodilution method. Additionally, 10 healthy donors were entered as a control. The patients were divided into nonsepsis, severe sepsis and septic shock using the ACCP/SCCM guidelines. University of Turku, Finland Critical Care 2015, 19(Suppl 1):P234 (doi: 10.1186/cc14314) The overall mortality of the 37 patients treated with FP-1201 S82 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 permeability in lung injury via paracrine hepatocyte growth factor (HGF). Recently, it has been indicated that MSCs secreted more factors by MSC–endothelial cell (MSC-EC) interaction. We hypothesized that MSC-EC interaction restored endothelial permeability induced by lipopolysaccharide (LPS) via paracrine HGF. was only 8.1%, fourfold to fi vefold less than the expected rate based on APACHE II score values of 21.9. The control group (n = 59), which was eligible for the trial but not possible to recruit, had mortality of 32.2% (P = 0.01) and APACHE II score 23.0. Conclusion Restriction of vascular leakage with FP-1201 seems to signifi cantly benefi t ALI/ARDS patients. Our results suggest that FP- 1201 could be the fi rst eff ective, mechanistically targeted, disease- specifi c pharmacotherapy for patients with ARDS. However, these fi ndings warrant conduction of a large multicenter study to establish safe and eff ective FP-1201 treatment of ARDS. p p y p Methods We investigated the endothelial permeability induced by LPS under two co-culture conditions in transwells. HPMECs were added into the upper chambers of cell-culture inserts, while there two diff erent co-culture conditions in the lower side of transwells as follows: MSC-EC interaction group: MSCs and HPMEC contact co- culture in the lower chambers; and MSC groups: MSCs only in the lower chambers. The endothelial permeability in the upper side of transwells was detected. Then the concentration of HGF was measured in the culture medium using an enzyme-linked immunosorbent assay kit, following by neutralizing HGF with anti-HGF antibody in the co-culture medium. In addition, VE-cadherin protein expression were measured under the co-culture conditions by western blot, adherens junctions (AJs) protein including F-actin and VE-cadherin were detected by immunofl uorescence technique as well.i P240 y Results Of those 4,652 patients, there were 2,947 nonsmokers, 1,148 exsmokers, and 557 active smokers. There was no diff erence of APACHE II score between three groups of patients. The active smokers exhibited the highest incidence of ARDS (active smokers 5.4%, exsmokers 4.8%, and nonsmokers 3%, P  = 0.003). There was no diff erence of 28-day mortality between the three groups of patients. Active smokers had the highest incidence of SIRS (active smokers 41%, exsmokers 37%, and nonsmokers 34%, P  = 0.006). Compared with nonsmokers and exsmokers, active smokers had a longer SICU LOS (P <0.01) and higher total SICU cost (P = 0.02). Patients who smoked more than 15 pack- years were 2.5 times more likely to develop ARDS than patients who smoked ≤15 pack-years (95% CI: 1.65 to 3.66, P <0.001). In multivariate analysis we found that every 1 pack-year of cigarette smoking before References 1. Hoshino Y, et al. Am J Physiol Lung Cell Mol Physiol. 2001;281:L509-16. 2. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 (Suppl 1):S45-54. P238 Smoking increased risk of ARDS in surgical critically ill patients: results from the multicenter THAI-SICU study P Wacharasint1, P Fuengfoo1, N Sukcharoen1, R Rangsin2, THAI-SICU Trial Group3 1Phramongkutklao Hospital, Bangkok, Thailand; 2Phramongkutklao College of Medicine, Bangkok, Thailand; 3THAI-SICU Collaborating Centre, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P238 (doi: 10.1186/cc14318) 2 Results A total of 101 ARDS patients was enrolled; 44 (43.6%) patients were normal weight, 36 (35.6%) overweight and 21 (20.8%) obese. Lung and chest wall elastance were not diff erent between groups, both at PEEP levels of 5  cmH2O and 15  cmH2O (P  = 0.580 and P  = 0.113, respectively), and the end-inspiratory transpulmonary pressure was also similar. We did not observe any diff erence between groups regarding PaO2/FiO2 ratio (P = 0.178 at PEEP 5 cmH2O; P = 0.073 at PEEP 15 cmH2O) and PaCO2 (P = 0.491 at PEEP 5 cmH2O; P = 0.237 at PEEP 15 cmH2O). Introduction Cigarette smoking slowly and progressively damages the respiratory system [1]. In surgical critically ill patients, whether active cigarette smoking until admission to the surgical intensive care unit (SICU) is associated with increased risk of acute respiratory distress syndrome (ARDS) is not clearly identifi ed. 2 Conclusion In ARDS obese patients the chest wall elastance and the end-inspiratory transpulmonary pressure are not aff ected by the body weight, suggesting that normal weight and obese patients present similar risks for lung stress and VILI onset. The severity of hypoxemia was not diff erent between groups. i Methods We conducted a cohort study using the THAI-Surgical Intensive Care Unit (THAI-SICU) study databases [2], which recruited 4,652 Thai patients admitted to the SICUs from nine university-based hospitals in Thailand (April 2011 to November 2012). The enrolled patients were divided into three groups (active smokers, exsmokers, and nonsmokers). Primary outcome was the incidence of patients diagnosed with ARDS and the secondary outcomes included 28-day mortality, incidence of systemic infl ammatory response syndrome (SIRS), SICU length of stay (LOS), and total SICU cost. f References 1. Pelosi et al. Chest. 1996;109:144-51. 1. Pelosi et al. Chest. 1996;109:144-51. . Suratt et al. J Appl Physiol Respir Environ Exerc Physiol. 1984;57:403 2. Suratt et al. J Appl Physiol Respir Environ Exerc Physiol. 1984;57:403-7. P239l P239 Infl uence of body weight on lung mechanics and respiratory function in ARDS patients I Algieri1, D Chiumello2, C Mietto1, E Carlesso1, A Colombo1, G Babini1, M Cressoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS, ‘Ospedale Maggiore Policlinico Mangiagalli Regina Elena’ di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P239 (doi: 10.1186/cc14319) Figure 1 (abstract P237). Relation between AFC rates and sRAGE plasma levels. (P = 0.03) and day 2 (P = 0.02). The rate of AFC was inversely correlated with sRAGE levels in the plasma (Spearman’s ρ = –0.73, P <0.001). See Figure 1. Introduction Traditionally, ARDS obese patients are ventilated with higher tidal volumes and higher PEEP due to expected increased in pleural pressure. However, data from the literature regarding the infl uence of body mass on lung mechanics and, particularly, on chest wall elastance are not univocal [1,2]. Failure to account for how the increase in body weight could aff ect the respiratory function can result in injurious mechanical ventilation and in the onset of VILI. The aim of this study was to evaluate the role of the body weight on respiratory mechanics in ARDS patients. g Conclusion The highest impairment in AFC is reported on day 1. sRAGE levels are also higher in injured mice and may be a good surrogate marker of AT I cell injury. This is a newly described relationship between AFC rates and sRAGE plasma level in a mouse model of direct epithelial injury. Our results support further translational investigation on the role of RAGE in alveolar injury and recovery. 1. Eur Respir J. 2012;39:1162-70. Methods A group of deeply sedated and paralyzed ARDS patients was divided into three classes according to the body mass index: normal weight (between 18.5 and 24.9 kg/m2), overweight (between 25.0 and 29.9 kg/m2) and obese (between 30.0 and 39.9 kg/m2). They were mechanically ventilated in volume-controlled mode with a tidal volume of 6 to 8 ml/kg according to predicted body weight. Respiratory mechanics and gas exchange were assessed at PEEP levels of 5 and 15 cmH2O. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Mesenchymal stem cell and endothelial cell interaction restores endothelial permeability via paracrine hepatocyte growth factor in vitro Q Chen, A Liu, H Qiu, Y Yang Results Basal AFC rate was 35% over 30 minutes in HCl-injured mice, but it was signifi cantly depressed on day 1 (16% over 30 minutes; P = 0.02). Over time, AFC reached basal levels again. Plasma levels of sRAGE were higher in HCl-treated animals than in control animals on day 1 Introduction Mesenchymal stem cells (MSCs) have potent stabilizing eff ects on the vascular endothelium injury, inhibiting endothelial Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S83 Figure 1 (abstract P237). Relation between AFC rates and sRAGE plasma levels. admission to the SICU is associated with increased risk of new ARDS with a hazard ratio of 1.02 (95% CI: 1.01 to 1.02, P  = 0.001) after adjustment for APACHE II score, age, gender, and chronic obstructive pulmonary disease. Conclusion In surgical critically ill patients, active smokers are associated with increased risk of new ARDS, longer SICU LOS, and higher total ICU cost, compared with exsmokers and nonsmokers. Our fi ndings emphasize the essential need for a smoking cessation program. f Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Introduction Acute respiratory distress syndrome (ARDS) develops on the basis of nosocomial pneumonia (NP) in 30 to 35% of cases. A complex clinical approach is required for diagnosing it. The aim of this study was to investigate into the role of the PaO2/FiO2 ratio (P/F), extravascular lung water index (EVLWI) and surfactant protein D (SPD) as a complex diagnostic tool for ARDS in NP. S84 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The observational study in ICU ventilated septic patients with peritonitis (70%), pancreonecrosis (25%) and mediastinitis (5%) was done in 2010 and 2014. ARDS was diagnosed and staged according to the V.A. Negovsky Research Institute criteria and the Berlin defi nition. Plasma SPD was measured on ARDS diagnosis (day 0) and days 3 and 5 by the immunoenzyme essay (BioVendor, USA). Patients were treated according to the international guidelines. Data were statistically analyzed by STATISTICA 7.0, ANOVA and presented as median and 25 to 75th percentiles (ng/ml); P  <0.05 was considered statistically signifi cant. Areas under the receiver operating curves were calculated. Results Sixty-fi ve patients (out of 450 screened) were enrolled in the study according to the inclusion/exclusion criteria. Patients were assigned into groups: NP + ARDS (n = 43, 43 ± 4.9 years old, M/F 39/4, mortality 23%) and NP (n = 22, 40 ± 5.1 years old, M/F 20/2, mortality 18%). Groups were comparable in APACHE II and SOFA scores on the baseline. In the NP + ARDS group SPD was higher at all points than in the NP group. Plasma SPD on day 0 >111.2 ng/ml yielded a sensitivity of 68.2% and specifi city of 92.3% (AUC 0.85; 95% CI 0.684 to 0.945; P <0.0001) for diagnosing ARDS in NP. P241f Eff ects of a recruitment maneuver on plasma soluble rage in patients with diff use ARDS: a prospective randomized crossover study M Jabaudon, N Hamroun, L Roszyk, R Blondonnet, R Guerin, JE Bazin, V Sapin, B Pereira, JM Constantin CHU Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P241 (doi: 10.1186/cc14321) y M Jabaudon, N Hamroun, L Roszyk, R Blondonnet, R Guerin, JE Bazin, V Sapin, B Pereira, JM Constantin CHU Clermont-Ferrand, France Critical Care 2015, 19(Suppl 1):P241 (doi: 10.1186/cc14321) Introduction The soluble form of the receptor for advanced glycation endproducts (sRAGE) is a promising marker for epithelial dysfunction, but it has not been fully characterized as a biomarker during ARDS. Whether sRAGE could inform on the response to ventilator settings has been poorly investigated, and whether recruitment maneuver (RM) may infl uence plasma sRAGE remains unknown. p p gl Results Forty-fi ve oesophageal balloon pressure–volume curves were obtained in 31 patients undergoing controlled mechanical ventilation (PEEP 12 ± 5 cmH2O, FiO2 0.7 ± 0.2, tidal volume/ideal body weight 8.0 ± 1.6 ml/kg). According to the graphically detected minimum slope section of the curve, the minimum and maximum appropriate balloon volumes were 1.5 ± 0.6 ml and 5.3 ± 0.9 ml, respectively. Between these two volumes, the slope of the curve was 1.1 ± 0.5 cmH2O/ml, ranging from 0.3 to 3.1 cmH2O/ml. l Methods Twenty-four patients with moderate/severe, nonfocal ARDS were enrolled in this prospective monocentric crossover study and randomized into a ‘RM-SHAM’ group when a 6-hour-long RM sequence preceded a 6-hour-long sham evaluation period, or a ‘SHAM-RM’ Figure 1 (abstract P241). Evolution of plasma levels of sRAGE (pg/ml) in both randomization sequences. 2 Conclusion The oesophageal artefact  – that is, the reaction of the oesophageal wall to balloon infl ation – may be clinically signifi cant, being on average 1  cmH2O for each millilitre of volume injected in the catheter, but reaching values as high as 3 cmH2O/ml. The pressure generated by the oesophageal reaction leads to overestimation of pleural pressure. Therefore, the oesophageal artefact may signifi cantly aff ect clinical decision-making based on absolute values of oesophageal pressure. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) P/F ratio on day 0 <280 yielded a sensitivity of 94.1% and specifi city of 76.9% (AUC 0.89; 95% CI 0.744 to 0.952; P <0.0001) and EVLWI on day 0 >8.3 ml/kg yielded a sensitivity of 94.1% and specifi city of 92.3% (AUC 0.92; 95% CI 0.810 to 0.982; P <0.0001) for the diagnosis of ARDS in NP. A complex ROC analysis (for SPD in the group of patients with P/F <280 and EVLWI >8.3) yielded a much better diagnostic accuracy of SPD: cutoff >93.7 ng/ml, sensitivity 81.0%, specifi city 100.0% (AUC 0.96; 95% CI 0.817 to 0.998; P <0.0001). Conclusion A complex approach (P/F <280, EVLWI >8.3, SPD >93.7) presents as a sensitive and highly specifi c method for diagnosing ARDS in NP patients. group (inverted sequences). Protective ventilation was applied, and RM consisted of the application of 40  cmH2O airway pressure for 40 seconds. Arterial blood was sampled for gas analyses and sRAGE measurements, 5  minutes pre RM (or 40-second-long sham period), 5 minutes, 30 minutes, 1 hour, 4 hours and 6 hours after the RM (or 40-second-long sham period). g p ) Results Mean PaO2/FiO2, tidal volume, PEEP and plateau pressure were 125  mmHg, 6.8  ml/kg (ideal body weight), 13 and 26  cmH2O, respectively. Median baseline plasma sRAGE levels were 3,232 pg/ml. RM induced a signifi cant decrease in sRAGE (–1,598 ± 859 pg/ml) in 1 hour (P = 0.043). At 4 and 6 hours post RM, sRAGE levels increased back toward baseline values. Pre-RM sRAGE was associated with RM- induced oxygenation improvement (AUC 0.87). See Figure 1. Conclusion We report the fi rst kinetics study of plasma sRAGE after RM in ARDS. Our fi ndings could help to design future studies of sRAGE as a marker of response to therapeutic interventions during ARDS. Results Mean PaO2/FiO2, tidal volume, PEEP and plateau pressure were 125  mmHg, 6.8  ml/kg (ideal body weight), 13 and 26  cmH2O, respectively. Median baseline plasma sRAGE levels were 3,232 pg/ml. RM induced a signifi cant decrease in sRAGE (–1,598 ± 859 pg/ml) in 1 hour (P = 0.043). At 4 and 6 hours post RM, sRAGE levels increased back toward baseline values. Pre-RM sRAGE was associated with RM- induced oxygenation improvement (AUC 0.87). See Figure 1. Conclusion We report the fi rst kinetics study of plasma sRAGE after RM in ARDS. P241f i Methods An oesophageal balloon catheter (NutriVent; Sidam, Mirandola, Italy) was introduced in mid/distal thoracic position in 31 patients under invasive mechanical ventilation for acute respiratory failure. At ambient pressure, the balloon of the NutriVent catheter can be infl ated up to 6 ml without generation of recoil pressure. The balloon was progressively infl ated in 0.5  ml steps up to 9  ml and end-expiratory values of balloon pressure were used to assemble the balloon pressure–volume curve. The minimum slope section of the curve was graphically detected and infl ation volumes corresponding to this part of the curve were considered appropriate. Overdistension of the balloon being excluded by defi nition in this section of the curve, its slope was attributed to the oesophageal reaction to balloon infl ation.i Methods An oesophageal balloon catheter (NutriVent; Sidam, Mirandola, Italy) was introduced in mid/distal thoracic position in 31 patients under invasive mechanical ventilation for acute respiratory failure. At ambient pressure, the balloon of the NutriVent catheter can be infl ated up to 6 ml without generation of recoil pressure. The balloon was progressively infl ated in 0.5  ml steps up to 9  ml and end-expiratory values of balloon pressure were used to assemble the balloon pressure–volume curve. The minimum slope section of the curve was graphically detected and infl ation volumes corresponding to this part of the curve were considered appropriate. Overdistension of the balloon being excluded by defi nition in this section of the curve, its slope was attributed to the oesophageal reaction to balloon infl ation. Results Forty-fi ve oesophageal balloon pressure–volume curves were obtained in 31 patients undergoing controlled mechanical ventilation (PEEP 12 ± 5 cmH2O, FiO2 0.7 ± 0.2, tidal volume/ideal body weight 8.0 ± 1.6 ml/kg). According to the graphically detected minimum slope section of the curve, the minimum and maximum appropriate balloon volumes were 1.5 ± 0.6 ml and 5.3 ± 0.9 ml, respectively. Between these two volumes, the slope of the curve was 1.1 ± 0.5 cmH2O/ml, ranging from 0.3 to 3.1 cmH2O/ml. Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia A Kuzovlev, V Moroz, A Goloubev, S Polovnikov, V Stec V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2015, 19(Suppl 1):P240 (doi: 10.1186/cc14320) Our fi ndings could help to design future studies of sRAGE as a marker of response to therapeutic interventions during ARDS. P242 Oesophageal artefact may signifi cantly aff ect oesophageal pressure measurement in mechanically ventilated patients F Mojoli, F Torriglia, M Pozzi, S Bianzina, G Tavazzi, A Orlando, A Braschi Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy Critical Care 2015, 19(Suppl 1):P242 (doi: 10.1186/cc14322) Introduction Oesophageal pressure is increasingly used to monitor and manage mechanically ventilated patients. Even if the oesophageal balloon catheter is correctly positioned, the measurement can be aff ected by inappropriate balloon fi lling and/or oesophageal reaction to balloon infl ation. We aimed to assess the oesophageal reaction to oesophageal balloon fi lling in mechanically ventilated patients. P244 P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation Regional distribution of excess tissue mass in ARDS lung I Algieri1, D Massari1, A Colombo1, G Babini1, F Crimella1, M Brioni1, A Cammaroto1, K Nikolla1, C Montaruli1, M Guanziroli1, M Gotti1, C Chiurazzi1, M Amini1, M Chiodi1, M Cressoni1, D Chiumello2, L Gattinoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS, ‘Ospedale Maggiore Policlinico Mangiagalli Regina Elena’ di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P244 (doi: 10.1186/cc14324) y p g y T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) Introduction ARDS has a wide variability of lung morphological charac- teristics. Both alveolar collapse and airway narrowing or closing are present, often heterogeneously. Despite advances in ARDS imaging, we have thus far been unable to distinguish regional airway opening from airway dilatation in PEEP-induced lung recruitment. We demonstrate the technique of functional respiratory imaging (FRI) to diff erentiate these two entities. Introduction ARDS is characterized by edema diff use to all lung fi elds. Distribution of excess tissue mass had been studied with CT scan in a few patients on a single slice, comparing with data obtained in healthy controls. Methods ARDS patients underwent CT scan imaging during their ICU stay at 45  cmH2O end-inspiratory pressure. After hospital discharge, patients underwent a follow-up CT scan performed at end inspiration. Each lung was divided into three sections along the apex–base axis and into three sections along the sternum–vertebral axis (nine regions per lung). Excess tissue mass in each lung region was defi ned as the diff erence in lung tissue (grams) between the CT scan performed during ARDS course and the follow-up CT scan. Results are presented as mean ± SD. Methods Six patients with early-stage ARDS were included in this prospective single-centre cohort trial. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P245 Functional respiratory imaging of airways in ventilated ARDS patients: revealing the regional relation between PEEP-induced airway opening and airway dilatation T Schepens1, C Vanholsbeke2, W Vos2, J De Backer2, P Parizel1, PG Jorens1 1Antwerp University Hospital, Edegem, Belgium; 2FLUIDDA, Kontich, Belgium Critical Care 2015, 19(Suppl 1):P245 (doi: 10.1186/cc14325) Figure 1 (abstract P244). Figure 1 (abstract P244). demonstrated recently its eff ectiveness on prognosis. Extrapulmonary etiologies of ARDS include abdominal emergencies. In cases of severe hypoxemia in the early postoperative period, intensivists discuss prone positioning based on the risk/benefi t ratio. demonstrated recently its eff ectiveness on prognosis. Extrapulmonary etiologies of ARDS include abdominal emergencies. In cases of severe hypoxemia in the early postoperative period, intensivists discuss prone positioning based on the risk/benefi t ratio. p gi Methods We conducted a retrospective two-center study of 5 years. The aim was to compare the prevalence of surgical complication potentially related to prone positioning between patients who had at least one prone positioning session and patients that remained in a supine position after abdominal surgery. Patients with ARDS in a context of recent (<7  days) abdominal surgery (except laparoscopy) were included. The primary outcome was the number of patients who had at least one surgical complication potentially related to prone positioning. We defi ned a priori these complications: scar dehiscence, abdominal compartment syndrome, stoma leakage, stoma necrosis, scar necrosis, wound infection, displacing of a drainage system, removal of gastro- or jejunostomy feeding, digestive fi stula, evisceration.i g j j y g gi Results We identifi ed 43 patients with postoperative ARDS (62  ± 8 years, SAPS II 50 ± 13), among whom 34 (79%) had emergent surgery. Fifteen patients had at least one stoma after surgery. Nineteen patients (44%) had at least one prone positioning session (number of sessions: 2 (1 to 3)). At baseline, prone group patients had minimum PaO2/FiO2 ratio lower than the supine group (77 ± 23 vs. 110 ± 46 mmHg, P = 0.005). Plateau pressure was higher in the prone group (28 ± 4 vs. 23 ± 5 cmH2O, P = 0.002). The fi rst prone positioning session signifi cantly increased the PaO2/FiO2 ratio: 106  ± 52 vs. 192  ± 90  mmHg (P  = 0.001). Mean duration of the fi rst prone positioning session was 20 ± 10 hours. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 In the prone group, 11 patients (58%) had at least one surgical complication, in comparison with nine (38%) in the supine group (P = 0.2). These complications resulted in revision surgery for two (10%) patients in the prone group and two (8%) in the supine group (P = 0.8). Mortality in the ICU was respectively 42% and 38% in prone group and supine group (P = 0.8).if Conclusion These preliminary results confi rm the eff ectiveness of prone positioning in terms of oxygenation in ARDS after abdominal surgery without signifi cant increase in surgical complications and no eff ect on the need for surgical revisions. Hence, if necessary, clinicians should not refrain from proning patients with postabdominal surgery ARDS. P243 P243 Prone positioning in acute respiratory distress syndrome after abdominal surgery S Gaudry1, S Tuff et1, AC Anne-Claire2, N Zucman1, S Msika1, M Pocard2, D Payen2, D Dreyfuss1, JD Ricard1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Lariboisière, Paris, France Critical Care 2015, 19(Suppl 1):P243 (doi: 10.1186/cc14323) Introduction Prone positioning has been used for many years as an alveolar recruitment strategy in acute respiratory distress syndrome (ARDS). Prone positioning in ARDS improves oxygenation and Introduction Prone positioning has been used for many years as an alveolar recruitment strategy in acute respiratory distress syndrome (ARDS). Prone positioning in ARDS improves oxygenation and Figure 1 (abstract P241). Evolution of plasma levels of sRAGE (pg/ml) in both randomization sequences. S85 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Effi cacy and safety of open lung ventilation in patients with impaired peripheral chemorefl ex sensitivity N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Kuban State Medical University, Krasnodar, Russia Critical Care 2015, 19(Suppl 1):P246 (doi: 10.1186/cc14326) g Results Piglets ventilated with higher energy loads (RR 15 and 12) developed ventilator-induced lung injury (VILI), while piglets ventilated with lower energy loads (RR 9, 6 and 3) did not. The energy dissipated in the lung parenchyma at each breath was 0.56  J (0.52 to 0.62). Dissipated energy increased with time in piglets that developed VILI, while it remained near-constant in all the other piglets. Recruitability was 6% (3 to 8) of lung parenchyma, strain was 3.1 (2.6 to 3.5) and lung inhomogeneity extent (that is, the percentage of lung parenchyma that is inhomogeneous) was 10% (9 to 11). The energy dissipated in the lung parenchyma was well related to lung recruitability (r2 = 0.50, P <0.0001), strain (r2 = 0.57, P <0.0001) and lung inhomogeneity extent (r2 = 0.42, P <0.0001). Multiple linear regression showed that dissipated energy was independently related to all of the three determinants of energy dissipation: lung opening and closing (P = 0.025), strain (P <0.0001) and lung inhomogeneities (P <0.01). Introduction Mechanical ventilation during anesthesia leads to the development of atelectasis, poor oxygenation and postoperative pulmonary complications. Application of PEEP and recruitment maneuver (RM) can signifi cantly reduce the severity of atelectasis and improve lung function. But the application of this strategy often leads to hemodynamic instability, which may be associated with impaired reactivity of the cardiovascular system. The purpose of this study was to evaluate the effi cacy and safety of RM in patients with increased sensitivity of peripheral chemoreceptors (SPCR), which refl ects the decreasing reactivity of the cardiovascular system. g y y Methods We conducted a prospective study in 116 patients with high SPCR, evaluated using the breath-holding test. The test was performed by measuring of voluntary breath-holding duration (BHD) after two- thirds of maximal inspiration. The end of breath-hold was determined by a palpation of contraction of the diaphragm. BHD <38 seconds was the marker of high SPCR [1]. All patients received a major abdominal surgery and were randomized into an open lung ventilation group or a PEEP group. P244 The lower infl iction point on a pressure–volume curve was considered as the clinically acceptable minimal PEEP value. Subsequently, four distinct PEEP levels were chosen to perform CT scans: at 20 cmH2O; median value between 1 and 3; clinically acceptable minimal; and 0 cmH2O. FRI methods as described by De Backer and colleagues [1] were used to evaluate airway opening and airway dilatation. Results We studied eight ARDS patients (55  ± 18  years) with a BMI of 27 ± 6 kg/m2. At ICU admission, patients had the following clinical parameters: PaO2/FiO2 106 ± 33 with PEEP 15 ± 5 cmH2O; PaCO2 43 ± 10 mmHg; pH 7.35 ± 0.05. The average increase in lung weight during ARDS compared with follow-up CT scan was 68 ± 40% (680 ± 320 g). Figure 1 presents the tissue volume during ARDS (white bars) and after ARDS resolution (black bars) and compares the ratio between the two (*P <0.01 vs. dependent region). Results Airway stretching (that is, bronchodilatation) could be quantifi ed and distinguished from airway recruitment with this technique. Higher PEEP pressures not only recruit, but also expand the bronchi. The ratio of dilation/recruitment of bronchi was higher in the upper lobes than in the lower lobes, as illustrated in Figure 1. We were able to phenotype each patient, allowing a prediction on when an increase in PEEP further recruits atelectasis/bronchi or distends certain airway regions. Conclusion The excess tissue mass was not diff erent between apex, hilum and base, but was increased in the dependent lung regions at apex and hilum, being uniformly distributed at the lung base. y g Conclusion The novel technique of FRI can be used to visualise the airway structures in ARDS and distinguish airway stretching from Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S86 more pronounced. So CI on average decreased by 34% (from 3.7 to 2.5 l/minute/m2) compared with 10% with no RM (P <0.05), and SVR decreased by 19% (from 1,310 to 1,150 dyn × sec–1 × cm–5, P <0.05), while in the PEEP group it did not change. No signifi cant diff erences between groups in the incidence of complications, length of stay in the ICU and in the hospital were noted. Figure 1 (abstract P245). Bronchodilation and bronchial recruitment split per lobe for one patient. Effi cacy and safety of open lung ventilation in patients with impaired peripheral chemorefl ex sensitivity The concept of open lung ventilation was performed as follows: PEEP was increased from 4 to 10 cmH2O for three breaths, from 10 to 15 cmH2O for three breaths, and from 15 to 20 cmH2O for 10 breaths [2]. Then PEEP was reduced to 12 cmH2O. This RM was repeated every hour. In the PEEP group PEEP was maintained at 12 cmH2O during the whole anesthesia. Hemodynamics, blood gases and dynamic compliance were evaluated. Conclusion Energy dissipation was largely dependent on strain, obtained with very high tidal volumes, but also lung inhomogeneities and lung opening and closing played a signifi cant role. Reference 1. Cressoni M. et al. Am J Respir Crit Care Med. 2014;189:149-58. P244 Conclusion RM patients with high SPCR and with reduced reactivity of the cardiovascular system improve lung function, but this is associated with the risk of hemodynamic instability. References 1. Zabolotskikh I, et al. Eur J Anesth. 2014;31:62. 2. Weingarten TN, et al. Br J Anesth. 2010;104:16-22. Determinants of energy dissipation in the respiratory system during mechanical ventilation Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P247 (doi: 10.1186/cc14327) Introduction Mechanical ventilation performs at each breath mechanical work on the lung parenchyma. Part of this energy is recovered and part is dissipated into the respiratory system. The purpose of this study is to assess the respective role of three known determinants of energy dissipation: lung opening and closing, strain and lung inhomogeneities [1]. Figure 1 (abstract P245). Bronchodilation and bronchial recruitment split per lobe for one patient. g g Methods Thirteen female piglets (21 ± 2 kg) were ventilated with a strain (VT/FRC) greater than 2.5 (VT ~ 38 ± 3 ml/kg) for 54 hours or until massive lung edema developed. Piglets were divided into fi ve groups characterized by diff erent energy loads obtained varying the respiratory rate: 15 breaths/minute (n = 3), 12 (n = 3), 9 (n = 3), 6 (n = 2) and 3 (n = 2). Every 6 hours two CT scans were performed (end-expiration and end- inspiration) and a static pressure–volume curve was obtained. A total of 51 CT scan couples and 51 corresponding pressure–volume curves was analyzed. The energy dissipated in the lung parenchyma at each breath was determined as the hysteresis area of the pressure–volume curve. CT scans were quantitatively analyzed with dedicated software. Data are presented as median (interquartile range). airway recruitment. This pilot study shows that, in ARDS, the upper lung regions are subject to airway dilation, whereas the lower (atelectatic) lung lobes have more airway opening with higher PEEP levels. Reference 1. De Backer et al. Radiology. 2010;257:854-62. P246fi P248 P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) P248 Immune response after prolonged hyperoxic mechanical ventilation HJ Helmerhorst1, LR Schouten2, NP Juff ermans2, MJ Schultz2, E De Jonge1, DJ Van Westerloo1 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) 1Leiden University Medical Center, Leiden, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Results RM improved oxygenation compared with the PEEP group. The mean increase in the oxygenation index at the end of surgery was 31% (from 340 to 445 mmHg, P <0.05), in the PEEP group the increase was less signifi cant and amounted to 12% (from 330 to 370 mmHg, P <0.05). Dynamic compliance increased by 35% in the RM group and did not change in the PEEP group. Hemodynamic changes at RM were Critical Care 2015, 19(Suppl 1):P248 (doi: 10.1186/cc14328) Introduction Mechanical ventilation and hyperoxia independently promote pulmonary injury and infl ammation. However, the time course of the immune response following concurrent exposure is unclear. The Introduction Mechanical ventilation and hyperoxia independently promote pulmonary injury and infl ammation. However, the time course of the immune response following concurrent exposure is unclear. The Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S87 lungs. We used EIT with increased signal quality and spatial resolution to describe and quantify the regional changes in aeration caused by body position, both during spontaneous breathing and mechanical ventilation in pulmonary healthy patients undergoing laparoscopic prostatectomy. lungs. We used EIT with increased signal quality and spatial resolution to describe and quantify the regional changes in aeration caused by body position, both during spontaneous breathing and mechanical ventilation in pulmonary healthy patients undergoing laparoscopic prostatectomy. Figure 1 (abstract P248). Infl ammatory mediators independent of tidal volumes after 12 hours of MV. Methods In 40 patients we performed EIT measurements at fi ve points of time (Table 1) with the Swisstom BB2 prototype. Thirty-two electrodes were used to apply weak alternating currents to the thorax and to measure the resulting voltages, from which tomographic images of the changes in regional impedance caused by ventilation were created. Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) p g g Methods Healthy male C57Bl/6J mice, aged 9 to 10  weeks, were randomly assigned to experimental groups (n = 8), in which the applied fractions of oxygen (FiO2) were 30%, 50% or 90% and tidal volumes were either 7.5 or 15 ml/kg. Anesthetized mice were tracheotomized and ventilated for 8 or 12 hours. Infl ammatory cells and mediators were measured in bronchoalveolar lavage fl uid (BALf).i Introduction The still unclear mechanisms causing ventilator- induced diaphragm dysfunction (VIDD) are considered intrinsic to the diaphragm muscle fi bers. VIDD delays and complicates weaning from mechanical ventilation (MV) and accordingly contributes to prolonged ICU stay by 50%, with older patients being more aff ected than the young. The main aim of this study was to measure the eff ects of aging and 5 days of MV on rat diaphragm muscle fi ber structure and function. We also aimed to investigate the biological age of the old rats to obtain data useful to design future experimental studies focusing on the eff ects of age in an ICU setting. Results Mice exposed to higher FiO2 had signifi cantly higher PaO2 levels at the end of the experiment. The total number of infl ammatory cell in the BALf was not signifi cantly diff erent between the experimental groups (P = 0.28), yet an increasing trend in the percentage of neutrophils was observed with increasing FiO2 (P = 0.03). Cytokine and chemokine levels did not diff er between FiO2 groups at 8  hours of ventilation. In mice ventilated for 12 hours, a signifi cantly increasing trend in IFNγ, IL-1β, IL- 10, MCP-1 and TNFα (Fig. 1, P <0.01) was measured with increasing FiO2, whereas IL-6, KC, MIP-2, GM-CSF and VEGF remained virtually unchanged. Diff erences between the tidal volume groups were small and did not markedly infl uence the eff ects of hyperoxia. See Figure 1.f Methods We used a unique ICU rat model, which allows us to maintain the vital parameters stable under deep sedation and MV for long durations (several weeks). Diaphragm fi ber cross-sectional area (CSA) and force-generating capacity (specifi c force = absolute force / CSA) were measured in young (6 months) and old (28 to 32 months) F344/ BN hybrid rats in response to 5 days of deep sedation and volume- controlled MV. P250 Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) Structural and functional eff ects of mechanical ventilation and aging on single rat diaphragm muscle fi bers N Cacciani, H Ogilvie, L Larsson Karolinska Institutet, Solna – Stockholm, Sweden Critical Care 2015, 19(Suppl 1):P250 (doi: 10.1186/cc14330) To investigate the biological age of the old rats, we performed a second set of experiments, comparing muscle fi ber CSA and specifi c force in fast and slow-twitch distal hind limb muscles in three diff erent age groups: young adults (6  months), middle aged (18 months) and old rats (28 months). ylf yp g Conclusion Hyperoxia induced a time-dependent and diff erentiated immune response that was independent of tidal volumes in a model of mechanically ventilated mice. The presence of cytokines and chemokines in the pulmonary compartment was more pronounced with prolonged and severe hyperoxia. P248 We describe the ventilation distribution using a novel EIT lung function parameter called Silent Spaces that provides information about areas that do not receive much or any air during tidal breathing and are divided into nondependent (NSS) and dependent Silent Spaces (DSS) using a reference line that runs perpendicular to the gravity vector right through the centre of ventilation. NSS and DSS are expressed as a percentage of the total lung area. Results Perioperative changes of NSS and DSS are shown in Table 1 as mean ± SD. Statistically signifi cant diff erences marked by § when compared with 1 or by * when compared with 3 (P <0.05). y Conclusion We describe for the fi rst time the mapping of Silent Spaces during spontaneous breathing and changing ventilation conditions and body positions in patients with healthy lungs using EIT. This mapping of Silent Spaces might prove useful for developing perioperative protective ventilation strategies. Figure 1 (abstract P248). Infl ammatory mediators independent of tidal volumes after 12 hours of MV. aim of this preclinical study was to study both time-dependent and dose-dependent eff ects of supplemental oxygen during prolonged ventilatory support on pulmonary infl ammation in a well-established murine model of ventilation comparing low and high tidal volumes. aim of this preclinical study was to study both time-dependent and dose-dependent eff ects of supplemental oxygen during prolonged ventilatory support on pulmonary infl ammation in a well-established murine model of ventilation comparing low and high tidal volumes. Methods Healthy male C57Bl/6J mice, aged 9 to 10  weeks, were randomly assigned to experimental groups (n = 8), in which the applied fractions of oxygen (FiO2) were 30%, 50% or 90% and tidal volumes were either 7.5 or 15 ml/kg. Anesthetized mice were tracheotomized and ventilated for 8 or 12 hours. Infl ammatory cells and mediators were measured in bronchoalveolar lavage fl uid (BALf).i Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods All 89 inpatients on the CCU who received mechanical ventilation continuously for 7  days or more between October 2012 and December 2013 were initially included. Forty patients who were intubated prior to arrival at RCHT, had incomplete notes, or were extubated during end-of-life care were excluded. Patients were divided into groups by fi rst airway intervention; 31 TOE, 18 TR. fi bers from young, middle aged and old animals confi rmed the 28 to 32 month rats to be senescent from a skeletal muscle point of view. p Conclusion These results demonstrate intrinsic changes in diaphragm muscle fi bers of signifi cant importance for the prolonged and complicated weaning from MV. Moreover, the increased number of frail diaphragm muscle fi bers observed after MV in old age, both controls and mechanically ventilated, off ers a further age-related possible mechanism which may be of signifi cant clinical importance. These results also provided useful information to design future experimental studies focused on the eff ect of age in an ICU setting, pharmacological intervention strategies as well as mechanisms underlying rat strain diff erences. g p yi y ; , Results A total 52% (16/31) of patients had TOE, required no other airway intervention and survived to discharge from hospital, compared with 72% (13/18) of TR patients. Four patients from each group failed the fi rst intervention and died prior to a second intervention. In total, 8/11 patients who had a second intervention after failed TOE survived to discharge from hospital. One patient had a second TR but died before discharge. This gave an in-hospital mortality rate of 19% for the TOE group and 28% for the TR group. TOE was performed earlier, all 31 on days 7 to 15. TR was performed later; 14/18 on days 7 to 15, and 4/18 on days 17 to 23. Early TR was more successful; 11/11 survived to discharge without a second intervention who had TR on days 7 to 12, compared with 29% (2/7) after day 12. TOE was more successful when performed later; 64% (7/11) survived to discharge without a second airway intervention when TOE was after day 10, 45% (9/20) between days 7 and 10. Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Results Twenty-eight patients were included in G1 and 22 patients in G2. There was no signifi cant diff erence between the patients’ admission parameters and daily NIV usage times. PaCO2 decreased >5 mmHg in 93% of G1 patients and in 60% of G2 patients in the fi rst 6 hours (P = 0.044). A 10 mmHg reduction in PaCO2 occurred in more patients (93% vs. 60%, P = 0.004) and in a shorter time (1.8 ± 1.2 vs. 3 ± 3 days, P = 0.044) in G1. At the time of discharge, PaCO2 levels were <50 mmHg in 79% of G1 and 41% of G2 patients (P = 0.006). Both groups showed similar and signifi cant improvements in PaO2, PaCO2 and HCO3 – levels within the fi rst 4 days but only in G1 patients were HCO3 – levels decreased more rapidly than G2 patients (P = 0.007). Duration of NIV (6 ± 2 vs. 8 ± 3 days, P = 0.002) and the number of mode and pressures changes (0.3 ± 1.8 vs. 2 ± 2 times, P >0.0001) were signifi cantly less in G1. While mean IPAP was similar in both groups, maximum and minimum EPAP titrated automatically in G1 were signifi cantly diff erent from G2. Mean tidal volume and amount of leakage were also signifi cantly higher in G1. fi g pfi Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Introduction Bilevel non-invasive ventilation (NIV) is an established therapy in chronic obstructive pulmonary disease (COPD) but confl icting evidence exists for its use in patients with pneumonia. Initial arterial pH <7.25 is used as a marker of severity and need for admission to critical care (CC) [1]. We examined the impact of pH and condition on outcome in patients with acute respiratory failure (ARF) of mixed aetiology treated with NIV. Methods Data were collected retrospectively for a 5-year period from 2008 to 2013 using the Metavision electronic patient record. We identifi ed all patients admitted with ARF treated with bilevel NIV. Patients who received continuous positive airway pressure or had a primary surgical problem were excluded. We recorded primary cause of respiratory failure, arterial blood gas values and mortality.i g gi y g Conclusion These results suggest that the AVAPS-AE mode may provide some advantages in hypercapnic ICU patients such as rapid PaCO2 reduction, less NIV duration and workload. Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Results A total of 145 patients were identifi ed. Mean age was 64 and 51% were male. The primary diagnosis was pneumonia in 69 patients and exacerbation of COPD in 57. The overall mortality was 19% on CC and 39% at 1 year. In patients with COPD, infective exacerbations had a higher CC mortality (17%) compared with non-infective (0%). However, by 1 year the mortality was 28% in infective and 29% in non-infective. Patients with pneumonia had a higher mortality on CC (25%) and at 1 year (48%). Patients with an initial pH <7.25 were less likely to survive. The mortality at discharge from CC was 16% (pH ≥7.25) and 26% (pH <7.25) but narrowed to 38% and 39% by 1 year. When subdivided, it was found that patients with infective COPD and pH <7.25 had the lowest 1-year mortality (17%) while those with pneumonia and pH <7.25 had the highest mortality (67%). P251 5 Does it make a diff erence to add automatic EPAP titration to the volume-targeted pressure support mode in noninvasive ventilation of hypercapnic ICU patients? G Gursel, A Zerman, B Basarik, K Gonderen, M Aydogdu, S Memmedova Gazi University Medical Faculty, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P251 (doi: 10.1186/cc14331) Introduction Obese patients are increasing in number in ICUs and more than 90% of them also have sleep apnea syndrome. Variability in upper airway resistance during sleep and awakening periods makes it diffi cult to set EPAP in these patients. A new mode that automatically titrates EPAP according to upper airway resistance and IPAP according to target tidal volume may be more eff ective. The aim of this study is to evaluate whether adding automatic EPAP titration to the volume-targeted pressure support mode will provide any therapeutic advantages in hypercapnic ICU patients. Conclusion TOE is more common and is associated with shorter time spent ventilated, in the CCU and in hospital than TR. It is also associated with a lower in-hospital mortality rate. TOE is more successful when performed after day 10; TR is more successful when performed before day 13. After failed TOE, a second TOE is associated with longer time in hospital but a better mortality rate than secondary tracheostomy. y Methods The hypercapnic patients treated with average volume- assured pressure support-automatic EPAP (AVAPS-AE) mode (Group1 (G1)) were compared with those treated with AVAPS mode (Group 2 (G2)). G2 was recruited retrospectively and matched with G1 according to diagnoses, demographic characteristics, arterial blood gas values and daily noninvasive ventilation (NIV) usage times. Trilogy 100® devices and their software Directview® (Philips Respironics) were used to reveal the respiratory data such as pressures, volumes, and daily usage times. For statistical analyses, t test, chi-square test and repeated measures of ANOVA were used. P253 Initial pH and mortality in patients with exacerbations of COPD and pneumonia treated with NIV in a teaching hospital critical care unit IM Goodhart, MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2015, 19(Suppl 1):P253 (doi: 10.1186/cc14333) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 After fi rst failed TOE, four patients had a successful second TOE; all four survived to discharge resulting in a median CCU stay of 29 days and median hospital stay of 39 days (excluding prior to CCU admission). Seven patients had TR after the fi rst failed TOE, fi ve survived to discharge from the CCU and four to discharge from the hospital. This group had shorter median stays in both the CCU (27 days) and hospital (32 days). Overall, the median duration of time ventilated, in the CCU, and in hospital was shorter for the TOE group; 13, 17 and 24 days respectively, compared with 22, 27.5 and 34 days for the TR group. P249 Perioperative assessment of regional ventilation during changing body positions and ventilation conditions by electrical impedance tomography with increased spatial resolution and signal quality A März1, A Ukere1, K Wodack1, C Trepte1, A Waldmann2, S Böhm2, D Reuter1 1University Medical Center Hamburg-Eppendor, University Hamburg, Germany; 2Swisstom AG, Landquart, Switzerland Critical Care 2015, 19(Suppl 1):P249 (doi: 10.1186/cc14329) Perioperative assessment of regional ventilation during changing body positions and ventilation conditions by electrical impedance tomography with increased spatial resolution and signal quality A März1, A Ukere1, K Wodack1, C Trepte1, A Waldmann2, S Böhm2, D Reuter1 1University Medical Center Hamburg-Eppendor, University Hamburg, Germany; 2Swisstom AG, Landquart, Switzerland Critical Care 2015, 19(Suppl 1):P249 (doi: 10.1186/cc14329) Results This study demonstrated an unexpected increase in CSA (P  <0.001) of the diaphragm fi bers in response to 5  days of MV in both young and old animals. Maximum force decreased 39.8 to 45.2% (P  <0.001) in both young and old animals compared with controls, resulting in a dramatic loss of specifi c force. This increase in CSA and the concomitant decrease in specifi c force observed in both young and old diaphragm fi bers are compatible with an ineff ective compensatory hypertrophy in response to the MV. The comparison of the limb muscles Introduction Electrical impedance tomography (EIT) is a functional imaging technology allowing one to regionally monitor aeration of the Table 1 (abstract P249) 1 2 3 4 5 (sitting, (supine, (ventilated, (ventilated, (ventilated, spontaneous breathing) spontaneous breathing) supine position) 30° head down position) supine position) NSS (%) 5.25 ± 2.9 4.12 ± 1.89§ 3.05 ± 1.9§ 2.8 ± 2.7§ 2.67 ± 1.9§ DSS (%) 0.07 ± 0.3 2.29 ± 2.3§ 9.23 ± 6.3§ 11.5 ± 8.9§* 8.5 ± 5.8§ Table 1 (abstract P249) 1 2 3 4 5 (sitting, (supine, (ventilated, (ventilated, (ventilated, spontaneous breathing) spontaneous breathing) supine position) 30° head down position) supine position) NSS (%) 5.25 ± 2.9 4.12 ± 1.89§ 3.05 ± 1.9§ 2.8 ± 2.7§ 2.67 ± 1.9§ DSS (%) 0.07 ± 0.3 2.29 ± 2.3§ 9.23 ± 6.3§ 11.5 ± 8.9§* 8.5 ± 5.8§ S88 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P252 Retrospective study of patients receiving long-term mechanical ventilation I D d B H d R Si l i I Dunwoody, B Hopwood, R Sinclair Royal Cornwall Hospital, Truro, UK Critical Care 2015, 19(Suppl 1):P252 (doi: 10.1186/cc14332) Introduction This study analysed the practice of clinicians managing patients requiring long-term mechanical ventilation in the critical care unit (CCU) of the Royal Cornwall Hospital, Truro (RCHT), comparing outcomes of primary tracheostomy (TR) with trial of extubation (TOE). Conclusion NIV is used in our unit with comparable success rates to published series [2,3]. COPD patients responded well to NIV, while Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S89 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 patients with pneumonia treated with NIV have the highest mortality. A low presenting pH is associated with a higher mortality in patients with pneumonia treated with NIV. However, in COPD patients, pH <7.25 is not associated with higher mortality in CC or at 1 year. Further work defi ning the precise role of pH as a prognostic indicator is warranted. References patients with pneumonia treated with NIV have the highest mortality. A low presenting pH is associated with a higher mortality in patients with pneumonia treated with NIV. However, in COPD patients, pH <7.25 is not associated with higher mortality in CC or at 1 year. Further work defi ning the precise role of pH as a prognostic indicator is warranted. References care units within the NoECCN. Patient data collected included patient demographics, admission diagnosis and speciality, hospital length of stay (LOS) pre and post critical care admission, severity of illness scores, critical care LOS and status at hospital discharge. p g Results During the study period 134 patients met the criteria for PMV representing 1% of annual admissions and 6.9% NoECCN bed- days. The majority of patients receiving PMV were medical (50.7%), followed by emergency surgery (20.1%), elective surgery (16.4%) and specialist services such as spinal cord injury (8.2%) and cardiothoracic transplant (4.5%). The commonest admission diagnosis in the medical population was pulmonary infection followed by acute neurological disorders, while 89.4% of surgical patients were admitted to critical care during the perioperative period. At the end of the study period the highest hospital mortality was observed in the nonspecialist surgical population (26.5%). In contrast, the medical population had one of the lowest hospital mortality rates (11.8%), lower than predicted using the intensive care national audit research network illness severity score. P254 Lung protective ventilation with lower tidal volumes and development of pulmonary complications in critically ill patients without ARDS FD Simonis1, A Serpa Neto2, M Gama de Abreu3, P Pelosi4, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Isrealita Albert Einstein, São Paulo, Brazil; 3University Hospital Carl Gustav Carus, Dresden, Germany; 4IRCCS San Martino IST, Genoa, Italy Critical Care 2015, 19(Suppl 1):P254 (doi: 10.1186/cc14334) Reference 1. Pilcher DV, et al. Outcomes, cost and long term survival of patients referred to a regional weaning unit. Thorax. 2005;60:187-92. Methods An individual patient data meta-analysis of studies of ventilation in ICU patients without ARDS. Corresponding authors of retrieved studies provided individual patient data. The primary outcome, pulmonary complications, was a composite of development of ARDS or pneumonia during hospital stay. Secondary outcomes included ICU and hospital length of stay, and in-hospital mortality. Patients were assigned to three groups based on tidal volume size (≤7 ml/kg predicted body weight (PBW), 7 to 10 ml/kg PBW, or ≥10 ml/ kg PBW). P252 Retrospective study of patients receiving long-term mechanical ventilation I D d B H d R Si l i Comparable rates of hospital discharge were found in both medical (85%) and nonspecialist surgical patients (88.9%). 1. 2013 BTS NIV audit. https://www.brit-thoracic.org.uk/document-library/ audit-and-quality-improvement/audit-reports/ bts-adult-niv-audit-report-2013/ 2. Carillo et al. ICM. 2012;38:458-66. 3. Lightowler et al. BMJ. 2003;326:185-9. Eff ects of positive end-expiratory pressure on lung ventilation/ perfusion matching: a clinical study Results Patients were 62 ± 12 years old, mean PaO2/FiO2 was 197 ± 52, lower PEEP level was 7 (7 to 9) cmH2O, while higher PEEP was 12 (12 to 14) cmH2O (P <0.001). At higher PEEP, EELV increased (391 (334 to 555) ml vs. PEEP low, considered as baseline, P <0.001); VtHetend- insp was reduced (1.8 (1.5 to 2.4) vs. 2.2 (1.8 to 2.6), P <0.001) and HA/P increased (0.29 ± 0.19 vs. 0.2 ± 0.15, P <0.05). Interestingly, the increase of HA/P was signifi cantly correlated with the decrease of VtHetend- insp (r = –0.48, P <0.05). Moreover, patients with higher potential for improvement of ventilation/perfusion matching (that is, patients with increase of HA/P >16%) had higher baseline VtHetend-insp (2.6 (2.3 to p y p Methods We enrolled 20 intubated critically ill patients undergoing controlled mechanical ventilation, sedated, paralyzed and with PaO2/FiO2 ≤300 at PEEP ≥5  cmH2O. We started EIT monitoring (Pulmovista500®; Dräger Medical GmbH, Lübeck, Germany) and applied two PEEP levels (clinical and clinical + 5 cmH2O) for 20 minutes each. We collected ventilatory and EIT parameters and, by offl ine analysis, we calculated the increase of EELV at higher PEEP and the EIT- based indexes of ventilation heterogeneity (VtHetend-insp) and of the regional homogeneity of ventilation/perfusion matching (HA/P). Lung protective ventilation with lower tidal volumes and development of pulmonary complications in critically ill patients without ARDS FD Simonis1, A Serpa Neto2, M Gama de Abreu3, P Pelosi4, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Hospital Isrealita Albert Einstein, São Paulo, Brazil; 3University Hospital Carl Gustav Carus, Dresden, Germany; 4IRCCS San Martino IST, Genoa, Italy Critical Care 2015, 19(Suppl 1):P254 (doi: 10.1186/cc14334) p g p Conclusion The results of this study highlight an expanding proportion of NoECCN critical care bed-days occupied by stable patients undergoing PMV. In keeping with published UK data, elevated hospital mortality was observed in the nonspecialist surgical subpopulation. Although the literature suggests the medical cohort of patients has poorer prognosis, within our region all were liberated from mechanical ventilation and over 80% were discharged from hospital. f Introduction A large meta-analysis suggests that use of low tidal volumes benefi ts patients without ARDS [1] but most studies in this meta-analysis included patients receiving ventilation during general anesthesia for surgery. The aim of the present meta-analysis is to determine the association between tidal volume size and development of pulmonary complications in ICU patients. P255 Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 1Sunderland Royal Hospital, Sunderland, UK; 2James Cook University Hospital, Middleborough, UK Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 1Sunderland Royal Hospital, Sunderland, UK; 2James Cook University Hospital, Middleborough, UK Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) Eff ects of positive end-expiratory pressure on lung ventilation/ perfusion matching: a clinical study N Eronia1, T Mauri2, G Bellani1, R Marcolin1, S Marocco Arrigoni1, G Grasselli1, A Pesenti1 , 1San Gerardo Hospital, Monza, Italy; 2IRCC Ca’ Granda Maggiore Policlinico Hospital, Milan, Italy Critical Care 2015, 19(Suppl 1):P256 (doi: 10.1186/cc14336) Results Seven investigations (2,184 patients) were meta-analyzed. Pulmonary complications occurred in 23%, 28% and 31% respectively in the ≤7 ml/kg PBW, 7 to 10 ml/kg PBW and ≥10 ml/kg PBW group (adjusted RR, 0.72; 95% CI, 0.52 to 0.98; P  = 0.042). Occurrence of pulmonary complications was associated with a lower number of ICU- free days and alive at day 28, a lower number of hospital-free days and alive at day 28 and increased in-hospital mortality. Introduction Positive end-expiratory pressure (PEEP) exerts multiple protective eff ects in hypoxemic critically ill patients: PEEP can increase end-expiratory lung volume (EELV) and induce recruitment, thus reducing lung strain and opening and closing of alveoli and potentially improving the ventilation/perfusion matching. In particular, estimation of PEEP-induced ventilation/perfusion matching might help identify the optimal PEEP setting, but bedside non-invasive methods are few and complex to be applied in daily clinical practice. Electrical impedance tomography (EIT) is a non-invasive bedside technique that claims to track global and regional changes in perfusion and ventilation over time. In the present study we aimed at verifying the eff ects of PEEP on ventilation/perfusion matching, as assessed by EIT, in acute respiratory failure patients. Conclusion Ventilation with low tidal volumes is associated with a lower risk of development of pulmonary complications. Occurrence of pulmonary complications is associated with an increased ICU and hospital length of stay and in-hospital mortality in ICU patients without ARDS. Reference Reference 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. Reference 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. 1. Serpa Neto A, et al. JAMA. 2012;308:1651-9. p y p Methods We enrolled 20 intubated critically ill patients undergoing controlled mechanical ventilation, sedated, paralyzed and with PaO2/FiO2 ≤300 at PEEP ≥5  cmH2O. We started EIT monitoring (Pulmovista500®; Dräger Medical GmbH, Lübeck, Germany) and applied two PEEP levels (clinical and clinical + 5 cmH2O) for 20 minutes each. We collected ventilatory and EIT parameters and, by offl ine analysis, we calculated the increase of EELV at higher PEEP and the EIT- based indexes of ventilation heterogeneity (VtHetend-insp) and of the regional homogeneity of ventilation/perfusion matching (HA/P). Pressure reconstruction method for spontaneous breathing eff ort monitoring Pressure reconstruction method for spontaneous breathing eff ort monitoring N Damanhuri1, YS Chiew1, NA Othman1, PD Docherty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P259 (doi: 10.1186/cc14339) N Damanhuri1, YS Chiew1, NA Othman1, PD Docherty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2015, 19(Suppl 1):P259 (doi: 10.1186/cc14339) Introduction Estimating respiratory mechanics of mechanically venti lated patients is unreliable when patients exhibit spontaneous breathing (SB) eff orts on top of ventilator support. This reverse triggering eff ect [1] results in an M-wave shaped pressure wave. A model-based method to reconstruct the aff ected airway pressure curve is presented to enable estimation of the true underlying respiratory mechanics of these patients. Table 1 (abstract P257) Ki <27.8 ml/ Ki ≥27.8 ml/ minute/ml minute/ml × 104 (n = 7) × 104 (n = 8) P value PaO2/FiO2 280 (261 to 346) 239 (212 to 271) 0.31 pH 7.46 (7.43 to 7.48) 7.41 (7.39 to 7.44) 0.15 PaCO2 (mmHg) 37 (34 to 44) 50 (46 to 53) 0.01 WBCs (103 cell/mm3) 8 (8 to 9) 9 (9 to 16) 0.34 Total lung volume (ml) 1,298 (1,092 to 1,494) 1,516 (1,408 to 1,665) 0.18 Total lung weight (g) 592 (488 to 741) 732 (597 to 774) 0.39 Total lung gas (ml) 706 (551 to 843) 804 (755 to 1,080) 0.15 Not-infl ated lung tissue (%) 25 (12 to 30) 23 (17 to 30) 0.95 Poorly infl ated lung tissue (%) 34 (42 to 42) 31 (29 to 34) 0.39 Well-infl ated lung tissue (%) 36 (27 to 47) 47 (35 to 52) 0.53 Conclusion Patients clinically defi ned as having primary graft dysfunction did not have an increased rate of 18-FDG uptake. 18-FDG uptake was not diff erent in single-lung versus bilateral transplantation and, in single-lung procedures, the native lung showed elevated infl ammatory activity. Methods Airway pressure and fl ow data from 72 breaths of a pneu- monia patient were used for proof of concept. A pressure wave reconstruction method fi lls parts of the missing area caused by SB eff orts and reverse triggering by connecting the peak pressure and end-inspiration slope (Figure  1). A time-varying elastance model [2] was then used to identify underlying respiratory elastance (AUCEdrs). P258 4.8) vs. 1.9 (1.5 to 2.1), P <0.01) and lower compliance of dependent lung regions (Crsdep, 13 ± 3 ml/cmH2O vs. 18 ± 6 ml/cmH2O, P <0.05), as compared with patients with smaller improvement. P257 18-FDG PET in lung transplantation I Al 1 F V l 1 M G l 1 B S f P257 18-FDG PET in lung transplantation l 1 l 1 G l 1 S f 18-FDG PET in lung transplantation I Algieri1, F Valenza1, M Guanziroli1, B Safaee Fakhr1, M Cressoni1, M Brioni1, A Colombo1, G Babini1, F Crimella1, D Massari1, K Nikolla1, G Crisafulli1, S Paladini1, L Rosso2, A Palleschi2, F Zito2, D Chiumello1, L Gattinoni1 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Ca’ Granda- Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2015, 19(Suppl 1):P257 (doi: 10.1186/cc14337) p g p g p g p g p Results The median value of LU comets was signifi cantly lower in Group A compared with Group B. There was a signifi cant increase in hypoxic index in Group A compared with Group B. There was no signifi cant diff erence between both groups as regards PIP, while Pplat showed a signifi cant increase in Group B compared with Group A and Cs showed a signifi cant decrease in Group B. Introduction Lung transplantation is associated with an infl ammatory reaction known as primary graft dysfunction. This clinical syndrome occurs within the fi rst 72  hours after transplantation and is characterized by hypoxemia (PaO2/FiO2 <300) and bilateral infi ltrates not secondary to cardiac dysfunction, viral or bacterial pneumonia and venous anastomotic obstruction. gi p Conclusion LU is a useful to quantify EVLW; it is a good predictor of weaning. References Methods 18-FDG PET scan was used to study 15 lung transplantation patients. The rate of 18-FDG uptake (Ki) was computed voxel by voxel with the Patlak method. Patients were divided according to the median Ki (27.8 (20.3 to 34.6) ml/minute/ml × 104). Data are reported as median and interquartile range. 1. Nektaria X, Eumorfi a K, George P, et al. Impact of lung ultrasound on clinical decision making in critically ill patients. Intensive Care Med. 2014;40:57-65. 2. Jambrik Z, Monti S, Coppola V, et al. Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water. Am J Cardiol. 2004;93:1265-70. 2. Jambrik Z, Monti S, Coppola V, et al. Usefulness of ultrasound lung comets as a nonradiologic sign of extravascular lung water. Am J Cardiol. 2004;93:1265-70. Results Five patients developed primary graft dysfunction; median Ki in these patients was not diff erent from patients who did not (24.5 (18.2 to 33.6)  ml/minute/ml  × 104 vs. 29.1 (23 to 35.4)  ml/minute/ ml × 104 respectively, P = 0.64). Bilateral lung transplantation patients were characterized by a median Ki of 30.5 (22.9 to 34.5) ml/minute/ ml  × 104, while patients undergoing single-lung transplantation presented a median Ki of 24.4 (21 to 34.1) ml/minute/ml × 104 (P = 0.61). Considering single-lung transplantation, graft and native lung had similar Ki: 24.4 (21 to 34.1) ml/minute/ml × 104 versus 24.2 (17.7 to 30.1) ml/minute/ml × 104 respectively (P = 0.64). When patients were divided according to the median Ki value, higher Ki was associated with higher PaCO2 values (50 (46 to 53) mmHg vs. 37 (34 to 44) mmHg, P = 0.01). See Table 1. Usefulness of extravascular lung water assessment as a predictor of weaning from mechanical ventilation Usefulness of extravascular lung water assessment as a predictor of weaning from mechanical ventilation Conclusion EIT might represent a feasible, bedside method to estimate PEEP-induced improvement in ventilation/perfusion matching. Assessing regional ventilation and mechanical lung properties might help identify patients who would benefi t more from higher PEEP. g Alexandria University Hospital, Alexandria, Egypt Critical Care 2015, 19(Suppl 1):P258 (doi: 10.1186/cc14338) Introduction Quantitative measurement of extravascular lung water (EVLW) would be extremely useful for clinical management, both as an index of severity and to guide treatment lung ultrasonography (LU) as a tool to quantify EVLW. Pressure reconstruction method for spontaneous breathing eff ort monitoring The area of the unreconstructed M-wave has less pressure, resulting in a lower overall AUCEdrs without reconstruction. The missing area of the airway pressure or AUCEdrs is hypothesized to be a surrogate of patient- specifi c inspiratory to assess the strength of SB eff orts. AUCEdrs and missing area A2 are compared with/without reconstruction.if 2 Results Median AUCEdrs and breath-specifi c eff ort using reconstruction were 24.99 (IQR: 22.90 to 25.98) cmH2O/l and 3.64 (IQR: 0.00 to 3.87)% versus AUCEdrs of 20.87 (IQR: 15.24 to 27.48) cmH2O/l for unreconstructed M-wave data, indicating signifi cant patient and breath-specifi c SB eff ort, and the expected higher elastance (P <0.05). Conclusion A simple reconstruction method enables the real-time measurement of respiratory system properties of a SB patient and measures the surrogate of the SB eff ort, that latter of which has clinical use in deciding whether to extubate or re-sedate the patient. Conclusion Patients clinically defi ned as having primary graft dysfunction did not have an increased rate of 18-FDG uptake. 18-FDG uptake was not diff erent in single-lung versus bilateral transplantation and, in single-lung procedures, the native lung showed elevated infl ammatory activity. Factors associated with survival and hospital discharge amongst critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network L O’Connor1, I Gonzalez2, L Garcia2 Critical Care 2015, 19(Suppl 1):P255 (doi: 10.1186/cc14335) g g y p g Results Patients were 62 ± 12 years old, mean PaO2/FiO2 was 197 ± 52, lower PEEP level was 7 (7 to 9) cmH2O, while higher PEEP was 12 (12 to 14) cmH2O (P <0.001). At higher PEEP, EELV increased (391 (334 to 555) ml vs. PEEP low, considered as baseline, P <0.001); VtHetend- insp was reduced (1.8 (1.5 to 2.4) vs. 2.2 (1.8 to 2.6), P <0.001) and HA/P increased (0.29 ± 0.19 vs. 0.2 ± 0.15, P <0.05). Interestingly, the increase of HA/P was signifi cantly correlated with the decrease of VtHetend- insp (r = –0.48, P <0.05). Moreover, patients with higher potential for improvement of ventilation/perfusion matching (that is, patients with increase of HA/P >16%) had higher baseline VtHetend-insp (2.6 (2.3 to Introduction The combination of a global demographic shift and increased survival following critical illness has led to an increasing number of patients requiring prolonged mechanical ventilation (PMV) and longer critical care stay. This is a prospective observational study evaluating the characteristics and speciality-based outcome of critically ill patients undergoing prolonged mechanical ventilation in the North of England Critical Care Network (NoECCN). Methods A weekly survey was conducted over a 1-year period screening patients older than 16 years of age requiring PMV in all 18 adult critical S90 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P257 q y Methods Forty mechanically ventilated patients were examined for 5  successive days after being eligible for weaning; counting B-lines (comets) in both lung fi elds by LU, peak airway pressure, plateau airway pressure, static compliance and ABG analysis every 6 hours. Patients were divided into two groups according to success of the weaning process: group A (weaned group), and group B (nonweaned group).i Breath-to-breath respiratory mechanics variation: how much variation should we expect? Each patient was sedated to prevent spontaneous breathing eff ort, and ventilated using the volume control mode with a square fl ow profi le. Varied PEEP levels were maintained for 30 minutes before 1 minute of data were collected for analysis. This dataset provides a wide range of respiratory mechanics values, and the clinical protocol detail is in [1]. A clinically proven, single compartment model respiratory system elastance (Ers) is identifi ed from data for every breathing cycle at each PEEP level using a linear regression method. The dynamic elastance (Edynamic = (peak airway pressure – positive end-expiratory pressure) / tidal volume) of the corresponding breathing cycle is calculated for comparison.fii p g g y p Results The coeffi cient of variation (CV) of identifi ed Ers across all patients was low (<0.005), as expected, as the 30-minute period allows time-dependent alveolar recruitment to fully occur and stabilise. However, even with substantial stabilisation periods, there remains a diff erence between the minimum and maximum estimated Ers within each 1-minute period of analysed data. The diff erences were median 2.8% (interquartile range (IQR): 1.5 to 4.6%, 90% range (90R): 0.8 to 13.4) for Ers and 3.6% (IQR: 2.3 to 5.2, 90R: 0.9 to 10.8) for Edynamic, and in extreme cases, can be up to 16%. Results Over 275 paired samples were analysed over the following concentration ranges: pH 7.249 to 7.545; pCO2 3.32 to 11.01 kPa; pO2 5.59 to 21.80 kPa; Hct 23.6 to 41.4%; K+ 3.3 to 4.79 mM. The imprecision for each sensor was calculated to be: pH –0.00 ± 0.03; pCO2 –0.48 ± 0.34 kPa; pO2 –0.85 ± 0.96 kPa; Hct –4.49 ± 3.42%; K+ 0.08 ± 0.15 mM, respectively. The data collected on the new analyser for Hct showed relative imprecision of 3.42%. The pooled SD was calculated to be 1.21% and the mean SD of each of the novel devices used was 1.14%. This indicates that scatter observed on the Hct sensor was largely due to inter-device rather than intra-device factors. Conclusion This study quantifi ed the variability (over short periods) of identifi ed and estimated respiratory mechanics properties used to (potentially) guide ventilation care in sedated patients. It is also important to note that this minimum level of variability occurs even when stabilisation is achieved. Breath-to-breath respiratory mechanics variation: how much variation should we expect? 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) KT Kim1, YS Chiew1, C Pretty1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P260 (doi: 10.1186/cc14340) KT Kim1, YS Chiew1, C Pretty1, GM Shaw2, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2015, 19(Suppl 1):P260 (doi: 10.1186/cc14340) Introduction Patient-dedicated arterial blood analysers off er the potential to allow close monitoring of critically ill patients without leaving the patient’s bedside or net loss of blood for the patient. A new patient-attached blood gas analyser was evaluated in a clinical environment to compare the results against the reference bench-top analyser. Introduction Model-based respiratory mechanics can be used to guide mechanical ventilation therapy. However, identifi ed mechanical properties vary breath to breath, leading to potential treatment errors when using model-based care that requires accuracy. This study investigates and quantifi es this variability to improve its application in guiding clinical interventions. Methods Twenty ICU patients with a range of clinical conditions, including trauma, head injury, post-surgical recovery and sepsis, were included in the study. The new analyser was operated by clinical staff at the Queen Elizabeth Hospital, Birmingham, who each underwent a 90-minute training programme. Patients were monitored using the patient-dedicated analyser (Proxima; Sphere Medical) for up to 3 days. Each time a sample was tested on the patient-dedicated analyser, a concurrent sample was drawn and tested on the hospital’s bench- top analyser (Cobas b221; Roche Diagnostics). Samples were assessed using methods described by Bland and Altman for repeated measures. Performance of the novel device was analysed by calculating bias as the mean diff erence between the new analyser and the comparator device, imprecision as ±1 standard deviation (SD) from the mean and limits of agreement as ±1.96 SD from the mean. Percentage values for bias and precision were calculated from analysis of the percentage diff erence between the two methods of analysis. Intra-device imprecision for the haematocrit (Hct) sensor was calculated using a pooled variance calculation. Methods Retrospective data from 12 acute respiratory distress syndrome (ARDS) patients were used [1]. 1. Bersten. Eur Respir J. 1998;12:526-32. References 1. Akoumianaki E, et al. Chest. 2013;143:927-38. 2. Chiew YS, et al. Biomed Eng Online. 2011;10:111. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S91 Figure 1 (abstract P259). Edrs for M-wave and reconstructed airway pressure at PEEP = 15 cmH2O. PEEP H O Figure 1 (abstract P259). Edrs for M-wave and reconstructed airway pressure at PEEP = 15 cmH2O. P261 P261 Evaluation of a patient-dedicated blood gas analyser JJ Fox1, TH Clutton-Brock2 1Sphere Medical, Cambridge, UK; 2University of Birmingham, UK Critical Care 2015, 19(Suppl 1):P261 (doi: 10.1186/cc14341) Breath-to-breath respiratory mechanics variation: how much variation should we expect? Thus, clinically, if this information was to be used to guide ventilation in real time, such as titrating PEEP to minimal elastance, larger errors, at least up to 15% variation in Ers, could be expected, which could well aff ect care. Such levels thus also begin to defi ne the minimum levels of change necessary to be larger than natural variation. Conclusion The patient-dedicated blood gas analysers demonstrated excellent agreement with the bench-top analyser for pH, pCO2, pO2 and K+, while Hct provides a reasonable trend monitor with variable bias. Proxima is well suited to enable staff to more closely manage unstable and critically ill patients. This device may be of signifi cant benefi t to patients at risk of iatrogenic anaemia or those being treated in side rooms/isolation rooms. Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) Introduction Maintaining the appropriate tidal volume (VT) is important for success of lung protective ventilation. However, in neonates, the presence of airway leaks may increase the errors in the delivery of tiny VT, which raises a concern of ventilator-induced lung injury. This study is to investigate the accuracy of VT delivery during non-invasive ventilation (NIV) with modern neonatal ventilators. Results There was a signifi cant correlation between the indexed EE and indexed VE in both groups (R = 0.51, P <0.0001 in the control group; R = 0.63, P <0.0001 in the MV group). The VR was signifi cantly suppressed in the MV group compared with the control group (0.041 ± 0.003/minute/ BEE vs. 0.069 ± 0.003/minute/BEE, P = 0.012; respectively). Although the indexed VE was comparable in the MV and control groups (0.19 ± 0.07 l/ kg vs. 0.17 ± 0.04 l/kg, P = 0.23; respectively), the indexed EE was shifted to a higher range in the MV group than in the control group (maximal indexed EE; 2.26 ± 0.68 vs. 1.74 ± 0.20, P = 0.008; respectively). The TV was smaller (maximal TV; 985 ± 592 ml vs. 1,410 ± 299 ml, P = 0.018; respectively) and the RR was more frequent (maximal RR; 30 ± 8/minute vs. 16 ± 4/minute, P <0.0001; respectively) in the MV group than in the control group. Methods Using a lung simulator for a patient body weight of 3  kg, we measured the actual delivered VT in the lung and compared it with the value displayed on the ventilator in six ventilators. We tested 18 conditions with various combinations of respiratory mechanics (normal, restrictive, obstructive), leak levels (0, 1.0, 1.5 l/minute), and PEEP settings (5, 10 cmH2O). All conditions were tested in NIV mode. The pressure level was set to achieve VT to the lung at 6 to 7 ml/kg. All other settings were: FIO2 0.21, Itime 0.6 seconds, f 25/minute, and default rise time. We calculated the mean errors of the ventilator-displayed VT at various levels of airway leak. Eff ective capnography training in the ICU using the ‘hats and caps’ training tool Eff ective capnography training in the ICU using the ‘hats and caps training tool CA Lobo, FE Kelly, S Steynberg, G Thomas, C Pope, M Eveleigh, M Charlton, TM Cook Royal United Hospital, Bath, UK Critical Care 2015, 19(Suppl 1):P264 (doi: 10.1186/cc14344) Reference Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S92 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P262 The VE was indexed by body weight and the EE was also indexed by the basal energy expenditure (BEE) estimated by the Harris–Benedict formula. VR was defi ned as the slope in the indexed VE–indexed EE plot with an assumption of those relationship in the linear manner. We examined the correlation between indexed EE and indexed VE in both groups, and the diff erences of the maximal indexed EE, the maximal indexed MV, and the others between both groups were investigated using the unpaired t test. For all the data, signifi cance was accepted at values of P <0.05. P262 Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators K Moon, S Mizuguchi, K Tachibana, M Takeuchi Osaka Medical Center and Research Institute for Maternal and Child Health, Izumi City, Osaka, Japan Critical Care 2015, 19(Suppl 1):P262 (doi: 10.1186/cc14342) Table 1 (abstract P262) Leak amount Hamilton Hamilton (l/minute) C3 G5 Servo i PB840 PB980 VN500 0 26 (15) 4 (6) 3 (2) 2 (7) –4 (2) 2 (2) 1.0 –9 (5) 9 (5) –2 (2) –5 (4) –2 (4) 20 (5) 1.5 16 (8) –14 (8) –2 (2) –6 (4) –2 (3) 33 (6)a Data presented as mean (SD), %. aUnable to measure in one case. Introduction Failure to use or correctly interpret capnography in patients dependent on an artifi cial airway in ICUs was thought to have contributed to 74% of ICU airway-related deaths in the NAP4 study [1]. However, capnography is only of value if those using it can interpret it correctly, with recommendations for training all ICU staff in capnography [1,2]. A recent UK survey identifi ed that only 48% of ICUs have trained all staff in capnography interpretation (TM Cook, personal communication). In this study, we used a capnography teaching aid (‘hats and caps’) to educate all ICU staff during a 1-month period, and evaluated its eff ectiveness. Conclusion Tidal volume accuracy during neonatal NIV varies greatly among diff erent ventilators and leak conditions. This must be considered in neonatal ventilation management to avoid over- ventilation or underventilation. Figure 1 (abstract P264). ‘Hats and caps’ capnography training guide. Tidal volume accuracy during non-invasive ventilation with modern neonatal mechanical ventilators Conclusion The VR to external stress with mechanical ventilation is more suppressed than in healthy volunteers. The VE in the mechanical ventilation was earned by a higher RR rather than by increased TV. Careful monitoring of VE or RR would be benefi cial in early rehabilitation with mechanical ventilation. y Results The VT mean error values are presented in Table 1. When no leak existed, the mean error was less than 5% in all ventilators except one (C3) which showed a mean error of 26%. As the leak level increased, three ventilators (C3, G5, and VN500) showed marked diff erences between the delivered and displayed VT. In particular, the VN500 could not operate in the large leak condition. The other three ventilators (PB840, PB980, Servo i) showed acceptable VT accuracy across all conditions tested. Comparison of three methods of applying high fl ow nasal oxygen: in vitro study p pp y g gl yg in vitro study M Muñoz Garach1, O Olga1, ME Yuste Osorio1, R Fernandez Fernandez2, R Ramirez Puerta1, F Acosta Díaz1, AM Perez Bailón1, S Narbona Galdó1, L Peñas Maldonado1 1Hospital Universitario San Cecilio, Granada, Spain; 2Hospital Nuestra Señora del Prado, Talavera de la Reina, Spain Critical Care 2015, 19(Suppl 1):P265 (doi: 10.1186/cc14345) Methods Twenty-one adult and clinically stable patients undergoing assisted mechanical ventilation for more than 48  hours were investigated during pressure support (baseline) and during a 2-hour CPAP trial. sEMG of diaphragm (costmar), intercostal and sternocleidal (accessory muscles) was recorded with a dedicated device (Dipha16; Inbiolab, Groningen, the Netherlands) simultaneously with airway waveforms and expressed as the ratio of the signal during baseline. Diaphragmatic electrical activity from a nasogastric tube (EAdi) of 14 of those patients was also measured. 1Hospital Universitario San Cecilio, Granada, Spain; 2Hospital Nuestra Señora del Prado, Talavera de la Reina, Spain Critical Care 2015, 19(Suppl 1):P265 (doi: 10.1186/cc14345) Introduction High fl ow nasal (HNF) requires precise control of the fraction of inspired oxygen (FiO2) and fl ow contributed as well as an adequate adjustment of temperature and humidity of the gas provided. There are several equipments for HNF. We evaluated the FiO2 and fl ow supplied with three diff erent systems Results The rapid shallow breathing index was lower than 105 in all patients and only one patient failed the trial. We observed that the mean inspiratory value of costmar increased immediately after switch to CPAP but did not signifi cantly vary during the CPAP trial (ANOVA, P = 0.7). On the other hand, the activation of accessory muscles increased signifi cantly during the same period (P  = 0.01) and was strongly correlated with respiratory rate (r = 0.41, P <0.001) and inversely with f y Methods There have been analyzed: (1)  ‘Oxygen Therapy’ from Dräger Evita-XL®; (2)  Fisher & Paykel Airvo® option; and (3)  pack of fl owmeters Debson®. Measurements were made in the distal part of the circuit that is used in clinical practice. Variables: programmed and measured FiO2, programmed and measured fl ow. We used the Oxygen Monitor Ohmeda 5120® and Flow Meter® Fisher-Porter. Before each measurement we checked and/or calibrated each of them. All measurements were performed at room temperature in the ICU of our hospital (23 to 26º).The data were processed using SPSS v.15.0.1, accepting a signifi cance level of 95%. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods ‘Hats and caps’ was devised on our ICU [3] and used for the training: this teaches that capnography traces on the left signify the airway is functional, in contrast to the traces on the right which indicate immediate attention is required (Figure 1). This was presented to staff working on the ICU in individual bedside teaching sessions with feedback obtained and evaluated. percentage variation –1.4040 ± 4.73 (–2.15 to –0.67); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.82 ± 3.85 (3.04 to 4.69); fl ow percentage variation 9.76  ± 8.08 (8.11 to 11.41); r  = 0.969 and r2 = 0.939 (P <0.000). (3) FiO2 variation –0.005 ± 0.26 (–0001 to 0009); FiO2 percentage variation –0.72 ± 5.2 (–1.5 to 0.1); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.91 ± 1.26 (3.69 to 4.13); fl ow percentage variation 12.77 ± 5.33 (11.84 to 13.7); r = 0.996 and r2 = 0.992 (P <0.000). See Figure 1. percentage variation –1.4040 ± 4.73 (–2.15 to –0.67); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.82 ± 3.85 (3.04 to 4.69); fl ow percentage variation 9.76  ± 8.08 (8.11 to 11.41); r  = 0.969 and r2 = 0.939 (P <0.000). (3) FiO2 variation –0.005 ± 0.26 (–0001 to 0009); FiO2 percentage variation –0.72 ± 5.2 (–1.5 to 0.1); r = 0.996 and r2 = 0.992 (P <0.000). Flow variation (l/minute) 3.91 ± 1.26 (3.69 to 4.13); fl ow percentage variation 12.77 ± 5.33 (11.84 to 13.7); r = 0.996 and r2 = 0.992 (P <0.000). See Figure 1. Results We delivered teaching sessions to 100% (9/9) of junior doctors, 100% (71/71) of nursing staff and other health professionals. We obtained feedback from 90% (76/84), showing an improvement in understanding of capnography from 73% of respondents to 100%, with 87% reporting that the teaching aid made capnography interpretation much easier. All felt the training would improve patient safety, and 97% felt it would be worthwhile training in other ICUs. g Conclusion The FiO2 percentage variation in the Airvo® is higher than the other two devices, with no clinical relevance. The fl ow percentage variation of Evita XL® is superior to the other two devices; this may have some clinical relevance. Surface electromyography of respiratory muscles during a CPAP trial for weaning g S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) 1. Cook TM, et al. BJA. 2011;106:617-31. 2. McGrath BA. BJA. 2014;113:521. 3. Cook TM, et al. Anaesthesia. 2013;68:421. Introduction Weaning from mechanical ventilation is an important concern in ICU clinical practice. Surface electromyography (sEMG) [1] is a non-invasive tool to assess activity of diff erent muscles. We describe sEMG patterns of respiratory muscles during a CPAP trial [2] in patients undergoing pressure support ventilation. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Use of ‘hats and caps’ enabled delivery of short bedside teaching sessions to clinical staff in ICU during everyday work. Feedback shows a marked improvement in confi dence around capnography interpretation. It may have value in other ICUs to improve staff understanding of capnography and improve patient safety. References P266 Surface electromyography of respiratory muscles during a CPAP trial for weaning S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) Surface electromyography of respiratory muscles during a CPAP trial for weaning S Arrigoni Marocco, G Bellani, A Bronco, M Pozzi, F Rabboni, G Villa, N Eronia, A Pesenti San Gerardo Hospital, Monza, Italy Critical Care 2015, 19(Suppl 1):P266 (doi: 10.1186/cc14346) Ventilatory response during intentional early rehabilitation in patients with mechanical ventilation K Obata1, N Shiba2, T Takahashi3, S Ichiba2, Y Ujike2 1 d C Ok l Ok , , , , j 1Japanese Red Cross Okayama Hospital, Okayama, Japan; 2Okayama University Graduate School of Medicine, Okayama, Japan; 3Tokyo University of Technology, Tokyo, Japan Critical Care 2015, 19(Suppl 1):P263 (doi: 10.1186/cc14343) Introduction Intentional early rehabilitation with mechanical ventilation in ICUs is performed in clinical settings. However, strict indexes for safe rehabilitation have not been fully elucidated. The purpose of this study is to analyze ventilator response (VR) between healthy volunteers and patients with mechanical ventilation. Methods Sixteen healthy volunteers (Control group) and 13 patients on mechanical ventilation (MV group) were enrolled in this study. Both groups were positioned in a variety of postures (baseline in a supine position, settled back 30°, settled back 60°, and sitting position) and measured with an indirect calorimeter. The instantaneous energy expenditure (EE), tidal volume (TV), respiratory rate (RR) and minute expiratory volume (VE) were non-invasively measured in each posture. Figure 1 (abstract P264). ‘Hats and caps’ capnography training guide. S93 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Systematic procedures including non-invasive ventilation improve morbidity in sleeve gastrectomy I Auriant, N Devos, S Rossi I Auriant, N Devos, S Rossi Critical Care 2015, 19(Suppl 1):P269 (doi: 10.1186/cc14349) Table 1 (abstract P267). Mean ventilator-days/reintubation rates Table 1 (abstract P267). Mean ventilator-days/reintubation rates 2009 2010 2011 2012 2013 2014 MICU mean ventilator-days 7.71 6.81 5.90 5.88 5.50 5.50* MICU reintubation rates 14.5% 9.8% 9.7% 9.8% 13.9% 8.3% SICU mean ventilator-days 5.91 5.93 5.68 5.93 4.93** 5.20* SICU reintubation rates 5.2% 3.0% 4.1% 4.3% 4.7% 7.5% *, **Signifi cance as described in Results. Introduction Postoperative morbidity after sleeve gastrectomy is decreasing, but remains signifi cant. Bleeding and surgical fi stules remain the leading causes of morbidity and mortality. In several studies in postoperative care of obese patients, non-invasive positive pressure ventilation (NPPV) reduced the risk of lower respiratory tract infection and pneumonia [1], thereby reducing in-hospital morbidity. The aim of study was to describe whether systematic use of NPPV improves morbidity in the postoperative care of sleeve gastrectomy. Methods A 4-year before–after study was conducted in a 19-bed intermediate care unit of a private hospital. Before period: standard treatment – all patients received oxygen supplementation to achieve SaO2 above 90%. After period: standard treatment plus NPPV  – all patients were submitted to a systematic postoperative protocol: NPPV was provided using an oxygen CIPAP system with 5 cmH2O. Statistical analysis: complication rates were compared using the chi-square test. P <0.05 was considered statistically signifi cant. Conclusion Initiatives to reduce ventilator-days per patient realized signifi cant reductions from 2009 to 2014 while reintubation rates were unaff ected. One component of the bundle, early mobilization, introduced in the SICU in 2013 was associated with an additional reduction in mean ventilator-days. y gi Results A total of 857 patients were included. Inclusion characteristics were similar in the two groups: Before group – noNPPV: 352 patients, 2010 to 2011. Age: 40.58 ± 10.94, BMI: 42.79 ± 5.51, sex ratio F/M: 0.81. After group – NPPV: 504 patients, 2012 to 2013. Age: 40.81 ± 11.24, BMI: 42.92 ± 5.09, sex ratio F/M: 0.77. There is a signifi cant between- group diff erence in the complication rate: Before group – noNPPV: 10 surgical fi stula (2.84%) and six postoperative bleeding (1.70%); After group – NPPV: seven surgical fi stula (1.39%) and three postoperative bleeding (0.6%). The overall complication rate fell from 4.54% to 1.98%. The chi-square statistic = 4.58. The number of degrees of freedom is 1. References 1. Chittawatanarat K, Thongchai C. Spontaneous breathing trial with low pressure support protocol for weaning respirator in surgical ICU. J Med Assoc Thai. 2009;92:1306-12. 1. Chittawatanarat K, Thongchai C. Spontaneous breathing trial with low pressure support protocol for weaning respirator in surgical ICU. J Med Assoc Thai. 2009;92:1306-12. Methods Ventilator-day data were compiled from 2009 to 2014 for MICU and SICU in our facility. Reintubation rates were calculated when intubations were required >1 day after an extubation. 2. Esteban A, Alia I, Gordo F, et al. Extubation outcome after spontaneous breathing trials with T-tube or pressure support ventilation. The Spanish Lung Failure Collaborative Group. Am J Respir Crit Care Med. 1997;156:459-65. 2. Esteban A, Alia I, Gordo F, et al. Extubation outcome after spontaneous breathing trials with T-tube or pressure support ventilation. The Spanish Lung Failure Collaborative Group. Am J Respir Crit Care Med. 1997;156:459-65. Results Ventilator volume ranged from 639 to 766 distinct patients/ year in the MICU and from 555 to 687 for the SICU. Ventilator-day reduction was signifi cant (P <0.01) for the MICU* (7.7 to 5.5, –29%) and the SICU* (5.91 to 5.20,  –12%). Reduction patterns diff ered between the units as the SICU had a distinct reduction (**P = 0.007) between 2012 and 2013 coinciding with implementation of an early mobilization program. Reintubation rates diff ered between the units and rates did not increase with decreasing mean patient ventilator- days. See Table 1. Comparison of three methods of applying high fl ow nasal oxygen: in vitro study Figure 1 (abstract P266). p g gi Results (1) FiO2 variation –0.001 ± 0.09 (–0.01 to 0.002); FiO2 percentage variation –0.012  ± 1.88 (–0.27 to 0.25); r2  = 0.999 and r  = 0.998 (P  <0.000). Flow variation (l/minute) 5.45  ± 3.23 (4.94 to 5.96); fl ow percentage variation 19.59 ± 11.63 (17.75 to 21.43); r = 0. 997 and r2 = 0.994 (P <0.000). (2) FiO2 variation –0.007 ± 0.26 (–0.011/–0.003); FiO2 Figure 1 (abstract P265). Programmed and measured fl ow. Figure 1 (abstract P265) Programmed and measured flow Figure 1 (abstract P266). Figure 1 (abstract P265). Programmed and measured fl ow. S94 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 before discontinue mechanical ventilation and extubation in the surgical intensive care unit (SICU). tidal volume (r = –0.16, P = 0.02). In patients with EAdi we confi rmed a tight correlation between costmar and EAdi (r = 0.62, P <0.001). See Figure 1. tidal volume (r = –0.16, P = 0.02). In patients with EAdi we confi rmed a tight correlation between costmar and EAdi (r = 0.62, P <0.001). See Figure 1. Methods We performed a prospective open-label randomized control study (non-inferiority trial) in SICU patients who were intubated and used mechanical ventilation, and appropriated discontinuation of the ventilator between June 2011 and November 2013. All patients underwent the same weaning protocol. The appropriated patients for discontinuation of MV were randomized into low pressure support up to 7  cmH2O and T-piece method. Reintubation within 72  hours, pneumonia after extubation, and hospital mortality were recorded. The statistical signifi cant diff erence was considered when P <0.05. Conclusion sEMG indicated that while diaphragm activation remains constant during the CPAP period, accessory muscles were progressively recruited and particularly in the conditions of increased respiratory rate and lower tidal volumes. Ventilator-day reductions not associated with reintubations and further reduced by an early mobilization program Ventilator-day reductions not associated with reintubations and further reduced by an early mobilization program M Palmquist-Tess, A Adams, J Mangan, D Owen, M LeClaire HCMC, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P267 (doi: 10.1186/cc14347) y y p g M Palmquist-Tess, A Adams, J Mangan, D Owen, M LeClaire HCMC, Minneapolis, MN, USA Introduction Mechanical ventilation is associated with increased risk of pneumonia, barotrauma, VILI, VAP, ARDS and mortality. From 2009 to 2014 in the MICU/SICU of our facility, eff orts to reduce ventilator-days included: noninvasive ventilation, sedation reduction, daily sedation vacations and weaning protocols. In 2013, an early mobilization of ventilated patients in the SICU was initiated. Aggressive ventilator-day reduction eff orts may be expected to lead to premature extubations and reintubations. Conclusion The outcomes after discontinuation of the mechanical ventilator between low pressure support and T-piece was not diff erence in term of reintubation, pneumonia after extubation and hospital mortality. References 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. 1. Yokoba M, et al. Respir Physiol Neurobiol. 2003;137:51-60. 2. Girard TD, et al. Lancet. 2008;371:126-34. gif Results A total of 520 patients were randomized into two groups: low pressure support group (260 patients) and T-piece group (260 patients). There was no diff erence in age, gender, body mass index, comorbidity, site of surgery, Charlson Comorbidity Index and Acute Physiologic and Chronic Health II score between groups (P >0.05). Regarding the intention to treat analysis, there were no diff erences between groups in reintubation rate (PSV 10% vs. T 14.6%; P = 0.109), pneumonia after extubation (PSV 14.2% vs. T 11.9%; P = 0.435) and hospital mortality rate (PSV 3.1% vs. T 3.5%; P = 0.805). Systematic procedures including non-invasive ventilation improve morbidity in sleeve gastrectomy The value returned from the chi-square statistic is P <5%.i Chiumello D, et al. Non-invasive ventilation in postoperative patients: a sytematic review. Intensive Care Med. 2011;37:918-29. P271 Systematic alveolar recruitment after cardiac surgery AL Lafarge, CK Kerneis, F Scalbert, LL Larnier, AB Brusset, PE Estagnasie, PS Squara Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P271 (doi: 10.1186/cc14351) Introduction The aim of our study was to investigate the value of C-reactive protein (CRP) and procalcitonin (PCT) in identifi cation of the systemic infl ammatory response syndrome (SIRS) and other complications in the early postoperative period after cardiac surgery with cardiopulmonary bypass (CPB) [1]. Methods In 93 patients undergoing coronary artery bypass graft surgery with CPB, after Ethical Committee approval in a prospective study, serum PCT and CRP values were collected before operation and daily until postoperative day 5. All patients were divided post hoc into patients with SIRS (n = 42) and patients without SIRS (n = 51). Student’s t test, the Mann–Whitney U test and receiver operating characteristic (ROC) curves were used. Introduction We designed a pilot study to evaluate the interest of an early systematic acute recruitment maneuver (ARM) in postcardiac surgery hypoxemic patients in order to properly design a larger trial. Methods This randomized controlled trial included consecutive patients operated on in our institution. Three hours after surgery, hypoxemic patients (PaO2 <300  mmHg, FIO2  = 1) were randomly assigned to ARM or control (H0). ARM was performed by applying once a positive end-expiratory pressure of 35  cmH2O during 45  seconds. Blood gases and hemodynamic variables were collected at H1, H8, H24 and H48. The primary endpoint was the duration of mechanical ventilation (MV). Secondary endpoints were survival rate, ICU length of stay and the occurrence of pneumonia. Results The comparison of serum CRP values in patients with or without SIRS on postoperative day 1 until postoperative day 5 demonstrated an increase in both groups without signifi cant diff erences (P >0.05). The PCT levels increased more signifi cantly in SIRS patients (5.78  ± 3.21 ng/ml vs. 1.23 ± 0.31 ng/ml) compared with patients without SIRS (P  = 0.0001) on postoperative day 1. In patients with postoperative complications (21/93, 22%) (circulatory failure  = 10, pneumonia  = 2, respiratory insuffi ciency  = 9, sepsis  = 0), PCT levels remained elevated until postoperative day 5 (6.11 ± 2.87 ng/ml) but diminished in patients with SIRS (0.96 ± 0.23 ng/ml) (P <0.0001). Early postoperative pulmonary complications following heart transplantation A Camkiran Firat, Ö Kömürcü, P Zeyneloglu, M Türker, A Sezgin, A Pirat Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P270 (doi: 10.1186/cc14350) Introduction The aim of this study was to determine the types, incidence, and risk factors for early postoperative pulmonary complications in heart transplantation recipients. Methods We retrospectively collected data from the records of consecutive heart transplantations from January 2003 to December 2013. A total of 83 patients underwent heart transplantation. Those patients younger than 10  years (n  = 9) and the patients who died intraoperatively (n = 1) or during the fi rst postoperative day (n = 1) were not included in the analyses. The data collected for each case were demographic features, duration of mechanical ventilation, respiratory problems that developed during the ICU stay, and early postoperative mortality (<30  days). The respiratory complications that we sought were pleural eff usion, pneumonia, pulmonary atelectasis, pulmonary edema, pneumothorax, and acute respiratory failure. Figure 1 (abstract P271). p p y Results Of the 72 patients considered, 52 (72.2%) were male. The mean age at the time of transplantation was 32.1  ± 16.6  years. The mean duration of postoperative mechanical ventilation was 71.8  ± 126.6 hours. The mean length of ICU stay was 13.5 ± 18.0 days. Two patients (2.8%) and one patient (1.4%) required extracorporeal membrane oxygenation support and intra-aortic balloon pump support, respectively, due to low cardiac output or primary graft failure postoperatively. Twenty-fi ve patients (34.7%) developed early postoperative respiratory complications. The most frequent problem was pleural eff usion (n = 19, 26.4%) followed by atelectasis (n = 6, 8.3%), acute respiratory distress syndrome (n = 5, 6.9%), pulmonary edema (n = 4, 5.6%), and pneumonia (n = 3, 4.2%). Postoperative duration of mechanical ventilation (44.2 ± 59.2 hours vs. 123.8 ± 190.8 hours, P = 0.005) and the length of ICU stay postoperatively (10.1  ± 5.8  hours vs. 19.8 ± 28.9 hours, P = 0.03) were longer among patients who had respiratory problems. Postoperative length of stay in the hospital (22.3 ± 12.5 days vs. 30.3 ± 38.3 days, P = 0.75) was similar in the two groups. The overall mortality rate was 12.5% (n  = 9 patients). The patients who had respiratory problems did not show higher mortality than those who did not have respiratory problems (16.0% vs. 10.6%, P = 0.71). the two groups. Early postoperative pulmonary complications following heart transplantation At H1, PaO2/FIO2 was 367 ± 15 in the recruited group versus 299 ± 15 mmHg in the control group, P = 0.002. At H8 and 24 the diff erence was not signifi cant. At H48, the PaO2/FIO2 was lower in the recruited group (296 ± 10 vs. 343 ± 11 mmHg, P = 0.003) (Figure 1). The duration of mechanical ventilation (invasive + non-invasive) was lower in the recruited group (total 6.4 ± 1.4 vs. 8.4 ± 1.4 hours, P = 0.02). The survival rate, the length of stay in the ICU and the occurrence of pneumonia were similar in the two groups (P >0.2). Conclusion We can speculate that the inverse evolution of the blood oxygenation between the ARM group versus control may be due to: barotraumatism of normal alveoli during the ARM and/or a higher de- recruitment rate after ARM due to the shorter mechanical ventilation support. This pilot study shows that a unique ARM decreased the duration of MV in cardiac surgery patients but this may have subsequent detrimental eff ects on blood oxygenation. P272 Open-label randomized control trial between low pressure support and T-piece method for discontinuation from mechanical ventilator and extubation in general surgical ICUs g g K Chittawatanarat, S Orrapin, S Orrapin Chiang Mai University, Chiang Mai, Thailand Chiang Mai University, Chiang Mai, Thailand g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) g y g Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) y Critical Care 2015, 19(Suppl 1):P268 (doi: 10.1186/cc14348) Introduction In routine practice for most surgical patients in Thailand, all appropriated patients with planned discontinuation of mechanical ventilator (MV) are routinely changed to T-piece before extubation. However, this method needs to alter the instrument for testing tolerability of the patient. The objective of this study was to compare continuous low pressure support (PSV) and the T-piece method (T) Conclusion Systematic use of NPPV signifi cantly improves morbidity in the postoperative care of sleeve gastrectomy. Reference Conclusion Systematic use of NPPV signifi cantly improves morbidity in the postoperative care of sleeve gastrectomy. Chiumello D, et al. Non-invasive ventilation in postoperative patients: a sytematic review. Intensive Care Med. 2011;37:918-29. S95 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P271). P270 Figure 1 (abstract P271). P270 Early postoperative pulmonary complications following heart transplantation A Camkiran Firat, Ö Kömürcü, P Zeyneloglu, M Türker, A Sezgin, A Pirat Baskent University, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P270 (doi: 10.1186/cc14350) Is procalcitonin a valuable marker for identifi cation of postoperative complications after coronary artery bypass graft surgery with cardiopulmonary bypass? y y A Baysal1, M Dogukan2, H Toman3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Adiyaman University Research and Training Hospital, Adiyaman, Turkey; 3Canakkale 18 Mart University Hospital, Canakkale, Turkey Critical Care 2015, 19(Suppl 1):P272 (doi: 10.1186/cc14352) A Baysal1, M Dogukan2, H Toman3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Adiyaman University Research and Training Hospital, Adiyaman, Turkey; 3Canakkale 18 Mart University Hospital, Canakkale, Turkey Critical Care 2015, 19(Suppl 1):P272 (doi: 10.1186/cc14352) Conclusion Respiratory complications were relatively common in our cohort of heart transplant recipients. However, these complications were mostly self-limiting and did not result in increased mortality. P271 A PCT threshold value of 2.79 ng/ml was able to discriminate between postoperative complications in patients with or without SIRS with a sensitivity of y Results We included 124 patients, age 67.5 ± 10.6 years, M/F sex ratio 95/29, left ventricle ejection fraction 58.8 ± 10.6%, forced expiratory volume 94 ± 23% of the predicted value, bypass/valve ratio 82/53. The preoperative and postoperative PaO2/FIO2 were 401 ± 66 and 204 ± 66 mmHg, respectively (P <0.0001). The hemodynamic and ventilation status as well as the fl uid and inotrope supports were comparable in S96 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 developed miniaturized device consisting of a centrifugal pump and a membrane ventilator (iLA Activve®; Novalung, Germany) allows eff ective decarboxylation via a jugular double lumen cannula. So far no data on gas exchange in this setting exist to date. 82.5% and a specifi city of 70% (area under the curve: 0.76  ± 0.05; P <0.01) on postoperative day 1. Conclusion After cardiac surgery with CPB, PCT values increased signifi cantly in the SIRS group of patients compared with patients without SIRS on postoperative day 1 and remained elevated until postoperative day 5. In the early postoperative period, early rise of PCT values may help to discriminate the development of postoperative complications in patients with or without SIRS. Methods We included 10 patients receiving iLA Activve® due to hypercapnic respiratory failure as bridge-to-transplant or obstructive lung disease. Sweep gas fl ow was increased in steps from 1 to 14 l/ minute at constant blood fl ow (phase 1). Similarly, blood fl ow was gradually increased at constant sweep gas fl ow (phase 2). At each step, gas transfer via the membrane as well as arterial blood gas samples were obtained. 1. Delannoy B, Guye ML, Slaiman DH, Lehot JJ, Cannesson M. Eff ect of cardiopulmonary bypass on activated partial thromboplastin time waveform analysis, serum procalcitonin and C-reactive protein concentrations. Crit Care. 2009;13:R180. 1. Delannoy B, Guye ML, Slaiman DH, Lehot JJ, Cannesson M. Eff ect of cardiopulmonary bypass on activated partial thromboplastin time waveform analysis, serum procalcitonin and C-reactive protein concentrations. Crit Care. 2009;13:R180. Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery p p dysfunction following open heart surgery A Baysal1, I Ozkaynak2, M Dogukan3 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Kahramanmaraþ Sütçü Ýmam University Hospital, Kahramanmaraþ, Turkey; 3Adiyaman University Research and Training Hospital, Adiyaman, Turkey Critical Care 2015, 19(Suppl 1):P273 (doi: 10.1186/cc14353) A Baysal1, I Ozkaynak2, M Dogukan3 A Baysal , I Ozkaynak , M Dogukan 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 1Kartal Kouyolu Research and Training Hospital, Istanbul, Turkey; 2Kahramanmaraþ Sütçü Ýmam University Hospital, Kahramanmaraþ, Turkey; 3Adiyaman University Research and Training Hospital, Adiyaman, Turkey Critical Care 2015, 19(Suppl 1):P273 (doi: 10.1186/cc14353) Conclusion Increasing sweep gas fl ow results in eff ective CO2 removal which can be further reinforced by raising blood fl ow. The clinically relevant oxygenation eff ect even in this setting of low invasivity could broaden the range of indications towards hypercapnic lung failure with mild to moderate hypoxia. Figure 1 (abstract P274). PaO2 with increasing blood fl ow (*P <0.0001). Introduction Our goal was to investigate the incidence of postoperative jaundice in open heart surgery patients and to determine the risk factors associated with hepatic dysfunction. Methods A total of 292 patients were included in a prospective study design. Patients undergoing on-pump coronary artery bypass graft surgery (CABG) (n = 154) and valve repair surgery (mitral, mitral and aortic valve and/or tricuspid valve) (n  = 138) were included. Postoperative hyperbilirubinemia was defi ned as occurrence of a plasma total bilirubin concentration of more than 34  μmol/l (2  mg/ dl) in any measurement during the postoperative period. All patients were divided into groups with or without hyperbilirubinemia. Liver enzymes were collected on postoperative days 1, 7, 14 and 30. The risk factors including age, cardiopulmonary bypass time, number of blood transfusions, inotropic support, use of intra-aortic balloon pump and ICU stay were evaluated with logistic regression. Figure 1 (abstract P274). PaO2 with increasing blood fl ow (*P <0.0001). y g g Results Postoperative hyperbilirubinemia was observed in 27 of 292 patients (9.3%). The numbers of valves replaced, preoperative total bilirubin concentration, increased cardiopulmonary bypass time, higher number of inotropic support agents, and use of intra-aortic balloon pump correlate with hyperbilirubinemia on postoperative day  7 (P  <0.05). Independent risk factors of early postoperative jaundice are; multiple valve replacement surgery, ejection fraction and use of intraaortic balloon pump (R = 0.58, R2 = 0.33, F = 26.44, P <0.001). P275 P275 Safety and effi cacy of extracorporeal CO2 removal combined with continuous renal replacement therapy in patients presenting both acute respiratory distress syndrome and acute kidney injury J Allardet-Servent, M Castanier, T Signouret, A Lepidi, R Soundaravelou, J Seghboyan Hopital Européen Marseille, France Critical Care 2015, 19(Suppl 1):P275 (doi: 10.1186/cc14355) P275 Safety and effi cacy of extracorporeal CO2 removal combined with continuous renal replacement therapy in patients presenting both acute respiratory distress syndrome and acute kidney injury J Allardet-Servent, M Castanier, T Signouret, A Lepidi, R Soundaravelou, J Seghboyan Hopital Européen Marseille, France Critical Care 2015, 19(Suppl 1):P275 (doi: 10.1186/cc14355) P271 Results During phase 1, we observed a signifi cant increase in CO2 transfer together with a decrease in PaCO2 levels from a median of 66 mmHg (range 46 to 85) to 49 (31 to 65) mmHg from 1 to 14 l/ minute sweep gas fl ow, while arterial oxygenation deteriorated with high sweep gas fl ow rates. During phase 2, oxygen transfer signifi cantly increased leading to an increase in PaO2 from 67 (49 to 87) at 0.5 l/ minute to 117 (66 to 305) mmHg at 2.0 l/minute. Higher blood fl ow rates also signifi cantly enhanced decarboxylation. Increasing blood fl ow to 2.0 l/minute led to negative suction pressures of more than –100 mmHg and signs of hemolysis. See Figure 1. Prediction of risk factors related to the development of hepatic dysfunction following open heart surgery The ICU stay was signifi cantly longer in group 2 (11.52 ± 3.76 days) as compared with group 1 (2.79 ± 1.36 days) (P <0.001). g y Conclusion Patients undergoing multiple valve replacement procedures are at greater risk for the development of postoperative hyperbilirubinemia and an association with prolonged ICU stay was observed. Other risk factors including ejection fraction, increased cardiopulmonary bypass time and use of intra-aortic balloon pump are also important as they have been reported to increase postoperative complications [1]. Conclusion Increasing sweep gas fl ow results in eff ective CO2 removal which can be further reinforced by raising blood fl ow. The clinically relevant oxygenation eff ect even in this setting of low invasivity could broaden the range of indications towards hypercapnic lung failure with mild to moderate hypoxia. p Reference 1. Nishi H, Sakaguchi T, Miyagawa S, et al. Frequency, risk factors and prognosis of postoperative hyperbilirubinemia after heart valve surgery. Cardiology. 2012;122:12-9. Interhospital transfer of patients in extracorporeal membrane oxygenation F Socci, S Di Valvasone, M Ciapetti, A Franci, M Bonizzoli, G Cianchi, S Batacchi, A Peris Careggi Hospital, Firenze, Italy Critical Care 2015 19(Suppl 1):P276 (doi: 10 1186/cc14356) gg y Critical Care 2015, 19(Suppl 1):P276 (doi: 10.1186/cc14356) gg y Critical Care 2015, 19(Suppl 1):P276 (doi: 10.1186/cc14356) Introduction The transfer of patients in extracorporeal membrane oxygenation (ECMO) from a peripheral hospital to a tertiary center represents a high-risk situation of adverse events [1]. The aim of this retrospective study is to determine the feasibility and safety of interhospital transfer for critically ill patients with ECMO support. Methods We collected data for the ECMO Regional Reference Centre Careggi Hospital activity from September 2009 to June 2014. In this study, 57 transfers were examined. The ECMO service is activated by a telephone call from a peripheral hospital. The team is represented by an intensivist, a heart surgeon, a cardiologist, a perfusionist and an intensive care nurse, all previously trained in the management of patients with ECMO. Medical personnel and the necessary equipment are transported by an ambulance and a van, specially designed and equipped for the transfer of patients with ECMO. Methods Retrospectively, we examined 20 patients with cerebral MRI (including SWI) who had suff ered from severe ARDS and received ECMO therapy. The MRI slides were anonymized and analyzed by two experienced neuroradiologists. Based on the distribution pattern and characteristic, a modifi ed HACE score (mHCS) was surveyed [2]. i y Results Six of 20 patients (30%) showed multiple MH with emphasis in the splenium of the corpus callosum. Eight patients had sporadic MH in the parenchyma of the brain but not in the corpus callosum. The remaining six patients had no intracerebral alterations. The distribution of MH with involvement of the splenium resembled that seen in HACE survivors. Results In this study, 57 patients transferred from the peripheral hospital to the ECMO center were examined; in all cases the ECMO system was implanted in the peripheral hospital (54 venovenous ECMO and three venoarterious ECMO). On average, trails were 271  km  ± 304 (round trip) (minimum 14 km to maximum 939 km). The activation time from the call to the ambulance departure from our hospital was an average of 2 hours 27 minutes 13 seconds ± 1 hour 25 minutes 35 seconds. Determinants of gas exchange during extracorporeal CO2 removal using a novel pump-driven venovenous gas exchange system in a minimally invasive setting y g A Hermann, K Riss, P Schellongowski, A Bojic, P Wohlfarth, O Robak, W Sperr, T Staudinger Medical University of Vienna, Austria Critical Care 2015, 19(Suppl 1):P274 (doi: 10.1186/cc14354) Introduction Pump-driven venovenous extracorporeal CO2 removal (ECCO2-R) increasingly takes root in hypercapnic lung failure to minimize ventilation invasiveness or to avoid intubation. A recently Introduction Pulmonary overdistension has been observed in 33% of patients with acute respiratory distress syndrome (ARDS) despite low tidal volume (6 ml/kg ideal body weight) ventilation [1]. Tidal volume S97 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (VT) reduction from 6 to 4  ml/kg attenuates overdistension but is associated with hypercarbia [2]. We thought to combine extracorporeal CO2 removal (ECCO2R) with continuous renal replacement therapy (CRRT) through the insertion of an oxygenator membrane within the hemofi ltration circuit in patients presenting both ARDS and acute kidney injury (AKI).i the devices during transport were not recorded; in some cases it was necessary to manage minor complications (circuit cavitation, minor vascula accesses bleeding). Conclusion Some studies have found several complications during transfer of patients in ECMO [2]. In our experience, there were no complications during the transfer of ECMO patients, even for longer trips. A wide and thorough clinical evaluation and multidisciplinary ECMO team allowed the optimization of clinical parameters before transport and a safely transfer. The start of ECMO treatment at peripheral hospitals and the transfer of patients in ECMO may be a viable option compared with conventional ventilation. Our data suggest that ECMO can be set up safely in peripheral hospitals by a multidisciplinary highly specialized ECMO team [3]. y j y Methods A fi rst set of measurement was performed at 6 ml/kg before and after ECCO2R. Twenty minutes later, VT was reduced to 4 ml/kg for the remainder of the study period (72 hours). Ventilator settings were those of the ARMA trial. The CRRT mode was hemofi ltration with 33% of predilution. Ultrafi ltration was adjusted to achieve a fi ltration fraction of 15%. Sweep gas fl ow was constant at 8 l/minute. The primary endpoint was a 20% reduction of PaCO2 at 20 minutes after initiation of ECCO2R. Results Eight patients were studied. Age was 69 ± 11 years, SAPS II was 68 ± 9 and SOFA score was 13 ± 4 at inclusion. P276 I h Introduction Cerebral microhemorrhages (MH) are diminutive focal bleedings which can be detected best by MRI using susceptibility- weighted imaging sequences (SWI). They can be found in a variety of neurologic diseases. The pattern of distribution can lead to the underlying pathomechanism [1]. Survivors of high-altitude cerebral edema (HACE) showed multiple MH, predominantly in the splenium of the corpus callosum. Mountaineers with a lack of acclimatization to high altitudes tend to suff er from HACE. Hypoxemia in great heights is discussed to be the main trigger of HACE [2]. Acute respiratory distress syndrome (ADRS) is characterized by oxygenation failure in mechanically ventilated patients. The severity is classifi ed by the ratio of arterial oxygen tension to fraction of inspired oxygen [3]. In some patients suff ering from severe ARDS, refractory to conventional therapy, venovenous extracorporeal membrane oxygenation therapy is the therapeutic option to ensure oxygenation. Determinants of gas exchange during extracorporeal CO2 removal using a novel pump-driven venovenous gas exchange system in a minimally invasive setting Blood fl ow, at the inlet of the oxygenator membrane, was 400 ± 4 ml/minute. CO2 removal rate was 84 ± 4 ml/minute. Initiating ECCO2R, at 6 ml/kg, induced a mean PaCO2 reduction of 17% (41 ± 5.5 to 33.9 ± 5.6 mmHg, P <0.001). Then, lowering the VT to 4  ml/kg induced a mean PaCO2 increase of 25% (33.9 ± 5.6 to 42.6 ± 8 mmHg) and a mean PaO2/FIO2 ratio increase of 8% (176 ± 63 to 190 ± 61). Minute ventilation decrease from 7.4 ± 1.6 to 5 ± 1.2 l/minute. Respiratory system compliance did not vary. No major complications were observed. y j y Methods A fi rst set of measurement was performed at 6 ml/kg before and after ECCO2R. Twenty minutes later, VT was reduced to 4 ml/kg for the remainder of the study period (72 hours). Ventilator settings were those of the ARMA trial. The CRRT mode was hemofi ltration with 33% of predilution. Ultrafi ltration was adjusted to achieve a fi ltration fraction of 15%. Sweep gas fl ow was constant at 8 l/minute. The primary endpoint was a 20% reduction of PaCO2 at 20 minutes after initiation of ECCO2R. Interhospital transfer of patients in extracorporeal membrane oxygenation Transfer duration (from activation to return to the ECMO center) was an average of 8  hours 25  minutes 6  seconds  ± 3  hours 27  minutes 58 seconds (minimum 3 hours to maximum 16 hours 55 minutes). The stop time (necessary for evaluation of the patient and for placement of the ECMO system) was an average of 3 hours 53 minutes 40 seconds ± 1 hour 6 minutes 35 seconds (minimum 2 hours 5 minutes to maximum 7 hours 30 minutes). Major complications related to malfunctions of Conclusion Based on these results, we postulate that hypoxemia is one of the main players in the development of splenium-associated MH, not only in HACE but also in severe ARDS and other diseases accompanied with severe hypoxemia. Further investigations have to examine potential triggers and special circumstances concerning ARDS treatment which lead to MH in this distinctive pattern. 1. Renard D, et al. J Neurol Neurosurg Psychiatry. 2014;85:1041-8. 2. Schommer K, et al. Neurology. 2013;81:1776-9. 3. Force ADT, et al. JAMA. 2012;307:2526-33. References 2 2 Results Eight patients were studied. Age was 69 ± 11 years, SAPS II was 68 ± 9 and SOFA score was 13 ± 4 at inclusion. Blood fl ow, at the inlet of the oxygenator membrane, was 400 ± 4 ml/minute. CO2 removal rate was 84 ± 4 ml/minute. Initiating ECCO2R, at 6 ml/kg, induced a mean PaCO2 reduction of 17% (41 ± 5.5 to 33.9 ± 5.6 mmHg, P <0.001). Then, lowering the VT to 4  ml/kg induced a mean PaCO2 increase of 25% (33.9 ± 5.6 to 42.6 ± 8 mmHg) and a mean PaO2/FIO2 ratio increase of 8% (176 ± 63 to 190 ± 61). Minute ventilation decrease from 7.4 ± 1.6 to 5 ± 1.2 l/minute. Respiratory system compliance did not vary. No major complications were observed. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 1. Lindén V, Palmér K, Reinhard J, Westman R, Ehrén H, Granholm T, et al. Inter- hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation-national and international experience. Intensive Care Med. 2001;27:1643-8. 2. Haneya A, Philipp A, Foltan M, Mueller T, Camboni D, Rupprecht L, et al. Extracorporeal circulatory systems in the interhospital transfer of critically ill patients: experience of a single institution. Ann Saudi Med. 2009;29:110-4. p p g 3. Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. p p g 3. Ciapetti M, Cianchi G, Zagli G, Greco C, Pasquini A, Spina R, et al. Feasibility of inter-hospital transportation using extra-corporeal membrane oxygenation (ECMO) support of patients aff ected by severe swine-fl u(H1N1)-related ARDS. Scand J Trauma Resusc Emerg Med. 2011;19:32. Conclusion Combining ECCO2R and CRRT in patients with ARDS and AKI is safe and feasible through the insertion of an oxygenator membrane within a RRT circuit. References 1. Terragni PP, Rosboch G, Tealdi A, et al. Tidal hyperinfl ation during low tidal volume ventilation in acute respiratory distress syndrome. Am J Respir Crit Care Med. 2007;175:160-6. P277 Microhemorrhages in the corpus callosum after treatment with extracorporeal membrane oxygenation S Riech, P Hellen, O Moerer, K Kallenberg, M Müller, M Quintel, M Knauth University Medical Center Goettingen, Germany Critical Care 2015, 19(Suppl 1):P277 (doi: 10.1186/cc14357) 2. Terragni PP, Del Sorbo L, Mascia L, et al. Tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal CO2 removal. Anesthesiology. 2009;111:826-35. 2. Terragni PP, Del Sorbo L, Mascia L, et al. Tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal CO2 removal. Anesthesiology. 2009;111:826-35. P279 Introduction Diff erential hypoxia is a pivotal problem in cardio- pulmonary failure patients with femoral venoarterial extracorporeal membrane oxygenation (VA ECMO) support. Although there was some attempt to deliver more oxygenated blood to the upper body, the mechanism of diff erential hypoxia has not been well investigated. Successful approach for emergent consent for ECMO research J Board1, S Vallance1, C Aubron2, D Pilcher1, V Pellegrino1, DJ Cooper1 1The Alfred Hospital, Melbourne, Australia; 2Monash University, Melbourne, Australia Critical Care 2015, 19(Suppl 1):P279 (doi: 10.1186/cc14359) f y Methods We used a sheep model of acute respiratory failure that was supported with femoral VA ECMO (from inferior vena cava to femoral artery (IVC-FA)), ECMO from superior vena cava to FA (SVC-FA), ECMO from IVC to carotid artery (IVC-CA) and ECMO adding an additional return cannula to internal jugular vein based on femoral VA ECMO (FA- IJV). Angiography and blood gas analysis were performed. Introduction The HELP-ECMO pilot study (Heparin low dose protocol versus standard care in critically ill patients undergoing ECMO; ACTRN12613001324707) is a randomised controlled phase II study evaluating two levels of heparin anticoagulation in patients with no requirement for full anticoagulation. This work is a substudy of the HELP-ECMO trial and describes the consent process of the parent study. At our site, consent for research is often obtained by the research coordinator with little involvement from investigators. However, the nature of the ECMO population required a modifi ed consent approach to be implemented given that ECMO is often inserted emergently. It required a model that would be successful out of hours and could be delivered by any member of the treating team. g g p y g y p Results Blood oxygen saturation (SO2) of IVC (83.6 ± 0.8%) was higher than that of SVC (40.3  ± 1.0%) in sheep with IVC-FA. Oxygen-rich blood was drained back to the ECMO circuit and poorly oxygenated blood in the SVC entered the right atrium (RA). SVC-FA achieved the oxygen-rich blood return from IVC to RA without shifting the arterial cannulation. SO2 of SVC and pulmonary artery increased (70.4 ± 1.0% and 73.4 ± 1.1%, respectively) subsequently. Compared with IVC-FA, Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. re 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. References 1. Renard D, et al. J Neurol Neurosurg Psychiatry. 2014;85:1041-8. 2. Schommer K, et al. Neurology. 2013;81:1776-9. 3. Force ADT, et al. JAMA. 2012;307:2526-33. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S98 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P278f less diff erences of venous oxygen return and attenuated diff erential hypoxia were also observed in IVC-CA and FA-IJV. See Figure 1.f P278 Diff erential venous oxygen return: a key factor of diff erential hypoxia in venoarterial extracorporeal membrane oxygenation X Hou1, X Yang1, Z Du1, J Xing1, C Jiang1, J Wang1, Z Xing1, H Wang1, H Zeng2 1Beijing Anzhen Hospital, Beijing, China; 2Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China Critical Care 2015, 19(Suppl 1):P278 (doi: 10.1186/cc14358) Conclusion Diff erential venous oxygen return is a key factor of diff erential hypoxia in VA ECMO. We can take advantage of the notion of diff erential venous oxygen return to choose better cannula confi guration in clinical practice. In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock M Bottiroli, T Maraffi , D Decaria, S Nonini, E Montrasio, K Gervasio, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P281 (doi: 10.1186/cc14361) y g M Bottiroli, T Maraffi , D Decaria, S Nonini, E Montrasio, K Gervasio, F Milazzo, R Paino Anestesia Rianimazione 3, A.O. Niguarda, Milan, Italy Critical Care 2015, 19(Suppl 1):P281 (doi: 10.1186/cc14361) Introduction Venoarterial (VA) ECMO is used for mechanical support in patients with cardiogenic shock (CS) unresponsive to medical therapy. Long-term survival and quality of life after hospital discharge have not yet been well analyzed. Methods We performed a retrospective observational study of patients admitted to the ICU for refractory CS from January 2010 to November 2014. Patients with postcardiotomy and/or post-transplant CS were excluded. Demographic, clinical and biochemical variables were collected. Continuous variables are presented as mean (standard deviation) and categorical variables as percentage. Long-term outcome and quality of life were assessed during scheduled follow-up evaluations or telephonic interviews. g p Results From April to December 2014, 30 patients were screened. Fourteen met the eligibility criteria and were approached for consent. Twelve patients were enrolled and randomised to receive either standard anticoagulation or low-dose heparin as per the study protocol. Consent was provided by the person responsible for 10 patients. One patient was competent to consent for themselves and one was enrolled under legislation allowing enrolment into research in the absence of a person responsible. There were two refusals. Seventy- one per cent of participants were approached out of hours. Eighty-six per cent were consented by clinicians. Twenty-one per cent of patients were consented by a non-investigator. Results We analyzed 23 consecutive patients undergoing VA ECMO for refractory CS. Etiologies of cardiac collapse were: 11 acute myocarditis, fi ve acute myocardial infarctions and seven acute decompensation of chronic cardiomyopathies (CCM). Thirteen patients died during the hospital stay and 10 survived. The main cause of ICU death was progressive multiple organ dysfunction (12/13). Baseline variables are presented in Table 1. All patients discharged from the hospital are still alive at follow-up (median 27 months, range 4 to 56) with a median NYHA class of 1 (range 1 to 2). All patients except one returned to an active style of life. P280 P280 Extracorporeal membrane oxygenator and ventricular assist device activity of a tertiary cardiothoracic centre: survival rates and length of ITU stay D Sarridou1, CP Walker1, N Rashid1, D Heaton1, I McGovern1, N Marczin2, J Mitchell1 1The Royal Brompton & Harefi eld NHS Foundation Trust, London, UK; 2Imperial College, London, UK Critical Care 2015, 19(Suppl 1):P280 (doi: 10.1186/cc14360) Extracorporeal membrane oxygenator and ventricular assist device activity of a tertiary cardiothoracic centre: survival rates and length of ITU stay y D Sarridou1, CP Walker1, N Rashid1, D Heaton1, I McGovern1, N Marczin2, J Mitchell1 1The Royal Brompton & Harefi eld NHS Foundation Trust, London, UK; 2Imperial College, London, UK Critical Care 2015, 19(Suppl 1):P280 (doi: 10.1186/cc14360) Introduction Harefi eld Hospital accepts patients as a tertiary centre for end-stage heart and respiratory failure for the south of England. Interventions include VA-ECMO, VV-ECMO as a bridge to lung transplantation, left ventricular assist devices (LVAD) as a bridge for heart transplantation, right ventricular assist devices (RVAD) and BIVADs. The aim of this review was to identify the total number of such patients, analyse the individual length of ITU stay and calculate survival and mortality rates for each intervention. Conclusion VA-ECMO is an eff ective treatment tool for refractory CS in patients with acute life-threatening heart failure. Patients aff ected by acute decompensation of CCM had poorer outcomes characterized by multiple organ dysfunction, as already known in the literature. y Methods Patients consisted of six groups: Group 1: VA ECMO, Group 2: VV-ECMO, Group 3: LVAD, Group 4: RVAD, Group 5: BIVAD, Group 6: combination of devices. Data were extracted from the Perfusion Department records and the intensive care dataset from 2011 to 2013. Data included length of ITU stay, outcome, indication for device insertion and device-related major complications.i In-hospital and long-term mortality after venoarterial ECMO for refractory cardiogenic shock Multivariate analysis (Cox) revealed pre-ECMO SOFA score (HR = 2.18, 95% CI = 1.016 to 4.6) and history of CCM (HR = 19, 95% CI = 2 to 178) and pre-ECMO lactate (HR = 1.2, 95% CI = 1.02 to 1.4) as independent risk factors for hospital mortality. Conclusion The model of consent described has proven to be successful in this challenging patient population. The ability of all staff to perform consent for the study has been a signifi cant factor in the success of the pilot. The review of study processes by research coordinators has supported this model. Table 1 (abstract P281) Alive Dead P value Age (years) 30 ± 18 43 ± 14 0.012 CCM 0 (0%) 7 (100%) 0.014 SOFA 8 ± 1.4 10 ± 1.9 0.002 Creatinine (mg/dl) 1.1 ± 0.3 2.2 ± 0.7 0.001 Bilirubin (mg/dl) 1 ± 0.4 2 ± 1.8 0.09 Lactate (mmol/l) 5 ± 2.1 8 ± 5.3 0.021 Platelets (103/μl) 257 ± 79 162 ± 99 0.03 Conclusion VA-ECMO is an eff ective treatment tool for refractory CS in patients with acute life-threatening heart failure. Patients aff ected by acute decompensation of CCM had poorer outcomes characterized by multiple organ dysfunction as already known in the literature P282 h P282 Characteristics of trauma patients with creatine kinase elevation N Assanangkornchai, O Akaraborworn, C Kongkamol, K Kaewsaengrueang Prince of Songkla University, Hatyai, Thailand Critical Care 2015, 19(Suppl 1):P282 (doi: 10.1186/cc14362) j p Results Forty patients were identifi ed. Twenty-nine were male and 11 female. Group 1 included 22 patients, Group 2: two patients, Group 3: four patients, Group 4: four patients, Group 5: zero, Group 6: eight patients treated with various combinations of ECMO or ventricular assist devices. ITU stay varied from 1 day to a maximum of 6 months intermittently for one patient. Duration of ITU stay for all 40 patients was 1,052  days with an average of 26.3  days per patient. Sixteen patients survived and were discharged to the Transplant Unit. Twenty- four patients died, putting the survival rate at 40% for this group. Conclusion This review demonstrates that the majority of these patients occupied intensive care beds for a prolonged period of time and despite the use of advanced support devices survival rates were signifi cantly lower than mortality rates. Results Forty patients were identifi ed. Twenty-nine were male and 11 female. Group 1 included 22 patients, Group 2: two patients, Group 3: four patients, Group 4: four patients, Group 5: zero, Group 6: eight patients treated with various combinations of ECMO or ventricular assist devices. ITU stay varied from 1 day to a maximum of 6 months intermittently for one patient. Duration of ITU stay for all 40 patients was 1,052  days with an average of 26.3  days per patient. Sixteen patients survived and were discharged to the Transplant Unit. Twenty- four patients died, putting the survival rate at 40% for this group. Introduction Rhabdomyolysis is a condition that results in the release of mainly creatine kinase (CK) and myoglobin from the breakdown of myocytes. Myoglobin has been known to cause renal failure (RF) and the CK level is routinely used as an indicator. A CK level >5,000  U/l was found to be associated with the risk of RF [1]. However, data are lacking on the level of CK to predict RF, especially in general trauma patients. The purpose of this study was to determine the initial CK level that predicts markedly elevated CK and the characteristics of trauma patients with elevated CK. Introduction Rhabdomyolysis is a condition that results in the release of mainly creatine kinase (CK) and myoglobin from the breakdown of myocytes. P279 Figure 1 (abstract P278). SO2 values from blood gas analysis and vena cava angiography. S99 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The HELP-ECMO pilot study is enrolling patients admitted to a large metropolitan ICU who require ECMO. Education on eligibility criteria and study processes was given to all ICU senior medical staff . Consent must be performed prior to the commencement of anticoagulation, often only a short time after ECMO cannulation. To facilitate recruitment at all hours, a HELP-ECMO study box is located in the ICU. Within the box is an instruction page outlining the screening, consent and randomisation process, as well as administrative tasks. Plain language statements, randomisation envelopes and consent documentation stickers are provided. A consent script is provided to ensure consistency across consenting personnel. The process is reviewed by the research coordinator the following day to confi rm local governance compliance. References Methods A retrospective observational study of adults with confi rmed rhabdomyolysis admitted to the Neurosciences Critical Care Unit between 2002 and 2012. Data collection included APACHE score, daily CK (with PEAK CK defi ned as the maximum CK recorded throughout ICU stay), creatinine, calcium, phosphate and bicarbonate levels, AKI, RRT, ICU length of stay and mortality. 1. Cartin Ceba R, et al. Risk factors for development of acute kidney injury in critically ill patients: a systematic review and meta-analysis of observational studies. Crit Care Res Pract. 2012:691013. 1. Cartin Ceba R, et al. Risk factors for development of acute kidney injury in critically ill patients: a systematic review and meta-analysis of observational studies. Crit Care Res Pract. 2012:691013. 2. Suh SH, et al. Acute kidney injury in patients with sepsis and septic shock: risk factors and clinical outcomes. Yonsei Med J. 2013;54:965-72. 2. Suh SH, et al. Acute kidney injury in patients with sepsis and septic shock: risk factors and clinical outcomes. Yonsei Med J. 2013;54:965-72. g y y Results A total of 232 patients met the inclusion criteria. Rhabdo- myolysis was associated with trauma (76%), medical (15%) and surgical (9%) admission diagnoses. Forty-fi ve (19%) patients developed AKI, with 29 (12.5%) requiring RRT. Mortality was signifi cantly higher in patients who developed AKI (62% vs. 18%, P <0.001). Average CK on admission was 5,009 IU/l (SD 12,403 IU/l). CK values remained greater than 2,000 IU/l for an average of 3.3 days (range 1 to 10 days). Although PEAK CK was greater in patients requiring RRT compared with those that did not (PEAK CK: 32,354 IU/l vs. 7,353 IU/l, P = 0.001), receiver operator characteristic curves revealed that a threshold for PEAK CK >5,000 IU/l is only 55% specifi c and 83% sensitive for the prediction of need for RRT. CK peaks on the day of admission in 46% of patients, on day 2 in 37%, and on day 3 or later in 17% of cases. A McMahon Score >6 calculated on admission is 68% specifi c and 86% sensitive for RRT. P284 Methods Data from the Songklanagarind Hospital trauma registry were reviewed over 1 year (January 2013 to December 2013). Patients with at least two records of CK and creatinine (Cr) levels were included. Creatine kinase levels were analyzed during the fi rst 3 days of hospital admission. RF was defi ned as a Cr increment >0.3 mg/dl within 48 hours. Results Of the 1,491 patients admitted to the trauma service, 47 patients had CK levels drawn twice. These patients had a mean age of 32  years and a median Injury Severity Score (ISS) of 14. The predominant mechanism of injury was motorcycle crash. Only three patients developed RF. The median CK during the fi rst 3  days after admission was 3,088 (IQR 1,327, 6,072) U/l. The CK peaked at 11 hours after admission at a mean value of 16,114.167 (SD 34,010.80) U/l. There were no signifi cant diff erences in demographic data, ISS scores and fl uid balance between the groups of CK level over or below 5,000 U/l. A mean positive fl uid balance observed; however, initial CK was signifi cantly diff erent between the two groups. None of the patients with initial CK of <900 U/l had a peak of CK >5,000 U/l. Risk factors for acute kidney injury in patients with complicated intra-abdominal infection A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) P283 Rhabdomyolysis: early prognostication of renal failure and other adverse outcomes J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) Rhabdomyolysis: early prognostication of renal failure and other adverse outcomes J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) J Simpson1, A Taylor2, DK Menon2, N Sudhan2, A Lavinio2 1University of Cambridge, UK 2Cambridge University Hospitals, Cambridge, UK Critical Care 2015, 19(Suppl 1):P283 (doi: 10.1186/cc14363) Introduction The clinical diagnosis of rhabdomyolysis is confi rmed by creatine kinase (CK) levels >1,000 IU/l [1]. A local therapeutic protocol triggers aggressive renoprotective treatment in all patients with CK >2,000 IU/l. To evaluate local practice and refi ne CK thresholds for the instigation of renoprotective treatment, we studied the correlation between CK time trends and adverse outcomes such as acute kidney injury (AKI), the need for emergency renal replacement therapy (RRT) and mortality. We also evaluated the McMahon Score, a risk prediction model based on demographic, clinical, and laboratory variables available on admission [2].i p y Conclusion Severe AKI with RRT need is highly associated with previous HTN. The number of previous medications is related to severe AKI too. HTN has been described as a risk factor for developing AKI in critically ill patients [1]. ACEI and ARB use has been associated with AKI development in septic patients [2]. To our knowledge, this is the fi rst study that investigates risk factors associated with AKI in surgical septic patients with CIAI. 2. McMahon GM, et al. JAMA Intern Med. 2013;73:1821-8. 1. Khan FY. Neth J Med. 2009;67:272-83. Reference 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. 1. Brown CVR, Rhee P, Chan L, Evans K, Demetriades D, Velmahos GC. Preventing renal failure in patients with rhabdomyolysis: do bicarbonate and mannitol make a diff erence? J Trauma. 2004;56:1191-6. Results A total of 114 patients were included, with a mean SAPS II of 42.14. Sixty-seven patients (58.8%) developed AKI and 33 (28.9%) required RRT. Development of AKI (R2 = 0.498; P <0.0001; AUC = 0.926) was independently associated with SOFA (OR = 1.57; 95% CI = 1.29, 2.02) and creatinine at admission (OR for 0.1 units = 1.56; 95% CI = 1.30, 1.99). RRT need (R2 = 0.382; P <0.0001; AUC = 0.892) was independently associated with arterial hypertension (HTN) (OR  = 4.90; 95% CI  = 1.50, 15.97) and SOFA score (OR = 1.71). In another model with more predictive capacity (R2 = 0.433; P <0.0001; AUC = 0.918) the number of previous medications (OR = 3.73; 95% CI = 1.92, 8.38) and SOFA score (OR = 1.86; 95% CI = 1.47, 2.54) were related to RRT need. Both AKI and RRT need were related to ICU (RR = 8.41, 95% CI = 1.14, 62.5; and RR = 8, 95% CI = 2.40, 27.85 respectively) and 28-day mortality (RR = 2.8, 95% CI = 1.00, 7.86; and RR = 4.65, 95% CI =1.99, 10.40 respectively). Risk factors for acute kidney injury in patients with complicated intra-abdominal infection Risk factors for acute kidney injury in patients with complicated intra-abdominal infection A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) A Suarez de la Rica, E Maseda, V Anillo, C Hernandez-Gancedo, A Lopez-Tofi ño, M Villagran, F Gilsanz Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P284 (doi: 10.1186/cc14364) Introduction AKI has been poorly studied in surgical septic patients. The aim of our study was to determine the factors related to AKI in surgical septic patients with complicated intra-abdominal infection (CIAI) and mortality associated with AKI in this setting. Methods An observational study was performed of all adult patients with CIAI requiring surgery and ICU admission from June 2011 to June 2013. We recorded demographic data, SAPS II, SOFA score at admission, presence of septic shock, history of pre-existing comorbidities, angiotensin-converting enzyme inhibitor (ACEI), angiotensin receptor blocker (ARB), NSAIDs, statins or diuretics consumption, baseline creatinine and at admission, and standard biomarkers. Factors associated with developing AKI and renal replacement therapy (RRT) were studied using a multivariate analysis. Association between mortality and AKI and RRT need was also analyzed. Conclusion Trauma patients had varying levels of elevated CK. Initial CK shows a promising result as a predictor of high peak CK levels. A larger sample size is needed to demonstrate the predictors of RF in trauma patients with elevated CK levels. P282 h Myoglobin has been known to cause renal failure (RF) and the CK level is routinely used as an indicator. A CK level >5,000  U/l was found to be associated with the risk of RF [1]. However, data are lacking on the level of CK to predict RF, especially in general trauma patients. The purpose of this study was to determine the initial CK level that predicts markedly elevated CK and the characteristics of trauma patients with elevated CK. Conclusion This review demonstrates that the majority of these patients occupied intensive care beds for a prolonged period of time and despite the use of advanced support devices survival rates were signifi cantly lower than mortality rates. S100 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Early detection of acute kidney injury during the fi rst week of the ICU M Flechet, F Güiza, M Schetz, P Wouters, I Vanhorebeek, I Derese, J Gunst, G Van den Berghe, G Meyfroidt KU Leuven, Belgium Critical Care 2015, 19(Suppl 1):P285 (doi: 10.1186/cc14365) Introduction Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients [1]. Early detection and treatment may improve outcome. Introduction Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients [1]. Early detection and treatment may improve outcome. y Methods A retrospective analysis of prospectively collected data from 2,158 patients without end-stage renal disease from the EPaNIC trial [2]. For early detection of AKI, defi ned according to the creatinine-based KDIGO guidelines [3], three multivariate logistic regression models (LR) were developed using data available at baseline (LR_B), upon ICU admission (LR_BA), and at the end of the fi rst day in the ICU (LR_BAD1). In a subpopulation (n  = 580) where plasma neutrophil gelatinase- associated lipocalin (pNGAL), an early biomarker of AKI, was measured at ICU admission, the value of adding pNGAL to LR_BA and LR_BAD1 was assessed. The models were evaluated via bootstrapping, by comparing receiver operator characteristic (ROC) and decision curves. i Conclusion Although higher CK levels are associated with adverse outcomes, instigation of renoprotective treatment should not be based solely on CK levels. A McMahon Score >6 on admission allows for a more sensitive, specifi c and timely identifi cation of patients at risk of renal failure requiring RRT. References S101 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P285). Area under ROC curves for the diff erent models LR_BA + pNGAL on LR_BA on pNGAL on subpopulation subpopulation subpopulation LR_B LR_BA LR_BAD1 with pNGAL with pNGAL with pNGAL (n = 2,158) (n = 2,067) (n = 1,808) (n = 528) (n = 528) (n = 528) 0.73 0.76 0.82 0.64 0.70 0.76 with the AMPK activator AICAR (100 mg/kg intraperitoneal, 24 hours before CLP), and sacrifi ced 24 hours after CLP. Blood and tissue samples were collected for all animals. AMPK activation (pThr172), B-ENaC, and mitophagy (LC3 II/I) were examined by western blot of kidney lysates. Plasma creatinine (Scr) was assessed using ELISA. Results Table 1 presents the performance of the models and admission pNGAL. Performance improved when predictions were made at a later time point, and was highest for LR_BAD1. Similar results were obtained in subgroups of septic and cardiac surgery patients. As an independent predictor, pNGAL alone did not perform better than a model using routine clinical data available upon admission. However, when combining pNGAL with LR_BA, predictive performance improved. The performance of LR_BAD1 was not improved by including pNGAL. with the AMPK activator AICAR (100 mg/kg intraperitoneal, 24 hours before CLP), and sacrifi ced 24 hours after CLP. Blood and tissue samples were collected for all animals. AMPK activation (pThr172), B-ENaC, and mitophagy (LC3 II/I) were examined by western blot of kidney lysates. Plasma creatinine (Scr) was assessed using ELISA. g Results The acute response to sepsis was characterized by early activation of AMPK which increased from 6 to 18  hours, peaked at 24 hours, and decreased by 48 hours (Figure 1A). This activation was associated with a consistent decrease in B-ENaC expression. In AICAR pretreated animals, AMPK was only activated in WT mice, which was associated with a decrease in the expression of B-ENaC as compared with AMPK KO mice (Figure 1B). p p y g p Conclusion This study shows the potential of data-driven models based on routinely collected patient information for early detection of AKI during the fi rst week of ICU stay. Although adding admission pNGAL to admission data improved early detection of AKI, this added value is lost upon inclusion of data from the fi rst day of ICU. References R f Conclusion AMPK was activated early after induction of sepsis, and was associated with a consistent decrease in Beta-ENaC expression in the apical membrane of tubular epithelial cells. In addition, absence of AMPK activation in KO animals was associated with increased expression of Beta-ENaC at 24 hours after CLP. These data support the hypothesis that early activation of AMPK decreases energy consumption through ion channel downregulation. 1. De Geus H, et al. Am J Respir Crit Care Med. 2011;183:907-14. 2. Casaer M, et al. N Engl J Med. 2011;365:506-17. 3. Acute Kidney Injury Work Group. Kidney Int. 2012;2:1-138. 1. De Geus H, et al. Am J Respir Crit Care Med. 2011;183:907-14. 2. Casaer M, et al. N Engl J Med. 2011;365:506-17. 3. Acute Kidney Injury Work Group. Kidney Int. 2012;2:1-138. 1. De Geus H, et al. Am J Respir Crit Care Med. 2011;183:907-14. 2. Casaer M, et al. N Engl J Med. 2011;365:506-17. 3. Acute Kidney Injury Work Group. Kidney Int. 2012;2:1-138. P286 Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? K Jin, H Li, J Volpe, D Emlet, N Pastor-Soler, MR Pinsky, BS Zuckerbraun, K Hallows, JA Kellum, H Gomez University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P286 (doi: 10.1186/cc14366) Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? S102 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 AKI as per the AKIN KDIGO defi nition, as well as their worst level of TIMP2*IGFBP7 during their fi rst 2-day stay. Values of mean and 25th to 75th percentile for the worst value of TIMP2*IGFBP7 were 0.24 (0.11 to 0.46), 0.50 (0.28 to 1.24), 0.94 (0.34 to 3.28) and 3.34 (1.47 to 6.22) for no AKI, AKIN I, II and II respectively (P <0.0001). The worst values for no AKI/no sepsis, no AKI/sepsis, AKI/no sepsis and AKI/sepsis were 0.21 (0.10 to 0.4), 0.32 (0.15 to 0.63), 1.05 (0.41 to 2.31) and 0.98 (0.36 to 3.94) respectively, with P <0.05 for AKI and P = NS for sepsis. The AUC ROC curve for prediction of AKI of the worst value was 0.80 with sensitivity of 73.5% and specifi city of 71.4% (P <0.0001). In contrast to the Sapphire study, in our population cutoff values of 0.4 and 0.8 (ng/ml)2/1,000 predict AKI and AKIN ≥II respectively, regardless of the presence of sepsis. See Figure 1. Results There were 101 patients without AKI, 95 patients with KDIGO 1 AKI, and 42 patients with KDIGO 2 to 3 AKI within 48 hours of the start of surgery. In patients without AKI, median TIMP-2·IGFBP7 values were less than 0.3 (ng/ml)2/1,000 (dashed line in Figure 1) for all time points. In patients with KDIGO 1 AKI, median [TIMP-2·IGFBP7] signifi cantly exceeded this cutoff at 24 and 36 hours following the start of surgery (*one-sided P  <0.025). Median [TIMP-2·IGFBP7] increased earlier in KDIGO 2 to 3 AKI patients, remaining signifi cantly elevated relative to the cutoff from 12 to 60 hours after the start of surgery. The highest median [TIMP-2·IGFBP7] was observed at 24 hours for KDIGO 2 to 3 AKI patients and was nearly fi ve times the 0.3 (ng/ml)2/1,000 cutoff .i p yi gf Conclusion Urinary [TIMP-2·IGFBP7] was signifi cantly elevated as early as 12 to 24 hours from the start of surgery in patients who developed AKI within 48 hours. Monitoring of these biomarkers in the immediate postsurgical period might enable improved management of patients at risk for AKI. Conclusion TIMP-2 and IGFBP-7 can predict AKI in both septic and nonseptic critically ill patients. Further pragmatic randomised controlled trials are needed to prove their role on clinical basis. P288 Urinary TIMP2 and IGFBP7 as early biomarkers of acute kidney injury in septic and nonseptic critically ill patients M Cuartero 1, A Betbesé 1, J Sabater2, J Ballús2, J Ordóñez1 1Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Spain; 2Hospital Universitari Bellvitge, Hospitalet Llobregat, Barcelona, Spain Critical Care 2015, 19(Suppl 1):P288 (doi: 10.1186/cc14368) Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? Reference 1. Kashani et al. Crit Care. 2013;17:R25. 1. Kashani et al. Crit Care. 2013;17:R25. Is acute kidney injury in the early phase of sepsis a sign of metabolic downregulation in tubular epithelial cells? P287 Urinary TIMP-2 and IGFBP7 elevate early in critically ill postoperative patients that develop AKI P Honore1, LS Chauwla2, A Bihorac3, AD Shaw4, J Shi5, JA Kellum6 1VUB, Brussels, Belgium; 2VAMC, Washington, DC, USA; 3UF, Gainesville, FL, USA; 4VUSM, Nashville, TN, USA; 5WB, Carlsbad, CA, USA; 6PiU, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P287 (doi: 10.1186/cc14367) Introduction This study tested the hypothesis that the cellular response in the kidney to sepsis is characterized by early activation of AMP activated protein kinase (AMPK), and that such activation is temporally associated with downregulation of the epithelial sodium channel (B-ENaC). Introduction Little is known about temporal changes in [TIMP- 2·IGFBP7] relative to injury in patients who develop AKI. In this analysis, we examined [TIMP-2·IGFBP7] in serial urine collections from the subset of Sapphire patients who were admitted to the ICU after major surgery. Methods We stratifi ed 238 Sapphire patients into three groups by their maximum AKI stage within 48 hours of the start of surgery using KDIGO criteria (No AKI, KDIGO 1, and KDIGO 2 to 3). Median TIMP-2·IGFBP7 values were calculated from all samples collected at 12 (±6)-hour intervals for 4 days following the start of surgery. Methods Fifteen C57BL/6 wildtype (WT) mice were subjected to cecal ligation and puncture (CLP), and sacrifi ced at 2, 6, 18, 24, or 48 hours. In addition, we pretreated three WT and three AMPK Beta 1 knockout mice Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. intervals for 4 days following the start of surgery. Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Figure 1 (abstract P287). Figure 1 (abstract P287). Figure 1 (abstract P287). Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Figure 1 (abstract P286). (A) AMPK activation (p-Thr172) and Beta-ENAC expression during early CLP. (B) Diff erence in Beta-ENAC expression between AICAR pretreated WT versus KO at 24 hours after CLP. Single point measurement of cystatin C has similar performance as serum creatinine for assessment of kidney function in critically ill patients J Houthoofd1, M Carlier2, J De Waele2, R Vanholder2, J Delanghe2, J Decruyenaere2, E Hoste2 Introduction Sepsis and acute kidney injury (AKI) have a high prevalence in the ICU population. The aim of this study is to describe the composite of tissue inhibitor of metalloproteinases-2 (TIMP2) and insulin-like growth factor-binding protein-7 (IGFBP7) as novel urinary renal biomarkers in both septic and nonseptic patients. Introduction The gold standard for routine evaluation of kidney function is measurement of serum creatinine concentration (Scr). In ICU patients, muscle loss and dilution leads to decreased Scr. Scr is distributed in total body water, resulting in delay of Scr changes when the glomerular fi ltration rate (GFR) changes (lag time). Cystatin C (CysC) is a protein produced by all cells with a nucleus and therefore less aff ected by muscle mass. Also, the lag time may be shorter as the volume of distribution is only extracellular fl uid. The objective of this study was to evaluate whether single point measurement of CysC is more adequate than Scr for monitoring of kidney function in ICU patients. Methods We conducted a prospective, observational study in two university hospitals. Patients were admitted in ICU either from the emergency department or after undergoing an acute surgery at hospital admission. Two months prior to the admission, recruited patients had not been admitted to hospital. We collected epidemiological, clinical and laboratory data at admission, 24 and 48 hours. TIMP2*IGFBP7 was analysed in urine samples by a point-of-care device (Nephrocheck®; Astute Medical). Results The sample included 98 patients (65 men) with mean age 55 ± 17.3 years, length of ICU stay 11.1 ± 14.6 days. In total, 41.4% had sepsis at ICU admission; 59.2% were diagnosed of sepsis within the fi rst 48 hours of stay. We stratifi ed patients based on the presence of Methods Data were collected in two prospective single-center studies on a convenience sample of ICU patients. During the 24-hour study period, we measured CysC, Scr, and urinary inulin clearance (Cinu) as a Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2]*[IGFBP7] value within the fi rst 48 hours of ICU admission to predict (a) AKI and (b) AKIN KDIGO ≥2. Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2]*[IGFBP7] value within the fi rst 48 h predict (a) AKI and (b) AKIN KDIGO ≥2. P291 gold standard for assessment of GFR. We compared Scr and CysC, with Cinu. Also, we assessed Cinu in patients who had CysC and Scr within the normal sex and age corrected limits. Finally, we determined the ability of CysC and Scr to detect normal range and decreased GFR (80 to 120 ml/minute/1.73m2 resp. <60). Association between urinary TIMP-2 and IGFBP7 as early biomarkers of AKI and oliguria during liver surgery: a prospective pilot study F Desmet1, M D’Hondt1, H Pottel2, S Carlier1, E Hoste3, J Kellum4, W De Corte1 Association between urinary TIMP-2 and IGFBP7 as early biomarkers of AKI and oliguria during liver surgery: a prospective pilot study F Desmet1, M D’Hondt1, H Pottel2, S Carlier1, E Hoste3, J Kellum4, W De Corte1 1AZ Groeninge, Kortrijk, Belgium; 2Catholic University Leuven, Kortrijk, Belgium; 3Ghent University Hospital, Ghent University, Ghent, Belgium; 4University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P291 (doi: 10.1186/cc14371) 1AZ Groeninge, Kortrijk, Belgium; 2Catholic University Leuven, Kortrijk, Belgium; 3Ghent University Hospital, Ghent University, Ghent, Belgium; 4University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P291 (doi: 10.1186/cc14371) p Results We included 68 patients, with median age 58  years (IQR 29 years), length of stay in the hospital before study 11 days (IQR 16), and APACHE II score 19 (IQR 9). Scr was 1.12 (IQR 1.55), CysC 0.64 (IQR 0.73), and Cinu 80  ml/minute/1.73 m2 (IQR 82). Cinu was markedly decreased in patients with Scr in normal range (n  = 12) compared with patients with CysC in normal range (n = 22) (55 ml/minute/1.73 m2 (IQR 57.4) vs. 100 (IQR 42.2), P <0.001). Patients with normal range Scr had similar proportion of patients with Cinu in the normal range compared with normal range CysC patients (33.3% vs. 45.5%, P = 0.23). ROC analysis showed that Scr and CysC had similar performance for detection of normal range Cinu (AUC: 0.66; 95% CI = 0.53 to 0.77 vs. 0.77; 95% CI = 0.65 to 0.87; P = 0.118), and decreased Cinu (AUC: 0.86; 95% CI = 0.76 to 0.97 vs. 0.94; 95% CI = 0.88 to 1; P = 0.113). Introduction Patients undergoing elective liver surgery have an increased risk for developing AKI [1]. This study was intended to assess [TIMP-2]*[IGFBP7] and its possible association with urine output (UO) in this population. References 1. Slankamenac et al. Development and validation of a prediction score for postoperative ARF following liver resection. Ann Surg. 2009;250:720-8. 2. Hoste et al. Derivation and validation cutoff s for clinical use of cell cycle arrest biomarkers. Nephrol Dial Transplant. 2014;29:2054-61. 2. Hoste et al. Derivation and validation cutoff s for clinical use of cell cycle arrest biomarkers. Nephrol Dial Transplant. 2014;29:2054-61. g Results Mean age was 81  ± 5.6  years (16 male, 40.0%). Thirty-fi ve patients underwent TA-TAVI and fi ve patients TAo-TAVI. AKI developed in 17 patients (42.5%); seven patients (17.5%) suff ered from AKI 3 and required renal replacement therapy (RRT). Mean maximum value of UTI within 24 hours after TAVI was signifi cantly higher in patients with AKI compared with patients without renal impairment (2.19 ± 3.11 vs. 0.67 ± 0.816, P = 0.037) and higher in patients with AKI 3 compared with patients with AKI 2 (4.73 ± 3.58 vs. 0.59 ± 0.71, P = 0.022). In contrast, preoperative creatinine (AKI (mg/dl) 1.22 ± 0.41 vs. no AKI 1.30 ± 0.59; AKI 3 1.32 ± 0.49 vs. AKI 2 1.26 ± 0.53, P = NS) and early postoperative serum creatinine levels (maximum within 24 hours after TAVI: AKI 1.41 ± 0.50 vs. no AKI 1.34 ± 0.60; AKI 3 1.69 ± 0.56 vs. AKI 2 1.32 ± 0.54, P = NS) did not show any association with the development of AKI. ROC analyses revealed a very good predictive value of early UTI levels for the development of AKI 3 within the next 72 hours after TAVI with a sensitivity of 100% and a specifi city of 80% for a cutoff value of 0.815 (AUC = 0.919, 95% CI = 0.824 to 1.0, SE 0.048, P = 0.001). Single point measurement of cystatin C has similar performance as serum creatinine for assessment of kidney function in critically ill patients Figure 1 (abstract P288). ROC curve and area under the curve (AUC) for the worst [TIMP2]*[IGFBP7] value within the fi rst 48 hours of ICU admission to predict (a) AKI and (b) AKIN KDIGO ≥2. S103 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Early prediction of acute kidney injury after transcatheter aortic valve implantation with urinary G1 cell cycle arrest biomarkers F Dusse, M Dudásová, D Wendt, M Thielmann, E Demircioglu, HG Jakob, K Pilarczyk Early prediction of acute kidney injury after transcatheter aortic valve implantation with urinary G1 cell cycle arrest biomarkers F Dusse, M Dudásová, D Wendt, M Thielmann, E Demircioglu, HG Jakob, K Pilarczyk University Hospital Essen, Germany Critical Care 2015, 19(Suppl 1):P290 (doi: 10.1186/cc14370) Introduction Acute kidney injury (AKI) is a common complication following transcatheter aortic valve implantation (TAVI) and has been shown to increase mortality. The concentration of the G1 cell cycle arrest proteins TIMP-2 and IGFBP7 in the urine have recently been suggested as sensitive biomarkers for early detection of AKI in critically ill patients. Whether postoperatively elevated levels of urinary [TIMP-2][IGFBP7] (UTI) predict the development of an AKI in patients undergoing TAVI is currently unknown. Conclusion This pilot study demonstrated the association between [TIMP-2]*[IGFBP7] increase and oliguria and may therefore indicate kidney damage during liver surgery. As Scr could not diff erentiate for these changes, patients did not meet the classical biomarker criteria for AKI. y Methods In a prospective cohort study, 40 patients undergoing TAVI, either trans-apical (TA) or trans-aortic (TAo), were enrolled. Serial measurements of UTI were performed every 12  hours in the postoperative course. Results were calculated for their multiplication and presented as arbitrary values. Urinary output and serum creatinine were recorded simultaneously. The primary clinical endpoint was the occurrence of AKI according to the AKI Network. P291 Secondly we sought to compare [TIMP-2]*[IGFBP7] with serum creatinine concentration (Scr). Methods A prospective single-center pilot study performed on 12 patients undergoing elective liver surgery. Serial urine samples were analyzed for [TIMP-2]*[IGFBP7] measured with the Nephrocheck device (Astute Medical, San Diego, CA, USA). Serial Scr was analyzed, UO, blood losses, and mean arterial pressure (MAP) were recorded. Fluid management was standardized, oliguria defi ned as a UO <0.5 ml/kg/ hour. [TIMP-2]*[IGFBP7] values of >0.3 identify patients at high risk and >2 at highest risk for AKI [2]. Conclusion Single point measurement of Scr and CysC has similar performance for detection of normal and decreased GFR in this cohort of ICU patients. Performance was weak for detection of normal GFR, but both biomarkers had moderate good performance for detection of decreased GFR. g [ ] Results Males comprised 66.7%, median age was 72  years. Median surgical time was 195  minutes. Peroperative median MAP was 71  mmHg (IQR 69; 77). Baseline median GFR was normal in eight patients and decreased in four patients (eGFR >90 and 66.5  ml/ minute/1.73 m2 respectively). Median baseline Scr was 0.75 mg/dl (IQR 0.61; 1.10), 0.74 mg/dl (IQR 0.64; 1.04) at ICU admission and 0.74 mg/ dl (0.64; 1.04) on day 1 postoperatively. No diff erence in Scr and eGFR was seen between these time points (P = 0.457 and P = 0.517 respectively; repeated-measures ANOVA). Median peroperative and postoperative UO was 0.18 ml/kg/hour (IQR 0.13; 0.23) and 0.93 ml/kg/ hour (IQR 0.79; 1.49) respectively. Median baseline [TIMP-2]*[IGFBP7] was 0.10 (IQR 0.04; 0.34), 2.02 (1.44; 6.23) during surgery, 0.61 (IQR 0.27; 1.22) at ICU admission and 0.74 (0.67; 0.97) on day 1 postoperatively. [TIMP-2]*[IGFBP7] diff ered at these time points (P <0.0001; repeated- measures ANOVA). Peroperative oliguria was associated with increased [TIMP-2]*[IGFBP7] (P = 0.018, chi-squared test). Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data y J Dixon1, K Lane1, R Dalton2, I MacPhee1, B Philips1 J Dixon1, K Lane1, R Dalton2, I MacPhee1, B Philips1 p 1St George’s Hospital and University of London, UK; 2King’s College, London, UK Critical Care 2015, 19(Suppl 1):P292 (doi: 10.1186/cc14372) 1St George’s Hospital and University of London, UK; 2King’s College, London, UK Critical Care 2015, 19(Suppl 1):P292 (doi: 10.1186/cc14372) Introduction There is currently no accurate method of measuring the glomerular fi ltration rate (GFR) during acute kidney injury (AKI). Four- hour creatinine clearance (4-CrCl) is often used. We have previously validated a method of measuring the GFR using a continuous infusion of low-dose iohexol (CILDI) in patients with stable renal function (GFR from normal to <30  ml/minute/1.73  m2). Steady state was achieved in <10 hours in all subjects and we calculate that variations >10.3% suggest an AKI. In this study we compare GFR measured by CILDI with 4-CrCl and 1-hour creatinine clearance (1-CrCl). Conclusion Early elevation of urinary [TIMP-2][IGFBP7] after TAVI is associated with the development of postoperative AKI. These biomarkers have an excellent diagnostic accuracy in the prediction of severe AKI requiring RRT that is superior to that of serum creatinine. Further studies are necessary to prove whether UTI-guided therapy of patients with AKI can reduce morbidity and mortality. Methods Critically ill patients with evolving AKI and patients following nephrectomy were recruited. Demographics were compared using the t test. CIDLI was connected for up to 72 hours. Plasma and renal iohexol and creatinine concentrations were measured by tandem mass spectrometry four times daily. Iohexol renal clearance (IRC) and S104 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 1-CrCl and 4-CrCl were calculated and compared using Bland–Altman analysis. P294 Impact of kidney injury on fl uid overload and impaired oxygenation A Akcan Arikan, LL Loftis, MA Arnold, CE Kennedy Baylor College of Medicine, Houston, TX, USA Critical Care 2015, 19(Suppl 1):P294 (doi: 10.1186/cc14374) y Results Baseline estimated GFR was similar in the postnephrectomy (88 ± 28) to the evolving AKI group (92 ± 23), P = 0.70. The evolving AKI group had a higher APACHE score (17.8 ± 5.1 vs. 10.6 ± 3.9; P <0.001). Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data When 1-CrCl was compared with IRC, a bias of 0.8% (SD 26%, limits of agreement –52 to 50%; Pearson’s r = 0.90) was observed in the evolving AKI group, whereas bias was –3.3% (SD 16, limits of agreement –35 to 29%; Pearson’s r = 0.95) in the postnephrectomy group. When 4-CrCl was compared with IRC, bias was 5.1% (SD 54, limits of agreement –102 to 112%, Pearson’s r = 0.45) in the established AKI group and bias was –4.5% (SD 38, limits of agreement –79 to 70%; Pearson’s r = 0.78) in the postnephrectomy group. Introduction Severity of acute kidney injury (AKI) and fl uid overload (FO) are not incorporated into current severity of illness measures and are invisible to the practitioner. The causal relationship and timing between AKI and FO and oxygenation is not clear. The Fluid Overload Kidney Injury Score (FOKIS) is a daily score incorporating subscores for AKI (pRIFLE (creatinine (Cr) and urine output (UOP))), FO (total fl uid (in – out) / ICU admission weight) >15% in fi ve percentile increments, and exposure to nephrotoxic medications. We previously reported that FOKIS outperforms PRISM in mortality prediction in our pediatric intensive care unit (PICU). We hypothesized that patients with AKI on admission to the PICU developed worse fl uid overload and in turn worse oxygenation. y g Conclusion Our data suggest that 4-CrCl is not as accurate and precise as 1-CrCl in patients with AKI and following nephrectomy. IRC appears to be more accurate and precise in patients with a predicted AKI risk and outcome (post nephrectomy) than in patients with evolving AKI. We hypothesise that IRC will be useful alternative to creatinine-based measures of AKI. Methods We prospectively calculated daily FOKIS scores and subscores (Cr, UOP, FO) in PICU patients. We excluded patients with <7 day stays in order to properly explore the association between timing of AKI and FO and oxygenation by oxygenation index (OI). y y y Results Over 18 months, there were 2,830 patients, 436 patients with PICU stay >7  days, 361 patients had complete data for all 7  days. Mortality was 4.5% overall and 11% cohort. A total of 246 patients (68%) had AKI (by FOKISCr or FOKISuop); 205 patients (57%) on day 1, 85 patients (24%) on day 3. Admission or day 3 AKI by either FOKIS subscore (FOKISCr or FOKISuop) did not predict maxFO or mortality. P295 P295 Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs M Ostermann1, L Forni2, K Kashani3, M Joannidis4, A Shaw5, M Chawla6, JA Kellum7, on Behalf of Sapphire Investigators7 1Guy’s & St Thomas Foundation Hospital, London, UK; 2Royal Surrey County Hospital, Guilford, UK; 3Mayo Clinic, Rochester, MN, USA; 4University Hospital Innsbruck, Austria; 5Vanderbilt University Hospital, Nashville, TN, USA; 6George Washington University Medical Center, Washington, DC, USA; 7University of Pittsburgh, PA, USA Critical Care 2015, 19(Suppl 1):P295 (doi: 10.1186/cc14375) FOKIS f Methods Retrospective data from all nondialysed patients undergoing isolated CABG at our institution were collected for the year 2013. Propensity scoring using the software platform MatchIt® was used to match subjects from ONCAB and OPCAB groups with regard to preoperative variables: logistic EuroSCORE, creatinine clearance (CrCl), gender and operative urgency. Postoperative AKI was defi ned as a rise of 50% or more in baseline serum creatinine [2]. Chi-square analysis was used to determine statistically signifi cant diff erences. Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. yif Results From 500 cases (369 OPCAB, 131 ONCAB), 262 subjects were included in the fi nal analysis, with 131 in each group. There was a higher incidence of AKI and renal replacement therapy (RRT) in the ONCAB group, although this was not signifi cantly greater than in the OPCAB group (Table 1). The mortality rate was identical with three deaths in each group. The average length of ICU stay for the OPCAB group was 1.96 days versus 2.49 days for the ONCAB group. 2. Acute Kidney Injury Network criterion. http://www.akinet.org. Continuous infusion of low-dose iohexol confi rms 1-hour creatinine clearance is more accurate in acute kidney injury than 4-hour creatinine clearance: preliminary data Increasing total FOKIS score was associated with increasing mortality and increasing OI (Table  1). FOKIS, controlled for PRISM, was an independent predictor of OI (P = 0.03). 1. Lamy A, et al. N Engl J Med. 2012;366:1489-97. Does cardiopulmonary bypass increase the risk of postoperative acute kidney injury after coronary artery bypass grafting? A Karmali, C Walker, L Kuppurao Harefi eld Hospital, London, UK Critical Care 2015, 19(Suppl 1):P293 (doi: 10.1186/cc14373) Does cardiopulmonary bypass increase the risk of postoperative acute kidney injury after coronary artery bypass grafting? A Karmali, C Walker, L Kuppurao Harefi eld Hospital, London, UK Critical Care 2015, 19(Suppl 1):P293 (doi: 10.1186/cc14373) Introduction Acute kidney injury (AKI) following coronary artery bypass grafting (CABG) results in signifi cant morbidity, mortality and prolonged stay on the cardiothoracic ICU. We sought to determine the incidence of AKI in our cardiothoracic centre, hypothesizing a higher occurrence in patients undergoing CABG using cardiopulmonary bypass (ONCAB) compared with off -pump (OPCAB) surgery [1]. Table 1 (abstract P294) P FOKIS 0 <4 4 to 7 >7 value maxOI, median (IQR) 7.4 11.1 16.4 14.2 0.03 (5.9 to 16.4) (6.2 to 23.6) (7.3 to 29.6) (10 to 38.7) Mortality, % 3.6 7.7 13 38 <0.001 Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. Table 1 (abstract P294) P FOKIS 0 <4 4 to 7 >7 value maxOI, median (IQR) 7.4 11.1 16.4 14.2 0.03 (5.9 to 16.4) (6.2 to 23.6) (7.3 to 29.6) (10 to 38.7) Mortality, % 3.6 7.7 13 38 <0.001 Conclusion In PICU patients, admission or day 3 AKI alone did not predict maxFO. A composite score that includes both AKI and FO parameters correlated with OI and discriminated survivors from nonsurvivors. FO seems to result from combination of increased fl uid exposure with underlying AKI but cannot fully be explained by oliguria in pediatric patients. Table 1 (abstract P294) Table 1 (abstract P294) Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK g Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) Introduction This study investigated renal outcomes following radio- contrast (RC) administration in patients from two intensive care units (ITUs), where one gave RC-induced nephropathy (RCIN) prophylaxis, while the other did not. Acute kidney injury (AKI) during critical illness increases morbidity and mortality. ITU patients, who already suff er a variety of renal insults, often require RC, increasing their risk of developing AKI, and requiring renal replacement therapy (RRT). Evidence suggests that hydration alone is inadequate for the prevention of RCIN in ITU patients, and is contraindicated in some disease states [1]. The European Society of Intensive Care Medicine (ESICM) provides recommendations for prophylaxis [2]. The current study aimed to establish the effi cacy of the ESICM recommended prophylaxis. p p y Conclusion Both SOFA score and BD may be used to predict AKI in surgical oncology patients at ICU admission. These variables allow physicians to recognize early patients who might be under risk, and anticipate measures to avoid further renal impairment. References 1. Rocktaeschel J, Morimatsu H, Uchino S, Goldsmith D, Poustie S, Story D, et al. Acid–base status of critically ill patients with acute renal failure: analysis based on Stewart–Figge methodology. Crit Care. 2003;7:R60. 1. Rocktaeschel J, Morimatsu H, Uchino S, Goldsmith D, Poustie S, Story D, et al. Acid–base status of critically ill patients with acute renal failure: analysis based on Stewart–Figge methodology. Crit Care. 2003;7:R60. gg gy 2. Smith I, Kumar P, Molloy S, Rhodes A, Newman PJ, Grounds RM, et al. Base excess and lactate as prognostic indicators for patients admitted to intensive care. Intensive Care Med. 2001;27:74-83. fi Methods Retrospective data from 140 Maidstone (M) ITU patients (men 101, women 39, mean age 63.5, mean APACHE 15.3) and 73 Tunbridge Wells (T) ITU patients (men 41, women 32, mean age 60.2, mean APACHE 20.2) admitted between 22 September 2011 and 22 September 2013, who underwent RC-enhanced CT, were collected. Patients on MITU received ESICM-recommended RCIN prophylaxis: 200 mg aminophylline i.v. over 30 minutes prior to RC, 1.26% sodium bicarbonate 3 ml/kg/hour for 1 hour prior to RC and 1 ml/kg/hour for 6  hours post RC. pp References 1. Kashani K, et al. Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury. Crit Care. 2013;17:R25. 2. Bihorac A, et al. Validation of cell-cycle arrest biomarkers for acute kidney injury: using clinical adjudication. Am J Resp Crit Care Med. 2014;189:932-9. 2. Bihorac A, et al. Validation of cell-cycle arrest biomarkers for acute kidney injury: using clinical adjudication. Am J Resp Crit Care Med. 2014;189:932-9. Results There were 76 (26.7%) patients who developed AKI within the fi rst 48 hours after ICU admission. In a univariate analysis, patients with AKI were more likely to be male, had higher Sequential Organ Failure Assessment (SOFA) score, higher baseline serum creatinine and urea levels, higher serum lactate levels and had more metabolic academia at admission. These patients also had a higher 24-hour Simplifi ed Acute Physiology III score and higher length of mechanical ventilation as compared with non-AKI patients. There were no diff erences between patients regarding intraoperative vasopressors, type and amount of fl uids, diuresis and blood transfusion. In a multivariate analysis we identifi ed admission base defi cit (BD) (OR = 1.13, 95% CI = 1.02 to 1.24, P = 0.017) and SOFA score (OR = 1.35, 95% CI = 1.2 to 1.51, P <0.001) as independent predictive factors of early AKI. Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs 1 2 h 3 d 4 h 5 h l 6 Table 1 (abstract P293) OPCAB ONCAB P value AKI 14 (10.69%) 19 (14.50%) 0.35 RRT 6 (4.58%) 9 (6.87%) 0.43 Table 1 (abstract P293) g Critical Care 2015, 19(Suppl 1):P295 (doi: 10.1186/cc14375) Conclusion Our study is limited by its size and the use of logistic EuroSCORE as a composite measure of risk factors. However, it has demonstrated that in our predominantly off -pump cardiac unit, OPCAB conferred no statistically signifi cant advantage over ONCAB with regard to postoperative AKI. Studies to date have standardised patient-specifi c variables, but not the conduct of the procedure itself; for example, minimising renal hypoperfusion during OPCAB. This may be a reason for the lack of clear superiority in the continuing debate over choice of CABG procedure and reducing the risk of AKI. References Introduction Tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) are specifi c urinary biomarkers which can predict acute kidney injury (AKI) in critically ill patients within 12 hours [1]. A [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 is associated with seven times the risk of AKI compared with a test result ≤0.3 [2]. The aim of our study was to explore the use of potentially nephrotoxic medications within the window between a positive biomarker test and the diagnosis of AKI stage 2 or 3.i Introduction Tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) are specifi c urinary biomarkers which can predict acute kidney injury (AKI) in critically ill patients within 12 hours [1]. A [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 is associated with seven times the risk of AKI compared with a test result ≤0.3 [2]. The aim of our study was to explore the use of potentially nephrotoxic medications within the window between a positive biomarker test and the diagnosis of AKI stage 2 or 3. Methods We identifi ed all patients enrolled into the Sapphire study [1] who received at least one potentially nephrotoxic drug on the day of AKI (defi ned by stage 2 or 3 as per KDIGO classifi cation). We subsequently Methods We identifi ed all patients enrolled into the Sapphire study [1] who received at least one potentially nephrotoxic drug on the day of AKI (defi ned by stage 2 or 3 as per KDIGO classifi cation). We subsequently 2. Acute Kidney Injury Network criterion. http://www.akinet.org. 2. Joannidis M, et al. Intensive Care Med. 2010;36:392-411. 1. Huber W, et al. Radiology. 2006;239:793-804. Acute kidney injury biomarkers off er the opportunity to reduce exposure to nephrotoxic drugs 1 2 h 3 d 4 h 5 h l 6 S105 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 determined the proportion of patients who had a [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 before meeting the criteria for AKI. determined the proportion of patients who had a [TIMP-2]·[IGFBP7] result >0.3 (ng/ml)2/1,000 before meeting the criteria for AKI. Predictors of renal recovery in critically ill patients with AKI: observations from the ongoing FBI clinical trial f S Robinson, U Larsen, A Zincuk, S Zwisler, P Toft , , , , Odense University Hospital, Odense, Denmark , , , , Odense University Hospital, Odense, Denmark y p Critical Care 2015, 19(Suppl 1):P298 (doi: 10.1186/cc14378) y Critical Care 2015, 19(Suppl 1):P298 (doi: 10.1186/cc14378) Introduction The predictive value of NGAL for renal recovery is not established. Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients Results Of 184 patients who developed AKI, 58% received one or more potentially nephrotoxic drug on the day of AKI. Eighty-nine percent of these patients had a positive biomarker test ≥12 hours earlier. In 41% of patients receiving one or more nephrotoxic drug on the day of AKI, at least one nephrotoxic medication was stopped within 1 day of AKI, and in 24% of patients all nephrotoxic drugs were stopped within 1 day of AKI, which implies that these medications were not absolutely necessary. Introduction Patients who undergo major oncology surgery are under high risk to develop postoperative acute kidney injury (AKI), mainly due to infl ammatory and ischemic insults. This complication results in worse outcomes. The aim of this study is to identify predictive factors of AKI in this population. Conclusion Nephrotoxic medications are commonly used in patients who develop moderate or severe AKI. The [TIMP-2]·[IGFBP7] test could have identifi ed many of these patients earlier and would have off ered an opportunity to reduce exposure to non-essential nephrotoxic drugs. References Methods We performed an observational study in 285 consecutive patients admitted to a surgical ICU after major abdominal oncology surgery. Baseline characteristics, laboratorial, clinical and intraoperative data, such as type of fl uids, blood transfusion, bleeding and use of vasopressor, were collected at ICU admission. Early acute kidney injury was defi ned according to the Acute Kidney Injury Network classifi cation at 48 hours of ICU admission. Logistic regression model was performed using AKI as the outcome. Introduction The predictive value of NGAL for renal recovery is not established. Methods Data from the fi rst 19 patients were assessed during a multicentre low molecular weight heparin trial (FBI, EudraCT Number: 2012-004368-23). Critically ill patients with AKI are randomly assigned into either a treatment arm (1  mg/kg enoxaparin) or a control arm (40  mg enoxaparin) upon commencement of CRRT. The primary outcome is the occurrence of venous thromboembolism. NGAL was measured at baseline and during CRRT-free intervals. Results The total number of patients developing renal injury falling into any RIFLE category for MITU at 24, 28 and 72 hours was eight (0.06%), 12 (0.09%) and 14 (0.1%), while for TITU it was fi ve (0.07%), six (0.08%), and four (0.05%) respectively. A repeated-measures ANOVA revealed no signifi cant diff erences in outcomes between the two groups overall (F = 2.35, P = 0.127) or at each time point (F = 1.93, P = 0.123). Results Patients were comparable at baseline with respects to demographics, APACHE II, creatinine, NGAL, start of dialysis, and the duration of dialysis. The main cause of AKI was sepsis (42%). In 63% of the patients, the reason for starting dialysis was a combination of anuria and electrolyte disturbances. Twenty-six percent of patients were dialysis dependent after the fi rst CRRT-free interval. Plasma NGAL levels were higher in nonrenal recovery patients (1,074 (±694) ng/ml) compared with renal recovery patients (296 (±197) ng/ml; P = 0.01). Urine NGAL levels were higher in nonrenal recovery patients (3,885 Conclusion While RCIN is a recognised problem within the critical care population, there is little clear evidence for any prophylactic strategy to reduce this risk. This study suggests that a RCIN prophylaxis protocol based on the ESICM recommendations has no eff ect on the incidence of RCIN. However, further studies are needed. P297 Base defi cit and SOFA score are predictive factors of early acute kidney injury in oncologic surgical patients A Gerent, J Almeida, E Almeida, A Lousada, C Park, J Ribeiro, J Fukushima, A Leme, E Osawa, A Rezende, I Bispo, F Galas, L Hajjar Instituto do Cancer do Estado de São Paulo – ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P297 (doi: 10.1186/cc14377) Retrospective analysis of the effi cacy of radio-contrast-induced nephropathy prophylaxis J Wood, N Shields, KV Wood Maidstone & Tunbridge Wells NHS Trust, Maidstone, UK Critical Care 2015, 19(Suppl 1):P296 (doi: 10.1186/cc14376) TITU patients received standard critical care alone. Exclusion criteria were: those undergoing RRT prior to CT, >1 CT in 48 hours, no creatinine (Cr) data available post scan. The Cr prior to CT (baseline), at 24, 48 and 72 hours post CT scan were identifi ed. The RIFLE criteria was used to classify the changes of Cr from baseline into low risk (% change >1.25), risk (% change >1.5), injury (% change >2) or failure (% change >3, or Cr>355 and increase of >44). P299 Comparison of two strategies for initiating renal replacement therapy in the ICU: study protocol plan for a multicenter, randomized, controlled trial from the AKIKI research group S Gaudry1, D Hajage2, F Schortgen3, L Martin-Lefevre4, J Ricard1, D Dreyfuss1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Bichat, Paris, France; 3Hôpital Henri Mondor, Créteil, France; 4CHD La Roche sur Yon, France Critical Care 2015, 19(Suppl 1):P299 (doi: 10.1186/cc14379) Results The total baseline plasma concentration of all standard amino acids was similar between IHD versus SLEDf groups (1,812  ± 517 vs. 2,675 ± 527 μmol/l, respectively) but were higher in the CVVH group (3,194  ± 564  μmol/l). RRT reduced the plasma concentration of amino acids in the SLEDf group (to 1,732 ± 529 μmol/l; P = 0.02), but had no eff ect in the IHD or CVVH groups (IHD; 1,853 ± 523, CVVH; 2,845 ± 512 μmol/l). The average, unadjusted loss of amino acids was signifi cantly infl uenced by mode of RRT (IHD, 5.13  ± 3.1 vs. SLEDf, 8.21 ± 4.07 vs. CVVH, 18.69 ± 3.04 g; P <0.01). The total baseline plasma concentration of trace elements was similar in the IHD, SLEDf and CVVH groups (3,797 ± 827, 3,667 ± 791, 3,642 ± 481 μg/l, respectively). By the end of the RRT session, the plasma concentration of trace elements had reduced (IHD, to 3,103 ± 827; SLEDf, to 2,805 ± 797; CVVH, to 3,433 ± 481 μg/l; P = 0.01). By the end of each RRT session, total losses of trace elements were estimated at IHD, 5,051 ± 2,312; SLEDf, 8,751 ± 2,421; CVVH, 11,258 ± 2,547 μg/l; P = 0.02 for treatment. Introduction There is currently no validated strategy for the timing of renal replacement therapy (RRT) for acute kidney injury (AKI) in the ICU when short-term life-threatening metabolic abnormalities are absent. No adequately powered prospective randomized study has to date addressed this issue. As a result, signifi cant practice heterogeneity exists and may expose patients either to unnecessary hazardous procedures or to undue delay in RRT. Methods This is a multicenter, prospective, randomized, open- label parallel-group clinical trial that compares the eff ect of two RRT initiation strategies on overall survival of critically ill patients receiving intravenous catecholamines and/or invasive mechanical ventilation and presenting with RIFLE F stage of AKI. In the early strategy, RRT is initiated immediately. References Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. Conclusion The AKIKI study will be one of the very few large randomized controlled trials evaluating mortality according to the timing of RRT in critically ill patients with RIFLE F stage of AKI. Results should help clinicians better decide when to initiate RRT. Figure 2 (abstract P298). Urine neutrophil gelatinase-associated lipocalin concentration by recovery. References Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S106 (±2,722) ng/ml) compared with renal recovery patients (597 (±565) ng/ml; P = 0.006). See Figures 1 and 2. Infl ammatory parameters (WBCs, CRP, and procalcitonin) did not diff er signifi cantly between the groups. Conclusion NGAL may be able to predict renal recovery, and allow proper utilization of resources. Figure 2 (abstract P298). Urine neutrophil gelatinase-associated lipocalin concentration by recovery. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. Figure 1 (abstract P298). Plasma neutrophil gelatinase-associated lipocalin concentration by recovery. more than 72  hours after randomization, serum urea concentration >40  mmol/l, serum potassium concentration >6  mmol/l, serum potassium concentration >5.5  mmol/l persisting despite medical treatment, arterial blood pH <7.15 in a context of pure metabolic acidosis (PaCO2 <35 mmHg) or in a context of mixed acidosis with a PaCO2 >50 mmHg without possibility of increasing alveolar ventilation, acute pulmonary edema due to fl uid overload despite diuretic therapy leading to severe hypoxemia requiring oxygen fl ow rate >5 l/minute to maintain SpO2 >95% or FiO2 >50% under invasive or non-invasive mechanical ventilation. The primary endpoint is overall survival, measured from randomization (D0) until death, regardless of the cause. The minimum follow-up duration for each patient will be 60 days. To demonstrate a 14% decrease in mortality, a total of 546 subjects (273 per group) should be randomized.i more than 72  hours after randomization, serum urea concentration >40  mmol/l, serum potassium concentration >6  mmol/l, serum potassium concentration >5.5  mmol/l persisting despite medical treatment, arterial blood pH <7.15 in a context of pure metabolic acidosis (PaCO2 <35 mmHg) or in a context of mixed acidosis with a PaCO2 >50 mmHg without possibility of increasing alveolar ventilation, acute pulmonary edema due to fl uid overload despite diuretic therapy leading to severe hypoxemia requiring oxygen fl ow rate >5 l/minute to maintain SpO2 >95% or FiO2 >50% under invasive or non-invasive mechanical ventilation. The primary endpoint is overall survival, measured from randomization (D0) until death, regardless of the cause. The minimum follow-up duration for each patient will be 60 days. To demonstrate a 14% decrease in mortality, a total of 546 subjects (273 per group) should be randomized.i p g p Results Enrollment is ongoing. After the fi rst interim analysis, the DSMB recommended to continue the study. On 5 December 2014, 318 patients were included in the trial. Figure 1 (abstract P298). P300 Micronutrient loss in renal replacement therapy for acute kidney injury W Oh1, M Devonald1, D Gardner2, R Mahajan3, D Harvey3, A Sharman3, B Mafrici1, M Rigby1, S Welham2 1Nottingham University Hospitals NHS Trust, Nottingham, UK; 2University of Nottingham, Sutton Bonington, UK; 3University of Nottingham, UK Critical Care 2015, 19(Suppl 1):P300 (doi: 10.1186/cc14380) Micronutrient loss in renal replacement therapy for acute kidney injury j y W Oh1, M Devonald1, D Gardner2, R Mahajan3, D Harvey3, A Sharman3, B Mafrici1, M Rigby1, S Welham2 1Nottingham University Hospitals NHS Trust, Nottingham, UK; 2University of Nottingham, Sutton Bonington, UK; 3University of Nottingham, UK Critical Care 2015, 19(Suppl 1):P300 (doi: 10.1186/cc14380) Introduction The prevalence of malnutrition in acute kidney injury (AKI) is high. Patients with AKI may require renal replacement therapy (RRT), which could result in loss of water-soluble micronutrients. Little is known about these losses in RRT and whether they diff er between types of RRT. This study aims to quantify micronutrient losses during RRT in patients with AKI and to compare them in three diff erent RRT modalities: continuous venovenous haemofi ltration (CVVH), intermittent haemodialysis (IHD) and sustained low-effi ciency diafi ltration (SLEDf). (±2,722) ng/ml) compared with renal recovery patients (597 (±565) ng/ml; P = 0.006). See Figures 1 and 2. Infl ammatory parameters (WBCs, CRP, and procalcitonin) did not diff er signifi cantly between the groups. Conclusion NGAL may be able to predict renal recovery, and allow proper utilization of resources. Methods A prospective observational study is being conducted at NUH. Thirty-three adult patients with AKI requiring RRT (13 IHD, 10 SLEDf, 10 CVVH) have been recruited. Samples of blood and RRT effl uent were obtained at baseline, mid and end-session from each participant during their fi rst RRT treatment. Samples were processed and stored at –80°C for subsequent analysis of amino acids by high- performance liquid chromatography and trace elements by inductively coupled mass spectrometry after derivatization from physiological fl uids. Micronutrient losses were calculated by multiplying mass- corrected concentrations by total volume of RRT effl uent, adjusted for baseline plasma concentrations and RRT dose. Data were analysed by restricted maximum likelihood estimating equations. P299 In the delayed strategy, clinical and metabolic conditions are closely monitored and RRT is initiated only when one or more events (severity criteria) occur, including: oliguria or anuria for Conclusion Micronutrients are lost during RRT in AKI. The degree of micronutrient loss is infl uenced by the type of RRT used. S107 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P301 Methods A comparison of patients receiving CVVHDF on the 11-bed critical care unit at Conquest Hospital, Hastings was undertaken, before and after the implementation of new CVVHDF protocols. All patients receiving CVVHDF were identifi ed from the electronic patient record system between March 2012 to 2013 and September 2013 to 2014. Patient demographics, the duration of CVVHDF and sodium bicarbonate supplementation were analysed between the groups to assess the impact of the new protocols. P301 Super high-fl ux CVVHD using regional citrate anticoagulation: long-term stability of middle molecule clearance M Siebeck, D Kindgen-Milles University Hospital, Düsseldorf, Germany Critical Care 2015, 19(Suppl 1):P301 (doi: 10.1186/cc14381) Introduction Conventional membranes used for CRRT have a limited middle molecule clearance. New membranes called super high-fl ux (SHF) or high cutoff membranes have been investigated. The loss of albumin with hemofi ltration is a major drawback, but these membranes can be used in CVVHD with regional citrate anticoagulation (Ci-Ca® CVVHD), which may limit albumin loss, and contribute to a prolonged fi lter patency and an improved and stable middle molecule clearance. We evaluated saturation coeffi cients (SC), plasma clearances (PCL) and serum levels of eight small and middle molecules during 72 hours of Ci-Ca® CVVHD with a SHF membrane (Ultrafl ux®EMiC®2). p p Results Sixty-four patients received CVVHDF in 2012 to 2013, 61 receiving citrate and three receiving unfractionated heparin due to fulminant liver failure. Forty-seven patients received CVVHDF in 2013 to 2014, two receiving no anticoagulation due to severe coagulopathy and one receiving unfractionated heparin. The two patient cohorts assessed were similar in age (median 65.5 for March 2012 to 2013 cohort vs. 66 for September 2013 to 2014 cohort), gender mix (64% male vs. 57% male) and severity of illness as assessed by APACHE II score (23 vs. 24). Mean duration of CVVHDF was also similar (71.5 hours vs. 75 hours). P299 A total 30/64 of 2012 to 2013 patients did not require a fi lter change prior to completion of RRT, compared with 23/47 of 2013 to 2014 patients. Sodium bicarbonate was added to the dialysate fl uid in 29/64 2012 to 2013 patients, compared with just 2/47 2013 to 2014 patients.i l Methods After approval of the local committee of medical ethics and written informed consent we enrolled patients on a surgical ICU with AKI RIFLE-F who were treated with a Ci-Ca® CVVHD with a SHF membrane for 72  hours. We measured urea (0.006  kDa), creatinine (0.113  kDa), osteocalcin (5.8  kDa), B2MG (12  kDa), myoglobin (17.2 kDa), FreeLightChains (FLC) kappa (25 kDa) and lambda (50 kDa) and albumin (66 kDa) at 0 hours, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours. PCL, SC and serum levels during 72 hours were compared, using the Wilcoxon signed-rank test with P <0.05. Conclusion Changing protocols resulted in a signifi cant reduction in off -license addition of sodium bicarbonate to dialysate bags without impacting on fi lter life, thus reducing nursing workload and removing a potential source of adverse events in this high-risk group of patients. Results Four females and 10 males (mean age 68.1 ±15.1 years; mean APACHE II score 13.7 ± 14.7; mean SAPS II 38.7 ± 12.7) were included. The SC and the PCL (ml/minute) of small solutes like creatinine at 1 hour (1.0 ± 0.0/23.72 ± 1.04) and 72 hours (0.95 ± 0.16/22.19 ± 3.99) were not statistically signifi cantly diff erent (P = 0.5/P = 0.42), the PCL was slightly reduced by 6%. The creatinine serum level was reduced by 42%. The SC and PCL of B2MG from 1 hour (0.61 ± 0.09/14.49 ± 2.5) to 72 hours (0.48 ± 0.13/11.6 ± 2.96) were signifi cantly decreased (P = 0.0024/P = 0.0061). The reduction was 23% only; the overall clearance still was high. There was almost no reduction in SC or PCL for FLC kappa from 1 hour (0.176 ± 0.047/4.14 ± 1.08) to 72 hours (0.164 ± 0.078/3.854 ± 1.87), not reaching statistical signifi cance (P = 0.94/P = 0.81). The serum levels of B2MG and FLC kappa were decreased by 39% and 23%. The SC of albumin was low (1 hour: 0.0009 ± 0.0004) and clearance decreased rapidly within the fi rst 6  hours from 0.021  ± 0.01 to 0.011  ± 0.009. Descriptive study of the haematological management of adult patients with severe respiratory failure receiving venovenous extracorporeal membrane oxygenation p yg O Tavabie, R Pocock, N Barrett, A Retter Introduction Venovenous extracorporeal membrane oxygenation (VV-ECMO) is a novel therapy for severe respiratory failure (SRF). Its introduction has reduced mortality; however, patients require substantial blood product support and between 10 and 20% of cases develop a life-threatening haemorrhage. p g g Methods We contacted 336 practitioners at 135 centres, examining their haematological management. Conclusion This study shows high middle molecular clearances using a SHF membrane with Ci-Ca® CVVHD for 72 hours with no loss of albumin. This set-up may improve blood purifi cation in critically ill patients with acute kidney injury. Results In total 25% of practitioners contacted responded; 85% were attending physicians, predominantly based in North America and Europe, 41 and 32% respectively. Ninety-six per cent of units used a polymethylpentene membrane oxygenator and all used a centrifugal pump. Thirty-four per cent of responders managed <10 cases a year and 60% worked in units handling <20 annually, 6% saw >50 patients. One centre did not use unfractionated heparin. Monitoring of anticoagulation varied; 52% used the APTT, 43% the ACT and 5% the APTTr. Sixty per cent did not routinely measure antithrombin. Results In total 25% of practitioners contacted responded; 85% were attending physicians, predominantly based in North America and Europe, 41 and 32% respectively. Ninety-six per cent of units used a polymethylpentene membrane oxygenator and all used a centrifugal pump. Thirty-four per cent of responders managed <10 cases a year and 60% worked in units handling <20 annually, 6% saw >50 patients. One centre did not use unfractionated heparin. Monitoring of anticoagulation varied; 52% used the APTT, 43% the ACT and 5% the APTTr. Sixty per cent did not routinely measure antithrombin. Scenario 1 was based on a patient with H1N1. Practitioners targeted a haemoglobin (Hb) of 80 to 100 g/l; however, 20% targeted a Hb outside this range; 38% favouring a transfusion threshold of <80  g/l when the patient was improving compared with 32% when the patient had just started on ECMO. Seventeen per cent of practitioners transfused platelets when the count was <30 × 109/l whilst 21% maintained the platelet count >100 × 109/l. Scenario 2 described a patient with SRF secondary to a hospital-acquired pneumonia. The patient developed a haemothorax, with persistent blood loss of 200 ml/hour. Descriptive study of the haematological management of adult patients with severe respiratory failure receiving venovenous extracorporeal membrane oxygenation Practitioners targeted a higher haemoglobin concentration of 100 g/l and targeted a higher platelet count of >100 × 109/l when compared with the patient in scenario 1, neither of these diff erences was statistically signifi cant. Seventy-one per cent stated they would manage the patient off anticoagulation. There was no agreement as to the length of time off anticoagulation; 26% restarted anticoagulation in <12  hours, compared with 22% who advised no anticoagulation for >5  days. Scenario 3 examined the management of an incidental intracranial haemorrhage. There was a lack of consensus regarding the duration off anticoagulation; 14% of responders held anticoagulation for less than P299 Serum levels of albumin did not decrease (1 hour: 2.64 ± 0.51; 72 hours: 2.63 ± 0.25). P302 30 Citrate anticoagulation for continuous venovenous haemodiafi ltration: the impact of a novel protocol on patients receiving therapy in one regional hospital J Highgate1, G Escott2, A Lowe2, F Stedman2, N McNeillis2 1Worthing Hospital, Worthing, UK; 2Conquest Hospital, Hastings, UK Critical Care 2015, 19(Suppl 1):P302 (doi: 10.1186/cc14382) Introduction Citrate has been used to anticoagulate extracorporeal haemofi ltration circuits since the 1960s, and has been used as the fi rst-line anticoagulant for continuous venovenous haemodiafi ltration (CVVHDF) at Conquest Hospital since 2009. Benefi ts of citrate demonstrated in clinical trials include increased fi lter life and increased bicarbonate formation from metabolism of the citrate complex; citrate also lacks the increased bleeding risk associated with unfractionated heparin use. One of the main issues with new renal replacement therapies is the development of ideal dialysate fl uids. During the initial period of citrate use at Conquest, hyponatraemia was identifi ed as an issue, with off -license supplementation of dialysate fl uid with sodium bicarbonate often necessary to prevent this. New protocols were therefore developed, designed to maximise the fi ltration dose and maintain normal electrolyte balance. S108 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Data were collected from our computerised information system on all AKI patients receiving RRT on our ICU, between October 2008 and October 2013. This included demographics, APACHE II and SOFA scores, modality and dose of RRT and ICU length of stay (LOS). Renal and patient survival at ICU discharge was collected, in addition to outcome data at 28 and 90 days and 12 months. Data were examined 12 hours whilst 37% held anticoagulation for >5 days and tranexamic was considered useful by 25%. y Conclusion There was wide variation in the use of blood products and the intensity of anticoagulation. This is not surprising given the current lack of evidence. Further work is required to provide a standardised approach. using Cox proportional hazard multivariate analysis, with Stata 10.1. Results A total of 620 patients with AKI received RRT on our ICU between October 2008 and October 2013. Sixty-one per cent were males. Median age was 65 years (IQR 54 to 74). Median APACHE II score was 23 (IQR 18 to 27). Median SOFA score was 11 (IQR 8 to 13). Fifty-fi ve per cent were mechanically ventilated. Comparison between nafamostat mesilate and unfractionated heparin as anticoagulant during continuous renal replacement therapy Comparison between nafamostat mesilate and unfractionated heparin as anticoagulant during continuous renal replacement therapy S Makino, H Kita, Y Miyatake, T Yokoyama, K Kubota, N Obata, M Egi, T Misumi, S Izuta, S Mizobuchi Kobe University Hospital, Kobe, Japan Critical Care 2015, 19(Suppl 1):P304 (doi: 10.1186/cc14384) Introduction For continuous renal replacement therapy (CRRT), continuous administration of anticoagulant would be necessary to prevent the circuit clotting. Nafamostat mesilate (NM) is commonly used as its anticoagulant in Japan, although unfractionated heparin (UFH) is the most frequently used anticoagulant internationally. There is little study to compare the risk and benefi t of NM with UFH as an anticoagulant during CRRT. Methods We conducted a single-center retrospective observational study to compare NM with UFH as anticoagulant during CRRT. We screened subsequent critically ill patients requiring CRRT in our ICU from January 2011 to December 2013. We excluded patients who required any other extracorporeal circuit including extracorporeal membrane oxygenation, who used both NM and UFH simultaneously, or who were administered any other anticoagulant including gabexate mesilate or urokinase. The primary outcome of this analysis was fi lter life, and the secondary outcome was the incidence of bleeding complications during CRRT. As an initial dose, NM and UFH were given pre fi lter at 15 to 25 mg/hour and 1,500 to 3,000 IU/hour, respectively. The dose of both drugs was adjusted to maintain activated clotting time at post fi lter between 150 and 200 seconds. Filter life was assessed using the Kaplan–Meier method and the incident of bleeding complications was compared using the chi-square test. P <0.05 was considered to be statically signifi cant. Conclusion Results from our cohort suggest that, in patients with AKI presenting to ICU for RRT, long-term patient survival is signifi cantly impaired. Renal outcomes are poor with 35% being either dialysis dependent or having severe chronic kidney disease (eGFR <15  ml/ minute), at 1 year from ICU discharge. Our data do not suggest a benefi t of using HVHF in AKI patients presenting to ICU for RRT. P306 Plasma antioxidant capacity in critical traumatized patients: severity and anatomical location y G Papakitsos1, A Kapsali1, T Papakitsou2, A Roimba3 1GHA, Arta, Greece; 2GHM, Messologi, Greece; 3General Practitioner, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P306 (doi: 10.1186/cc14386) i Results We included 101 patients in this study. Among them, 76 patients were with NM and 25 patients were with UFH. They used 239 fi lters in total; 173 with NM, 66 with UFH. There were signifi cantly more post- surgical patients in the NM group (P = 0.001). There was no diff erence in age, APACHE II score, days from ICU admission to commencement of CRRT, length of ICU stay and mortality between two groups. There was no diff erence in median number of fi lters used by one patient (NM vs. heparin; median of 1.5 (IQR) vs. 2 (IQR), P = 0.27). Filter life in the UFH group was signifi cantly longer than those in the NM group (NM vs. UFH; median of 24 hours vs. 36 hours; P = 0.01). The incidence of bleeding complications was not signifi cantly diff erent between two groups (P = 0.15). Introduction Oxidative stress (OS) has been invoked as a relevant factor in the evolution and outcome of critical care patients. Indeed, antioxidant therapies have been used in critical care patients, but with controversial results. This may be explained by assuming OS as a homeostatically regulated parameter and both its excess and its defi cit infl uencing severity progression. Nonetheless, antioxidant agents could mask an OS signaling role, blocking otherwise physiological responses aimed at recovery of homeostasis. We have evaluated plasma total antioxidant capacity (TAC) in traumatized patients in the emergency department (ED) and we determined its potential relationship with severity and trauma location. Conclusion In our retrospective analysis with 101 patients, fi lter life with UFH was signifi cantly longer than those with NM. The incidence of bleeding complications was not signifi cantly diff ered between patients with NM and UFH. y Methods In a prospective observational study of ED polytraumatized patients (n = 23, mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score of 11 ± 6) we measured (in the fi rst 24 hours) plasma TAC by the ferric reducing activity/antioxidant power (FRAP). For control subjects, we used age-matched and gender-matched volunteers (n = 32). We also evaluated the contribution of antioxidant molecules (uric acid, bilirubin, and albumin) to these values. P302 A total of 96.7% received CVVH as the principal RRT modality. Twenty-one per cent received a period of high-volume haemofi ltration (HVHF) (80 ml/kg/hour), median LOS was 6 days (IQR 3 to 14). In total, 331 (53.4%) patients recovered their renal function at ICU discharge, whilst 237 (38.2%), 220 (35.4%), and 220 (35.4%) patients did not at 28 and 90 days and 12 months respectively. A total of 414 (66.7%) patients survived to ICU discharge, with 368 (59.3%), 341 (55%) and 308 (49.6%) patients being alive at 28 and 90 days and 12 months respectively. Overall patient survival at the end of follow-up was 43%. Adjusting for age and sex; APACHE II score, SOFA score and use of HVHF were associated with worse patient survival at ICU discharge (HR: 1.07, 95% CI: 1.03 to 1.11, P <0.001, HR: 1.11, 95% CI: 1.03 to 1.19, P = 0.006 and HR: 2.27, 95% CI: 1.4 to 3.66, P = 0.001, respectively). Adjusting for age and sex; APACHE II score and use of HVHF were associated with worse renal recovery at ICU discharge (HR: 1.06, 95% CI: 1.03 to 1.09, P <0.001 and HR: 1.55, 95% CI: 1.03 to 2.3, P = 0.032 respectively). SOFA score did not appear to signifi cantly impact renal recovery (HR: 0.99, 95% CI: 0.94 to 1.04, P = 0.81). Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu Suzhou Municipal Hospital, Nanjing Medical University, Suzhou, China Critical Care 2015, 19(Suppl 1):P309 (doi: 10.1186/cc14389) Introduction The aim was to analyze the injury features and treatment strategies of surviving casualties in the explosion accident on 2 August 2014 in Kunshan city (Kunshan ‘August 2’ Explosion Accident). Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. Introduction The aim was to analyze the injury features and treatment strategies of surviving casualties in the explosion accident on 2 August 2014 in Kunshan city (Kunshan ‘August 2’ Explosion Accident). Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. p p p p Results In total, 722 trauma patients were included, of which 300  patients were hypothermic. The mortality in the hypothermia group was signifi cantly higher than in normotherm patients (OR = 3.73, 95% CI = 2.02 to 7.13, P <0.001). A cutoff point of 36°C was observed as the best threshold for hypothermia (sensitivity 74%, specifi city 56%). Besides hypothermia, other predictors found for 28-day mortality were APACHE II score corrected for temperature, minimum thrombocytes in fi rst 24 hours and urea and included in the fi nal model with an AUC of 0.89 (95% CI = 0.85 to 0.92). External validation of the model was associated with a predicted probability of an AUC of 0.64 (95% CI = 0.51 to 0.77).i Methods We retrospectively studied 40 cases of surviving casualties in the Kunshan ‘August 2’ Explosion Accident, and systemically reviewed the relevant literature. Results A total of 40  cases were admitted to our hospital within 6.3  ± 0.8  hours after the explosion accident on 2 August 2014 in Kunshan city, including 28 males and 12 females. The major injury types included burn injury, inhalation injury, blast injury (lung, eye, eardrum, and so forth), traumatic brain injury and bone fractures. All cases suff ered from burn injury caused by the explosion. The mean burned area in the surviving casualties accounted for 92  ± 14% total body surface area (TBSA) and those with third-degree burns for 77 ± 19% TBSA. Additionally, incidence rate of inhalation injuries was 97.5%. There were 34 cases complicated by multiple organ dysfunction syndrome, which accounted for 85.0%. Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu The common organ dysfunction of surviving casualties included the lungs, circulation, liver, gastrointestinal tract, kidney, brain, and coagulation. During hospitalization, the most common infectious site in surviving casualties was a burn wound, followed by blood and lung. The most common infectious strain of bacteria was Gram- negative bacteria, which accounted for 91.3%. Further analysis showed that Proteus mirabilis ranked fi rst in occurrence, followed by Acinetobacter baumannii and Pesudomonas aeruginosa, and Klebsiella pneumonia and Enterobacter cloacae ranked fourth and fi fth. After intensive treatment, the mean 28-day mortality was 20.0% and 90- day mortality was 62.5%, mainly due to septic shock and multiple organ dysfunction syndrome. Conclusion Hypothermia, defi ned as <36°C, is associated with an increased 28-day mortality. The discriminative ability of the developed model for predicting mortality in a new patient population is moderate. P305 Long-term renal and survival outcomes in acute kidney injury patients receiving renal replacement therapy in intensive care I Elsayed1, N Pawley1, J Rosser1, MJ Heap1, GH Mills1, A Tridente2, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P305 (doi: 10.1186/cc14385) Results Polytraumatized patients show diff erences in TAC with reference to control subjects. ED polytraumatized patients show high FRAP values. We found that FRAP values were inversely correlated with APACHE II score (r = –0.266, P <0.01) suggesting that, in trauma patients, increased antioxidant response, as measured by FRAP assay, could be a pathophysiological response to stress. Albumin and uric acid concentrations reproduced the FRAP trend with severity. Introduction Acute kidney injury (AKI) aff ects 40% of critically ill patients, with UK data reporting 5% needing renal replacement therapy (RRT). Hospital mortality is reported as being up to 60%. We sought to evaluate renal and long-term patient survival outcomes in AKI patients receiving RRT on our ICU. Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, S109 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion An increase in StO2 and lower tissue oxygen extraction rates were associated with H-survival in polytrauma patients. Figure 1 (abstract P308). P  <0.002) and head trauma (β  = –0.219, P  <0.045). These results accentuate the infl uence of trauma location and severity in TAC changes. TAC response in ED patients reinforces the need for an adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. Figure 1 (abstract P308). P  <0.002) and head trauma (β  = –0.219, P  <0.045). These results accentuate the infl uence of trauma location and severity in TAC changes. TAC response in ED patients reinforces the need for an adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. P309 Methods A retrospective cohort study was performed in adult trauma patients admitted to a level 1 trauma center and who were transferred to the ICU between 2007 and 2012. Diff erent cutoff points for hypothermia were compared to fi nd the best defi nition for hypothermia. Logistic regression analysis was performed to quantify the net eff ect of hypothermia on admission to the ICU on 28-day mortality and to develop a model with predictors. The developed model was externally validated in data from another level 1 trauma center with a comparable patient population. Features and treatment of surviving casualties in the Kunshan ‘August 2’ Explosion Accident: 40 case reports and literature review J Liu, WU Wu Suzhou Municipal Hospital, Nanjing Medical University, Suzhou, China Critical Care 2015, 19(Suppl 1):P309 (doi: 10.1186/cc14389) P307 Hypothermia as a predictor for mortality in trauma patients K Balvers1, JM Binnekade1, C Boer2, JC Goslings1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU University Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P307 (doi: 10.1186/cc14387) Introduction Previous studies reported hypothermia as an independent predictor for mortality. However, diff erent cutoff points were used in these studies and external validation has never been applied. The aim of this study was to quantify the net eff ect of hypothermia on admission to the ICU on the 28-day mortality and to test the predictors from the developed model in another level 1 trauma center with a comparable patient population to validate the model. Figure 1 (abstract P308). Conclusion An increase in StO2 and lower tissue oxygen extraction rates were associated with H-survival in polytrauma patients. Near-infrared spectroscopy to assess tissue oxygenation in patients with polytrauma: relationship with outcome A Donati, E Damiani, R Domizi, S Pierantozzi, S Calcinaro, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) Università Politecnica delle Marche, Ancona, Italy , , y Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) y Critical Care 2015, 19(Suppl 1):P308 (doi: 10.1186/cc14388) Introduction We evaluated tissue oxygenation by means of near- infrared spectroscopy (NIRS) and explored its relationship with outcome in polytrauma patients. Methods A prospective observational study; 37 polytrauma patients underwent NIRS monitoring (thenar eminence) every day during their stay in the ICU. A VOT was performed with a 40% tissue oxygen saturation (StO2) target. Healthy volunteers (n = 27) were studied as controls. y y Conclusion During the Kunshan ‘August 2’ Explosion Accident, burn injury was the leading cause of injuries. Most surviving casualties are accompanied by multiple organ dysfunction syndrome and infection. Results StO2 increased over the fi rst 7 days only in hospital survivors (n = 29), who showed higher values as compared with healthy volunteers at days 5 and 7 (Figure 1). StO2 downslope and upslope tended to be lower in H-nonsurvivors (n = 8) (P <0.05 at days 2 and 4) as compared with H-survivors. Tissue hemoglobin index was lower in H-no survivors over the fi rst 7  days and tended to normalize only in H-survivors (P >0.05 vs. healthy at day 7). Five patients were discharged from the ICU but did not survive until H-discharge. At discharge from the ICU, these patients were similar to H-survivors in SOFA score, heart rate, mean arterial pressure and lactate, but showed lower StO2 downslope (–13 (–16.5, –11.7)%/minute vs. –8.6 (–11.7, –6.5)%/minute, P = 0.01). P310 In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users A Franci1, S Piantini2, M Pierini2, A Peris1, M Mangini1 1A.O.U. Careggi, Firenze, Italy; 2University of Florence, Italy Critical Care 2015, 19(Suppl 1):P311 (doi: 10.1186/cc14391) Introduction The purpose of this study is to analyze anatomic distributions, diagnostic methods, and prognosis of missed fractures in patients with severe injury. Introduction The purpose of this study is to analyze anatomic distributions, diagnostic methods, and prognosis of missed fractures in patients with severe injury. 1A.O.U. Careggi, Firenze, Italy; 2University of Florence, Italy Critical Care 2015, 19(Suppl 1):P311 (doi: 10.1186/cc14391) gg y y y Critical Care 2015, 19(Suppl 1):P311 (doi: 10.1186/cc14391) Methods A review of single-institutional medical records between January 2001 and May 2012 identifi ed 58 patients with 62 delayed diagnoses of fractures among 4,643 severely injured patients older than 20 years with Injury Severity Scores higher than 16. We evaluated combined injuries, location of fractures, diagnostic methods, and reasons for missed diagnosis at initial examination. Introduction Road accidents are the leading cause of death for young people, 50% being represented by vulnerable road users (VRU) (pedestrians, cyclists). In-depth accident studies assess the consequences of lack of use of safety devices and the need to develop new ones. Since 2009 a permanent team (physicians and engineers) has performed in-depth studies on road trauma admitted to our ICU [1]. Results Among 62 missed fractures, there were eight cases of spine fracture, 10 cases of peri-shoulder joint fracture, eight cases of upper extremity fracture, 10 cases of pelvis of acetabulum fracture, and 26 cases of lower extremity fracture. Head injury was the most common concomitant injury (23 cases). Initially missed fractures were most commonly discovered by offi cial reading by radiologists. The most common reasons for misdiagnosis were the use of improper radiologic study and missed-reading of proper radiologic studies. Methods The team studied 52 VRU crashes that occurred in an urban area. The clinical data included an injury assessment using total body CT scan, Injury Severity Score (ISS), Abbreviated Injury Score (AIS), ICU and hospital length of stay and outcome score. Engineers collect data onsite with the partnership of the police, and assess the dynamics of the vehicles with the most advanced reconstruction techniques. Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) I Turriziani, A Cecchi, A Giugni, L Copertino Introduction In the literature, duration of mechanical ventilation (DMV) is often considered an important risk factor (RF) [1] for VAP [2] in critical patients; generally the whole duration of MV is taken into S110 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P311). Frequency of lesions. account, including days before and after infection onset. We tried to assess whether, counting only MV days prior to VAP development (MVp), something would change. Methods We considered, in a 10-year period, data prospectively collected in our database (4D solution, V11) on trauma patients admitted to the ICU directly from the emergency department. Inclusion criteria were: age ≥16 years, ICU length of stay (ICUlos) ≥4 days, DMV ≥48 hours; we excluded patients who received antibiotics before VAP (or during the whole stay, for patients without VAP) and with incomplete data. Data were: age, sex, prehospital GCS <9, prehospital intubation (preHTI), admission base excess (BE), Injury Severity Score (ISS), surgery, massive transfusion, feeding, antacids, nursing, DMV, ICUlos and MVp. MVp was calculated as the diff erence between the fi rst day of VAP and the fi rst day of MV in patients who developed VAP (vapY) and whole DMV in patients that did not (vapN). We only considered the fi rst infectious episode. The outcome was VAP onset. Group comparison was made with Fisher’s exact test and Student’s t test. Signifi cant variables were evaluated in a logistic regression (LR) model; the Hosmer–Lemeshow test (HL) was used as the post-estimation test. Odds ratio (OR) and 95% confi dence interval (95% CI) were calculated. Statistical signifi cance for P <0.05. We used Stata/IC 10.1 for analysis. Figure 1 (abstract P311). Frequency of lesions. Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Dynamic N injured Speed (SD) Car vs. pedestrian 29 41.1 (13.9) Motorcycle vs. pedestrian 7 40.6 (4.3) Car vs. cyclist 10 40.7 (15.9) Other 6 49.0 (5.4) Table 1 (abstract P311). Accident dynamics: medium speed (km/hour) Dynamic N injured Speed (SD) Table 1 (abstract P311). Duration of mechanical ventilation in trauma patients: risk factor for VAP? I Turriziani, A Cecchi, A Giugni, L Copertino Ospedale Maggiore, Bologna, Italy Critical Care 2015, 19(Suppl 1):P310 (doi: 10.1186/cc14390) Accident dynamics: medium speed (km/hour) y Results A total of 541 patients met the inclusion criteria, 378 (69.9%) developed VAP. MVp does not seem to be a RF for VAP because they are longer in vapN than in vapY (mean MVp 5.5 vs. 4.41, P = 0.001). PreHTI (vapY/N: 49.74%/38.65%; OR: 1.57; 95% CI: 1.08 to 2.28), ISS (mean vapY/N: 28.4/25.55; P = 0.0018), BE (mean vapY/N: –3.76/–3.04; P = 0.03) were signifi cantly diff erent between the two groups. In LR only preHTI (OR: 1.47; 95% CI: 1.01 to 2.15) and ISS (OR: 1.03; 95% CI: 1.01 to 1.05) are RF for VAP (HL: P = 0.133). Conclusion In our study MVp are not a RF for VAP in trauma patients, although the whole DMV is longer in patients with VAP (mean DMV vapY/N: 13.57/6.09; P = 0.0001). Further studies could confi rm whether the whole DMV in trauma patients with VAP is a consequence of infection. Conclusion The head is still the most frequently and severely injured region. The severity of injuries increases in the most rigid part of the car. Improving VRUs’ safety devices (active and passive) to reduce impact speed and severity of the primary impact has to be the next step. Reference ferences Richmond J, Egol KA, Koval KJ. Management of orthopaedic injuries in polytrauma patients. Bull Hosp Jt Dis. 2001-2002;60:162-7. References 1. Charles MP, et al. Ventilator-associated pneumonia. Australas Med J. 2014;7:334-44. 1. Charles MP, et al. Ventilator-associated pneumonia. Australas Med J. 20 33 1. Piantini et al. BMC Emerg Med. 2013;13:3. 2. CDC ventilator-associated event (January 2014). www.cdc.gov. 2. CDC ventilator-associated event (January 2014). www.cdc.gov. P311 Dankook University Hospital, Cheonan City, South Korea Critical Care 2015, 19(Suppl 1):P312 (doi: 10.1186/cc14392) Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) Introduction Severe trauma aff ects the immune system, which in its turn is associated with poor outcome. The mediators driving the immune responses in trauma are largely unknown. The aim of this study was to investigate the role of endogenous microparticles (MPs) in mediating the immune response following severe trauma. Figure 1 (abstract P314). sRAGE kinetics (days). on days 1, 3 and 5. sRAGE was measured by ELISA and cfDNA was measured by UV absorbance after plasma isolation. g p g Methods A prospective, observational substudy of the Acute Coagulopathy and Infl ammation in Trauma (ACIT) II study was performed at our academic level 1 trauma center. Adult multiple trauma patients with an Injury Severity Score of 15 or higher were included between May 2012 and June 2013. Ex vivo whole blood stimulation with lipopolysaccharide was performed on aseptically collected patient plasma containing MPs and in plasma depleted of MPs. Flow cytometry and transmission electronic microscopy were performed on plasma samples to investigate the numbers and cellular origin of MPs. Healthy individuals served as a control group. Results Median ISS was 39 and mortality was 21% (8/38). During the fi rst 5 days after trauma, the median concentration of sRAGE (Figure 1) decreased signifi cantly over time (P <0.0001) while median levels of DNA did not (P = 0.73), and remained elevated compared with normal control. No correlation was found with ISS. Patients initially in shock had lower levels of sRAGE or cfDNA (P <0.05) and had received more fl uid (10.6 l vs. 5.25 l) or blood (6 l vs. 0.5 l). Day 3 and day 5 sRAGE levels were inversely correlated with PRBC received. Medians of sRAGE on days 0 (1,301 vs. 730 pg/ml) and day 1 (925 vs. 760 pg/ml) were signifi cantly higher in nonsurvivors (P <0.01). Finally, day 0 sRAGE was correlated with the maximal (r = 0.44; P = 0.007) and the cumulative renal failure component of the MODS, over the 10 days (r = 0.48; P = 0.005).f Results Ten trauma patients and 10 healthy individuals were included. Trauma patients were signifi cantly injured with a median ISS of 19 (17 to 45). On admission to the hospital, the host response to bacterial stimulation was blunted in trauma patients compared with healthy individuals, as refl ected by decreased production of IL-6, IL-10 and TNFα (P <0.001). In-depth study of road accidents in Florence: understanding the biomechanical eff ects in major trauma involving vulnerable road users Medical and engineering data were cross-matched during the correlation process. Injuries suff ered by each person were related to specifi c impact objects. y g p p g Conclusion In order to prevent misdiagnosis of fractures in patients with severe injury, meticulous physical examination with suspicion of fractures should come fi rst. In addition, obtaining proper radiologic study and thorough evaluation of radiologic images are important to decreasing the rates of missed fracture diagnoses. In addition, thorough surveillance for ipsilateral fractures is important in extremities with identifi ed fractures. i Results The average ISS is 21.5 (SD 10.9). Cars are the most involved in serious urban VRU crashes. Car-to-pedestrian crashes are the most frequent (50%). The impact speed is always over 40 km/hour (Table 1). The head and face are the most frequently injured part (48% of the 571 injuries collected), followed by lower extremities (15%). In terms of maximum AIS (MAIS), the head is the most severely injured region with 42% of MAIS 3+ (Figure 1). i References S111 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P314). sRAGE kinetics (days). 2. Houshian S, Larsen MS, Holm C. Missed injuries in a level I trauma center. J Trauma. 2002;52:715-9. 3. Janjua KJ, Sugrue M, Deane SA. Prospective evaluation of early missed injuries and the role of tertiary trauma Survey. J Trauma. 1998;44:1000-6; discussion 1006-7. 3. Janjua KJ, Sugrue M, Deane SA. Prospective evaluation of early missed injuries and the role of tertiary trauma Survey. J Trauma. 1998;44:1000-6; discussion 1006-7. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. 4. Kalemoglu M, Demirbas S, Akin ML, et al. Missed injuries in military patients with major trauma: original study. Mil Med. 2006;171:598-602. Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients In trauma patients, MP-positive plasma was associated with a signifi cantly higher synthesis of IL-6 and TNFα compared with plasma depleted from MPs (P = 0.047 and 0.002 respectively). Compared with healthy individuals the number of circulating MPs was signifi cantly decreased in trauma patients (P = 0.009). Most MPs originated from platelets. Multiple cellular protrusions, which result in MP formation, were observed in plasma from trauma patients, but not in healthy controls. Conclusion DNA and sRAGE kinetics diff er following trauma. Early elevation of sRAGE predicts mortality in univariate analysis and correlates with subsequent renal failure. P313 P313 Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients N Curry1, K Balvers2, DJ Kleinveld2, AN Boïng2, R Nieuwland2, JC Goslings2, NP Juff ermans2 1John Radcliff e Hospital, Oxford, UK; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) P313 Endogenous microparticles drive the proinfl ammatory host immune response in severely injured trauma patients N Curry1, K Balvers2, DJ Kleinveld2, AN Boïng2, R Nieuwland2, JC Goslings2, NP Juff ermans2 1John Radcliff e Hospital, Oxford, UK; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P313 (doi: 10.1186/cc14393) Clinical decision rule for cervical magnetic resonance imaging in suspected cervical spinal cord injury without bony injury is useful in predicting severity of cervical stenosis p g y T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 Conclusion On admission, trauma patients have a reduced immune response towards endotoxin challenge which is, at least in part, mediated by MPs, which circulate in low numbers and in early stages. Most MPs originate from platelets, which indicates that these cells may be the most important source of MPs involved in initiating an infl ammatory host response post injury. p g y T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 1National Center for Global Health and Medicine Hospital, Tokyo, Japan; 2Seirei Hamamatsu General Hospital, Shizuoka, Japan Critical Care 2015, 19(Suppl 1):P315 (doi: 10.1186/cc14395) Introduction Cervical spinal cord injury (CCI) without bony injury (CCIWOBI) is more frequent among Asian than among Caucasian populations and shows various extents of severity. Cervical magnetic resonance imaging (MRI) is useful for detecting intramedullary lesions, ligament injuries and intervertebral disk hernias, but some patients with mild CCIWOBI do not show clinically signifi cant abnormalities on MRI. To date, the cost–benefi t ratio of performing MRI in addition to computed tomography (CT) is unclear. We have developed a clinical decision rule for cervical MRI (MR-CDR), indicating MRI for patients >70 years old with ossifi cation of the posterior longitudinal ligament on CT or injury in a ground-level fall or a fall down stairs. The objective of the present study was to prospectively validate this MR-CDR for cervical MRI in patients with suspected mild CCIWOBI. References 1. Cohen MJ. J Trauma. 2010;68:1273-8. 1. Cohen MJ. J Trauma. 2010;68:1273-8. . Cohen MJ. J Trauma. 2010;68:1273-8. 1. Cohen MJ. J Trauma. 2010;68:1273 8. 2. Simmons JD. Ann Surg. 2013;258:591-6. 2. Simmons JD. Ann Surg. 2013;258:591-6. 2. Simmons JD. Ann Surg. 2013;258:591-6. Clinical decision rule for cervical magnetic resonance imaging in suspected cervical spinal cord injury without bony injury is useful in predicting severity of cervical stenosis T Inagaki1, A Kimura1, A Hagiwara1, R Sasaki1, K Kobayashi1, A Inaka1, G Makishi2 1National Center for Global Health and Medicine Hospital, Tokyo, Japan; 2Seirei Hamamatsu General Hospital, Shizuoka, Japan Critical Care 2015, 19(Suppl 1):P315 (doi: 10.1186/cc14395) Clinical decision rule for cervical magnetic resonance imaging in suspected cervical spinal cord injury without bony injury is useful in predicting severity of cervical stenosis P314 DNA and sRAGE circulation in the early phase after polytrauma P Joly1, C Massé2, D Dwivedi2, P Liaw2, J Marshall3, Y Berthiaume2, E Charbonney4 1University of Montreal, QC, Canada; 2McMaster University, Hamilton, ON, Canada; 3University of Toronto, ON, Canada; 4CRHSCM-University of Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P314 (doi: 10.1186/cc14394) Introduction Various DAMPS, alarmins are released after trauma. The soluble receptor for advanced glycation endproducts (sRAGE) was reported to be associated with acute renal failure and duration of ventilation [1]. Cell-free DNA (cfDNA) has been associated with prognosis in trauma patients [2]. We studied the kinetics of these two biomarkers over the fi rst 5 days, in a cohort of severely ill trauma patients. Methods We have been conducting a prospective observational study in two institutions in Japan since September 2012, enrolling patients with CCIWOBI among head or neck trauma patients >16  years old brought in by ambulance. We collect data about patient characteristics, injury profi les, neurological fi ndings, results of radiological examinations, and medical courses. We then analyze the sensitivity and specifi city of MR-CDR for detecting intramedullary lesions on MRI and conduct further analysis. Methods Patients who had sustained serious traumatic injury, within 24  hours of trauma, were recruited in a level I trauma center. We collected ISS, baseline demographic characteristics, resuscitation information and daily organ dysfunction (MOD) scores, over 10 days. Blood samples were collected within 24 hours of trauma (day 0) and S112 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results During the study period, 63 patients were brought in with CCIWOBI. Mean age was 60.6  years (standard deviation, 17.9  years) and 76% were male. Forty-fi ve patients presented with mild symptoms (Frankel Grade D). Cervical MRI was performed for 23 patients. Sensitivity and specifi city of MR-CDR in detecting intramedullary lesions on T2- weighted imaging among cases of suspected mild CCIWOBI were 85.7% (95% confi dence interval (CI), 60.1 to 96.0%) and 33.3% (95% CI, 12.1 to 35.4%). Further analysis showed a signifi cant diff erence in minimal spinal canal diameter as measured on sagittal T2-weighted imaging between the MR-CDR-positive and MR-CDR-negative groups (5.0 mm vs. 8.3 mm, P = 0.0003). One patient underwent surgery during hospitalization and no patients experienced exacerbated neurological fi ndings. P316 Accuracy of targeted wire-guided tube thoracostomy in comparison with classical surgical chest tube placement: a clinical study A Protic, I Barkovic, A Sustic University Hospital Rijeka, Croatia Critical Care 2015, 19(Suppl 1):P316 (doi: 10.1186/cc14396) Introduction Chest tube malfunction, after the tube thoracostomy, is often the result of an inappropriate chest tube tip position. The aim of this study was to analyze the precision of chest tube placement using the targeted wire guide technique (TWG technique) with a curve dilator and to compare it with the classical surgical technique (CS technique). Methods In this clinical study 80 patients with an indication for thoracic drainage due to pneumothorax or pleural eff usion were included. The experimental group contained 39 patients whose chest tube was placed using the TWG technique. The control group contained 41 patients whose chest tube was placed using the CS technique. Introduction Chest tube malfunction, after the tube thoracostomy, is often the result of an inappropriate chest tube tip position. The aim of this study was to analyze the precision of chest tube placement using the targeted wire guide technique (TWG technique) with a curve dilator and to compare it with the classical surgical technique (CS technique). Results There is positive signifi cant correlation between T day and LOS of injured patients (P <0.001, Spearman coeffi cient = 0.1672). Statistical analysis by Mann–Whitney test, between groups A and B, showed signifi cant diff erences in ICU LOS (P <0.001); no signifi cant diff erences (P <0.05) were found for age, ISS and outcome (Table 1). p g q q Methods In this clinical study 80 patients with an indication for thoracic drainage due to pneumothorax or pleural eff usion were included. The experimental group contained 39 patients whose chest tube was placed using the TWG technique. The control group contained 41 patients whose chest tube was placed using the CS technique. Conclusion The optimum and early time point of tracheostomy seems to be directly related with LOS in the ICU, independently of the rate of ISS, patient’s age and outcome. These results could account for ICU cost-eff ectiveness, as diminished LOS decreased the overall cost. Reference Results The comparison of the outcomes of the two techniques applied suggests that the TWG technique was signifi cantly more successful, irrespective of patient diagnosis (TWG vs. CS in all patients, 78.4% vs. 36.6%, P <0.001). See Table 1. 1. Alali et al. P317 Evaluating trauma care: comparison of early versus late tracheostomy ICU data outcome on injured patients V Kaldis1, N Mourelatos1, D Markopoulou1, K Venetsanou2, E Diogou1, E Papadaki1, D Chroni1, I Alamanos1 1General Hospital KAT-EKA, Kifi sia Athens, Greece; 2ICU Research Unit KAT, Kifi ssia Athens, Greece Critical Care 2015, 19(Suppl 1):P317 (doi: 10.1186/cc14397) Introduction In the surgical ICU, bedside tracheostomy (T) is one of the most frequently applied surgical techniques for multi-injured patients mainly with TBI [1]. The optimum surgical time decision for T still remains a contradiction in trauma. This retrospective study was designed to register all trauma patients who underwent T, during 60 months of observation (2009 to 2013), in order to identify factors associated with their ICU outcome on the basis of the T day (A <10th day >B) after tracheal intubation. g Conclusion MR-CDR was not validated for predicting the existence of intramedullary lesions on cervical MRI. MR-CDR is useful in predicting the severity of cervical stenosis. Methods Seventy-eight injured patients in the SICU underwent T, from a total of 403 issues; 58 male and 20 female, with mean age 59.3 and 74.7 years respectively. The total length of ICU stay recorded was 2,098 days, nursing time 26.55 (4/93), whereas the T time was adjusted between the 6th and 16th day (mean 11th). Mean ISS score was 22.59 (9 to 50). Classifi cation according to trauma type was TBI (n = 44) followed by thoracic trauma. Thirty-one male survivors were discharged from the ICU, to the ward. The mortality rate amounts to 47 cases due to infectious/non-infectious nosocomial complications and multiorgan dysfunction syndrome. Clinical ISS, the type of injury, ICU length of stay (LOS), T day, demographic (gender, age) data and ICU outcome were registered. Statistical analysis was performed with GraphPad 5.0.i Factors related to sepsis and outcome in multiple trauma patients H Pavlou, E Pappa, M Eforakopoulou KAT-EKA General Hospital, Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) pi Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398 Introduction The outcome of multiple trauma patients is related to a number of diagnostic and therapeutic interventions during hospitali- zation. ICU patients with severe trauma are susceptible to sepsis leading to poor outcome. Factors associated with the occurrence of sepsis and the outcome of these patients were investigated. Conclusion Using a curved dilator and the TWG technique for the thoracic drainage procedure we found statistically signifi cant advantage to the TWG technique in comparison with the CS technique regarding precise chest tube placement within the pleural cavity. References Methods We studied retrospectively all trauma patients admitted to the A’ ICU of KAT General Hospital in Athens during the last 3 years and were treated for more than 5 days. Age, gender, the type of injury, the severity of injury (Injury Severity Score), the length of ICU stay, severe sepsis, coexisting diseases, the outcome and the cause of death were recorded. Logistic regression and chi-square tests were used for statistical analysis. 1. Chen F, Yamada T, Aoyama A, et al. Position of a chest tube at video-assisted thoracoscopic surgery for spontaneous pneumothorax. Respiration. 2006;73:329-33. 2. Protic A, Barkovic I, Bralic M, et al. Targeted wire-guided chest tube placement: a cadaver study. Eur J Emerg Med. 2010;17:146-9. P314 No signifi cant diff erences were evident between groups in discharge status, duration of hospitalization, or neurological fi ndings at discharge. P316 Tracheostomy timing in traumatic brain injury: a propensity- matched cohort study. J Trauma Acute Care Surg. 2014;76:70-8. Table 1 (abstract P316) CS technique TWG technique Diagnosis N (% of success) (% of success) P value Pleural eff usion 47 37.5 78.2 0.005 Pneumothorax 31 35.3 78.6 0.029 Total 78 36.6 78.4 <0.001 P318 Factors related to sepsis and outcome in multiple trauma patients H Pavlou, E Pappa, M Eforakopoulou KAT-EKA General Hospital, Kifi sia, Athens, Greece Critical Care 2015, 19(Suppl 1):P318 (doi: 10.1186/cc14398) Ventilator-associated pneumonia in a trauma ICU M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399 Figure 1 (abstract P320). M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Royal London Hospital, London, UK normoxia will create a condition of relative hypoxia, which acts in turn as a stimulus for erythropoietin (EPO) production [1]. Variations in GSH and oxygen free radical (ROS) levels may be involved in this process. We tested the NOP in critically ill patients. y p Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399) Critical Care 2015, 19(Suppl 1):P319 ( Introduction Ventilator-associated pneumonia (VAP) is associated with increased length of ventilation, ICU stay, mortality, cost and antibiotic burden [1]. There is a large variation in reported rates of VAP, partly as a result of inconsistencies in defi nition [2]. We explored a more pragmatic defi nition to describe the VAP rate, antibiotic burden and outcome of VAP in a 44-bed adult critical care unit in a level 1 trauma centre. Methods A prospective observational study on 38 mechanically ventilated (FiO2 <50%) patients with no active bleeding, no blood transfusion needed, and no kidney failure. Eighteen patients underwent a 2-hour period of normobaric hyperoxia (FiO2  = 100%), 20 patients were evaluated as controls (no FiO2 variations). EPO was assayed at baseline (t0), 24 hours (D1) and 48 hours (D2). Serum GSH and ROS were assayed at t0 (baseline), t1 (2-hour FiO2 100%) and t2 (2- hour return to normoxia) in 12 patients in the hyperoxia group. Methods A retrospective review of all adult patients admitted to the ICU at The Royal London Hospital over a 6-month period (February to August 2014). The diagnosis of VAP was based on the Clinical Pulmonary Infection Score. Patients were identifi ed with VAP if they were started on antibiotics for chest sepsis 48  hours after start of mechanical ventilation. Demographic, clinical, microbiological and radiological data were collected to identify risk factors, and compare VAP and non- VAP groups. Chi-squared and ANOVA tests were performed using the SOFA statistics package. Results EPO tended to increase in the hyperoxia group over time (P = 0.05), while it remained stable in the control group (P = 0.53) (Figure 1). ROS levels increased at t1 and decreased at t2, GSH tended to decrease at t1 and increased at t2 in the hyperoxia group. Conclusion Relative hypoxia after a transient period of normobaric hyperoxia induces an increase in GSH levels, thus enhancing ROS scavenging. Table 1 (abstract P317). SICU data Table 1 (abstract P317). SICU data Table 1 (abstract P317). SICU data Outcome ICU data ISS Age ICU LOS (days) Tracheostomy day Survival Mortality A <10 16 ± 87 (16/45) 66 ± 94 (28/87) 602 17.2 ± 21.5 (4/47) 5.5 ± 6.5 (1/10) n = 13 (43.3%) n = 17 (57.7%) B >10 16 ± 25 (9/50) 64 ± 87 (23/93) 1,496 25 ± 34 (9/95) 15 ± 26 (11/23) n = 18 (40%) n = 27 (60%) t test NS NS <0.001 <0.001 NS NS Data presented as median ± IQR S113 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results A total of 106 multiple trauma patients, 85 men and 21 women, met the inclusion criteria. Depending on their age, patients were divided into two groups: <60  years old and >60  years old. In both groups, gender, the type and severity of injuries and the length of ICU stay were not associated with outcome. The length of ICU stay was correlated with severe sepsis and coexisting diseases (P <0.01) in both groups. Mortality was not diff erent in the two groups. The presence of at least one coexisting disease was signifi cantly associated with mortality (P <0.007). Sepsis was signifi cant cause of death in trauma patients >60 years (P <0.05). Figure 1 (abstract P320). Figure 1 (abstract P320). Conclusion In multiple trauma patients, the length of ICU stay and comorbidities infl uence the occurrence of severe sepsis, comorbidities increase mortality, and sepsis is the leading cause of death in trauma patients >60 years old. Ventilator-associated pneumonia in a trauma ICU M Raja, A Ely, P Zolfaghari Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P319 (doi: 10.1186/cc14399 This may act as a stimulus for EPO production. Reference Results A total of 535 mechanically ventilated patients were admitted in the study period, with 281 ventilated for more than 48 hours. The incidence of VAP was 11% in all ventilated patients and 19.6% in those ventilated more than 48 hours. VAP rates were 31% in polytrauma, 25% in neurotrauma and 18% in the neuromedical/surgical cohort. Early and late onset VAP were equal in number. Patients with VAP spent longer on mechanical ventilation (9 ± 9 in no VAP vs. 18 ± 18 days in VAP patients; P <0.001), and had longer ICU and hospital LOS (11 ± 10 vs. 22 ± 20 days; P <0.001). However, APACHE II scores and hospital mortality were unaff ected (VAP 33.3% vs. no VAP 37.6%; P = 0.173). Despite rising infl ammatory markers and secretion load, many patients did not exhibit oxygenation defi cits. Sputum microbiology showed S. aureus, H. Infl uenza, Klebsiella and Enterobacter as predominant pathogens with low rates of Pseudomonas, Acinectobacter and other resistant organisms. Average length of antibiotic use was 6 (3 to 18) days. Conclusion Chest sepsis after 48  hours of mechanical ventilation commonly complicates neurocritical illness and polytrauma requiring signifi cant ICU resources and antibiotic burden. However, it does not aff ect mortality. Further research should focus on pathophysiology and new preventative measures to reduce VAP in the at-risk population. References 1. Balestra et al. J Appl Physiol. 2006;100:512-8. p References 1. Taylor MD, et al. J Trauma. 2002;53:407-14. 2. Osborn TM, et al. Crit Care Med. 2004;32:2234-40. References References 1. Safdar N, et al. Crit Care Med. 2005;33:2184-93. 2. Nair GB, et al. Intensive Care Med. 2014 [Epub ahead of print]. 1. Safdar N, et al. Crit Care Med. 2005;33:2184-93. 2. Nair GB, et al. Intensive Care Med. 2014 [Epub ahead of print]. Results Mean D-dimer concentration of the control group and of the patient group with PE was 0.28 (95% CI: 0.25 to 0.31)  mg/l and 1.45 (95% CI: 1.23 to 1.72)  mg/l, respectively. Receiver operator characteristics analysis revealed an optimized cutoff value of 0.466 mg/l for the PATHFAST D-dimer assay (AUC = 0.975 (95% CI: 0.938 to 0.993); sensitivity: 95% (95% CI: 86 to 99%); specifi city: 89% (95% CI: 82 to 95%)). Therefore we used a rounded up cutoff value of 0.5 mg/l to examine the diagnostic accuracy of PATHFAST D-dimer to exclude PE. The correlation between PATHFAST and VIDAS results was particularly close for concentrations at or around the critical cutoff value of P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, R Castagnani, N Mininno, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P320 (doi: 10.1186/cc14400) P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study S Zuccari, A Donati, E Damiani, R Castagnani, N Mininno, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2015, 19(Suppl 1):P320 (doi: 10.1186/cc14400) P321 Comparison of the PATHFAST D-dimer assay with two POC D-dimer assays E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) Comparison of the PATHFAST D-dimer assay with two POC D-dimer assays E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) E Spanuth1, B Ivandic1, R Thomae2, E Giannitsis3 1DIAneering GmbH, Heidelberg, Germany; 2Mitsubishi Chemical Europe, Düsseldorf, Germany; 3University of Heidelberg, Germany Critical Care 2015, 19(Suppl 1):P321 (doi: 10.1186/cc14401) Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Methods A total of 272 patients with symptoms of PE and VTE were included. The diagnoses of VTE and PE were established by duplex ultrasound, venography and spiral CT. D-dimer values were determined in the patients and in plasma samples obtained from 102 healthy individuals who served as the control group. Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Introduction The early exclusion of PE is a major precondition for goal- oriented diagnostic and therapeutic measures. The aim of the study was to evaluate the new point-of-care assay PATHFAST D-dimer in comparison with VIDAS D-Dimer Exclusion and STRATUS CS D-dimer. Methods A total of 272 patients with symptoms of PE and VTE were included. The diagnoses of VTE and PE were established by duplex ultrasound, venography and spiral CT. D-dimer values were determined in the patients and in plasma samples obtained from 102 healthy individuals who served as the control group. Impact of introducing guidelines for thrombolysis of submassive pulmonary embolism at a large UK teaching hospital GP Misselbrook g p p Results The peak action of enoxaparin and rivaroxaban was observed at 4 hours post administration. LPTEG indicators that determine the coagulation state after 4 hours in the fi rst group: CTA was decreased by 72.12% (P <0.05), ICD was decreased by 68.44% (P <0.05), GP was increased by 17.9%; in the second group: CTA was decreased by 76.24% (P <0.05), ICD was decreased by 74.52% (P <0.05), GP was increased by 23.34%. After 12 hours, CTA in the fi rst group decreased by 22.41%, ICD decreased by 5.3%, GP increased by 8.12%, indicating reduction of hypocoagulation eff ect; in the second group, CTA decreased by 39.35% (P <0.05), ICD decreased by 40.24% (P <0.05), GP increased by 18.25%. After 24  hours in the fi rst group LPTEG indicators returned to the original value, and in the second group of patients CTA was decreased by 15.14%, ICD was decreased by 6.62%, GP increased by 14.22%.f University Hospitals Southampton NHS Foundation Trust, Southampton, UK Critical Care 2015, 19(Suppl 1):P324 (doi: 10.1186/cc14404) Introduction Pulmonary embolism (PE) is a signifi cant cause of death with 10% of patients dying within 3 months [1]. Multiple studies now advocate the use of thrombolysis (TPA) in both massive and submassive PE [1,2]. This audit assessed the impact of introducing a guideline allowing for thrombolysis of submassive and massive PE at a large UK teaching hospital. g p Methods Retrospective data collection using notes and imaging to risk-stratify patients. First audit ran from January to June 2012. New guidance was introduced in March 2013 (Figure 1) after which a second cycle ran for a further 6 months. y y y Conclusion Using LPTEG showed the hypocoagulation eff ect of continuous rivaroxaban 24  hours after oral administration compared with enoxaparin, which retains less hypocoagulation eff ect 12 hours after administration. LPTEG indicators in the second group were bigger than in the fi rst group after 12 hours: CTA 43.07%, ICD 69.72%, GP 54.12%. Results Re-audit revealed 46 patients with radiological evidence of massive or submassive PE on CTPA (32% of all PEs). Ten patients had clinical features of submassive PE and nine presented as massive PE. Previous guidelines suggested consideration of TPA in only seven patients in 6  months. TPA was given to two patients; however, six patients had no contraindications to treatment (Table 1). P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study Introduction The normobaric oxygen paradox (NOP) postulates that a period of normobaric hyperoxia followed by a rapid return to S114 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 0.5 mg/l. The correlation between PATHFAST and STRATUS results was particularly close in the patient group with VTE (r = 0.9694), whereas slightly lower results were obtained with STRATUS in the control group. With the widely used cutoff value 0.5 mg/l, PATHFAST demonstrated suitable sensitivity but not STRATUS. ROC analysis indicated that optimal cutoff values could be set at either 0.5 or 0.6 mg/l and at 0.3 or 0.4 mg/l for PATHFAST and STRATUS, respectively. clinical guidelines recommend the administration of pharmacological thromboprophylaxis (PTP) to reduce the risk of developing TEC [2]. However, it is unknown whether delayed PTP initiation increases risk of TEC. We hypothesize that delayed PTP initiation is associated with increased TEC rates. Methods A retrospective chart review (2010 to 2013) was conducted on adult trauma patients that were admitted into a level 1 trauma centre in Toronto. Demographics, date of PTP initiation, date of TEC diagnosis (CT-PE/US Doppler), injury type and severity were collected. A comparison between early and late PTP initiation has been made with regards to TEC development. Student’s t test, univariate and multivariate logistic regression analyses were performed. y Conclusion By use of the PATHFAST D-dimer assay only six of diagnoses were missed at the time of fi rst presentation compared with 10 diagnoses missed by the VIDAS D-dimer Exclusion assay, yielding higher sensitivity of the PATHFAST D-dimer assay compared with the VIDAS assay (90% vs. 83%). The STRATUS assays showed comparable performance and appeared to be suitable for the exclusion of VTE in the emergency room setting, whereas PATHFAST demonstrated superior sensitivity. Moreover, the PATHFAST analyzer allows simultaneous determination of D-dimer and cardiac troponin I within 16 minutes from whole blood samples. Therefore, this method might be useful at the point of care for early diagnostic assessment of patients with symptoms of PE or chest pain admitted to the ER or to the chest pain unit. g g y p Results A total of 1,312 patients received PTP, 821 (62.5%) initiated early PTP (within 48 hours) while 491 (37.5%) initiated after 48 hours. The group that initiated early prophylaxis was younger (mean: 46 vs. Using rivaroxoban in patients with venous thromboembolism I Tyutrin, O Tarabrin, B Todurov, S Shcherbakov, D Gavrychenko, G Mazurenko Odessa National Medical University, Odessa, Ukraine Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P322 (doi: 10.1186/cc14402) Critical Care 2015, 19(Suppl 1):P322 (doi: 10.1186/cc14402) Conclusion Mortality rates in patients with delayed PTP are higher. Our study shows LOS as the only independent predictor for TEC. However, this might not necessarily refl ect causation. Delayed PTP appears not to be an independent predictor to TEC events in trauma patients, which favours current clinical trends when it comes to contraindicating early PTP initiation. Introduction A prospective study was conducted in patients for treatment of venous thromboembolism (VTE) to compare the eff ect of enoxaparin and rivaroxaban using the method of low-frequency piezoelectric thromboelastography (LPTEG) for checking coagulation activation markers. P320 Normobaric oxygen paradox and erythropoietin production in critically ill patients: a prospective observational study 55, P <0.0005), had lower ISS (mean: 17 vs. 24, P <0.0005), shorter length of stay (LOS) (mean: 11 vs. 23, P <0.0005), more pelvic fractures (19% vs. 13%, P = 0.0058), more head injury (AIS Head ≥3, P <0.0005), less blunt trauma (85% vs. 95%, P <0.0005), lower incidence of TEC (5.3% (44) vs. 8.5% (42), P = 0.023), and lower mortality rate (1.5% vs. 7.5%). Univariate analysis showed LOS (P <0.0005), ISS (P <0.0005), time to PTP initiation (P  = 0.0018) and blunt MOI (P  = 0.0099) signifi cantly associated with TEC events. Multivariate analysis, however, showed TEC events correlated only to LOS (P = 0.0001). Stepwise multiple logistic regression confi rmed LOS as independently associated with TEC events (95% CI = 0.003, 0.006, P <0.0005). P323 P323 Is delaying pharmacological thromboprophylaxis associated with thromboembolic complications? P Padim1, A Alshafai2, S Canestrini3, S Rizoli2, J De Rezende Neto2, A McFarlan2 1Universidade de São Paulo, Ribeirao Preto, Brazil; 2St Michael’s Hospital, Toronto, ON, Canada; 3St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P323 (doi: 10.1186/cc14403) Is delaying pharmacological thromboprophylaxis associated with thromboembolic complications? P Padim1, A Alshafai2, S Canestrini3, S Rizoli2, J De Rezende Neto2, A McFarlan2 1Universidade de São Paulo, Ribeirao Preto, Brazil; 2St Michael’s Hospital, Toronto, ON, Canada; 3St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P323 (doi: 10.1186/cc14403) q g g g Conclusion Delivering a service that off ers TPA to patients with submassive PE signifi cantly increases the need to consider this therapy. Introducing this service is only eff ective if doctors initially assessing these patients are aware of recent changes to guidelines for PE. R f Impact of introducing guidelines for thrombolysis of submassive pulmonary embolism at a large UK teaching hospital GP Misselbrook Limitations to TPA administration were late recognition of submassive PE and inadequate knowledge of changes to guidelines.f References Methods A total of 60 patients entered the Odessa Clinical Regional Hospital for treatment venous thromboembolism. Patients were divided into two groups. The fi rst group (n  = 30) were receiving enoxaparin in dosage 1.5 mg/kg subcutaneously per day. The second group (n  = 30) were receiving rivaroxaban orally 15  mg/day. For checking the coagulation state we were using such indicators of LPTEG as constant thrombin activity (CTA), intensity of coagulation drive (ICD) and gel point (GP). We performed LPTEG three times per day: 4, 12 and 24 hours after taking the drug to check for changes in the coagulation state in both groups of patients. 1. Geerts W, Code K, Jay R, Chen E, Szalai J. A prospective study of venous thromboembolism after major trauma. N Engl J Med. 1994. 331;1601-6. 1. Geerts W, Code K, Jay R, Chen E, Szalai J. A prospective study of venous thromboembolism after major trauma. N Engl J Med. 1994. 331;1601-6. 2. Rogers FB, Cipolle MD, Velmahos G, et al. Practice management guidelines for the prevention of venous thromboembolism: the EAST Practice Management Guidelines Work Group. J Trauma. 2002;53:142-64. Using rivaroxoban in patients with venous thromboembolism I Tyutrin, O Tarabrin, B Todurov, S Shcherbakov, D Gavrychenko, G Mazurenko Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P322 (doi: 10.1186/cc14402) Using rivaroxoban in patients with venous thromboembolism I Tyutrin, O Tarabrin, B Todurov, S Shcherbakov, D Gavrychenko, G Mazurenko p References Introduction Thromboembolic complications (TEC) are very common and lethal in patients suff ering from traumatic injury [1]. The trauma Introduction Thromboembolic complications (TEC) are very common and lethal in patients suff ering from traumatic injury [1]. The trauma Meyer G, et al. N Engl J Med. 2014;370:1402-11. Kearon C, et al. Chest. 2012;141:419-96S. S115 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (P <0.001). The type of thrombosis was directly associated with poor outcome, especially one that resulted from central catheters (P <0.001) and pulmonary embolism (P <0.001). However, no correlations were found with temperature, white blood cells and platelet counts on admission (P > 0.05). (P <0.001). The type of thrombosis was directly associated with poor outcome, especially one that resulted from central catheters (P <0.001) and pulmonary embolism (P <0.001). However, no correlations were found with temperature, white blood cells and platelet counts on admission (P > 0.05). Figure 1 (abstract P324). New local guidelines. Conclusion Thrombosis aff ects the ICU patient’s fi nal outcome. The type of thrombosis contributes to a poor outcome and mainly the occurrence of pulmonary embolism signifi cantly increases the mortality rate. P325 Results A total of 51 patients were included. The median age and BMI were 73 years and 23 kg/m2, respectively; 31% were female and 69% were surgical critical care patients. The median APACHE II and SOFA scores were 20 and 8, respectively. Risk factors associated with DVT were presence of central venous catheter 63%, malignancy 9% and hemodialysis 14%. The rate of DVT was 18.6% and the rate of CRT was 13.7%. All of these were asymptomatic and seen in neck and upper extremities. There was no DVT-associated adverse event (pulmonary embolism, bleeding) during hospital stay. The 28-day all-cause mortality rate was 3.4%. Introduction Despite preventive anticoagulation therapy measures, venous thromboembolic disease is a major cause of morbidity and mortality among patients hospitalized in ICUs. In fact, pulmonary embolism is not only the most serious manifestation of the disease, but also one of the primary causes of sudden death. The aim of this study is to investigate the frequency of thromboembolism and pulmonary embolism in ICU hospitalized trauma and neurosurgical patients. Conclusion While incidence of asymptomatic DVT is relatively high in adult critically ill patients, they were found only in the neck and upper extremities without any adverse event. Further research is needed to evaluate the clinical signifi cance of this type of DVT. Methods One hundred ICU patients, 51 postoperative neurosurgical and 49 trauma, were included in the study. Patients’ demographic data as well as medical history, temperature, white blood cells and platelets counts were recorded on admission, the day of thrombosis diagnosis and the fi nal outcome of their treatment. Statistics were performed with SPSS-19. P <0.05 was considered signifi cant. P326 P326 Incidence and outcome of asymptomatic deep vein thrombosis in critically ill patients: a prospective cohort study R Echigoya, H Okamoto, H Uchino, A Kuriyama, N Tamura, K Sato, T Fukuoka Kurashiki Central Hospital, Okayama, Japan Critical Care 2015, 19(Suppl 1):P326 (doi: 10.1186/cc14406) Figure 1 (abstract P324). New local guidelines. Figure 1 (abstract P324). New local guidelines. Introduction Asymptomatic deep vein thrombosis (DVT) including catheter-related thrombosis (CRT) is an increasingly recognized disease entity in critically ill patients. However, the reported rate and outcome of DVT vary widely depending on study design, patient background and detecting method. The objective of this study is to evaluate the incidence and outcome of DVT in adult critically ill patients. Table 1 (abstract P324). TPA decisions High Intermediate Total risk risk Considered TPA and given 2 2 0 Considered TPA but contraindications 1 1 0 Considered TPA and not given on balance 3 0 3 Considered TPA and not given but fi t 3 2 1 criteria Not considered TPA but contraindicated 4 1 3 anyway Not considered TPA and on balance 5 1 4 would not be given Not considered TPA but fi t criteria 1 0 1 Table 1 (abstract P324). TPA decisions y Methods This study is a prospective cohort study of patients admitted to a medical and surgical ICU from 1 July 2014 to 15 October 2014. All consecutive patients over 18 years of age and with expected ICU stay over 72 hours were included. Patients who had previous history of DVTs were excluded. We examined internal jugular vein, subclavian vein, axillary vein, brachial vein, femoral vein, superfi cial femoral vein, and popliteal vein, on ICU admission and within 48 hours after ICU discharge. The DVT was diagnosed using compression ultrasonography with color Doppler. Images were interpreted by two independent investigators trained in ultrasonography. All patients received intermittent pneumatic compression and unfractionated heparin twice daily during their IUC stay. Once the DVT was detected, therapeutic anticoagulation was initiated. Contrast-enhanced CT was performed when the patients were suspected to have pulmonary embolism. The primary outcome was the incidence of DVT during the ICU stay. Patients were followed until their hospital discharge. P327 P327 Computed tomographic pulmonary angiographic fi ndings to predict adverse outcomes in acute pulmonary embolism P Tajarernmuang, J Euathrongchit, C Liwsrisakun, A Deesomchok, T Theerakittikul, C Bumroongkit, C Pothirat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2015, 19(Suppl 1):P327 (doi: 10.1186/cc14407) gi Results Thirty-eight out of 100  patients presented thrombosis, 14 trauma and 24 neurosurgical. We examined the correlation of thrombosis development during hospitalization with diagnosis, treatment allocated time and overall patient outcome. It was found that neurosurgical patients developed thrombosis more frequently than trauma patients (P <0.05). In relation to diagnosis, thrombosis was prevalent among patients with brain lesions (P = 0.018). Regarding the type of thrombosis, pulmonary embolism was also commonly apparent among individuals with brain lesion (P = 0.020). In addition, there was a statistically signifi cant correlation in thrombosis occurrence between hospitalization day (P  <0.01) and patients’ outcome on discharge Introduction Computed tomographic pulmonary angiography (CTPA) has been used as a standard tool for diagnosing an acute pulmonary embolism (APE). The right ventricular (RV) strain signs may be used to predict adverse outcomes. However, the results are still controversial. The primary objective of our study was to evaluate the relationship between the RV strain signs and respiratory failure requiring mechanical ventilation or death in APE. The secondary objective was to identify clinical factors which related to those outcomes. Introduction Computed tomographic pulmonary angiography (CTPA) has been used as a standard tool for diagnosing an acute pulmonary embolism (APE). The right ventricular (RV) strain signs may be used to predict adverse outcomes. However, the results are still controversial. The primary objective of our study was to evaluate the relationship between the RV strain signs and respiratory failure requiring mechanical ventilation or death in APE. The secondary objective was to identify clinical factors which related to those outcomes. S116 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P329). Kaplan–Meier analysis of ESRD progression for SC and Ecu treatment for all patients. Methods CTPA and the medical records of patients with suspected APE on admission from June 2011 to March 2013 were reviewed. Mean platelet volume and mean platelet volume/platelet count ratio in risk stratifi cation of pulmonary embolism T Yardan , M Meric , C Kati , Y Celenk , A Atici 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) T Yardan , M Meric , C Kati , Y Celenk , A Atici 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) Introduction Recently, mean platelet volume (MPV) was reported to predict early death in acute pulmonary embolism (PE). The aim of this study was to investigate the role of MPV and MPV/platelet count ratio (MPV/P) in risk stratifi cation of patients with acute PE. Conclusion Ecu treatment reduces the number of ESRD events and the rate of progression to ESRD; thus initiation of Ecu early after aHUS diagnosis may prevent cumulative kidney damage and progression to ESRD. i p Methods We retrospectively reviewed the medical records of patients with acute PE admitted to the emergency department. In addition to the clinical evaluation, platelet count and MPV were measured on admission. P328 Mean platelet volume and mean platelet volume/platelet count ratio in risk stratifi cation of pulmonary embolism T Yardan1, M Meric1, C Kati1, Y Celenk2, A Atici1 1Ondokuz Mayis University School of Medicine, Samsun, Turkey; 2Ministry of Health Van Education & Research Hospital, Van, Turkey Critical Care 2015, 19(Suppl 1):P328 (doi: 10.1186/cc14408) p Reference 1. Ghuysen A, Ghaye B, Willems V, Lambermont B, Gerard P, Dondelinger RF, et al. Computed tomographic pulmonary angiography and prognostic signifi cance in patients with acute pulmonary embolism. Thorax. 2005;60:956-61. p p Results The SC and Ecu treatment phases included 32 and 33 patients, respectively. With SC, during a median (range) of 211 (7 to 745) days, 13 (41%) patients had a total of 16 ESRD events. On Ecu treatment, during a median (range) of 924 (73 to 1,254) days, three (9%) patients had a total of fi ve ESRD events. The ESRD event rate was 92% lower during Ecu treatment versus the SC phase (0.36 vs. 0.07; P = 0.001); the incidence rate ratio was 0.08 (95% CI = 0.02 to 0.37; P = 0.001). HR for progression to ESRD for patients on Ecu versus SC was 0.03 (95% CI <0.01 to 0.34), a 97% reduction (Figure 1). Stratifi cation by baseline CKD stage showed no patients with CKD stage 2 or 3 at baseline progressed to ESRD over 3 years of Ecu treatment. P327 RV dysfunction signs included right ventricular to left ventricular (RV/LV) diameter ratio, interventricular septal shift, main pulmonary artery to ascending aorta (mPA/AA) diameter ratio, IVC contrast refl ux, SVC diameter, IVC diameter, PA diameter and azygos vein diameter. Clinical factors included cardiovascular, respiratory parameter and also time to diagnosis and treatment. g Results There were total of 36 cases with suspected APE on admission. Ten patients required mechanical ventilation (27.8%) and seven patients died (19.4%). Interventricular septal (IVS) shift was a signifi cant risk factor of in-hospital death (85.7% vs. 27.6%, P  = 0.008) and respiratory failure (70% vs. 26.9%, P = 0.026). The sensitivity, specifi city, positive predictive and negative predictive values of IVS shift to predict in-hospital death were 85.7%, 70%, 42.8% and 95.5%, respectively. The sensitivity, specifi city, positive predictive and negative predictive values of IVS shift to predict respiratory failure were 70%, 73.1%, 50% and 86.4%, respectively. The ratios of RV to LV diameter and the ratio of main pulmonary artery to ascending aorta diameter tended to be higher in the nonsurvivor group. The clinical factor that predicted mortality was the PaO2 to FiO2 ratio (P/F ratio). Mean P/F ratio in survivor and nonsurvivor groups was 246.1 ± 94.1 vs. 132.2 ± 78.1, respectively (P = 0.011). P/F ratio ≤150 was the best predictor of mortality (66.7% vs. 8.7%, P = 0.008). Figure 1 (abstract P329). Kaplan–Meier analysis of ESRD progression for SC and Ecu treatment for all patients. with aHUS progress to end-stage renal disease (ESRD) after the fi rst episode [1]. Two prospective clinical trials have assessed the effi cacy of eculizumab (Ecu) in patients with aHUS [2]. We now evaluate data on progression to ESRD before and during Ecu treatment. Methods Patients with chronic kidney disease (CKD) stage 1 to 4 were analysed for progression to an ESRD event (two consecutive glomerular fi ltration rate measurements <15 ml/minute/1.73m2 (CKD stage 5)). ESRD incidence rate ratios during supportive care (SC) and Ecu treatment phases were calculated using a negative binomial regression analysis. Kaplan–Meier analyses were calculated for all patients and stratifi ed by CKD stages 2 to 4 at baseline. Hazard ratios (HR) were calculated from Cox proportional hazard models. Conclusion The IVS shifting from CTPA and P/F ratio ≤150 helps predict poor outcomes in APE. Early initiation of eculizumab treatment in patients with atypical haemolytic uraemic syndrome improves long-term outcomes: a pooled analysis of clinical trials p y J Vande Walle1, Y Delmas2, G Ardissino J Vande Walle1, Y Delmas2, G Ardissino3, J Wang4, J Kincaid4, H Haller5 1University Hospital Ghent, Belgium; 2Centre Hospitalier Universitaire de Bordeaux, France; 3Ospedale Maggiore Policlinico, Milan, Italy; 4Alexion Pharmaceuticals, Cheshire, CT, USA; 5Medical School Hannover, Germany Critical Care 2015, 19(Suppl 1):P330 (doi: 10.1186/cc14410) Introduction Atypical haemolytic uraemic syndrome (aHUS) is a severe, life-threatening disease requiring rapid treatment to inhibit complement-mediated thrombotic microangiopathy (TMA) and avoid irreversible organ damage. Four prospective clinical trials have reported the safety and effi cacy of eculizumab (Ecu) in the treatment of aHUS [1,2]. We report data from a pooled analysis of these trials on renal function in patients starting Ecu within ≤7 days or >7 days after the current aHUS manifestation. Introduction Atypical haemolytic uraemic syndrome (aHUS) is a severe, life-threatening disease requiring rapid treatment to inhibit complement-mediated thrombotic microangiopathy (TMA) and avoid irreversible organ damage. Four prospective clinical trials have reported the safety and effi cacy of eculizumab (Ecu) in the treatment of aHUS [1,2]. We report data from a pooled analysis of these trials on renal function in patients starting Ecu within ≤7 days or >7 days after the current aHUS manifestation. References 1. Fremeaux-Bacchi V, et al. CJASN. 2013;8:554-62. Results One hundred and fi fty-two patients were included. Patients with right ventricular (RV) dysfunction had signifi cantly higher MPV levels and MPV/P than patients without RV dysfunction. Receiver operating characteristic analysis revealed that a MPV cutoff of 7.85 fl provided 53.3% sensitivity and 68.5% specifi city, and a MPV/P cutoff of 0.0339 fl /(109/l) provided 69.6% sensitivity and 65% specifi city for prediction of RV dysfunction. There was a positive correlation between MPV and systolic pulmonary artery pressure (SPAP) and between MPV and RV diameter. There was a positive correlation between MPV/P and SPAP and between MPV/P and RV diameter. The low-risk PE group had lower MPV and MPV/P than the massive PE and submassive PE groups. Conclusion MPV and MPV/P are associated with RV dysfunction and clinical severity in acute PE. Low MPV and MPV/P levels may be an indicator of low risk in patients with acute PE. 2. Legendre C, et al. N Engl J Med. 2013;368:2169-81. Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemiai Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemia A Taha, A Shafi e, M Mostafa, N Syed, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P331 (doi: 10.1186/cc14411) y Results We identifi ed 21 eligible studies (16,951 patients). After pooling data from the 20 included cohort studies (16,884 patients), at least around 33% (95% CI: 27 to 39; I2: 97.8%) of patients with TBI in published reports received transfusions at some point during their hospital stay. In a post hoc analysis of one RCT comparing transfusion strategies, 82% of patients were transfused RBCs. Thresholds for transfusion were rarely available and varied from 6 to 10 g/dl. From raw data, Glasgow Coma Scale scores were lower in patients who were transfused than those who were not (three cohort studies; n = 1,371; mean diff erence of 1.38 points (95% CI: 0.86 to 1.89); I2 = 12%). Mortality was not signifi cantly diff erent among transfused and nontransfused patients both in univariate and multivariate analyses. Hospital length of stay was longer among patients who were transfused (three studies; n = 455; mean diff erence 9.58 days (95% CI: 3.94 to 15.22); I2 = 74%). Due to the observational nature of included studies, results should be considered cautiously due to the high risk of confounding. i A Taha, A Shafi e, M Mostafa, N Syed, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P331 (doi: 10.1186/cc14411) Introduction Hemoglobin as the main trigger parameter for blood transfusion usually gives diminutive information about oxygen delivery and consumption. Although central venous oxygen saturation (ScvO2) is an alternative parameter, its changes are unable to detect regional hypoxia. Our aim was to evaluate the quotient of the central venous-to- arterial carbon dioxide gradient (δPCO2) and the arteriovenous oxygen content diff erence (Ca-cvO2) as a valid transfusion trigger parameter in hemodynamically stable anemic patients to reduce the amount of potentially counterproductive erythrocyte transfusions [1].i Methods Forty-fi ve postoperative patients admitted to our cardiac ICU were enrolled between January 2013 and September 2014. Three groups were defi ned according to the trend of blood loss over the surgical drains in the fi rst 24 postoperative hours. Red blood cell transfusion in patients with traumatic brain injury: a systematic review Red blood cell transfusion in patients with traumatic brain injury: a systematic review A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada Critical Care 2015, 19(Suppl 1):P332 (doi: 10.1186/cc14412) minute/1.73 m2 were included. Changes from baseline in eGFR were analysed at study visits using a one-sample t test. y A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada A Boutin1, M Chasse1, M Shemilt1, F Lauzier1, L Moore1, R Zarychanski2, J Lacroix3, DA Fergusson4, D Griesdale5, P Desjardins1, AF Turgeon1 1Université Laval, Quebec, QC, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3Université de Montreal, QC, Canada; 4Ottawa Hospital Research Institute, Ottawa, ON, Canada; 5University of British Columbia, Vancouver, BC, Canada Critical Care 2015 19(Suppl 1):P332 (doi: 10 1186/cc14412) Results Data from 97 patients were analysed: median (range) age at enrolment was 29 (0 to 80) years; 62% of patients were females; median (range) duration of current manifestation to start of Ecu treatment was 23 (1 to 1,447) days; median (range) baseline eGFR was 15.9 (5.6 to 76.1) ml/minute/1.73 m2. Ecu treatment was started in 21 patients in ≤7  days and 76 patients in >7  days after presentation with TMA. Median eGFR was 11 ml/minute/1.73 m2 for the patients started within 7 days and 16 ml/minute/1.73 m2 for those initiating >7 days. The mean change from baseline in eGFR for patients starting Ecu in ≤7 days and in >7 days after presentation with TMA were 57 and 23 ml/minute/1.73 m2 at 1 year, respectively (Figure 1). Critical Care 2015, 19(Suppl 1):P332 (doi: 10.1186/cc14412) Introduction We aimed to evaluate the frequency of red blood cell (RBC) transfusion in patients with traumatic brain injury (TBI) as well as determinants and outcomes associated with RBC transfusion in this population. Methods We conducted a systematic review of cohort studies and trials of patients with TBI. Evaluation of the quotient of the venoarterial carbon dioxide gradient and the arteriovenous oxygen content diff erence as a transfusion trigger parameter in hemodynamically stable patients with signifi cant anemiai Mild blood loss was defi ned as 500 to 1,000  ml/24  hours, moderate (1,000 to 1,500/24  hours) and severe (>1,500  ml/24  hours). In addition to the δPCO2 the following parameters were monitored: CI, CO, SVR, serum lactate, ScvO2 and hemoglobin. Ca-cvO2 was calculated and the δPCO2/ Ca-cvO2 quotient was assessed for a total of 400 paired blood samples. All enrolled patients were hemodynamically stable. A retrospective analysis of this data was performed.i Conclusion RBC transfusion is frequent in patients with TBI, but practices varied widely in cohort studies in this population. The paucity of data precludes defi nitive conclusions and highlights the lack of clinical evidence guiding transfusion strategies in TBI. P329 Progression to end-stage renal disease is reduced with eculizumab in patients with atypical haemolytic uraemic syndrome J Vande Walle1, S Johnson2, E Harvey3, J Kincaid3 1University Hospital Ghent, Belgium; 2Medicus Economics, LLC, Boston, MA, USA; 3Alexion Pharmaceuticals, Cheshire, CT, USA Critical Care 2015, 19(Suppl 1):P329 (doi: 10.1186/cc14409) Methods Data from four phase 2, open-label, single-arm trials including both paediatric and adult patients with aHUS were pooled. Patients with a documented date of onset of current TMA manifestation and a baseline estimated glomerular fi ltration rate (eGFR) of <90  ml/ Introduction Atypical haemolytic uraemic syndrome (aHUS) is associated with severe kidney damage; almost one-half of adults S117 Critical Care 2015, Volume 19 Suppl 1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P330). Mean change in eGFR from baseline over 1 year (standard error). hemoglobin capacity decline and signifi cantly improved after erythrocyte transfusions (P <0.005). Figure 1 (abstract P330). Mean change in eGFR from baseline over 1 year (standard error). Conclusion Blood transfusions carry risks of adverse eff ects and should be carried out responsibly. Our fi ndings suggest an additive and easy detectable transfusion trigger parameter (δPCO2/Ca-cvO2) providing physiological information on anemia-related altered oxygen extraction conditions and hence the indication for erythrocyte transfusions. However, additional studies are warranted to confi rm these fi ndings. Reference 1. Mekontso-Dessap A, Castelain V, Anguel N, Bahloul M, Schauvliege F, et al. Combination of venoarterial PCO2 diff erence with arteriovenous O2 content diff erence to detect anaerobic metabolism in patients. Intensive Care Med. 2002;28:272-7. 1. Mekontso-Dessap A, Castelain V, Anguel N, Bahloul M, Schauvliege F, et al. Combination of venoarterial PCO2 diff erence with arteriovenous O2 content diff erence to detect anaerobic metabolism in patients. Intensive Care Med. 2002;28:272-7. y References 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. 1. Legendre C, et al. N Engl J Med. 2013;368:2169-81. 2. Keating GM. Drugs. 2013;73:2053-66. Red blood cell transfusion in patients with traumatic brain injury: a systematic review We searched Medline, Embase, The Cochrane Library and BIOSIS databases from their inception up to 30 June 2014. We selected cohort studies and RCTs of adult patients with TBI reporting data on RBC transfusions. We extracted data related to demographics, baseline characteristics, blood product use and any relevant clinical patient-oriented outcome. Cumulative incidences of transfusion were pooled through random eff ect models with a DerSimonian approach, after a Freeman–Tukey transformation to stabilize variances. To evaluate the association between RBC transfusion and potential determinants as well as outcomes, we pooled risk ratios or mean diff erences with random eff ect models and the Mantel–Haenszel method. Sensitivity and subgroup analysis were planned a priori. y p y g Conclusion This pooled analysis indicates that patients treated with Ecu within 7 days of a TMA manifestation had a greater improvement in eGFR over time than patients in whom treatment was delayed. These data show the importance of rapid diagnosis and treatment of aHUS for recovery of renal function. Red blood cell transfusion is associated with an increased mortality in critically ill surgical patients y A Piriyapatsom, O Chaiwat, S Kongsayreepong Siriraj Hospital, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P334 (doi: 10.1186/cc14414) Results δPCO2/Ca-cvO2 showed signifi cant correlation with the moderate and severe blood loss groups (P <0.01), while no signifi cant correlation was detected in the mild blood loss group. The abnormality of the δPCO2/Ca-cvO2 was easy detectable and refl ected intracapillary Introduction The aim of this study is to explore the association between red blood cell transfusion (RBCT) and mortality in Thai critically ill surgical patients. Introduction The aim of this study is to explore the association between red blood cell transfusion (RBCT) and mortality in Thai critically ill surgical patients. S118 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods This study was a part of the multicenter, prospective, observational study performed in nine surgical intensive care units (SICUs) across the nation between April 2011 and November 2012 [1]. This study included adult patients admitted to the SICUs after surgery. Patients were categorized into transfusion and no transfusion groups according to whether or not they received RBCT at any time during SICU stay. Demographic data, clinical outcomes as well as SICU and hospital length of stay (LOS) and SICU and hospital mortality were collected. Patients were followed for up to 28 days or until discharge from the SICUs. The primary endpoint was hospital mortality. Data were compared between groups and logistic regression analysis was performed to determine whether RBCT was an independent risk factor of hospital mortality. In addition, patients were matched between groups based on the propensity score of the requirement of RBCT and were then compared. confounders, a Hb level of ≤9 g/dl was associated with a poor neurologic outcome (OR = 2.572, 95% CI = 1.058 to 6.250), whereas an Hb level of ≤10 g/dl was not. A Hb level of ≤10.3 g/dl had a sensitivity of 82% and a specifi city of 62% to predict poor neurologic outcome in TBI patients. Conclusion In TBI patients a Hb level of ≤9 g/dl is associated with poor neurologic outcome. Red blood cell transfusion is associated with an increased mortality in critically ill surgical patients A transfusion threshold of ≤10.3  g/dl may be a reasonable target to be tested in future transfusion trials aimed at improving neurologic outcome of TBI patients.fi Acknowledgements All of the author institutes are affi liated members of the International Trauma Research Network (INTRN) and as such this work represents a combined output resulting from this Network. Microparticles from red blood cell transfusion products induce a strong infl ammatory host response Results Overall, 968 of 2,374 (40.8%) patients received RBCT. Trans- fused patients, when compared with those without RBCT, had more frequency of admission after emergency surgery, higher APACHE II score, higher SOFA score, higher number of organ dysfunctions and lower hemoglobin level at admission. When compared with patients without RBCT, those with RBCT had more frequency of all adverse events including infection, AKI, ALI/ARDS and MI, and longer SICU and hospital LOS. Both SICU and hospital mortality were also higher in the transfusion group compared with the no transfusion group (9.4% vs. 1.6% and 13.7% vs. 3.6%, both P <0.001, respectively). The logistic regression analysis showed that RBCT was an independent risk factor of hospital mortality with odds ratio of 1.60 (95% CI 1.05 to 2.45). In the propensity-score matched cohort of 852 patients, when compared with patients without RBCT, transfused patients had more frequency of adverse events including infection and AKI, longer SICU and hospital LOS and higher hospital mortality (7.5% vs. 4.0%, P = 0.027). l M Straat1, M Van Hezel1, A Boing1, R Nieuwland1, R Van Bruggen2, N Juff ermans1 f 1Academic Medical Center, Amsterdam, the Netherlands; 2Sanquin Blood Cell Research, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P336 (doi: 10.1186/cc14416) Introduction Red blood cell (RBC) transfusion is associated with increased morbidity and mortality in the critically ill. Adverse eff ects of transfusion may be mediated by red blood cell storage lesion. In this study, we hypothesized that MPs from stored RBC bags would induce a more pronounced host response than MPs from fresh RBC bags and that this response is dose dependent. p p Methods MPs were isolated by high-speed centrifugation from red blood cell transfusion bags stored for 2 to 7 (fresh) or 25 to 35 (stored) days. Whole blood from healthy volunteers was incubated with supernatant from the bags either containing MPs or depleted from MPs (n = 12 bags per group). Controls were incubated with PBS as a negative and LPS (10 ng/ml) as a positive control. Cytokines in supernatant were measured by ELISA. Data are expressed as medians and interquartile ranges. y Conclusion This study showed that RBCT was associated with increased morbidity and mortality in critically ill surgical patients. These results supported the restrictive strategy of RBCT suggested by more recent studies. Reference y p q g Results Supernatant from blood bags containing MPs strongly induced production of all cytokines compared with supernatant without MPs, a reaction which equaled that of LPS stimulation. MPs from stored RBC bags induced higher production of TNF (868 (263 to 1,625) vs. 2,596 (407 to 3,040)  pg/ml, P  = 0.049), IL-6 (1,088 (234 to 3,716) vs. 6,952 (1,507 to 21,990), P = 0.042) and IL-8 (1,333 (535 to 3,569) vs. 5,562 (833 to 13,904), P = 0.081) compared with MPs from fresh RBC bags. There was no diff erence in IL-10 responses between groups (8.0 (3.9 to 32.1) vs. 3.9 (3.9 to 22.2), P = 0.390). The host response was dose dependent both for fresh and stored MPs. In addition, the same amount of older MPs induced a stronger host response compared with fresh MPs. Conclusion MPs from RBC transfusion bags induce a strong proinfl ammatory response, which is largely negated when MPs are removed. This MP-mediated response depends both on the amount of MPs as well as on alterations in MPs as a result of storage. Reference Reference 1. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 Suppl 1:S45-54. 1. Chittawatanarat K, et al. J Med Assoc Thai. 2014;97 Suppl 1:S45-54. Eff ect of the haemoglobin level on neurologic outcome in patients with severe traumatic brain injury Eff ect of the haemoglobin level on neurologic outcome in patients with severe traumatic brain injury K Balvers1, MR Wirtz1, C Rourke2, S Eaglestone2, K Brohi2, S Stanworth3, C Gaarder4, JC Goslings1, NP Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Blizard Institute, Queen Mary University of London, UK; 3John Radcliff e Hospital, Oxford, UK; 4Oslo University Hospital, Oslo, Norway Critical Care 2015, 19(Suppl 1):P335 (doi: 10.1186/cc14415) Introduction Anaemia in patients with severe traumatic brain injury (TBI) may worsen neurologic outcome. The aim of this study was to determine the association of haemoglobin level (Hb) with neurologic outcome and to determine a transfusion threshold which may be used in future transfusion trials aimed at improving neurologic outcome in TBI patients. Value of thromboelastography in managing hypercoagulopathy in intensive care Methods A retrospective audit over 2 months of all blood transfusion forms at St Francis Hospital, Eastern Province, Zambia. Respective patients’ notes were reviewed for: record of observations during transfusion; patient demographics; and length of stay. We surveyed nurses’ attitudes, confi dence and knowledge in relation to blood transfusion standards. J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P340 (doi: 10.1186/cc14420) J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK g p , , Critical Care 2015, 19(Suppl 1):P340 (doi: 10.1186/cc14420 Introduction This aim of this analysis is to explore the use of thromboelastography (TEG) in the management of hypercoagulation in the ICU. TEG allows the assessment of whole blood coagulation and fi brinolysis and hence can identify patients who are hypercoagulable. Methods A prospective audit of TEG tests performed on patients being treated on a general surgical and medical ICU was conducted over a 2-month period. Results In May and June 2014, 457 requests were made for blood, of which 157 (34%) received blood transfusion, of which 108 (69%) had records of observations available. The audit demonstrated that requests were mostly complete (90%), but urgency was indicated in only 32%. The matching of blood to patient by more than one nurse was recorded amongst 66% of cases. Only 2% of transfusions met minimal requirements for transfusion reaction monitoring. Of nurses surveyed (n = 20), most were experienced in their post (mean 7.3 years, range 2 weeks to 20 years). Nurses rated themselves as highly confi dent in handling blood transfusions and identifying and dealing with transfusion reactions. However, 90% believed they could identify all transfusion reactions by measuring temperature alone, and 25% would measure temperature only as a parameter to monitor the transfusion, even in ideal settings. Most knew to check observations before, 15  minutes after the start of transfusion and then hourly thereafter (88%); but only 10% would check at the start, at completion and 4 hours after completing transfusion. The most frequently reported reason for not doing observations was time pressure on the ward (85%). Results Twenty-one out of 78 patients (26.9%) had one or more TEG criteria consistent with hypercoagulopathy. Admission diagnoses included trauma (37%), haemorrhage (23%), postoperative (23%) and sepsis (14.3%). Sixty-two per cent of patients with a primary diagnosis of trauma were in a hypercoaguable state. P337 Improving blood transfusion safety in a low-resource setting: an audit of 1,163 transfusion requests S Kudsk-Iversen1, R Colhoun1, D Chama2, J Mulenga2, MD Bould1, J Kinnear1, D Snell1 1Zambia Anaesthesia Development Project, Lusaka, Zambia; 2Zambia National Blood Transfusion Service, Lusaka, Zambia Critical Care 2015, 19(Suppl 1):P337 (doi: 10.1186/cc14417) Methods A substudy of the prospective multicentre Activation of Coagulation and Infl ammation in Trauma (ACIT) II study was performed on subjects recruited between January 2008 and December 2014. All adult trauma patients admitted to a level 1 trauma centre with severe traumatic brain injury (AIS head ≥3), ICU admission and available Hb levels on admission were selected for analysis. The primary outcome was the cognitive functioning of patients as determined by an estimated Glasgow Outcome Scale (GOS) on discharge. Anaemia was defi ned as a Hb level of ≤9  g/dl (severe anaemia) or ≤10  g/dl (moderate anaemia) within the fi rst 24 hours post injury. Multivariate logistic regression models were used to determine the association between anaemia and neurologic outcome. The receiver operating characteristic curve and the Youden Index were used to determine an optimal transfusion threshold. Introduction Sub-Saharan Africa suff ers from more acute life- threatening indications for blood transfusion compared with high- income countries [1]. The commonest ‘systems failure’ contributing to perioperative death in low-resource settings is the timely availability of correctly cross-matched blood products [2]. Often this is not the result of an absolute shortage of blood products, but failure in the chain of supply and distribution. We audited an early step in this chain, the quality of blood requests, at the University Teaching Hospital (UTH) in Lusaka, Zambia. UTH does not have a formal blood request form, and only the cancer diseases hospital (CDH) has a blood request form developed by the blood bank. Results Of a total of 261 TBI patients, 61 patients (23%) fell below the threshold for severe anaemia and 101 patients (39%) had moderate anaemia within the fi rst 24 hours. In a model adjusted for all relevant S119 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We performed a 1-day retrospective review in June of blood request forms submitted to the cross-match laboratory, followed by a 14-day prospective review in September 2014. Group and save requests were excluded. Inadequate monitoring risks safety of blood transfusion in rural Zambia Results The mean age was 43.5 ± 5.7 years. Of 58 patients (38 male/20 female), 25 (43.1%) had iron defi ciency with outcomes of blood samples used at ICU admission. The overall transfusion rate was 32.8%, being higher in iron-defi ciency patients than in normal iron profi le patients (42.3 vs. 14.9%, P = 0.001). After adjusting for severity of illness and hemoglobin level, iron-defi ciency patients remained signifi cantly associated with transfusion, with a hazard ratio of 4.2 (95% CI, 1.3 to 12.9; P = 0.001). Zambia O Todd1, K Sikwewa1, J Kamp1, I Hodt Rasmussen1, K Mortensen1, S Kudsk-Iversen2 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) O Todd1, K Sikwewa1, J Kamp1, I Hodt Rasmussen1, K Mortensen1, S Kudsk-Iversen2 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) 1St Francis Hospital Katete, Zambia; 2Unversity College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P338 (doi: 10.1186/cc14418) Introduction In Zambia, supply of blood is insuffi cient to meet clinical need, on a national level. Paradoxically, blood is also more often transfused unnecessarily in this setting. The Zambian National Blood Transfusion Service is currently scaling up voluntary blood donation and supply systems, and requires hospitals to improve blood transfusion safety. At a rural district hospital in Zambia, we audited practice and surveyed knowledge amongst staff using standards established in national guidelines. Conclusion Iron defi ciency is common at ICU admission and is associated with higher transfusion requirements. These fi ndings have important implications for transfusion practices in ICU patients. P340 P339 Eff ects of iron defi ciency on transfusion requirements in critically ill patients: a preliminary observational study M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) P339 Eff ects of iron defi ciency on transfusion requirements in critically ill patients: a preliminary observational study M Aydogan, M Ucar, A Yücel, B Karakas, A Gok, T Togal Inonu University, Malatya, Turkey Critical Care 2015, 19(Suppl 1):P339 (doi: 10.1186/cc14419) Introduction Critically ill patients often need blood transfusion, but no reliable predictors of transfusion requirements are available at ICU admission. We hypothesized that ICU patients admitted with iron defi ciency may be at higher risk for developing anemia, requiring blood transfusion. The aims of this study were to determine the frequency of iron defi ciency in ICU patients at admission and to investigate its relationship with transfusion requirements in ICU patients. p Conclusion The audit revealed an important system failure impacting on effi cacy and safety of transfusion practice at UTH. Full patient identifi ers, as well as vital information such as the indication and urgency, were rarely fi lled in, which are crucial for the blood bank to prioritise the release of blood products. The audit shows that practice may be signifi cantly improved by a cheap intervention such as a standardised blood request form meeting international standards. Acknowledgements UK aid, THET. References 1 L d l T f A h S i 2013 49 416 21 Methods Eighty-fi ve patients admitted to the general ICU were enrolled in the prospective observational study. We studied 58 patients, after excluding those transfused on or before ICU admission. The patients’ age, gender, APACHE II score, diagnosis, severity score, presence of sepsis, ICU complications, ICU treatments, and transfusion-free interval were recorded. Iron defi ciency was assessed on the basis of several parameters, including hemoglobin, hematocrit, levels of serum iron, iron-binding capacity, transferrin saturation, levels of ferritin, soluble transferrin receptor, hepcidin, C-reactive protein, and peripheral blood smear. 1. Lund et al. Transfus Apher Sci. 2013;49:416-21. 2. Ohanaka et al. Turk J Med Sci. 2007;37:219-22. 1. Lund et al. Transfus Apher Sci. 2013;49:416-21. 2. Ohanaka et al. Turk J Med Sci. 2007;37:219-22. P337 Each form was audited against the American Association of Blood Banks (AABB) minimum standards for content of a blood request form. Analysis was performed with Fisher’s exact test for nominal data and t test for continuous data. Conclusion In this setting, current practice is evidently inadequate to identify and prevent blood transfusion reactions. The survey revealed high confi dence but patchy knowledge amongst nurses of the requirements for safe blood transfusion. Better timing to transfuse at times when nursing staff numbers are higher, alongside compulsory training, may together represent potential low-cost interventions to improve blood transfusion safety. Results A total of 1,163 blood requests were reviewed, 51 from CDH and 1,112 from other wards. Eighteen forms from CDH (35%) and 22 from other wards (2%) met all minimum AABB standards (P <0.0001). The mean number of standards met on the requests from CDH and the rest were 11.25 (SD 0.93) and 8.87 (SD 1.75) respectively (P <0.0001). Considering all blood requests, the standards met in order from least to most were: signature of requesting doctor (36%), urgency of request (43%), hospital number (59%), indication for transfusion (62%), type of product requested (72%), requesting doctor’s name (78%), age or date of birth of patient (84%), gender of patient (89%), quantity of products requested (90%), date form was completed (90%), patient’s ward (95%), and patient’s full name (100%). P339f P342 P342 In trauma, when used in the emergency department, do viscoelastic hemostatic tests decrease mortality? A systematic review J Cousineau, R Daoust, K Doyon, M Marquis, É Piette, JM Chauny, M Clar, D Rose, É Notebaert Hôpital du Sacré Coeur de Montréal, Montreal, QC, Canada Critical Care 2015, 19(Suppl 1):P342 (doi: 10.1186/cc14422) Introduction This systematic review done in March and updated in November 2014 has been conducted in accordance with the STARD, PRISMA and STROBE recommendations. Retrospective and prospective studies with a comparison group published in English were kept. Methods Two reviewers (JC and ÉN) and two librarians (MC and DR) independently conducted a systematic review and identifi ed abstracts. Full texts were read by two authors (ÉN and ÉP), and data were extracted. The following databases were searched: Cochrane CENTRAL, Medline, Embase, LILACS, Web of Science, Science.gov, SciFinder Scholar, WorldCat, the Transf Evid Lib Database, and proceedings of the congresses of the International Society on Thrombosis and Haemostosis and the American Society of Hematology. Figure 1 (abstract P340). Coagulation modifi cation prior to TEG analysis in hypercoagulable patients. as a result of performing TEG was documented in 14 of these 21 patients. No further blood products were administered in all cases and anticoagulation was commenced or increased in four cases. Conclusion Hypercoagulopathy was present in 27% of patients. One- third of these patients had recently received prothrombotic therapy indicating a possible iatrogenic aetiology. TEG analysis resulted in cessation of prothrombotic drug and blood product administration in all cases. Further research is required to determine whether titrated anticoagulation treatment to normalise the TEG profi le in these patients would be benefi cial. f y y Results We initially kept 2,870 references. In total, 453 articles had mortality in their keywords. After reading the abstracts, 37 papers were analysed, and three articles evaluating mortality in two groups of patients (using and not using a VHT) were identifi ed. Among these three studies, only one had raw data available. We did not succeed in getting this information from the two other authors. We asked the main authors of the 37 selected papers, and renowned authors in the fi eld, if they had studies with new data that could be included in our review. The answers were negative. Thromboelastography may detect hypercoagulation in early sepsis and improve anticoagulation during extracorporeal treatments F T S B R B h AB B ll S M M F l Aurelia and European Hospital, Rome, Italy p p y Critical Care 2015, 19(Suppl 1):P341 (doi: 10.1186/cc14421) Introduction During early sepsis, activation of the infl ammatory response and coagulation occurs. Extracorporeal therapies are used to adsorb mediators, but the coagulation of fi lters is a drawback [1,2]. The aim of this study is to evaluate whether thromboelastography (TEG) may detect hypercoagulation and may improve anticoagulation during extracorporeal treatments. Conclusion With the studies available as of the end of 2014, it is impossible to conclude whether the use of a VHT in the emergency department decreases mortality. Other studies are needed. References 1. Johansson PI, Stensballe J. Eff ect of haemostatic control resuscitation on mortality in massively bleeding patients: a before and after study. Vox Sang. 2009;96:111-8. p Methods Twenty-four patients with early severe sepsis had a TEG monitoring at basal time (T0) and during three diff erent extracorporeal treatments (T1): coupled plasma fi ltration (CPFA) with heparin infusion (Group A), CPFA with citrate infusion (Group B) and RRT with oXiris fi lter – heparin coated – and no heparin infusion (Group C). ANOVA test was used for the statistical analysis. 2. Messenger BM, et al. TEG-guided massive transfusion in trauma patients. Anesth Analg. 2011;112:S-9. 3. Kashuk K, et al. Initial experiences with POC rapid TEG for management of life-threatening postinjuty coagulopathy.Transfusion. 2012;52:23-33. 3. Kashuk K, et al. Initial experiences with POC rapid TEG for management of life-threatening postinjuty coagulopathy.Transfusion. 2012;52:23-33. y Results Table 1 presents the TEG values in early septic patients at T0. At T1, angle and MA decreased and r increased in Group A at diff erence with Group B and Group C (P <0.01). In group C, LY 30 was higher than in Group A and B (P <0.01). P342 The three studies were: Johansson and Stensballe, total 832 cases, 121 traumas, raw data unavailable for trauma [1]; Messenger and colleagues, prospective study, 50 cases, mortality similar, raw data unavailable [2]; and Kashuk and colleagues, prospective study, 68 cases, mortality 59% control group, 28% VHT group [3]. 1. Kashuk JL, et al. r-TEG identifi es hypercoagulability and predicts thromboembolic events in surgical patients. Surgery. 2009;146:764-72. 1. Kashuk JL, et al. r-TEG identifi es hypercoagulability and predicts thromboembolic events in surgical patients. Surgery. 2009;146:764-72. P341 Thromboelastography may detect hypercoagulation in early sepsis and improve anticoagulation during extracorporeal treatments F Turani, S Busatti, R Barchetta, AB Belli, S Martini, M Falco Aurelia and European Hospital, Rome, Italy Critical Care 2015, 19(Suppl 1):P341 (doi: 10.1186/cc14421) References Figure 1 (abstract P340). Coagulation modifi cation prior to TEG analysis in hypercoagulable patients. 1. Livigni S, Bertolini G, Rossi C, et al. BMJ Open. 2014;4:e003536. 2. Ronco C, et al. Crit Care. 2014, 18:309. Value of thromboelastography in managing hypercoagulopathy in intensive care Hypercoagulopathy was suggested by an abnormally short R time in 16 patients (76%), an abnormal alpha angle in 17 cases (81%), a maximum amplitude >74 mm in nine cases (43%) and a high LY30 in one case. Procoagulant treatment was given to seven patients and fi ve patients had received no coagulation modifi cation prior to testing (Figure 1). Eight patients were receiving prophylactic anticoagulation and only one was receiving treatment-dose anticoagulation. A change in management S120 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Role of thromboelastography in the management of haemorrhage: an observational analysis y C Ward, J Aron, A Gibbon, J Ball y C Ward, J Aron, A Gibbon, J Ball Table 1 (abstract P341) Control Sepsis r 8 ± 2 6 ± 3 k 4 ± 2 1.7 ± 0.5* Angle 47 ± 15 67 ± 7* MA 55 ± 8 72 ± 5* LYS 30 1.8 ± 1 0.95 ± 0.9 *P <0.01. St George’s Hospital, London, UK g p Critical Care 2015, 19(Suppl 1):P343 (doi: 10.1186/cc14423) Introduction The aim of this analysis is to explore and evaluate the role of thromboelastography (TEG) in aiding the management of patients admitted with haemorrhage to the ICU. TEG has been shown to rationalise and reduce transfusion requirements [1] but the precise role of TEG in the assessment and management of haemostasis in the bleeding patient is uncertain and has not been previously demonstrated. Methods A prospective audit of TEG analyses performed on patients in the general medical and surgical ICU was recorded over a 2-month period. Operators documented the reason for admission, demographic data, indication for TEG, laboratory results, blood gas analysis, TEG results, diagnosis and subsequent action from the TEG result. Only patients who had been admitted with haemorrhage were included in this analysis. Conclusion In early sepsis, TEG monitoring may detect hyper coagu la- bility. CPFA with heparin, but not CPFA with citrate and oXiris, is able to reverse hypercoagulability. OXiris may induce fi brinolysis. TEG detects alterations of coagulation during early sepsis and extracorporeal treatments. S121 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 TEG analysis Both TEG normal/ Standard normal/ abnormal standard abnormal TEG abnormal Clot factor defi cit 9 (11.5) 20 (25.6) 3 (3.8) Platelet defi cit 11 (14.1) 13 (16.6) 2 (2.6) Fibrinogen defi cit 5 (6.4) 4 (5.1) 5 (6.4) Data presented as n (%). Results Seventy-eight audit sheets were completed, of which 31 identifi ed haemorrhage as the reason for admission. The mean age was 59.3 (range 21 to 90) and the mean APACHE II score was 18.23 (range 11 to 37). The main indications for TEG analysis included coagulopathy (64%) and ongoing haemorrhage (45%). As a result of performing TEG analysis, 23 (74%) patients had a documented change in their management. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) M Popescu, D Tomescu M Popescu, D Tomescu Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P345 (doi: 10.1186/cc14425) g p Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) Introduction Our aim was to assess hemostasis, using ROTEM, in patients with end-stage liver disease (ESLD) undergoing liver transplantation (LT) and to develop a predictive model for patients prone to high intraoperative blood loss. Introduction This review aims to assess the value of thrombo- elastography (TEG) on the general ICU, which has not been previously demonstrated. TEG is a near-patient assessment of whole blood coagulation and fi brinolysis, which reduces transfusion requirements during cardiothoracic surgery and liver transplantation. Methods We retrospectively analyzed 122 patients who underwent LT between January and December 2013 in a single national center. Patients with acute liver failure or incomplete data were excluded. Demographic data, severity of liver disease assessed by MELD score (model for ESLD), presence of portal vein thrombosis, and laboratory data were recorded preoperatively. We performed concomitant ROTEM assay and standard coagulation tests (prothrombin time (PT), International Normalized Ratio (INR), fi brinogen) 1 hour before surgery and 15  minutes after the neohepatic phase. Intraoperative blood loss was recorded. High blood loss was defi ned as loss of one blood volume during surgery. Correlation between recorded data standard ROTEM parameters and derived thrombodynamic ROTEM parameters (potential index (TPI), maximum velocity of clot formation (MaxV), time to MaxV (MaxVt), AUC) were analyzed using SPSS 19.0. Methods A prospective audit of TEG tests performed on patients being treated on a general ICU was conducted over 2 months. Results A total of 332 TEG tests were performed with a failure rate of 29.8%. Seventy-eight audit sheets were collected. Mean patient age was 68.9 years and mean APACHE II score was 18.1. Admissions included trauma (33.0%), perioperative (43.6%), haemorrhage (42.3%) and sepsis (21%). Standard tests of coagulation demonstrated 22 defi cits in coagulation which were not identifi ed as functionally signifi cant with TEG. Of these, 20 had abnormal clotting factor activity as measured by the INR/APTTr and 13 patients were thrombocytopenic. In total, 52.6% documented that the TEG result changed the management of the patient. In 46.8% of these cases no further blood products were required. Role of thromboelastography in the management of haemorrhage: an observational analysis Ten patients did not require any further administration of blood products, which they would have received based on conventional laboratory results. The information gained from TEG also resulted in the omission of anticoagulation in three patients, and with a further two patients anticoagulation increased. Conclusion TEG analysis suggested that 22 patients who were identifi ed as coagulopathic with traditional measures of coagulation did not have a functional defi ciency. Over one-half of TEG studies resulted in a change in management and in 46.8% no further transfusions were required. There was a high technical failure rate and a low audit return rate, which may indicate the need for further training. Reference p g Conclusion TEG aids prompt rationalisation of blood products and titration of anticoagulation in the bleeding patient. TEG identifi es a number of patients who required administration of platelets and other procoagulants which would not have been identifi ed by conventional methods. Several patients would have also received inappropriate transfusions which has both cost and resource implications, alongside the potential adverse eff ects on patients. We recognise that further research is needed to clarify the overall effi cacy of TEG in the bleeding patient. 1. Da Luz et al. Eff ect of TEG and ROTEM on diagnosis of coagulopathy, transfusion guidance and mortality in trauma: descriptive systematic review. Crit Care. 2014;18:518. 1. Da Luz et al. Eff ect of TEG and ROTEM on diagnosis of coagulopathy, transfusion guidance and mortality in trauma: descriptive systematic review. Crit Care. 2014;18:518. Decreased coagulation kinetics is associated with high blood loss in patients with end-stage liver disease undergoing liver transplantation Decreased coagulation kinetics is associated with high blood loss in patients with end-stage liver disease undergoing liver transplantation M Popescu, D Tomescu Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P345 (doi: 10.1186/cc14425) Reference 1. Bollinger D, et al. Principles and practice of thromboelastography in clinical coagulation management and transfusion practice. Transfusion Med Rev. 2012;26:1-13. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) In 41% there was no documentation. See Table 1 and Figure 1. Table 1 (abstract P344). Summary of concordance with standard tests versus y g Results After applying exclusion criteria, 72 patients were analyzed with mean age of 54.5 years (SD 11.6) and a median MELD score of 17.4 (7 to 34). Preoperative MCE correlated with age (P = 0.044, 95% CI (–7.50, –0.12)) and MELD score (P = 0.009, 95% CI (–37.21, –6.69)), but not with PT (P = 0.557) or INR (P = 0.623). MaxV correlated with fi brinogen level (P = 0.005, 95% CI (0.01, 0.05)) and AUC correlated with age (P = 0.034, 95% CI (–257.74, –11.91)) and MELD score (P = 0.01, 95% CI (–1,233.14, –215.33)). Patients with portal vein thrombosis had an increase in InTEM CFT (P = 0.002, 95% CI (77.98, 317.97)) and MaxVt (P = 0.03, 95% CI (5.53, 105.63)). No correlation was found between preoperative ROTEM parameters and intraoperative blood loss. We calculated ΔMaxV, ΔMaxVt and ΔAUC as the mathematical diff erence between preoperative and intraoperative MaxV, MaxVt and AUC. High blood loss correlated with ΔAUC (P  = 0.005, 95% CI (15.69, 61.03)), ΔMaxV (P = 9=0.002, 95% CI (–20,413, 6,392)) and ΔMaxVt (P = 0.008, 95% CI (15.69, 61.07)). Figure 1 (abstract P344). Change in management due to TEG results. Conclusion MELD score correlated with a decrease in MaxV and AUC on preoperative ROTEM but not with INR. Patients with portal vein thrombosis have increased InTEM CFT and MaxVt. High blood loss was associated with a decrease in thrombodynamic parameters, but no correlations were found between blood loss and standard ROTEM parameters. Figure 1 (abstract P344). Change in management due to TEG results. S122 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P346 time (aPTT), and prothrombin time (PT). In addition, the plasma levels of vitamin K-dependent coagulation factors were determined. P346 Evaluation of fi xed dose four-factor prothrombin complex concentrate for warfarin reversal at a level 1 trauma center H Drone, J Jancik, J Gorlin, M McCarthy Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P346 (doi: 10.1186/cc14426) p g Results Acute coumarin anticoagulation of rats induced a rise in median bleeding time by ≥2-fold from an average of 823 to 1,800  seconds (maximum observation period) compared with untreated animals. Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation E Herzog, F Kaspereit, W Krege, P Niebl, G Dickneite g p g CSL Behring GmbH, Marburg, Germany g g y Critical Care 2015, 19(Suppl 1):P347 (doi: 10.1186/cc14427) Introduction The study was conducted as a head-to-head comparison of a four-factor prothrombin complex concentrate (4F-PCC) and two diff erent three-factor PCCs (3F-PCC) for eff ective reversal of vitamin K antagonist (VKA)-induced anticoagulation using an established rat model of acute bleeding [1]. The 4F-PCC (containing the human coagulation factors II, VII, IX and X) is indicated for the urgent reversal of acquired coagulation factor defi ciency induced by VKA therapy in adult patients with acute major bleeding. In contrast, the 3F-PCCs (containing factors II, IX, X and only minimal VII) are indicated for the prevention and control of hemorrhagic episodes in hemophilia B patients. Nevertheless, the use of 3F-PCC for correcting hemostasis following warfarin overdose has been discussed but the lack of factor VII in these 3F-PCC products has raised questions about effi cacy. Conclusion In conclusion, 4F-PCC treatment eff ectively decreased apixaban-induced hemorrhage at a clinically relevant dose range. References . Herzog et al. Anesthesiology. Forthcoming 2014. 1. Herzog et al. Anesthesiology. Forthcoming 2014. 1. Herzog et al. Anesthesiology. Forthcoming 2014. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. 2. Pragst et al. J Thromb Haemost. 2012;10:1841-8. References 1. Varga C, et al. Transfusion. 2013;53:1451-8. 2. Junagade P, et al. Hematology. 2007;12:439-40. g g p Results Dose-dependent increases in time to hemostasis and total blood loss were observed post apixaban administration with maximum bleeding signals seen at 1,200 μg/kg. Treatment with 4F-PCC resulted in a statistically signifi cant reversal in apixaban-induced bleeding time (all doses) and volume (doses ≥12.5 IU/kg). Of the coagulation parameters measured, thrombin generation initiated using phospholipids only was the in vitro coagulation parameter most sensitive to 4F-PCC-mediated bleeding reversal, although statistically signifi cant 4F-PCC-mediated reductions in the prothrombin time and whole blood clotting time were also observed.f P347 Four-factor prothrombin complex concentrate (Beriplex® P/N) is superior to three-factor prothrombin complex concentrate for reversal of coumarin anticoagulation E Herzog, F Kaspereit, W Krege, P Niebl, G Dickneite CSL Behring GmbH, Marburg, Germany Critical Care 2015, 19(Suppl 1):P347 (doi: 10.1186/cc14427) p Reference 1. Dickneite. Thromb Res. 2007;119:643-51. f Results The INR was reduced to <2 in 100% of patients in the 4F-PCC group versus 84.6% of patients in the factor IX group (P <0.05). The INR was reduced to <1.6 in 90.8% of patients in the 4F-PCC group versus 50% in the factor IX group (P <0.05). Mean pre-reversal INRs were 3.5 and 4 and ranged from 1.1 to 10 and from 1.3 to 10 in the 4F-PCC and factor IX group respectively (P = 0.29). On average, a medication cost savings of US$802.63 dollars per patient was calculated from using a fi xed 1,500 unit dose over traditional dosing of 4F-PCC. There was a trend toward a shorter mean ICU LOS in the PCC group when compared with the factor IX group (5.8 vs. 2.8 days) and shorter mean hospital LOS (10.7 vs. 5.7  days), although neither outcome was statistically signifi cant. No diff erence in adverse event rates was observed.ii P348 Four-factor prothrombin complex concentrate (Beriplex® P/N) mediated reversal of apixaban-induced bleeding in a rabbit model E Herzog, F Kaspereit, W Krege, J Mueller-Cohrs, B Doerr, P Niebl, G Dickneite CSL Behring GmbH, Marburg, Germany Critical Care 2015, 19(Suppl 1):P348 (doi: 10.1186/cc14428) Introduction This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex®/Kcentra®; CSL Behring) can eff ectively reverse bleeding associated with the direct oral factor Xa inhibitor apixaban in an established in vivo rabbit model [1,2].i Introduction This study assessed whether a four-factor prothrombin complex concentrate (4F-PCC; Beriplex®/Kcentra®; CSL Behring) can eff ectively reverse bleeding associated with the direct oral factor Xa inhibitor apixaban in an established in vivo rabbit model [1,2].i gif Conclusion A fi xed dose of 1,500 units of 4F-PCC was signifi cantly more eff ective at lowering the INR to a threshold of less than either 2 or 1.6 when compared with a combination of factor IX complex and vitamin K with or without FFP. Further research is needed to investigate clinical outcomes and a possible reduction in ICU and hospital LOS. References Methods For dose-fi nding purposes, anesthetized rabbits were treated with a single intravenous dose of apixaban (800 to 1,600  μg/kg). In a subsequent study phase, anesthetized rabbits were treated with apixaban (1,200 μg/kg) followed by 4F-PCC (6.25 to 100 IU/kg). Bleeding signals were quantifi ed following a standardized kidney incision by measurement of the volume of blood loss and time to hemostasis over an observation period of 30 minutes. Blood samples were collected for monitoring of coagulation parameters. Utilisation review of thromboelastography in intensive care J Aron, A Gibbon, C Ward, J Ball St George’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P344 (doi: 10.1186/cc14424) In parallel, PT and aPTT were prolonged from 8.9 to 29.9  seconds and from 14.5 to 25.5  seconds, respectively. Treatment with 4F-PCC was able to fully and statistically signifi cantly reverse bleeding, achieving average bleeding times of 676 seconds. In parallel, the elevation in PT was reduced to 15.1 seconds. In contrast, the two 3F-PCC products were not or only partially able to reduce coumarin-induced bleeding with average bleeding times of 1,398 and 1,708  seconds post treatment, respectively. This also correlated with inferior reductions in PT which achieved minimum levels of 23.8 and 29.5 seconds, respectively. There was no reduction in aPTT seen for any treatment option.i Introduction FDA-approved dosing of four-factor prothrombin complex concentrate (4F-PCC) in the USA is based on an INR and weight; however, there are data suggesting that a fi xed dose of 4F-PCC may be suffi cient for INR reversal and hemostasis [1,2]. The objective of this study was to assess the effi cacy and safety of a fi xed dose of 1,500 units of 4F-PCC. Historically, warfarin reversal included a combination of factor IX complex, vitamin K, and fresh frozen plasma (FFP). Using a fi xed dose of 4F-PCC may also provide signifi cant cost savings when compared with traditional dosing. y p Conclusion In conclusion, this fi rst direct comparison of 4F-PCC and 3F-PCCs for the reversal of VKA anticoagulation in a rat model of acute bleeding suggests that replenishment of all four vitamin K-dependent coagulation factors including factor VII as achieved using a 4F-PCC may result in superior effi cacy compared with the use of 3F-PCCs. R f Methods This retrospective chart review compared 26 admitted adults who received a fi xed dose of 1,500 units 4F-PCC with 26 patients who received a combination of factor IX complex and vitamin K, with or without FFP, for warfarin reversal from 1 January 2012 to 1 November 2014. Primary outcomes included reversal to an INR of <2 and reversal to an INR of <1.6. Secondary outcomes included ICU and hospital length of stay (LOS), change in INR, INR nadir, potential cost savings from 4F-PCC versus traditional dosing, and major adverse eff ects. Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model M Honickel1, T Braunschweig1, J Van Ryn2, R Rossaint1, O Grottke1 1RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co KG, Biberach, Germany Critical Care 2015, 19(Suppl 1):P351 (doi: 10.1186/cc14431) Results In the ShBl injury strand there was a signifi cant reduction in ATC in Early compared with Late PRBC:FFP treatment (TEG R and K times) in both the prehospital (P = 0.004 and P = 0.003 respectively, ANOVA) and early in-hospital (P = 0.002 and P = 0.005) phases, although clotting was normalised in the Late group within 60  minutes of initiating PRBC:FFP. Prehospital base defi cit (BD) was signifi cantly attenuated in ShBl Early versus Late (9.0 ± 2.1 vs. 14.4 ± 2.2 mM). BD improved in both Early and Late treatment groups during the in-hospital phase but remained greater in the Late group throughout (P <0.001). In the Bl injury strand the trend in coagulation was similar to that seen in the ShBl injury strand (but the diff erences between Early and Late did not attain statistical signifi cance). By contrast, Early versus Late PRBC:FFP treatment did not result in a diff erence in BD in the Bl strand. Finally, there was no diff erence in the total amount of PRBC:FFP used between the two treatments in either injury strand, but in both injury strands the Early treatment groups required signifi cantly less saline (P <0.001). Conclusion Prehospital use of PRBC:FFP may attenuate ATC and improve physiological status. Furthermore the amount of crystalloid may be reduced with potential benefi t of reducing the third-space eff ect and later tissue oedema. M Honickel1, T Braunschweig1, J Van Ryn2, R Rossaint1, O Grottke1 1RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co KG, Biberach, Germany Critical Care 2015, 19(Suppl 1):P351 (doi: 10.1186/cc14431) Introduction The anticoagulant eff ect of dabigatran can be reversed with idarucizumab or PCCs in porcine blood in vitro [1]. However, the impact on clinical parameters such as blood loss is not known. Thus, this study assessed the effi cacy of idarucizumab in comparison with PCC and aPCC in dabigatran-anticoagulated swine following polytrauma on clinically relevant endpoints. Reference 1. Grottke O, et al. Crit Care. 2014;18:R27. p p Results According to LFTEG, polytrauma patients had statistically signifi cant abnormalities in platelet aggregation (intensity of contact coagulation (ICC)), in coagulation (intensity of coagulation drive (ICD), clot maximum density (MA)) and in fi brinolytic activity (index of retraction and clot lysis (IRCL)). ICC in patients with multiple injuries was decreased by 27.51%, ICD was decreased by 34.68%, MA was decreased by 75.16%, IRCL was 91.06% above the norm. Patients of group 1 according to LFTEG had signifi cant changes in all parts of coagulation 24  hours after intensive care. Indicators of platelet hemostasis characterized by persistence of hypoaggregation: ICC was decreased by 24.51%, compared with the norm; parameters of coagulation and fi brinolysis had a reliable trend toward normal and decreasing the activity, the fi brinolysis index reached normal reference values. Effi cacy of idarucizumab, prothrombin complex concentrate (PCC) and activated PCC to reverse the anticoagulatory potential of dabigatran in a porcine polytrauma model y p Methods After ethical approval, 28 male pigs were administered dabigatran etexilate (30 mg/kg twice daily p.o.) for 3 days. Dabigatran was administered intravenously in anaesthetised animals on day 4 to achieve consistent high concentrations. Animals were randomised to receive idarucizumab (60 mg/kg, n = 7), PCC (50 U/kg; n = 7), aPCC (50 U/kg; n = 7) or placebo (n = 7). Intervention started 12 minutes after bilateral femur fractures and a standardised blunt liver injury. The primary endpoint was blood loss (observation period 300  minutes). Further, histopathology, haemodynamics and several coagulation variables were also assessed. Data were analysed by repeated-measures ANOVA (mean ± SD). Acknowledgements © Crown copyright 2014. Published with the permission of the Dstl on behalf of the Controller of HMSO. P350 Results Dabigatran levels were comparable between groups (571  ± 174  ng/ml) and resulted in altered coagulation variables. Blood loss was comparable 12 minutes post trauma between groups (801 ± 49 ml) and increased to 3,816 ± 236 ml in anticoagulated control animals post injury. Idarucizumab treatment reduced total blood loss to 1,086  ± 55 ml (P <0.005 vs. all), aPCC to 1,639 ± 104 ml (P <0.05 vs. control) and PCC to 1,797 ± 80 ml (P <0.05 vs. control) after 5 hours. All animals in the intervention groups survived, whereas control animals died within the observation period (mean survival: 89 minutes, range: 62 to 145 minutes). In histopathology no signs of thromboembolic events were present. Altered coagulation variables returned to baseline levels after idarucizumab application and were also signifi cantly, although inconsistently and to a lesser extent, ameliorated following PCCs. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of prehospital versus immediate in-hospital packed red blood cells and fresh frozen plasma (PRBC:FFP) in two models of severe battlefi eld injury. of prehospital versus immediate in-hospital packed red blood cells and fresh frozen plasma (PRBC:FFP) in two models of severe battlefi eld injury. Patients in group 2 had hypoaggregation and hypocoagulation state with increased activity of fi brinolysis: ICC was reduced by 25.62%, ICD decreased by 19.76%, MA was decreased by 22.34%, IRCL was increased by 24.52%. Clinically, patients of group 1 had reduced indicators for infectious complications, reducing the term of mechanical ventilation and reducing the volume of blood transfusions. j y Methods This is a prospective randomised controlled trial using in vivo models of injury conducted in accordance with the Animals (Scientifi c Procedures) Act, 1986. Two injury strands were investigated in 43 terminally anaesthetised Large White pigs: whole body blast exposure (Bl) or no blast (ShBl) plus soft tissue injury and haemorrhage. Thirty minutes later animals were randomly allocated to a 60-minute simulated prehospital hypotensive resuscitation with either PRBC:FFP (1:1 ratio) or 0.9% saline (Early and Late groups respectively). This was followed by 150 minutes of simulated in-hospital resuscitation with a revised normotensive target whereby PRBC:FFP was initiated in the Late group and continued in the Early group.i g Conclusion Patients with multiple injuries have violation in all parts of blood coagulation. The use of prothrombin complex concentrate can reduce the severity of pathological changes in the hemostatic system in patients with polytrauma. Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) Comparing prothrombin complex concentrate and fresh frozen plasma with blood viscosity characteristics in patients with trauma-induced coagulopathy O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) g p y O Tarabrin, I Tyutrin, S Shcherbakov, D Gavrychenko, G Mazurenko, V Ivanova, P Tarabrin Odessa National Medical University, Odessa, Ukraine Critical Care 2015, 19(Suppl 1):P350 (doi: 10.1186/cc14430) Introduction To compare the eff ectiveness of prothrombin complex concentrate and fresh frozen plasma (FFP) in patients with multiple injuries, complicated with coagulopathy bleeding. Introduction To compare the eff ectiveness of prothrombin complex concentrate and fresh frozen plasma (FFP) in patients with multiple injuries, complicated with coagulopathy bleeding. Methods The study involved 51 patients who entered Odessa Regional Hospital with traumatic injuries (concomitant skeletal trauma) complicated with hypocoagulation. Patients were divided into two groups: in the fi rst group (26 patients), as a treatment for coagulopathy, was administered PCC in a dose of 1 ml/kg (25 IU/kg); in the second group (25 patients) was administered FFP in a dose of 15  ml/kg. Evaluation of the functional state of the hemostasis system was carried out using low-frequency thromboelastography (LFTEG) on admission to hospital and 24 hours after the patient’s admission to the ICU. y g Conclusion All medical interventions were associated with reduced blood loss and increased survival. However, idarucizumab, a specifi c antidote to dabigatran, reduced total blood loss more prominently and normalised coagulation parameters to a greater degree as compared with either PCC or aPCC. Reference 1. Grottke O, et al. Crit Care. 2014;18:R27. Reference Benefi cial eff ects of prehospital versus immediate in-hospital blood products during resuscitation in two models of severe military injury S Watts, G Nordmann, C Wilson, A Carter, H Poon, E Kirkman Dstl, Salisbury, UK Critical Care 2015, 19(Suppl 1):P349 (doi: 10.1186/cc14429) Methods Rats received an oral dose of 2.5 mg/kg phenprocoumon. At 15.75 hours post dosing, animals were treated with a single intravenous dose of saline, 4F-PCC (Beriplex® P/N, Kcentra®; CSL Behring) or 3F-PCC (Bebulin® VH; Baxter and Profi lnine® SD; Grifols). Study endpoints included bleeding following tail clip, activated partial thromboplastin Introduction Acute trauma coagulopathy (ATC) is seen in 30 to 40% of severely injured trauma casualties. Early use of blood products is thought to attenuate ATC. This study determined the potential impact S123 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P352 Coagulation support algorithm with rapid TEG and functional fi brinogen TEG in critical bleeding: more results and less time E De Blasio, C Pellegrini, A Federico, V Rocco, M Fumi, Y Pancione, S Sale, D Liberti Hospital G. Rummo, Benevento, Italy Critical Care 2015, 19(Suppl 1):P352 (doi: 10.1186/cc14432) Hospital G. Rummo, Benevento, Italy Critical Care 2015, 19(Suppl 1):P352 (doi: 10.1186/cc14432) Introduction Early coagulation support is essential in massively bleeding patients. A Coagulation Support Algorithm (CSA), integrating rapid TEG (r-TEG) and functional fi brinogen TEG (ff -TEG) could shorten the time to a tailored treatment (Figure 1). Introduction Early coagulation support is essential in massively bleeding patients. A Coagulation Support Algorithm (CSA), integrating rapid TEG (r-TEG) and functional fi brinogen TEG (ff -TEG) could shorten the time to a tailored treatment (Figure 1). Critical Care 2015, Volume 19 Suppl 1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S124 Table 1 (abstract P353). Results of the cost-eff ectiveness model Total medical Total costs/ Treatment QALYs costs ($) QALY ($) Antibiotics + albumin 2.45 7,628 3,111 Antibiotics 1.48 7,682 5,182 Results Total costs were decreased when using albumin, and the improved survival resulted in an additional QALY for patients on albumin, decreasing the cost per QALY. See Table 1 and Figure 2. Conclusion The use of albumin in the treatment of SPB is cost-eff ective. References 1. Poca M, et al. Clin Gastroenterol Hepatol. 2012;10:309-15. 2. Wells CD, et al. Dig Dis Sci. 2004;49:453-8. P354 Estimation of the latent therapeutic demand for albumin in the USA: a focus on three indications Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 1 (abstract P352). Coagulation Support Algorithm. Figure 1 (abstract P352). Coagulation Support Algorithm. Table 1 (abstract P352). Comparison of time to results Test k-TEG r-TEG r-TEG + ff -TEF SCT r (minutes) 13.8 ± 7.1 2.6 ± 2 – ACT (seconds) 265.7 ± 171.9 – 105.2 ± 46.3 TMA (minutes) 42.6 ± 12.4 25.4 ± 14.1 – CSAT (minutes) 21 ± 7.4 Data presented as mean ± SD. ACT, activated clotting time; CSAT, Coagulation Support Algorithm total time; SCT, standard coagulation tests; TMA, time to maximum amplitude. P352 r: r-TEG versus k-TEG, P = 0.0000003; r-TEG versus SCT, P = 0.000000001; k-TEG versus SCT, P = 0.000000009; TMA: r-TEG versus k-TEG, P = 0.0001; r-TEG versus SCT, P = 0.00000004; k-TEG versus SCT, P = 0.000002; ACT versus r of k-TEG (seconds), P = 0.000004; CSAT versus k-TMA, P = 0.00000005. Table 1 (abstract P352). Comparison of time to results Figure 1 (abstract P353). Structure of decision tree for patients with SBP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Methods A retrospective comparison of the time to available TEG and Standard Coagulation Tests (SCT: INR, aPTTr, fi brinogen level) results in two groups of bleeding and coagulopathic patients using citrate kaolin-TEG (k-TEG) or the CSA protocol (r-TEG/ff -TEG). Statistical analysis was performed with Student’s t test for unpaired samples. Figure 2 (abstract P353). Cost-eff ectiveness acceptability across range of WTP. Results Twenty-three patients for each k-TEG and CSA group were compared. The time to available results was shorter using the CSA protocol in comparison with k-TEG (Table  1). The diff erences were both statistically (P  <0.00001) and clinically (mean reduction time 21 minutes) signifi cant. SCT needed the longest time to obtain the fi nal results. Table 1 (abstract P353). Results of the cost-eff ectiveness model Total medical Total costs/ Treatment QALYs costs ($) QALY ($) Antibiotics + albumin 2.45 7,628 3,111 Antibiotics 1.48 7,682 5,182 Conclusion The implementation of a CSA, including r-TEG and ff -TEG, could shorten the time to a targeted treatment in critically bleeding patients. References Results Total costs were decreased when using albumin, and the improved survival resulted in an additional QALY for patients on albumin, decreasing the cost per QALY. See Table 1 and Figure 2. C l i Th f lb i i th t t t f SPB i t ff ti 1. Stensballe J, et al. Curr Opin Anesthesiol. 2014;27:212-18. 3. Kashuk JL, et al. Ann Surg. 2010;251:604-14. g g Conclusion The use of albumin in the treatment of SPB is cost-eff ective. f P353 Use of albumin in spontaneous bacterial peritonitis is cost-eff ective A Farrugia1, M Bansal2, P Caraceni3 353 Use of albumin in spontaneous bacterial peritonitis is cost-eff ective A Farrugia1, M Bansal2, P Caraceni3 1University of Western Australia, Perth, Australia; 2Thought Semantics LLC, Sterling, VA, USA; 3University of Bologna, Italy Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) g 1University of Western Australia, Perth, Australia; 2Thought Semantics LLC, Sterling, VA, USA; 3University of Bologna, Italy Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) P353 1. Poca M, et al. Clin Gastroenterol Hepatol. 2012;10:309-15. 2. Wells CD, et al. Dig Dis Sci. 2004;49:453-8. Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) Introduction Adequate fl uid therapy is essential to the care of septic patients, aiming to optimize oxygen delivery without compromising microcirculation. In recent years, a few studies have suggested that albumin may be superior when compared with crystalloids in severe cases of septic shock. However, there are no data in the fi rst hours of resuscitation. The aim of this study is to evaluate whether albumin 4% solution compared with lactated Ringer decreases 30-day mortality in cancer patients with septic shock. p p Methods The Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study is a prospective, randomized, double-blind and controlled trial, with 360 patients. Until November 2014, at the Cancer Institute of University of São Paulo, we enrolled 110 patients with cancer and septic shock to receive as resuscitation fl uid in the fi rst 12 hours of ICU an admission bolus of albumin 4% solution or lactated Ringer. The primary outcome was 30-day mortality. Secondary outcomes include ICU mortality, ICU and hospital length of stay, 90-day mortality, daily SOFA score, rates and length of mechanical ventilation, renal replacement, needing of vasopressor drugs, status performance and fl uid balance. Results From 650 eligible patients, 110 patients were included in the study – 50 patients in the albumin group and 60 in the Ringer group. The mean age was 63 (57 to 70) years in the albumin group and 61 (51 to 71) in the Ringer group, P = 0.508. Most patients were male (58% in the albumin group vs. 56.1% in the Ringer group, P = 0.846). The ECOG was similar between the albumin and Ringer groups ((0) 26% vs. 8%, (1) 38% vs. 36.8%, (2) 20% vs. 38.6%, (3) 16% vs. 15.8%, P = 0.05). The SAPS 3 admission score was 51 ± 13 in the albumin group and 49 ± 10 in the Ringer group, P = 0.492. The total amount of administered fl uid in the fi rst 12 hours of resuscitation was 1,000 ml (1,000 to 1,500) in the albumin group and 1,000 ml (1,000 to 1,000) in the Ringer group, P = 0.59. The 12-hour fl uid balance was 1,053 ml (385 to 1,700) in the albumin group and 990 ml (200 to 1,525) in the Ringer group. The 30- day mortality was similar in both groups (60% in the albumin group and 50.9% in the Ringer group, P  = 0.34). Reference Reference 1. Caironi P, et al. N Engl J Med. 2014;370:1412-21. Methods A decision analysis model was constructed using Excel. The model is based on the relationships of the epidemiological and clinical factors shown in the infl uence diagram (exemplifi ed in Figure  1 for sepsis). Data for the individual factors were obtained from the literature. One-way sensitivity analysis was used to generate Tornado diagrams (exemplifi ed in Figure 2 for albumin use in sepsis) to determine the relative contribution of diff erent factors to the LTD. Probabilistic sensitivity analysis was used to generate a probability distribution and calculate a mean level for the LTD of each indication. p g p y p E Scotti, M Ferrari, M Chiodi, F Zadek, I Belloni, L Zazzeron, T Langer, L Gattinoni, P Caironi Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436) One-way sensitivity analysis was used to generate Tornado diagrams (exemplifi ed in Figure 2 for albumin use in sepsis) to determine the relative contribution of diff erent factors to the LTD. Probabilistic sensitivity analysis was used to generate a probability distribution and calculate a mean level for the LTD of each indication. Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436) g , , y Critical Care 2015, 19(Suppl 1):P356 (doi: 10.1186/cc14436 Results On average, albumin use was calculated as 104 g per 1,000 inhabitants in severe sepsis, 157 g per 1,000 inhabitants in liver diseases and 61 g per 1,000 inhabitants in CABG. This shows a total LTD of 322 g per 1,000 use of albumin in the US annually. Introduction The induction of ECMO may result in metabolic acidosis [1] due to circuit priming with chloride-rich fl uids, and the sudden decrease in plasma strong ion diff erence (SID). This eff ect can be attenuated using balanced solutions with a SID equal to the patient’s plasma bicarbonate concentration (HCO3 –) [2]. We aimed to compare the eff ects of a novel balanced solution (SID equal to patients’ HCO3 –) with those of commonly employed crystalloids for circuit priming in patients undergoing venovenous ECMO. y Conclusion Albumin consumption in the USA currently averages 479 g per 1,000 population [3]. Hence, the LTD of these three evidence-based indications represents 67% of current usage. Further work is needed to assess the LTD for albumin in other, less well-defi ned areas. f Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) No signifi cant diff erences in the other secondary outcomes were observed between the two groups. Methods The Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study is a prospective, randomized, double-blind and controlled trial, with 360 patients. Until November 2014, at the Cancer Institute of University of São Paulo, we enrolled 110 patients with cancer and septic shock to receive as resuscitation fl uid in the fi rst 12 hours of ICU an admission bolus of albumin 4% solution or lactated Ringer. The primary outcome was 30-day mortality. Secondary outcomes include ICU mortality, ICU and hospital length of stay, 90-day mortality, daily SOFA score, rates and length of mechanical ventilation, renal replacement, needing of vasopressor drugs, status performance and fl uid balance. Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Figure 2 (abstract P354). Relative importance of inputs into the LTD model for sepsis. g Conclusion In cancer patients with septic shock, resuscitation with albumin 4% as compared with lactated Ringer did not improve the rate of survival at 30 days. allocation, but many of the clinical and epidemiologic variables are subject to uncertainty. Decision analysis [2] may assist in generating an assessment of the demand for albumin. Estimation of the latent therapeutic demand for albumin in the USA: a focus on three indications g y g y Critical Care 2015, 19(Suppl 1):P353 (doi: 10.1186/cc14433) Introduction Assessing the cost-eff ectiveness of therapeutic interven- tions is increasingly crucial for health decision-making. Spontaneous bacterial peritonitis (SBP) is one of the major complications of liver cirrhosis. The use of albumin in conjunction with antibiotics has been shown to be eff ective through clinical trials [1]. Introduction The use of albumin in therapeutics is controversial in several areas and requires assessment based on evidence for eff ective resource allocation. Supported indications include sepsis, areas of hepatic diseases and coronary artery bypass grafts (CABG). Latent therapeutic demand (LTD) [1] is the underlying evidence-based demand ensuring ample supplies of drugs are available and aff ordable. Estimating the LTD would assist decision-making and resource f Methods A decision tree (TreeAge®) (Figure  1) was populated from published sources for clinical, cost and epidemiologic variables. The perspective taken was that of the US payer. The robustness of the model was checked using one-way and probabilistic sensitivity analyses. The clinical course was followed for 3 months or until death. Total medical costs and quality-adjusted life years (QALYs) [2] were calculated. S125 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P354). Variables used to construct the LTD model in sepsis. Lactated Ringer Versus Albumin in Early Sepsis Therapy (RASP) study: preliminary data of a randomized controlled trial C Park, E Osawa, J Almeida, R Nakamura, I Duayer, J Fukushima, G Queiroz, F Galas, L Hajjar ICESP, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P355 (doi: 10.1186/cc14435) 2. Goel V. CMAJ. 1992;147:413-7. Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury Conclusion During the resuscitation phase of the burn patients, the use of HES (130/0.4/6%) at low doses does not seem to cause more risk or injury according to RIFLE or AKIN criteria than those reported by studies in burn patients resuscitated without HES. However, the need for RRT is associated with a high mortality, although in many cases the display is terminal. Introduction The aim of our study is to investigate the eff ect of intraoperative use of gelatin in living donor liver transplantation on postoperative acute kidney injury (AKI). It has been demonstrated that ischemia and chloride-liberal fl uid management cause AKI in liver transplantation [1]. Gelatin has minimal side eff ects on renal functions [2]; however, it might be a reason for postoperative AKI. g Methods A total of 154 liver transplantation patients were retrospectively evaluated between September 2011 and September 2013, and among these, 128 patients were included in the study. The patients who were under 18 years old, transplanted from cadaveric donors and needed preoperative renal replacement therapy were excluded. The patients were divided into two groups as GI (without gelatin administration) and GII (with gelatin administration). The patient’s age, gender, actual body weight, diagnoses, MELD score, APACHE II score, duration of operation, total clamping time, noradrenalin infusion rate, amount of erythrocyte suspension, fresh frozen plasma (FFP) and thrombocyte suspension used, intraoperative fl uid balance, intraoperative and total clamping diuresis, serum creatinine levels on the postoperative 1st, 2nd, 4th and 7th days, duration of mechanical ventilation, length of ICU and hospital stay, hospital and 1-year mortality rate were recorded. The changes in creatinine levels on the 1st, 2nd, 4th and 7th days were evaluated according to the KDIGO guideline for AKI [3]. Infl uence of anaesthetic factors on skin graft viability in a burns ICU C Isitt, KA McCloskey, A Cabello, P Sharma, MP Vizcaychipi Chelsea and Westminster Hospital, London, UK Critical Care 2015, 19(Suppl 1):P359 (doi: 10.1186/cc14439) Introduction Graft failure is a major cause of morbidity in patients with burns, resulting in increased length of hospital stay and increased number of operations. At our regional burns unit we collated the data from anaesthetic charts of patients admitted to our burns ICU who required skin grafting. The aim was to analyse whether any anaesthetic variables contribute to graft failure. g Methods Thirty-fi ve patients were included in the analysis with a total of 191 operations. References Methods We randomly assigned patients with acute respiratory failure in need of ECMO to receive either NaCl 0.9% (NS, SID = 0), Ringer lactate (RL, SID = 28), or a novel balanced solution (Solution X, SID equal to the patient’s HCO3 –) for circuit priming solution. Arterial blood gases and laboratory parameters were collected at 0, 5, 30, 60, 90, and 1. Stonebraker J, et al. J Clin Immunol. 2014;34:233-44. 2. Goel V. CMAJ. 1992;147:413-7. 3. Market Research Bureau. The plasma proteins market in the USA (2013). http://marketingresearchbureau.com/list-of-reports/ the-plasma-proteins-market-in-the-united-states/. S126 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 120 minutes after pump start. SID, base excess (BE) and total weak acids (Atot) were calculated. total clamping time was longer and amount of blood products used during surgery was more than the other group. Which of these factors is associated with AKI has to be revealed with further studies. 120 minutes after pump start. SID, base excess (BE) and total weak acids (Atot) were calculated. total clamping time was longer and amount of blood products used during surgery was more than the other group. Which of these factors is associated with AKI has to be revealed with further studies. Results We enrolled 20 patients (23 priming procedures – RL, n = 8; NS, n = 8; Solution X, n = 7). ECMO was initiated for ARDS (45%), bridge to lung transplant (25%), acute graft failure after transplant (15%), and acute on chronic respiratory failure (15%). Average priming volume was 10 ± 5 ml/kg; patients’ baseline HCO3 – was 28 ± 6 mEq/l. During the fi rst 2 hours after ECMO initiation, arterial pH raised similarly in all groups (P = 0.39) due to CO2 removal. In contrast, BE decreased starting after 5 minutes in both the NS and RL groups (BE variation, –2.2 ± 1.7 and –1.9 ± 1.3 mEq/l, P <0.001 vs. baseline; P = 0.04 for interaction, two-way ANOVA, 2-hour period). No BE changes were observed in the Solution X group (0.3 ± 0.8 mEq/l). In the NS group, BE reduction was associated with a reduction in SID (from 39 ± 8 to 34 ± 6 mEq/l at 5 minutes, P = 0.008), entirely due to an increase in Cl (103 ± 7 vs. 108 ± 6 mEq/l, P = 0.001). References In the RL group, BE and SID reductions (40 ± 8 vs. 36 ± 8 mEq/l, P  = 0.008) were associated with an increase in both Cl (105  ± 7 vs. 107 ± 7 mEq/l, P = 0.01) and lactate (1.4 ± 0.6 vs. 2.2 ± 1.0 mEq/l, P = 0.008). No changes were observed in other electrolyte concentrations. Dilution did not diff er between groups (P = 0.25 for Atot variation). The acidifying eff ect of NS and RL was amplifi ed in patients with higher baseline HCO3 –. References 1. Nadeem A, et al. Crit Care. 2014;18:625. 2. Eremenko AA, et al. Anesteziol Reanimatol. 2001;3:58-61. 3. KDIGO AKI Work Group. Kidney Int. 2012;2 Suppl:1–138. 1. Nadeem A, et al. Crit Care. 2014;18:625. 2. Eremenko AA, et al. Anesteziol Reanimatol. 2001;3:58-61. 3. KDIGO AKI Work Group. Kidney Int. 2012;2 Suppl:1–138. Incidence of acute kidney injury in critically burned patients resuscitated with crystalloid and colloid according to parameters of transpulmonary thermodilution, diuresis and lactic acid P Extremera Navas, M Sanchez Sanchez, I Pozuelo Echegaray, A Agrifoglio Rotaeche, A Robles Caballero, A García de Lorenzo Hospital Universitario la Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P358 (doi: 10.1186/cc14438) Introduction The purpose was to study the incidence of acute kidney injury (AKI) according to RIFLE and AKIN criteria in critically ill burn patients resuscitated with Ringer’s solution and supplements of lower molecular weight hydroxyethyl starch (HES)130/0.4/6%, and to determine the relationship between RRT indication and mortality. 3 Conclusion As compared with NS and RL, the use of a novel balanced solution with a SID equal to the patient HCO3 – level for ECMO priming uniquely avoids the addition of metabolic acidosis to patients with uncompensated hypercapnia. Methods We studied 165 consecutive patients admitted to the critical care burn unit. Resuscitation was performed using lactated Ringer’s solution and HES at a low dose to achieve urine output, lactate levels, and transpulmonary thermodilution parameters. The contributions of colloids and crystalloids were measured, and renal function was evaluated. Statistical analysis was performed using the Spearman test. Results The average total body surface area (TBSA) burned was 30 ± 15%, and the median of the total volume needed in the fi rst 24 hours was 4.01  ml/kg/% TBSA burned. According to the RIFLE criteria, 10 (6.1%) patients presented with risk, 11 (6.7%) presented with injury, and 11 (6.7%) presented with failure. References 1. Liskaser, et al. Anesthesiology. 2000;93:1170-3. 2. Langer, et al. Intensive Care Med. 2012;38:686-93. 1. Liskaser, et al. Anesthesiology. 2000;93:1170-3. 2. Langer, et al. Intensive Care Med. 2012;38:686-93. References According to the AKIN criteria: 9.7% presented stage I, 3% stage II and 10.3% stage III. Replacement therapy (RRT) was performed in 15 patients (9.1%). In six of these patients RRT was employed in the fi nal stages of multiorgan failure. In the remaining nine patients, for various reasons only one survived. P357 Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury HK Atalan1, B Gucyetmez2, S Aslan1, M Berktas3, KY Polat1 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Kappa Consulting, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P357 (doi: 10.1186/cc14437) Association of elevated levels of plasma chloride, in severity and mortality, in adult patients in the ICU Introduction For a long time, many investigators have tried to demon- strate increased mortality associated with acid–base distur bances. In this study, we sought to determine the association of hyperchloremia measured at ICU admission and whether this electrolyte disturbance is associated with an increase in morbidity and mortality. Methods A retrospective study was conducted. Patients with a diagnosis of AKI according to KDIGO creatinine criteria and available urinary chemistry at one point during their ICU stay were evaluated. Day 0 was defi ned as the day when SIDu was calculated from urinary spot analysis (SIDu = Na+U + K+U – Cl–U). Patients were followed and staged for AKI in the next 3 days. AKI reversibility was defi ned according to the lack of criteria for AKI. y y Methods Data were retrospectively collected for consecutive adult patients admitted to Agustin O’Horan Hospital ICU, between January 2011 and July 2014, who underwent inpatient medical treatment using electronic fi les. Results In total, 143 critically ill patients with a diagnosis of AKI were included. SIDu at day 0 did not diff er between diff erent AKI stages at day 0. SIDu at day 0 was statistically diff erent between diff erent AKI stages at days 1, 2, 3 (Table 1). SIDu at day 0 was statistically diff erent between reversible and not reversible AKI at days 1, 2, 3 (Table 2). A conventional receiver-operating curve was generated to assess the accuracy of SIDu to predict AKI reversibility at day 1. AUC for SIDu was 0.82 (P <0.0001; 95% CI: 0.75 to 0.88). i Results The dataset consisted of 936 medical fi les and serum chloride concentration values on admission, 853 being eligible. Hyperchloremia (serum chloride >110 mmol/l) is quite common, with an incidence of 47.71%. Patients were propensity matched based on their association with death and hyperchloremia. Of the 853 patients collected, patients with hyperchloremia after admission (n = 446, 52.3%), patients were matched to patients who had normal serum chloride levels after admission. These two groups were well balanced with respect to all variables collected. The hyperchloremic group was at increased risk of mortality at ICU discharge, relative risk ratio = 1.81; 95% confi dence interval, 1.41 to 2.51 risk increase of 25.31%. Admission hyperchloremia was associated with increased morbidity, mortality and higher scores in severity scales; this association was statistically important. See Figure 1. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results There was no signifi cant diff erence in age, %total burn surface area or Belgian Outcome Burns Injury score between the groups. For all operation data, use of colloids was found to signifi cantly contribute towards poor graft viability (P  = 0.035, 95% CI). When analysis was performed on only SSG and debridement operations, colloids remained signifi cant (P = 0.034, 95% CI) and metarminol use was found to signifi cantly contribute (P = 0.028, 95% CI) to poor graft viability. Overall use of inotropes was not signifi cant between the two groups. Other variables including minimum and maximum temperature, preoperative haemoglobin and blood transfusion were not found to be signifi cant. References 1. Gauthier PM, Szerlip HM. Metabolic acidosis in the intensive care unit. Crit Care Clin. 2002;18:289-308. 1. Gauthier PM, Szerlip HM. Metabolic acidosis in the intensive care unit. Crit Care Clin. 2002;18:289-308. 2. Eisenhut M. Causes and eff ects of hyperchloremic acidosis. Crit Care. 2006;10:413. 2. Eisenhut M. Causes and eff ects of hyperchloremic acidosis. Crit Care. 2006;10:413. 3. Wooten EW. Science review: quantitative acid–base physiology using the Stewart model. Crit Care. 2004:8:448-52. 3. Wooten EW. Science review: quantitative acid–base physiology using the Stewart model. Crit Care. 2004:8:448-52. 36 Urinary strong ion diff erence and acute kidney injury: an early marker of renal dysfunction? i Conclusion Our results suggest that the use of colloids is a contributor to poor graft viability in burns. This was found to be independent of temperature and overall inotrope use; however, the use of metarminol may be a contributing factor. y P Balsorano1, A De Gaudio1, Stefano Romagnoli1, Ipsita Krishnan2 1AOUC Careggi, Florence, Italy; 2Rhode Island Hospital, Providence, RI, USA Critical Care 2015, 19(Suppl 1):P361 (doi: 10.1186/cc14441) Introduction Kidneys play a crucial role in the regulation of electrolytes and acid–base homeostasis. Impaired renal function is associated with greater urinary strong ion diff erence (SIDu) in patients with metabolic acidosis [1]. In critically ill patients, several factors, such as infused fl uids and acid endogenous production, would lead to changes in plasma SID and acid–base homeostasis without renal regulation of urinary electrolytes and SIDu [2]. Hence, AKI can be highlighted as an inability to address acid–base metabolic disturbances, which may be detected before major increases in creatinine or decreases in urine output. We evaluated the eff ects of renal function on urinary strong ion excretion using the Stewart approach to acid–base in critically ill patients with AKI. P360 Association of elevated levels of plasma chloride, in severity and mortality, in adult patients in the ICU Conclusion This retrospective cohort trial demonstrates an association between hyperchloremia and poor ICU admission outcome (death). Additional studies are required to demonstrate a causal relationship between these variables. Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission AKI stage 3 2 1 0 P value Day 1 48.1 (21) 46 (22) 37.9 (20) 17.3 (22) <0.001 Day 2 40.2 (23) 45.9 (20) 45 (23) 29 (22) 0.004 Day 3 40.3 (26) 47.2 (18) 53.2 (23) 31 (23) 0.006 Table 2 (abstract P361). SIDu (mEq/l) between reversible versus not reversible AKI at days 1, 2, 3 Reversible Not reversible P value Day1 16.8 (23) 43.9 (21) 0.0001 Day2 28.5 (24) 45.3 (22) 0.0001 Day3 30 (24) 47.3 (21) 0.0001 Conclusion SIDu identifi ed patients with reversible AKI with good accuracy. SIDu can be a promising, simple and cost-eff ective tool in AKI patient evaluation. Further research is needed to assess SIDu capability to early detect patients with renal dysfunction before increases in creatinine or decreases in urine output. References 1. Moviat M, et al. J Crit Care. 2012;27:255-60. 2. Masevicius FD, et al. Crit Care Resusc. 2010;12:248-54. Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission Table 1 (abstract P361). SIDu (mEq/l) between diff erent AKI stages at days 1, 2, 3 post admission AKI stage 3 2 1 0 P value Day 1 48.1 (21) 46 (22) 37.9 (20) 17.3 (22) <0.001 Day 2 40.2 (23) 45.9 (20) 45 (23) 29 (22) 0.004 Day 3 40.3 (26) 47.2 (18) 53.2 (23) 31 (23) 0.006 Table 2 (abstract P361). SIDu (mEq/l) between reversible versus not reversible AKI at days 1, 2, 3 Figure 1 (abstract P360). Group mortality, high and low chlorine. Intraoperative use of gelatin in living donor liver transplantation and postoperative acute kidney injury These were a combination of debridement, split skin grafts (SSG) and change of dressings. All patients were admitted to our burns ICU between January 2009 and October 2013. Exclusion criteria were death prior to discharge and initial surgery at a diff erent hospital. Sixteen patients had good graft viability (Group A) and 19 patients had poor graft viability (Group B). Logistical regression was performed using SPSS (Version 22.0). Hosmer and Lemeshow testing was used to confi rm goodness of fi t. Independent variables were age, sex, preoperative haemoglobin, intraoperative fl uid resuscitation, blood products, inotropes, volatile agents and temperature. Poor graft viability was defi ned as requiring at least one additional skin graft. Analysis was performed on all operations and then by subtype of operation (that is, SSG and debridement, SSG only). Results In total, 128 patients were categorized as GI (58, 45%) or GII (70, 55%). Total clamping time, intraoperative diuresis, intraoperative crystalloid use, intraoperative fl uid balance, operation bleeding, erythrocyte suspension, FFP and thrombocyte suspension use and postoperative lactate levels of GII were statistically signifi cantly higher than GI (P <0.001 for each). According to the KDIGO guideline, AKI in GII on the 1st, 2nd, 4th and 7th days (11.4%; 20%; 24.3%; 17.1%) was statistically signifi cantly higher than GI (P <0.001 for each). Conclusion In patients who received gelatin, kidney dysfunction in the postoperative period was observed more frequently. Also in this group, S127 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P366 Incidence and predisposing factors for the development of disturbed glucose metabolism and diabetes mellitus after intensive care admission: the DIAFIC study S Van Ackerbroeck, K Janssens, P Jorens, T Schepens, W Verbrugghe, V Van Hoof, L Van Gaal, C De Block University Hospital Antwerp, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P366 (doi: 10.1186/cc14446) Incidence and predisposing factors for the development of disturbed glucose metabolism and diabetes mellitus after intensive care admission: the DIAFIC study S Van Ackerbroeck, K Janssens, P Jorens, T Schepens, W Verbrugghe, V Van Hoof, L Van Gaal, C De Block University Hospital Antwerp, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P366 (doi: 10.1186/cc14446) Low serum 25-hydroxyvitamin D at critical care initiation is associated with sepsis and morbidity in Dutch critically ill patients K De Haan Erasmus MC, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P365 (doi: 10.1186/cc14445) Low serum 25-hydroxyvitamin D at critical care initiation is associated with sepsis and morbidity in Dutch critically ill patients K De Haan Erasmus MC, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P365 (doi: 10.1186/cc14445) Introduction Vitamin D defi ciency may frequently occur in critically ill patients and may be associated with sepsis and increased mortality. We therefore evaluated the prevalence of 25-hydroxyvitamin D defi ciency in a Dutch ICU, and its relationship with sepsis, morbidity and mortality. Methods We conducted a prospective observational study in a 10-bed mixed ICU. A total of 1,372 patients were admitted between July 2011 and June 2013 including 198 readmissions, of which 940 patients were studied. 25-Hydroxyvitamin D levels were determined within 24 hours after admission. 25-Hydroxyvitamin D levels were judged as suffi ciency (>50 nmol/l), insuffi ciency (30 to 50 nmol/l) and defi ciency (<30 nmol/l). Results The prevalence of defi ciency and insuffi ciency was 36% and 38%, respectively. Only 26% of the patients had suffi cient vitamin D levels. Vitamin D defi ciency is associated with sepsis (P <0.001) at ICU admission. Patients with defi cient levels had higher mean APACHE IV scores, 64 versus 52 (P <0.001), and longer length of hospital stay, 12 versus 9 days (P <0.001), respectively, as compared with patients with suffi cient levels. Patients with defi cient vitamin D levels had an odds ratio for in-hospital mortality of 1.4 (95% confi dence interval of 0.84 to 2.29, P = 0.2) relative to patients with suffi cient vitamin D levels.i Conclusion Implementation of i.v. potassium replacement guidelines improved the use of i.v. potassium in the ICU by reducing the requirement for i.v. potassium supplementation and increasing the overall time patients spent without hypokalaemia. Whilst nursing staff found the guideline useful and felt it increased safe use of i.v. potassium, more work is needed to ensure nurse workload is not increased signifi cantly. fi Conclusion 25-Hydroxyvitamin D defi ciency frequently occurs in Dutch critically ill patients. Although relating to sepsis, disease severity and morbidity, vitamin D defi ciency is not an independent predictor of mortality in these patients, which was otherwise relatively low. References 1. Moviat M, et al. J Crit Care. 2012;27:255-60. Masevicius FD, et al. Crit Care Resusc. 2010;12:248-54. S128 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P362 almost every patient and at every studied time point. Moreover, these levels were signifi cantly higher than in controls or compared with referenced literature. The chronology of exposure was demonstrated: the preoperative urine and serum levels of the DEHP metabolites were often below the detection limit. Medical devices are the source of these chemicals: patients on hemofi ltration, extracorporeal membrane oxygenation or both showed serum levels 100-fold or 1,000-fold higher than the general population or workers in plastic industry. The serum and some of the urinary levels of the DEHP metabolites are the highest ever reported in humans; some at biologically highly relevant concentrations of even ≥10 to 50 μM. almost every patient and at every studied time point. Moreover, these levels were signifi cantly higher than in controls or compared with referenced literature. The chronology of exposure was demonstrated: the preoperative urine and serum levels of the DEHP metabolites were often below the detection limit. Medical devices are the source of these chemicals: patients on hemofi ltration, extracorporeal membrane oxygenation or both showed serum levels 100-fold or 1,000-fold higher than the general population or workers in plastic industry. The serum and some of the urinary levels of the DEHP metabolites are the highest ever reported in humans; some at biologically highly relevant concentrations of even ≥10 to 50 μM. P362 Evaluation of the eff ect of guidelines to reduce intravenous potassium infusions in ICU patients MC Law Min1, RS Bourne1, S Burd2, M Stone2 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2De Montfort University, Leicester, UK Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) P362 Evaluation of the eff ect of guidelines to reduce intravenous potassium infusions in ICU patients MC Law Min1, RS Bourne1, S Burd2, M Stone2 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2De Montfort University, Leicester, UK Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) Critical Care 2015, 19(Suppl 1):P362 (doi: 10.1186/cc14442) Introduction The aim was to evaluate whether guidelines for intra- venous (i.v.) potassium replacement improved plasma potassium homeostasis in ICU patients. Prompt and eff ective treatment of hypokalaemia is an important intervention in the ICU, but concentrated i.v. potassium solutions may cause serious harm if used inappropriately [1]. There were previously no formalised guidelines on i.v. potassium supplementation in the ICU at Sheffi eld Teaching Hospitals. Practice was reviewed and guidelines were introduced to improve patient safety, plasma potassium homeostasis and reduce i.v. potassium supplementation requirements. Conclusion Adult ICU patients are exposed to plastic softeners, in particular PMs. Despite the continuously tightening regulations, BPA and DEHP are still present in medical devices. Because patient safety is a concern in the ICU, further research into the (possibly toxic and clinical) eff ects of chemicals released from medical devices should be undertaken. pp q Methods A before and after evaluation of plasma potassium homeo- stasis in ICU patients requiring i.v. potassium supplementation was conducted over a period of 8 months (August 2013 to May 2014). Patient data on plasma potassium levels, i.v. and oral potassium supplements administered were obtained from the clinical information system. Clinical appropriateness of i.v. potassium acetate prescriptions, fl uid and chloride intake related to potassium infusions and cost linked to the guidelines were also compared pre/post implementation. Impact of the guidelines on nurses’ practice was assessed using questionnaires. Results Median i.v. potassium replacement dose per patient was signifi cantly reduced in the post-guidelines group from 215 (IQR: 94; 485) to 80 (IQR: 40; 160) mmol; P <0.001. Although the percentage time per group for patients who were hypokalaemic was less in the post group (18.2% vs. 14.8%), there was no diff erence in mean patient values (24.2 (20.3)% vs. 22.1 (17.5)%; P = 0.228). The duration of hyperkalaemia was increased. Prescribing of i.v. potassium acetate was not always appropriate. Median patient fl uid-related dose was increased (107.5 (IQR: 47.1; 242.4) vs. 250 (IQR: 100; 600) ml; P <0.001), whilst chloride doses were reduced (170.7 (IQR: 91.3; 438.3) vs. 110 (IQR: 55; 250) mmol; P <0.009). Nurses were satisfi ed with the new practice, reporting it was safe, eff ective and clinically useful. However, compared with baseline practice, they perceived the guidelines as less eff ective and felt the workload was higher. Reference 1. Ridley SA, et al. Anaesthesia. 2004;59:1193-200. 1. Ridley SA, et al. Anaesthesia. 2004;59:1193-200. P366 Associations between the degree of correction of hypoglycemia and ICU mortality g g Results The median glucose in the EndoTool group (141.5  mg/dl) was lower than in the Adult ICU group (159.9  mg/dl) (P  <0.0001). The standard deviation of glucose in the EndoTool group (32.3  mg/ dl) was lower than the Adult ICU group (39.5  mg/dl) (P  = 0.0001). The proportion of patients in each group with 10% or higher of measurements at a severe hyperglycemia level (≥200  mg/dl) in the EndoTool group (35.2%) was lower than the Adult ICU group (64.1%) (P <0.0001). The proportion of patients who had at least one moderate hypoglycemic measurement (<70 mg/dl) was not signifi cantly diff erent between the EndoTool group versus the Adult ICU group (11.73% vs. 9.3%, respectively; P = 0.34). However, there was a higher overall incidence of hypoglycemia in the EndoTool group (5.65 hypoglycemic measurements/100 person-protocol days) compared with the Adult ICU group (3.43/100 person-protocol days) (RR = 1.65, 95% CI = 1.09 to 2.45, P = 0.014). Severe hypoglycemia (<40 mg/dl) was rare, only occurring in 1/179 (0.56%) in the EndoTool group and 4/580 (0.69%) in the Adult ICU group. R Van Hooijdonk1, JM Binnekade1, A Abu-Hanna1, F Van Braam Houckgeest2, LS Hofstra3, J Horn1, MA Kuiper4, f NP Juff ermans1, HL Van den Oever5, JP Van der Sluijs6, PE Spronk7, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2Tergooi Hospitals, Hilversum, the Netherlands; 3Scheper Hospital, Emmen, the Netherlands; 4Medical Centre Leeuwarden, the Netherlands; 5Deventer Hospital, Deventer, the Netherlands; 6Medical Center Haaglanden, The Hague, the Netherlands; 7Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P367 (doi: 10.1186/cc14447) Introduction It is conjectured that transition of hypoglycemia to hyperglycemia may be more harmful than hypoglycemia itself. We investigated the association between the degree of correction of hypoglycemia and ICU mortality in patients under moderately strict to strict glycemic control. Methods This is a retrospective analysis from a pooled cohort from seven ICUs in the Netherlands over 6 years. ICU patients who developed hypoglycemia (<70 mg/dl) were included. We excluded patients who were readmitted, and patients with hypoglycemia in whom no follow- up blood glucose measurement was performed within 8  hours. Marked exposure to the endocrine-disrupting chemicals phthalates and bisphenol A in the ICU J Huygh, P Jorens Antwerp University Hospital, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P364 (doi: 10.1186/cc14444) J Huygh, P Jorens J Huygh, P Jorens Introduction Care for ICU patients has benefi ted from medical devices. Bisphenol A (BPA) and phthalates can leach from the plastic matrix. We hypothesized that ICU patients are exposed to BPA and phthalates through medical devices. Introduction Stress hyperglycaemia (SH) is commonly observed during hospitalisation in the ICU and adversely infl uences outcome [1]. When SH occurs in previously nondiabetic patients, this might refl ect a latent disturbance of glucose metabolism and predict future risk of diabetes. We wanted to assess the incidence of disturbed glucose metabolism (DGM) and identify predictors for future diabetes risk. This could support timely diagnosis, prevention, and early treatment of impending diabetes mellitus (DM). Methods Serum (n = 118) and urinary (n = 102) samples of adult (n = 35) ICU patients were analyzed for total BPA and di(2-ethylhexyl)phthalate (DEHP) and other phthalate metabolites (PMs). We also enrolled patients preoperatively before scheduled thoracic surgery and repeat samples were taken on days 1 to 4 during the ICU stay. Control data came from 44 healthy controls or from referenced literature. Methods In this prospective observational study, we enrolled 338 patients without known DM, who were admitted for at least 36 hours to the ICU of the Antwerp University Hospital between September 2011 and March 2013. A 75  g oral glucose tolerance test was performed Results Our results show that adult ICU patients are continuously exposed to phthalates (that is, DEHP) as well as to BPA, albeit to a lesser extent, resulting in detectable serum and urinary levels in S129 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Not the transition to hyperglycemia, but insuffi cient recovery from hypoglycemia is associated with an increased ICU mortality in patients under moderately strict or strict glucose control with insulin. Conclusion Not the transition to hyperglycemia, but insuffi cient recovery from hypoglycemia is associated with an increased ICU mortality in patients under moderately strict or strict glucose control with insulin. 6 to 9  months post ICU admission to screen for disturbed glucose metabolism. Furthermore, we examined whether post-discharge glucose disturbances could be predicted by the FINDRISC questionnaire [2], patient demographics, comorbidities, HbA1c at ICU admission, and by parameters related to ICU stay (glucose parameters, insulin need, caloric intake, disease severity). Computer versus paper insulin protocol for managing hyperglycemia in three ICUs A Peckham Oregon Health & Science University, Portland, OR, USA Critical Care 2015, 19(Suppl 1):P368 (doi: 10.1186/cc14448) Computer versus paper insulin protocol for managing hyperglycemia in three ICUs A Peckham Oregon Health & Science University, Portland, OR, USA Critical Care 2015, 19(Suppl 1):P368 (doi: 10.1186/cc14448) Introduction The purpose of this study was to compare a computer protocol against a paper protocol in managing three domains of glucose control. Hyperglycemia is common in critically ill patients, and their risk of death is associated with hyperglycemia, hypoglycemia, and glucose variability. A safe and eff ective insulin protocol must minimize hyperglycemia and glucose variability while also avoiding hypoglycemia. Computer-based insulin protocols promise better performance by adjusting to each individual’s sensitivity to insulin. Conclusion Stress hyperglycaemia is frequent in nondiabetic patients and has a tendency towards future disturbances in glucose metabolism and DM. Glucose metabolism was disturbed in 35% of subjects 8 months post ICU admission, of whom 7% was diagnosed with diabetes mellitus. Predictors of elevated risk included a high FINDRISC score, high SAPS 3 score, and a lower daily caloric intake during ICU stay. References Methods This is a historical cohort study with 759 patients admitted to three ICUs (medical/cardiac, trauma, and neuroscience) at an academic tertiary care hospital. All adult patients from January 2012 to October 2013 on one of two continuous insulin protocols for at least 8 hours were included. At the start of the study period the paper protocol in use (Adult ICU) had a target glucose of 140 to 180 mg/dl and was used for any patient with a glucose higher than 180 mg/dl. In June 2013 this was replaced by a computer-based insulin protocol (EndoTool) that had the same criteria for initiation and had a target glucose of 150 mg/dl. The primary exposure was the insulin protocol, and the primary outcome was performance in maintaining glucose control. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Schwarz PE, et al. Horm Metab Res. 2009;41:86-97. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Schwarz PE, et al. Horm Metab Res. 2009;41:86-97. Associations between the degree of correction of hypoglycemia and ICU mortality We determined the association between three measures of correction of hypoglycemia within 8 hours after hypoglycemia and ICU mortality: predefi ned ranges of the ‘highest blood glucose level’ (<80 mg/dl; 80 to 110 mg/dl; 110 to 150 mg/dl (reference category); 150 to 180 mg/ dl; and >180  mg/dl); quartiles of the ‘delta glucose’, defi ned as the diff erence between minimum and maximum blood glucose level with the third quartile as reference category; and quartiles of the ‘standard deviation’ of the blood glucose level with the third quartile as reference category. Conclusion Patients on the computer protocol had a lower median glucose, less variability, and less hyperglycemia than patients on the paper protocol. There was a higher risk of moderate but not severe hypoglycemia in the computer group. Marked exposure to the endocrine-disrupting chemicals phthalates and bisphenol A in the ICU J Huygh, P Jorens Antwerp University Hospital, Edegem, Belgium Critical Care 2015, 19(Suppl 1):P364 (doi: 10.1186/cc14444) y Results In total, 246 patients (73%) experienced SH during their ICU stay. Eight months post ICU admission, glucose metabolism was disturbed in 119 (35%) subjects. Of these, 27 (8%) had impaired fasting glucose, 43 (13%) had impaired glucose tolerance, 25 (7%) had impaired fasting glucose and impaired glucose tolerance, and 24 (7%) were diagnosed with DM. A disturbed glucose metabolism tended to be more prevalent in subjects who experienced SH during ICU stay as compared with those without SH (38% vs. 28%, P = 0.065). HbA1c on admission correlated with the degree of SH (r = 0.308, P <0.001). The FINDRISC score (9.5 vs. 11, P = 0.001), SAPS 3 score (median of 42 in both groups, P = 0.003) and daily caloric intake during ICU stay (222 vs. 197, P = 0.011) were associated with a DGM. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf yp g y y Results Of the 1,059 patients admitted with sepsis, 526 (55.8%) had admission glucose levels within the normal range, 270 (25.5%) had mild hyperglycemia and 202 (19.1%) severe hyperglycemia. Patients with severe hyperglycemia were older, had higher APACHE IV scores and were more often diabetics compared with euglycemic patients. Shock on admission was more common in patients admitted with euglycemia. Crude mortality increased with increased admission glucose and a Cox regression analysis showed increased risk for 30- day (HR = 1.67, CI = 1.24 to 2.23), 60-day (HR = 1.42, CI = 1.08 to 1.87) and 90-day (1.31, CI = 1.02 to 1.70) mortality in patients admitted with severe hyperglycemia compared with euglycemia. The association between mortality and severe hyperglycemia on admission was only present in patients without known diabetes but not in patients with a history of diabetes (30-day mortality HR = 1.67, CI = 1.15 to 2.43 vs. 1.84, CI = 0.97 to 3.49). Severe hyperglycemia was associated with a blunted proinfl ammatory cytokine response (IL-6 and IL-8) on admission in patients without, but not in patients with diabetes. g y p R Van Hooijdonk, JH Leopold, T Winters, JM Binnekade, NP Juff ermans, J Horn, JC Fischer, EC Van Dongen-Lases, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P370 (doi: 10.1186/cc14450) Introduction There is a need for continuous glucose monitoring in critically ill patients. The objective of this trial was to determine the point accuracy and reliability of a device designed for continuous monitoring of interstitial glucose levels in ICU patients (Sentrino; Medtronic MiniMed, Northridge, CA, USA). Methods Critically ill patients with an anticipated life expectancy >96 hours were eligible for participation, if the platelet count was >30 × 1012 ml. Device readings were compared with glucose measurements in arterial blood using blood gas analyzers (RapidLab Siemens Healthcare Diagnostics, The Hague, the Netherlands). We used a linear mixed model to determine which factors aff ect point accuracy. In addition, we determined the reliability, including duration of device start-up and calibration, skips in data acquisition, and premature disconnections of sensors. Conclusion Severe hyperglycemia on admission is associated with increased 30-day, 60-day and 90-day mortality in sepsis patients without a history of diabetes mellitus. Results We included 50 patients, aged 65 (56 to 72) years with an APACHE II score of 23 (17 to 26). P370 Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patients R Van Hooijdonk, JH Leopold, T Winters, JM Binnekade, NP Juff ermans, J Horn, JC Fischer, EC Van Dongen-Lases, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P370 (doi: 10.1186/cc14450) Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 The number of skips in data acquisition was low, resulting in availability of real-time data during 95 (89 to 98)% of the connection time per sensor. glucose data prove in such routine use?’ Using actual case data, we have shown how comparing the mean absolute relative diff erence (MARD) and integration of the area under the curve (AUC) from the continuous glucose monitoring and intermittent measurement can be used to measure patient risk. Conclusion The point accuracy of the device was relatively low in critically ill patients. The device reliability was relatively good, although sensors were removed prematurely for a variety of reasons. p Methods The analysis used aggregated case data generated from our recent clinical trials, where a GlySure sterile, single-use sensor and dedicated monitoring system was used to measure the blood glucose concentration in patients continuously and in real time. The measurement of risk was compared using the MARD, an accepted error calculation tool, and the AUC was calculated using an AUC analysis software program. g References 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442.i 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442.i 2. Gopal PB, Mulavisala KP. ESICM 2014. Abstract 0264. http://react-profi le.org/ Download/ESICM2014_Abstract_Book_fi nal_version.pdf. Eff ect of admission hyperglycemia in sepsis patients with or without a history of diabetes g Results When MARD from the GlySure sensor and intermittent measurement using the hospital’s existing protocol was compared, the measure of risk to the patient (that is, the uncertainty regarding the patient’s absolute blood glucose status) for the GlySure sensor was 50.5% lower than the intermittent measurement. The results also showed that as the variability of the BG data increases, the benefi t of continuous monitoring increases by signifi cantly reducing patient risk. The continuous monitoring reduces the patient’s risk by 88%, 73%, and 69% respectively in high, medium and low variability situations. Introduction Hyperglycemia is common and often multifactorial in critically ill patients. The association of hyperglycemia with adverse outcome has repeatedly been established in a variety of settings. The objective of this study was to investigate whether hyperglycemia on admission to the ICU impacts presentation and outcome of sepsis patients and whether this eff ect is diff erent for patients with a history of diabetes mellitus. p y g y Conclusion It is more and more evident that continuous glucose technology will be instrumental in driving safe and eff ective glucose management protocols that will support more consistent glycemic management standards within ICUs and across institutions. Methods A two-center, prospective observational cohort study was conducted including all consecutive critically ill patients admitted to the ICU between January 2011 and July 2013. Sepsis patients were identifi ed using strict clinical and diagnostic criteria. The fi rst glucose measurement within a time window of 4 hours before up to 4 hours after ICU admission was categorized into euglycemia (71 to 140 mg/dl), mild hyperglycemia (141 to 200 mg/dl) or severe hyperglycemia (>200 mg/ dl), patients with hypoglycemia were excluded. A multivariable Cox proportional hazard model was used to determine the eff ect of admission hyperglycemia on mortality corrected for covariates. Continuous blood glucose monitoring reduces the risk to ICU patients KP Mulavisala1, J Norrie2, B Crane3, N Barwell3 1CARE Hospitals, Hyderabad, India; 2SumStats Ltd, Edinburgh, UK; 3GlySure Ltd, Abingdon, UK g Critical Care 2015, 19(Suppl 1):P369 (doi: 10.1186/cc14449) Results In total, 4,516 ICU patients developed at least one episode of hypoglycemia. In three separate multivariate analyses for each of the three measures we adjusted for the respective confounders. The category 80 to 110  mg/dl of the ‘highest blood glucose level’ was associated with increased mortality compared with the reference category (odds ratio (OR) = 1.31, 95% confi dence interval (CI) = 1.06 to 1.61). The lowest quartile of the ‘delta glucose’ (OR = 1.32, 95% CI = 1.03 to 1.69) and the lowest quartile of the ‘standard deviation’ (OR = 1.55, 95% CI = 1.23 to 1.96) were associated with higher ICU mortality than their reference categories. Introduction GlySure Limited (Abingdon, UK) has developed a continuous intravascular glucose monitoring system (CIGMS) to simplify the application of hospital protocols for optimal glucose control at the point of care. We have previously reported on the early results achieved in cardiac patients [1] and MICU patients [2]. This initial success has been sustained and demonstrated in further patient groups. We have now reached the point where we can conjecture upon the regular application of the GlySure CIGMS in day-to-day ICU practice. This in turn prompts the question, ‘How eff ective will continuous blood S130 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P371f Eff ect of admission hyperglycemia in sepsis patients with or without a history of diabetes LA Van Vught1, MA Wiewel1, PM Klein Klouwenberg2, AJ Hoogendijk1, DS Ong2, OL Cremer2, MJ Bonten2, MJ Schultz1, T Van der Poll1 1Academic Medical Center, Amsterdam, the Netherlands; 2University Medical Center Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P371 (doi: 10.1186/cc14451) 1. Mulavisala KP, Gopal PB, Crane B. ISICEM 2014. p. 442. http://ccforum.com/ content/18/S1/P442. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf Admission types were medical (62%), elective surgery (22%) and emergency surgery (16%), and 22% had diabetes. For the accuracy analyses we had 929 comparative samples from 100 sensors in 45 patients (11 (7 to 28) samples per patient) during 4,639 hours (46 (27 to 134) hours per patient and 46 (21 to 69) hours per sensor). The Bland–Altman plot showed a bias of –0.6 mg/dl with limit of agreement between –57.2 and 56 mg/dl. Glucose prediction error analysis showed 60% of the glucose values <75  mg/dl within ±15 mg/dl and 75.8% of the glucose values ≥75 mg/dl within 20% of the comparative RapidLab results. Clarke error grid analysis showed 75.3% in zones A and 23.5% of the paired measurements in zones B, 0.3% of the paired measurements in zones C and 0.9% of the paired measurements in zones D. Point accuracy did not meet the ISO14971 standard for dosing accuracy, but improved with increasing numbers of calibrations, and was better in patients who did not have diabetes mellitus. Sixty out of 105 sensors were removed prematurely for a variety of reasons. The device start-up time was 49 (43 to 58) minutes. Acknowledgement This research was performed within the framework the Center for Translational Molecular Medicine (http://www.ctmm.nl), project MARS (grant 04I-201). Point and trend accuracy of continuous glucose monitoring using intravenous microdialysis in critically ill patients JH Leopold, RT Van Hooijdonk, M Boshuizen, T Winters, LD Bos, A Abu-Hanna, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P372 (doi: 10.1186/cc14452) Introduction Insulin infusion in critically ill patients mandates frequent measurements of the blood glucose level [1]. Microdialysis is a well- established technology that off ers the opportunity to sample blood analytes with high accuracy, without the need for drawing blood samples [2,3]. We aimed to determine point and trend accuracy of S131 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 values and arterial glycemia correlated well with 98.6% of data falling in regions A and B of error grid analysis. microdialysis-based continuous glucose monitoring (CGM) (EIRUS®; Maquet Critical Care AB, Solna, Sweden). g g y Conclusion In our study, the use of RT-CGM neither improved glucose control and variability, nor did it reduce hypoglycemic events. y g References 1. Schultz MJ, et al. Crit Care. 2010;14:223. 2. Schierenbeck F, et al. Diabetes Technol Ther. 2015;15:26-31. y y p y Results Ninety-nine FP patients were admitted between April 2008 and January 2014. Median age was 73 (IQR 61 to 79), with a female preponderance (53.5%). The median ICU length of stay (LOS) was 5 days (IQR 2 to 16). On admission to critical care, clinical data included (all medians): temperature 36.6°C (IQR 36 to 37.2), systolic blood pressure (BP) 113 mmHg (IQR 104 to 136), diastolic BP 56 mmHg (IQR 49 to 67), lactate 2.3 mmol/l (IQR 1.5 to 3.7), bilirubin 12 μmol/l (IQR 9 to 20), haemoglobin 104 g/l (IQR 93 to 116), haematocrit 31 (IQR 28 to 36), creatinine 88 μmol/l (IQR 66 to 152), prothrombin time 13.1 seconds (IQR 11.9 to 14.4). In 86 patients the initial operation was an emergency laparotomy, with primary perforation in 53 cases. Subsequent anastomotic dehiscence and need for relaparotomy happened in 24 and 33 cases respectively. Forty per cent of patients underwent more than one surgical abdominal intervention. The most common antibiotic used was tazobactam and fl uconazole was the commonest antifungal. The percentages of patients receiving mechanical ventilation, renal replacement therapy and inotropic/vasopressor support during ICU stay were 72.7%, 25.3% and 84.8% respectively. The ICU and hospital mortality rates were 23.5% and 26.1%, respectively, increasing to 26.7% at 28 days, 28.4% at 90 days and 32.2% at 1 year. None of the surgical factors or diabetes infl uenced survival. The strongest independent risk factors associated for ICU mortality were systolic BP on ICU admission (OR = 1.05, 95% CI = 1.01 to 1.09, P = 0.015), acute kidney injury (AKI) within the fi rst 24 hours of ICU admission (OR = 0.15, 95% CI = 0.03 to 0.9, P = 0.026) and lactate on ICU admission (OR = 0.62, 95% CI = 0.39 to 1, P = 0.05). 3. Blixt C, et al. Crit Care. 2013;17:R87. 4. Krouwer JS, et al. J Diabetes Sci Technol. 2010;4:75-83. 5. Clarke WL, et al. Diabetes Care. 1987;10:622-8. 6. Kovatchev, et al. Diabetes Care. 2004;27:1922-8. P373 P373 Real-time continuous glucose monitoring in the ICU J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) P373 Real-time continuous glucose monitoring in the ICU J Gios1, B Manuel-y-Keenoy2, P Rogiers3 J Gios1, B Manuel-y-Keenoy2, P Rogiers3 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) J Gios , B Manuel y Keenoy , P Rogiers 1University Hospital Antwerp, Edegem, Belgium; 2University of Antwerp, Wilrijk, Belgium; 3Ziekenhuisnetwerk Antwerpen Middelheim General Hospital, Antwerp, Belgium Critical Care 2015, 19(Suppl 1):P373 (doi: 10.1186/cc14453) Introduction Hyperglycemia occurs in 50 to 85% of patients admitted to a medical ICU (MICU) and has been associated with poor prognosis [1,2]. Whether applying intensive insulin therapy to achieve tight glycemic control in critically ill patients is benefi cial remains controversial [2]. Another important observation is a link between glycemic variability and mortality [3]. We performed a pilot study hypothesizing that when implementing intensive insulin therapy, real-time continuous glucose monitoring (RT-CGM) may help to safely achieve tight glucose control, while avoiding hypoglycemia and reducing glycemic variability in MICU patients. Methods This two-center randomized controlled pilot study was performed during a 3-year period. To be included, patients had to be severely ill (APACHE II score ≥20) and CGM monitoring had to be started within 48 hours after admission in the MICU. Thirty-fi ve patients (age 66 ± 10 years; nondiabetic/diabetic patients 27/8; APACHE II score 28 ± 6) were randomly assigned to RT-CGM (n = 16) or to blinded CGM. In both groups a microdialysis-based glucose sensor (GlucoDay®S) was used during a 96-hour period of glucose monitoring. Insulin infusion was performed using a modifi ed Yale protocol. Outcome measures were percentage of time in normoglycemia and in hypoglycemia, glycemic variability, and CGM accuracy. Conclusion In this cohort of critically ill FP patients the ICU and 12-month mortality rates were 23.5% and 32.2%, respectively. The most consistent predictors of mortality across all time points were AKI within 24 hours of ICU admission and admission lactate.i 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. 1. Tridente A, et al. Intensive Care Med. 2014;40:202-10. References 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Kavanagh BP, et al. N Engl J Med. 2010;363:2540-6. 3. Hermanides J, et al. Crit Care Med. 2010;38:838-42. 1. De Block C, et al. Curr Diabetes Rev. 2008;4:234-44. 2. Kavanagh BP, et al. N Engl J Med. 2010;363:2540-6. 3. Hermanides J, et al. Crit Care Med. 2010;38:838-42. Results Three-hundred and fi fty-four paired samples were obtained from seven patients (66 (59 to 79) years old, APACHE II score 23 (20 to 28), 51 (19 to 77) samples per patient). Point accuracy: 91% of paired values were in zone A, with the remaining 9% of the values in zone B in the Clarke error grid. In the Bland–Altman, bias was 5.4 mg/dl with an upper limit of agreement of 32.5 mg/dl and a lower level of agreement of –21.8 mg/dl. Glucose prediction error analysis showed that 91% of the values ≥75 mg/dl within 20% of the values measured by the blood gas analyzer were within range. Trend accuracy: in the rate error grid of the continuous glucose error grid analysis, 96% of the paired values were in zone A, 3.7% were in zone B and 0.3% were in zone C. Point accuracy and reliability of an interstitial continuous glucose monitoring device in critically ill patientsf On the other hand we can state that our insulin infusion protocol already led to overall tight glucose control without a signifi cant hypoglycemia risk, leaving little space for improvement. R f Methods Patients with an expected stay in the ICU of >48  hours needing an arterial catheter and a central venous catheter (CVC) were eligible. For a maximum of 3 days, during 8 hours per day, 125 μl blood was drawn from the arterial line every 15 minutes. Point accuracy was expressed using Clarke error grids, Bland–Altman plots and glucose prediction error analysis [4,5]. Trend accuracy was expressed using continuous glucose error grid analysis [6].i Critically ill patients with faecal peritonitis: a 5-year review in a tertiary centre Critically ill patients with faecal peritonitis: a 5-year review in a tertiary centre V Paul1, A Tridente2, P Kaur1, M Mahmood1, R Mellors1, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454) V Paul1, A Tridente2, P Kaur1, M Mahmood1, R Mellors1, AH Raithatha1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Whiston Hospital, St Helens & Knowsley, UK Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454) y Critical Care 2015, 19(Suppl 1):P374 (doi: 10.1186/cc14454 Introduction Faecal peritonitis (FP) is a common cause of sepsis and admission to the ICU [1]. We report a review of all patients admitted to our ICU over 5 years with FP. The aim was to defi ne the clinical characteristics, outcomes and risk factors for mortality in ICU patients with FP. Introduction Faecal peritonitis (FP) is a common cause of sepsis and admission to the ICU [1]. We report a review of all patients admitted to our ICU over 5 years with FP. The aim was to defi ne the clinical characteristics, outcomes and risk factors for mortality in ICU patients with FP. Conclusion Point and trend accuracy of the tested microdialysis-based CGM are good in critically ill patients. Acknowledgement Maquet Critical Care AB provided two CGM systems and disposables for the duration of the study, but had no infl uence on study design or study reporting. Acknowledgement Maquet Critical Care AB provided two CGM systems and disposables for the duration of the study, but had no infl uence on study design or study reporting. y Methods Data were extracted retrospectively from electronic case fi les. The primary outcome was ICU mortality. Secondary outcomes were hospital, 28-day, 90-day and 1-year mortality. Logistic regression analysis was used to identify independent risk factors for mortality. P377 Results We included 349 patients: age 67.5 ± 10.8 years, M/F sex ratio 252/97, preoperative left ventricle ejection fraction 58.8  ± 10.6%, bypass/valve ratio 234/154, number of grafts 2.7 ± 0.9, mammal arteries 1.8 ± 0.5. In univariate analyses, bypasses received more anaesthetic drugs (P <0.01), had shorter extracorporeal circulation duration, 67 ± 27 versus 75 ± 24 minutes (P <0.01), and received less blood products (P <0.0001). Bypasses had lower postoperative levels of troponin (3.9 ± 7.6 vs. 8.1 ± 21 pg/ml, P <0.01) and LDH (330 ± 162 vs. 420 ± 175 pg/ ml). In contrast, the intra-abdominal pressure (IAP) was higher and related to the number of grafts at day 0 (Figure 1) and day 1 (P = 0.01 and 0.02 respectively), and to the number of mammal grafts at day 0 and day 1 (P = 0.01 and 0.04 respectively). The TTFE was longer but did not reach signifi cance (P = 0.13) as well as the occurrence of abdominal ischaemia (P = 0.22). The occurrence of pneumonia was higher (P = 0.01). In multivariate analysis, the IAP at day 0 and day 1 was related to propofol quantities only. The predictors of pneumonia were: duration of mechanical ventilation, peak lactate in the postoperative 24 hours, and coronary bypass: OR = 163, 2.6, and 4.2 respectively. P377 Disseminated intravascular coagulation score predicts mortality in critically ill patients with liver cirrhosis A Drolz, T Horvatits, K Rutter, K Roedl, S Kluge, V Fuhrmann University Medical Center Hamburg-Eppendorf, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P377 (doi: 10.1186/cc14457) Introduction The disseminated intravascular coagulation (DIC) score is a predictor of outcome in critically ill patients [1,2]. Yet disturbances of coagulation and hemostasis, as refl ected by the DIC score, are a common fi nding in patients with liver cirrhosis. Thus, it is unclear whether the DIC score has prognostic value in critically ill patients with liver cirrhosis. The aim of this study was to assess the applicability and prognostic impact of the DIC score in critically ill patients with liver cirrhosis. Methods Patients with liver cirrhosis admitted to the medical ICU were analyzed for this study. Detailed laboratory analyses including platelet count, D-dimer, fi brinogen and prothrombin index were performed on admission and the DIC score was calculated. Survival was assessed on site or by contacting the patients or the attending physician. Bowel and related complications after cardiac surgery Results In the RT-CGM group the percentage of time at the target glycemia (80 to 110 mg/dl) was 37 ± 12% versus 34 ± 10% in the control group (NS) and glycemia averaged 119 ± 17 mg/dl versus 122 ± 11 mg/ dl respectively (NS). Time spent in hypoglycemia (<60 mg/dl) was not statistically diff erent between the group assigned to RT-CGM (0.6  ± 1.6% of the time) versus those with blinded CGM registration (2.4 ± 4.3% of the time). Parameters of glucose variability (standard deviation of mean glucose value, coeffi cient of variation, mean amplitude of glucose excursions) did not diff er between the groups. The GlucoDay®S Bowel and related complications after cardiac surgery CK Kerneis, AL Lafarge, LL Larnier, F Scalbert, AB Brusset, PE Estagnasie, PS Squara Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P375 (doi: 10.1186/cc14455) CK Kerneis, AL Lafarge, LL Larnier, F Scalbert, AB Brusset, PE Estagnasie, PS Squara q Clinique Ambroise Paré, Neuilly-sur-Seine, France q Clinique Ambroise Paré, Neuilly-sur-Seine, France Clinique Ambroise Paré, Neuilly-sur-Seine, France Critical Care 2015, 19(Suppl 1):P375 (doi: 10.1186/cc14455) Introduction Postoperative ileus appears to be underestimated after cardiac surgery. We conducted this study to analyse the incidence, risk factors and outcomes of postoperative ileus. Introduction Postoperative ileus appears to be underestimated after cardiac surgery. We conducted this study to analyse the incidence, risk factors and outcomes of postoperative ileus. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S132 Figure 1 (abstract P375). Figure 1 (abstract P375). Results Eighty-seven patients (69.9%) were females. Mean age was 48.9  years. Primary cancer was colorectal in 42 patients (32.5%), ovarian in 39 (30%), appendiceal in 29 (22%), others in 15.5%. Average operative time was 11 ± 2.1 hours. Average intraoperative crystalloids given were 12,217 ± 4,359 ml, packed RBCs were 2 ± 2.3 units, colloids 1,083 ± 898 ml, average blood loss was 1,108 ± 785 ml. All patients were admitted to the ICU post procedure. The average fl uid balance during the OR was 9,481 ± 4,694 ml. Patients stayed in the ICU for an average of 6 ± 5.3 days. All patients survived the ICU stay. The duration of mechanical ventilation was 57 ± 83 hours, total fl uid balance while in the ICU was 1,467  ± 3,399  ml. Hypomagnesemia was the most frequent electrolyte abnormalities in 79 (61%). 1. Taylor FB, Toh CH, Hoots WK, Wada H, Levi M, Scientifi c Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Towards defi nition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost. 2001;86:1327-30. 2. Angstwurm MWA, Dempfl e C-E, Spannagl M. New disseminated intravascular coagulation score: a useful tool to predict mortality in comparison with Acute Physiology and Chronic Health Evaluation II and Logistic Organ Dysfunction scores. Crit Care Me. 2006;34:314-20; quiz 328. ICU outcome of patients undergoing cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy: a single-center study A Nadeem, A Al-Tarifi King Faisal Specialist Hospital, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P376 (doi: 10.1186/cc14456) g y A Nadeem, A Al-Tarifi King Faisal Specialist Hospital, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P376 (doi: 10.1186/cc14456) overt DIC was 70% compared with 40% in those with a DIC score <5. Conclusion Disturbances in coagulation and hemostasis are found in the majority of cirrhotic patients admitted to the ICU. The DIC score is a suitable predictor of 28-day mortality in critically ill patients with liver cirrhosis. Introduction Peritoneal carcinomatosis (PC) is associated with poor prognosis. The advent of complete cytoreductive surgery (CRRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has shown promise in improved survival for locally advanced intra- abdominal carcinomatosis. Such patients are routinely admitted to the ICU postoperatively. Little is known about the natural course of such patients while in the ICU. P377 Conclusion The number of coronary grafts and of mammal artery used in cardiac surgery is associated with higher IAP and higher risk of pneumonia. However, whether this is due to direct bowel ischaemia or longer anaesthesia remains to be studied in larger trials. Results In total, 150 admissions to the ICU with liver cirrhosis were analyzed. Thirty-nine percent were female. Median age was 56 (IQR 49 to 63) years. The median SOFA score on admission was 9 (6 to 13), median MELD score 26 (IQR 18 to 36). Twenty-eight-day mortality was 59%. Median DIC score on admission was 5 (IQR 4 to 6). Overt DIC (DIC score ≥5) was found in 65%. DIC score was signifi cantly higher in nonsurvivors compared with survivors (5 (IQR 4 to 7) vs. 4 (IQR 3 to 6); P <0.01). AUROC for the DIC score in prediction of 28-day mortality was 0.68 (95% CI = 0.59 to 0.77). Overt DIC on admission was signifi cantly associated with 28-day mortality (OR  = 3.4 (95% CI  = 1.69 to 6.84), P <0.01). The 28-day mortality rate in admissions with cirrhosis and overt DIC was 70% compared with 40% in those with a DIC score <5. Bowel and related complications after cardiac surgery Pleural eff usions in 48 (37%), of which three patients only required drainage, Seven patients (5.6%) developed pneumonia, no patient required renal replacement therapy. Average hospital LOS was 33.7 ± 29 days. Only two patients died in the hospital. When the fi rst 65 patients were compared with the last 64 patients, the duration of MV, ICU LOS and hospital LOS were all signifi cantly shorter in the latter group (72 vs. 43 hours, 6.8 vs. 5.0 and 40 vs. 27 days respectively; P <0.01 for all). Figure 1 (abstract P375). Methods In this single-centre observational study we prospectively enrolled all patients undergoing elective cardiac surgery. The primary output was the time to faeces (TTFE) as representing the postoperative ileus. Secondary outputs were the occurrence of ischaemic colitis and pneumonia. Quantitative variables were compared by ANOVA or Wilcoxon tests, qualitative variables by chi-square tests. Multivariate analyses were performed by logistic regression, P  <0.1 for inputs P <0.05 for outputs. Methods In this single-centre observational study we prospectively enrolled all patients undergoing elective cardiac surgery. The primary output was the time to faeces (TTFE) as representing the postoperative ileus. Secondary outputs were the occurrence of ischaemic colitis and pneumonia. Quantitative variables were compared by ANOVA or Wilcoxon tests, qualitative variables by chi-square tests. Multivariate analyses were performed by logistic regression, P  <0.1 for inputs P <0.05 for outputs. Conclusion With proper selection of patients, CRS with HIPEC can be done safely with no major complications. There is a signifi cant reduction in ICU utilization and shorter hospital LOS with more experience in such procedure, suggesting a learning curve as well as better utilization of resources by referring such patients to a high-volume center. P377 References 1. Taylor FB, Toh CH, Hoots WK, Wada H, Levi M, Scientifi c Subcommittee on Disseminated Intravascular Coagulation (DIC) of the International Society on Thrombosis and Haemostasis (ISTH). Towards defi nition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation. Thromb Haemost. 2001;86:1327-30. Methods The procedure was introduced in our hospital in 2008 as the fi rst regional center performing such therapy. A retrospective chart review of 129 cases of CRS-HIPEC admitted to a 22-bed surgical ICU in a tertiary care academic center between November 2008 and March 2014. Primary outcomes were ICU length of stay (LOS) and duration of mechanical ventilation (MV). Secondary outcomes were hospital LOS and hospital mortality. 2. Angstwurm MWA, Dempfl e C-E, Spannagl M. New disseminated intravascular coagulation score: a useful tool to predict mortality in comparison with Acute Physiology and Chronic Health Evaluation II and Logistic Organ Dysfunction scores. Crit Care Me. 2006;34:314-20; quiz 328. S133 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P378 Conclusion In the present study, it was observed that the sero- prevalences of HBsAg, anti-HCV and anti-HIV were not higher than in our city population. However, taking the safety precautions of the healthcare workers during surgical or invasive procedures such as catheterization, intubation or tracheostomy without any information about the serological test results of the patients will reduce the contamination of these agents. P378 Warm ischemia time, postreperfusion syndrome and initial poor function after liver transplantation: are they connected? E Scarlatescu, G Manga, G Droc, D Tomescu Fundeni Clinical Institute, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P378 (doi: 10.1186/cc14458) Introduction Factors associated with initial poor graft function (IPGF) after liver transplantation are still under debate. Although the initial insult to the graft begins during the cold ischemia time (CIT), recent studies showed that most injuries occur during rewarming. Ischemic– reperfusion (I/R) injuries are present in all grafts and may be responsible for postoperative graft dysfunction. Along with other factors, I/R injury may also play a role in the development of postreperfusion syndrome (PRS) after revascularization of the liver graft. The aim of this study was to assess whether longer warm ischemia time (WIT) is associated with PRS or with IPGF after liver transplant. Outcomes of decompensated chronic liver disease in a UK district general hospital critical care setting E Ahmadnia1, F Manneh2, K Raveendran2 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) , , 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) , , 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) p p Methods This retrospective observational study included 60 liver transplant patients. We excluded from the study group patients with retransplant procedures, and the recipients of divided grafts and of grafts from extended criteria donors. We recorded: demographic data, intraoperative PRS, CIT, WIT, ALT, AST levels and standard coagulation tests on postoperative days (POD) 1 to 5. Statistical analysis was performed using SPSS Statistics v.19.1 with signifi cant P value under 0.05. Results We used the criteria of Nanashima and colleagues for the diagnosis of IPGF (ALT and/or AST level above 1,500 IU/l within 72 hours after OLT). The study group included 33 men (55%) and 27 women. Mean (±SD) age was 50.56 (±13.26) years. WIT longer than 60 minutes correlated signifi cantly with ALT and AST levels in POD 1 to 3 (P <0.0001 for ALT in POD 1 to 3, P = 0.001 for AST in POD 1, P = 0.007 and 0.013 for AST in POD 2 and 3) and with prothrombin time (P = 0.008 in POD 1, P = 0.03 in POD 2 and P = 0.015 in POD 3). We could not fi nd a correlation between PRS and WIT (P = 0.566), CIT (P = 0.439) or transaminase levels on POD 1 to 3. The correlation between WIT >60 minutes and IPGF was confi rmed using the Pearson chi-square test (P <0.0001). The same test was used to correlate IPGF with PRS with nonsignifi cant results (P = 0.876). Introduction Patients with decompensated cirrhosis admitted to the ICU have historically had a very high mortality rate [1]. It has been suggested that improving patient selection can improve ICU outcomes in patients with cirrhosis [2]. The aim of this study was to determine the mortality and evaluate the risk factors that may infl uence the outcome of this group of patients in a large UK district general hospital with a view to introducing selection criteria for future ICU admission of patients with decompensated liver disease. p p Methods A retrospective analysis was performed of all adult patients with decompensated chronic liver disease admitted to a general (nontransplant) critical care unit between January 2012 and December 2013. References The secondary aim was to investigate whether patients with intraoperative PRS have a higher risk for postoperative IPGF. P379 Seroprevalence of hepatitis B, hepatitis C and HIV in ICU patients H Bayir, I Yildiz, E Kocoglu, A Kurt, H Kocoglu Abant Izzet Baysal University, Medical School, Bolu, Turkey Critical Care 2015, 19(Suppl 1):P379 (doi: 10.1186/cc14459) Conclusion Those with decompensated chronic liver disease admitted to the ICU have a signifi cant ICU/hospital mortality, which is increased in alcoholic liver disease. Sepsis and AKI were the most common acute diagnoses in this cohort. Alcoholic liver disease patients requiring organ support have a very high mortality, and the outlook for multiorgan failure requiring RRT in this group is dismal. Introduction Healthcare workers are at risk for infections caused by hepatitis B (HBV), hepatitis C (HCV) and human immunodefi ciency (HIV) viruses that transmit via blood and body fl uids. In the present study, it was aimed to investigate the seroprevalences of HBsAg, anti- HCV and anti-HIV in patients admitted to the ICU. Outcomes of decompensated chronic liver disease in a UK district general hospital critical care setting E Ahmadnia1, F Manneh2, K Raveendran2 1Homerton University Hospital, London, UK; 2Queen’s Hospital, London, UK Critical Care 2015, 19(Suppl 1):P380 (doi: 10.1186/cc14460) Data were collected regarding demographics, ICU mortality, hospital mortality, aetiology of chronic liver disease, severity scores, acute diagnoses, and organ support requirements.i Results Thirty-seven patients were identifi ed, with a median age of 57  years, predominantly male (62%). Seventy-six per cent had alcohol-related cirrhosis. Overall ICU mortality was 29.7% and hospital mortality was 48.6% – these values were higher in the alcoholic group (39.3% and 57.1% respectively). All ICU deaths were in those with alcoholic liver disease. Median scores were: APACHE III 93, SOFA (day 1) 9, Child–Pugh 11, MELD 21. Seventy per cent were treated for sepsis, 22% had a GI bleed, 57% had encephalopathy, 24% had suspected/ confi rmed spontaneous bacterial peritonitis, and 70% had an acute kidney injury. Organ support requirements were: 35% respiratory (non-invasive or invasive ventilation), 38% vasoactive agent support, 24% renal replacement therapy (RRT). Alcoholic liver disease patients requiring respiratory or cardiovascular support had an ICU mortality of 64%, and those requiring RRT had a mortality of 75%. Alcoholic liver disease patients requiring combined respiratory, cardiovascular, and RRT support had 100% mortality. Conclusion Our study showed that PRS is not a risk factor for IPGF after liver transplantation. WIT over 60  minutes does not infl uence the development of PRS, but is associated with IPGF after liver transplantation. Close monitoring of liver tests in the early postoperative period is very important especially in recipients of grafts with WIT over 60 minutes. Further eff orts to decrease WIT may prove useful for the reduction of IPGF in liver transplant patients. References References 1. Olson JC, et al. Hepatology. 2011;54:1864-72. 2. Sauneuf B, et al. Crit Care. 2013;17:R78. Methods HBsAg, anti-HCV and anti-HIV test results and demographical data of the patients admitted to the Reanimation ICU between January 2012 and December 2014 were evaluated retrospectively. HBsAg, anti-HCV and anti-HIV tests were assayed with a macro-ELISA method (Axsym-Abbott, Architect i2000; Abbott, USA). Statistical analysis was performed with the chi-square test. References 1. Erdem K, Tas T, Tekelioglu UY, Bugra O, Akkaya A, Demirhan A, et al. The hepatitis B, hepatitis C and human immunodefi ciency virus seroprevalence of cardiac surgery patients. SDÜ Tip Fak Derg. 2013;20:14-17 1. Erdem K, Tas T, Tekelioglu UY, Bugra O, Akkaya A, Demirhan A, et al. The hepatitis B, hepatitis C and human immunodefi ciency virus seroprevalence of cardiac surgery patients. SDÜ Tip Fak Derg. 2013;20:14-17 y 2. Tekin A, Deveci O. Seroprevalences of HBV, HCV and HIV among healthcare workers in a state hospital. J Clin Exp Invest. 2010;1:99-103. 2. Tekin A, Deveci O. Seroprevalences of HBV, HCV and HIV among healthcare workers in a state hospital. J Clin Exp Invest. 2010;1:99-103. First clinical experience with a new type of albumin dialysis: the HepaWash® system First clinical experience with a new type of albumin dialysis: the HepaWash® system First clinical experience with a new type of albumin dialysis the HepaWash® system B Henschel, R Schmid, W Huber TU-München Klinikum rechts der Isar, Munich, Germany Critical Care 2015, 19(Suppl 1):P383 (doi: 10.1186/cc14463) B Henschel, R Schmid, W Huber TU-München Klinikum rechts der Isar, Munich, Germany Critical Care 2015, 19(Suppl 1):P383 (doi: 10.1186/cc14463) Introduction Liver failure (LF) is associated with prolonged hospital stay, increased cost and substantial mortality. With regard to a limited number of donor organs, extracorporeal liver support is an appealing concept to bridge to transplant or to avoid transplant in case of recovery. A new type of albumin dialysis, the HepaWash® system, was recently introduced. The HepaWash® system provides rapid re- generation of toxin-binding albumin by secondary circuits altering binding capacities of albumin by biochemical (changing pH) and physical (changing temperature) modulation of the dialysate.i Results Eight children (fi ve male/three female), 8.6  ± 5.9  years old (range 2 to 15.6 years), BW 32 ± 21 kg, GFR 71 ± 20 ml/minute/1.73 m2, with an uncuff ed double lumen dialysis catheter (8 to 14 Fr Femoralis (n = 6) and 9 Fr Jugularis (n = 2)) were treated according to this protocol. In total, 19 sessions were executed using FX40 (n = 13), FX50 (n = 3), and FX60 (n = 3) dialysers during 6.5 ± 0.9 hours. Blood fl ow was 149 ± 45  ml/minute, albumin fl ow 226  ± 49  ml/minute, and ultrafi ltration fl ow 432 ± 517 ml. RRs were 70 ± 15% (urea), 34 ± 14% (Crea), 44 ± 16% (bili), and 36 ± 10% (NH3). Primary survival rate was 100%. Four patients were transplanted (bridge to transplant) of which, however, one died within 30 days after discharge from the ICU. The fi fth patient died due to primary disease 9 months after treatment, and the remaining three patients fully recovered (bridge to recovery). y y Methods We evaluated the fi rst 14 patients treated with the HepaWash® system with regard to safety and effi cacy. Seven patients were treated in the context of the run-in phase of the studies (HEPATICUS 1 and HEPATICUS 2) and seven patients were treated since the HepaWash® system received the CE certifi cate in July 2013. P381 Prometheus® liver therapy in children with acute liver failure J Vande Walle, S Claus, E Snauwaert, J De Rudder, A Raes, M Dick, A Prytula, W Van Biesen, S Eloot Ghent University Hospital, Gent, Belgium Critical Care 2015, 19(Suppl 1):P381 (doi: 10.1186/cc14461) Results The records of 462 patients admitted to our ICU were reviewed. The results of 36 patients could not be reached, so 426 patients were evaluated in the study. Among 426 patients, 169 (39.7%) were female and 257 (60.3%) were male. The mean age was 63.7 ± 18.7. HBsAg was positive in nine (2.1%) patients; all of these nine were male. Anti-HCV was positive in four (0.9%) patients; among these, three were male and one was female. Only one patient was positive for anti-HIV. Introduction The Fractionated Plasma Separation and Adsorption System Prometheus® (Fresenius Medical Care, Germany) aims at being S134 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Close observation and treatment of coagulopathy and electrolyte disturbances is essential when treating patients with MARS. MARS can reduce and stabilize ammonium and other biomedical markers in patients listed for urgent liver transplantation with high MELD score and liver encephalopathy. It seems that, in some cases and with our settings, the detoxifi cation properties of MARS may be insuffi cient. a supportive therapy as a bridge to transplantation or recovery in adults with liver failure. The system off ers specifi c challenges when applied in children due to the large extracorporeal volume (700 to 750 ml). We therefore developed an adapted protocol for the application in children. Methods Priming of the blood circuit is performed using 2 l isotonic saline, whereas the plasma circuit, containing both adsorption devices, is fi lled with 2 U fresh frozen plasma or 400 ml stabilized solution of human plasma proteins. Next, for children with body weight (BW) <25 kg, a solution of 60 to 65% packed cells (PC) is infused in the inlet blood line at 40  ml/minute. The volume of PC needed is calculated based on the circuit priming volume and the maximum allowed extracorporeal blood volume of the child (= 8 ml/kg × BW). After the priming phase, blood and plasma fl ow are increased to at least 100 ml/ minute and 200 ml/minute, respectively, and dialysate fl ow is set at 300 ml/minute. P381 Regional citrate anticoagulation is done with a calcium- free dialysate, while, eventually, heparin is added to the priming solution. Post treatment, the circuit volume is either not reinfused (BW <25 kg) or reinfused using isotonic saline (BW >25 kg), with a volume depending on the hydration status and the originally infused volume of PC. Reduction ratios (RRs, %) of urea, creatinine (Crea), bilirubin (bili), and ammonia (NH3) were calculated from pretreatment and posttreatment serum concentrations. Primary and secondary patient outcome was evaluated.i P383 Molecular adsorbent recirculating system treatment in 69 patients listed for liver transplantation P Oli H H Molecular adsorbent recirculating system treatment in 69 patients listed for liver transplantation P Olin, H Haugaa Oslo University Hospital, Oslo, Norway Critical Care 2015, 19(Suppl 1):P382 (doi: 10.1186/cc14462) Introduction The molecular adsorbent recirculating system (MARS) is used to remove circulating albumin-bound toxins in patients with liver failure. However, the application of MARS has not demonstrated improved survival in randomized clinical trials and the clinical utility has not been fi nally established. In our department, the use of MARS is now restricted to the most critically ill patients with acute or acute on chronic liver failure. We aimed to explore MARS effi cacy in removing toxicity parameters and the safety of the system. Conclusion So far the HepaWash® system has proven to be a safe and feasible procedure to eff ectively eliminate water and protein-bound toxins in humans with LF. First clinical experience with a new type of albumin dialysis: the HepaWash® system Patients treated suff ered under acute on chronic LF (n  = 9) or secondary LF which resulted from nonhepatic diseases such as sepsis (n  = 5). Primary endpoint: comparison of serum bilirubin, creatinine and serum BUN before and after the fi rst treatment with the HepaWash® system. Statistics: IBM SPSS Statistics version 22. The Wilcoxon test for paired samples was used to detect signifi cant treatment eff ects. gif Results A total of 254 treatments (1 to 101 per patient) were performed in 14 patients (six female, eight male). Mean age 54 ± 13. MELD score 33.7 ± 7.0, CLIF-SOFA 14.6 ± 2.7. Main underlying disease: nine acute- on-chronic LF; fi ve secondary LF. While bilirubin did not change signifi cantly on the day before HepaWash® treatment (26.2 ± 15.4 vs. 26.0 ± 15.4 mg/dl; P = 0.116), serum bilirubin levels were signifi cantly decreased by the HepaWash® procedure (26.0 ± 15.4 vs. 17.7 ± 10.5 mg/ dl; P = 0.001). Similarly, serum creatinine (2.2 ± 0.8 vs. 1.6 ± 0.7 mg/dl; P = 0.005) and serum BUN (49.4 ± 23.3 vs. 31.1 ± 19.7 mg/dl; P = 0.003) were signifi cantly lowered by the HepaWash® procedure. There were no serious adverse events observed in conjunction with the HepaWash® treatment. Conclusion This adapted Prometheus® protocol is promising for the treatment of children with liver failure. Intensive care referral and admission: do the criteria for liver disease match? J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P384 (doi: 10.1186/cc14464) Intensive care referral and admission: do the criteria for liver disease match? J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P384 (doi: 10.1186/cc14464) toxicity parameters and the safety of the system. Methods Since 2005, we have treated 69 patients (30 males/39 females with median age of 39 years ranging from 1 month to 70 years) listed for liver transplantation with MARS. The median Model of End Stage Liver Disease (MELD) score in patients older than 12 years of age (n = 56) was 33 (interquartile range 26 to 39). The fl ow rate was 35 to 40 ml/ kg/hour and treatment kits were changed every 8 to 12  hours. The patients were treated for a median of 31 hours (range 1 to 240 hours). Results Fifty-fi ve patients (79%) were successfully bridged to transplantation. Nine died before they could be transplanted, and fi ve patients recovered without liver transplantation. Forty-four (81%) of the transplanted patients were alive 30 days after transplantation. Ammonium decreased modestly from a median of 148 to 124 μM (P  = 0.03) during MARS treatment. We detected worsening of coagulopathy with signifi cant decreases in platelet count and fi brinogen concentrations, and increase in International Normalized Ratio. Phosphate and magnesium decreased signifi cantly during MARS treatment. J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow UK J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Introduction Hospital admission and mortality rates for patients with cirrhosis in the UK are rising [1]. Cirrhotic patients are physiologically challenged and at increased risk of sepsis and death [2]. Mortality rates for cirrhosis in nontransplant ICUs are up to 37% [3]. Increased availability of medical therapies and public expectation places pressure on limited intensive care resources. There is a lack of research into factors used to decide which patients to admit or refer to the ICU Introduction Hospital admission and mortality rates for patients with cirrhosis in the UK are rising [1]. Cirrhotic patients are physiologically challenged and at increased risk of sepsis and death [2]. Mortality rates for cirrhosis in nontransplant ICUs are up to 37% [3]. Increased availability of medical therapies and public expectation places pressure on limited intensive care resources. There is a lack of research into factors used to decide which patients to admit or refer to the ICU. Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? g A Vaz, M Eusebio, A Antunes, A Sousa, P Perez, R Ornelas, C Granja, H Guerreiro Centro Hospitalar do Algarve, Faro, Portugal Critical Care 2015, 19(Suppl 1):P385 (doi: 10.1186/cc14465) Methods A prospective study of 132 consecutive patients admitted to the Critical Burn Unit between October 2008 and October 2011. In all of them resuscitation was performed by objectives: blood pressure (>65  mmHg), hourly diuresis (0.5 to 1  cm3/kg), lactic acid clearance and thermodilution transpulmonary parameters (CI >2.5 l/minute/m2, ITBI: 600 ml/m2). We performed measurements of IAP with a bladder catheter every 8 hours in the fi rst 72 hours. Introduction Cirrhotic patients admitted to the ICU are usually regarded as having a particularly poor prognosis when compared with other groups of critically ill patients. The aim of our study was to evaluate the prevalence, case mix and outcomes of patients with cirrhosis admitted to the general ICU of a nontransplant center. yi Results Ninety-eight men and 34 women were studied. Mean age 48 ± 18 years and a TBSA of 35 ± 22%. The fl uid provided by %TBSA in the fi rst 8 hours was less than predicted by Parkland (4.05 ml/kg), although the total contribution in the fi rst 24 hours was similar. The evolution of the intra-abdominal pressure was: admission 9.7 mmHg, 8 hours 11, 16 hours 10.5, 24 hours 12.1, 32 hours 12.0, 40 hours 12.0, 48 hours 11.1, 56 hours 10.3, 64 hours 10.0 and 72 hours 10.0. A total of 44 patients (33.3%) had a determination higher than 12  mmHg, distributed: 15 patients between 12 and 15 mmHg (IAHT I grade), 14 between 16 and 20 mmHg (II), nine between 21 and 25 mmHg (III) and six >25 mmHg (IV). See Figures 1 and 2.l g p Methods Data were collected from a running ICU database. We studied cirrhotic patients admitted to the ICU between January 2013 and November 2014. Results A total of 30 patients with cirrhosis were admitted, accounting for 3% of total ICU admissions. Mean age was 54.5 years, with a male preponderance (76.7%). The main cause for cirrhosis was alcohol (53.3%), followed by alcohol plus chronic hepatitis C virus (HCV) infection (20%) and HCV virus infection alone (13.3%). The most common causes for admission were sepsis/septic shock (26.7%), surgical (23.4%), gastrointestinal bleeding and hepatic encephalopathy (16.7% each). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 decision. Recipients listed additional criteria used in their own practice and asked whether they would admit or refer individual grades of Child–Pugh cirrhosis with either a gastrointestinal bleed or sepsis.i decision. Recipients listed additional criteria used in their own practice and asked whether they would admit or refer individual grades of Child–Pugh cirrhosis with either a gastrointestinal bleed or sepsis.i C in the Child–Pugh score; mean Acute Physiology and Chronic Health Evaluation (APACHE II) 29.2 ± 8.7 and new Simplifi ed Acute Physiology Score (SAPS II) 62.7  ± 29. Regarding organ failure at admission, the mean Sequential Organ Failure Assessment score was 12.8 ± 4.5. The ICU mortality of these patients was 43.3% and hospital mortality was 53.3%. The variables at admission that related signifi cantly with ICU mortality were: all scores described except for Child–Pugh score, bilirubin, the International Normalized Ratio, creatinine, bicarbonate, lactate, pH and the use of renal replacement therapy during the ICU stay (P  <0.05). The mortality rate of cirrhotic patients was superior to the general ICU mortality (43% vs. 26%). However, patients with cirrhosis presented signifi cantly higher severity scoring systems (APACHE II; SAPS II) at admission compared with noncirrhotics, with high prevalence of organ dysfunction as assessed by SOFA score. Results Thirty-fi ve consultant gastroenterologists and 65 intensive care consultants responded, representing a response rate of 34% and 45% respectively. The only criterion given an average rating of 5 by both gastroenterologists and intensivists was Child–Pugh score when stable. Presence on the transplant list, referral secondary to bleeding varices, recent discharge from the ICU, abstinence from alcohol, nutritional status, age under 30 and more than one additional organ failure all scored 4 or 5 from both groups. Sex, employment, smoking or drug use, deprivation and positive virology status did not infl uence the decision to refer or admit patients. Clinicians reported compliance with medication and outpatient appointments plus an obvious precipitant factor as important features in their decision. The majority of respondents would refer or admit all grades of Child–Pugh cirrhosis with gastrointestinal bleeding. Most would refer or admit Child–Pugh A or B with sepsis. A total 76.5% of gastroenterologists would refer Child–Pugh C cirrhosis with sepsis but only 33.3% of intensivists would accept. P385 Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? A Vaz, M Eusebio, A Antunes, A Sousa, P Perez, R Ornelas, C Granja, H Guerreiro Centro Hospitalar do Algarve, Faro, Portugal Critical Care 2015, 19(Suppl 1):P385 (doi: 10.1186/cc14465) Introduction The aim was to study the evolution and incidence of intraabdominal hypertension in critical burn patients using a slightly restrictive fl uid therapy protocol based on monitoring transpulmonary thermodilution and lactic acid. Intraabdominal pressure in critical burn patients PM Millan 2. Foreman MG, et al. Chest. 2003;124:1016-20. 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. Hospital Universitario La Paz, Madrid, Spain Hospital Universitario La Paz, Madrid, Spain Critical Care 2015, 19(Suppl 1):P386 (doi: 10.1186/cc14466) p , , p Critical Care 2015, 19(Suppl 1):P386 (doi: 10.1186/cc14466) Intensive care referral and admission: do the criteria for liver disease match? J McPeake, CR Soulsby, T Quasim, J Kinsella University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P384 (doi: 10.1186/cc14464) Methods A prospective survey was sent to all consultant gastroenterologists and consultant intensivists in Scotland. Each recipient rated the signifi cance of 18 physiological and social criteria on their decision to refer or admit a patient to intensive care from 1 to 5, with 1 being no infl uence and 5 denoting signifi cant impact on the factors used to decide which patients to admit or refer to the ICU. Methods A prospective survey was sent to all consultant gastroenterologists and consultant intensivists in Scotland. Each recipient rated the signifi cance of 18 physiological and social criteria on their decision to refer or admit a patient to intensive care from 1 to 5, with 1 being no infl uence and 5 denoting signifi cant impact on the S135 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The high severity of disease in conjunction with the high mortality rate observed in this group of patients should make us consider the possible benefi ts of earlier referring/admission to the ICU, ideally before multiorgan failure arises. On the other hand, in nontransplant centers where cirrhotic patients constitute a small percentage of total ICU admissions, the complexity and peculiarities of the management of these patients should prompt their early transfer to a specialized center. Conclusion Referral and admission decisions for patients with cirrhosis are multifactorial. Child–Pugh status when stable appears to be of greatest signifi cance. The diff erence in opinion of admission of patients with Child–Pugh C with sepsis requires further evaluation. References 1. Thomson SJ, et al. Alcohol Alcohol. 2008;43:416-22. 2. Foreman MG, et al. Chest. 2003;124:1016-20. 3. Filloux B, et al. Eur J Gastroenterol Hepatol. 2010;22:1474-80. 1. Thomson SJ, et al. Alcohol Alcohol. 2008;43:416-22. P387 Intraabdominal hypertension in burn patients Results are depicted as mean values ± SD in Tables 1 and 2. y y Results Twenty patients were enrolled in the study. The mean age was 36 ± 13 years. There were 14 males and six females. The average TBSA was 44 ± 17%. Screening and monitoring of IAP were applied by: oliguria (42%), abdominal distension (31.5%) and gastrointestinal trouble (21%). IAH occurred between day 2 and day 3 after early burn resuscitation, respectively in 52% and 63%. IAH was observed in 69% of cases in patients admitted to the ICU with a delay of 1.6 days post burn injury. IAH was noted in 13 patients; of these, fi ve patients developed an abdominal compartment syndrome. The mean IAP was 16 ± 7 mmHg. Patients were assigned into two groups: G1 (IAH+; n = Conclusion Both sepsis and IAH have negative eff ects on respiratory mechanics. However, their combination has even more detrimental eff ects, which do not ameliorate after deinsuffl ation. Table 1 (abstract P388). P387 Intraabdominal hypertension in burn patients Methods Sixteen pigs were divided into two groups of eight (G-A/ G-B). All animals received general anesthesia and were mechanically ventilated. Parameters recorded included respiratory system, chest wall and lung compliance (CRS, CCW, CL) and respiratory system and chest wall inspiratory and expiratory resistances (RRSisp, RRSexp, RCWisp, RCWexp). After baseline measurements (0  minutes), intra- abdominal pressure IAP was raised by helium insuffl ation to 25 mmHg in both groups and remained at that level for the whole study. In G-B, sepsis was induced 60 minutes after IAP increase, by i.v. administration of Escherichia coli endotoxin. Parameters were recorded every 20 minutes. The last measurement was made at 180 minutes, right after deinsuffl ation, and IAP return to baseline levels.i Intraabdominal hypertension in burn patients A Mokline, I Rahmani, L Gharsallah, A Hachani, S Tlaili, R Hammouda, B Gasri, A Ksontini, AA Mesadi Trauma and Burn Centre of Tunis, Tunisia Critical Care 2015, 19(Suppl 1):P387 (doi: 10.1186/cc14467) Introduction Intra-abdominal hypertension (IAH) is frequent in the ICU and has been associated with adverse outcomes and worse prognosis. The purpose of our study was to assess risk factors for IAH and prognosis of major injured patients during burn resuscitation. Introduction Intra-abdominal hypertension (IAH) is frequent in the ICU and has been associated with adverse outcomes and worse prognosis. The purpose of our study was to assess risk factors for IAH and prognosis of major injured patients during burn resuscitation. j j p g Methods Adult burned patients with a burn injury exceeding 20% of total body surface area, from 1 April to 30 November 2013, were included. IAP was measured when IAH was suspected, according to the Kron method via the Foley catheter. Monitoring of IAP was performed every 6 hours during 5 days until normalization. fl Results CRS decreased statistically signifi cantly in both groups after IAP increase and increased after deinsuffl ation only in G-A. Similarly, CCW decreased in both groups but returned to baseline values in both groups after deinsuffl ation. CL decreased more signifi cantly in G-B and returned to baseline values only in G-A. RRSisp increased only in G-B and did not decrease after deinsuffl ation, whereas RRSexp increased in both groups, in a more signifi cant manner in G-B, and decreased only in G-A after deinsuffl ation. RCWisp and RCSesp did not show any alterations during the study period. P388 Eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension Figure 2 (abstract P386). IAH classifi cation. Introduction The aim of our study was to investigate the eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension (IAH). aSepsis induction. Comparison with baseline: *P <0.05, ** P<0.01. Mortality in patients with cirrhosis admitted to the ICU: time to rethink strategies? At admission, these patients presented an average Model for End- Stage Liver Disease score of 23.5 ± 10.4 with 70% classifi ed as grade Conclusion IAH incidence when a slightly restrictive fl uid protocol used is less than expected. Figure 1 (abstract P386). Intraabdominal pressure. S136 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 2 (abstract P386). IAH classifi cation. 13) and G2 (IAH–; n = 7). Comparative study of the two groups shows that HIA increases signifi cantly body weight gain within the fi rst 5 days after injury: 8 kg for G1 versus 2 kg for G2 (P = 0.04), occurrence of ARDS (70% for G1 vs. 16.7% for G2, P = 0.02), respiratory failure (77% for G1 vs. 28.5% for G2, P = 0.06), shock (70% for G1 vs. 16.7% for G2, P 0.02) and mortality (61.5% vs. 50%). Figure 2 (abstract P386). IAH classifi cation. 13) and G2 (IAH–; n = 7). Comparative study of the two groups shows that HIA increases signifi cantly body weight gain within the fi rst 5 days after injury: 8 kg for G1 versus 2 kg for G2 (P = 0.04), occurrence of ARDS (70% for G1 vs. 16.7% for G2, P = 0.02), respiratory failure (77% for G1 vs. 28.5% for G2, P = 0.06), shock (70% for G1 vs. 16.7% for G2, P 0.02) and mortality (61.5% vs. 50%). Conclusion IAH was frequent in early burn resuscitation of major injured patients. It seems to be associated with fl uid overload in burns and contributes to organ damage. P388 P388 Eff ects of sepsis on respiratory mechanics in a porcine model of intra-abdominal hypertension B Fyntanidou, K Kotzampasi, M Kyparissa, G Stavrou, E Oloktsidou, X Mpesi, K Papapostolou, V Grosomanidis Aristotle Medical School, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P388 (doi: 10.1186/cc14468) aSepsis induction. Comparison with baseline: *P <0.05, ** P<0.01. Severity markers in acute pancreatitis Introduction CD73/ecto-5’-nucleotidase is an enzyme that generates adenosine, which dampens infl ammation and improves vascular barrier function in several disease models. CD73 also circulates in a soluble form in the blood [1]. We studied whether levels of soluble form of CD73 and cytokines/chemokines predict the development of organ failure in acute pancreatitis [2,3]. Introduction Heterogeneous published results led us to conduct a clinical trial to assess the effi cacy of a new formulation of four probiotics (P) as prophylaxis for complications after colorectal surgery. p Methods Altogether, 161 patients with acute pancreatitis (107 were subclassifi ed according to the revised Atlanta criteria into mild, 29 into moderately severe and 25 into severe forms) were studied. Serum and blood cell samples were collected at admission. Protein levels of soluble form of CD73 in serum were determined using a novel enzyme- linked immunosorbent assay, activity of soluble form of CD73 using radioactive enzyme assays, and CD73 messenger RNA levels from leukocytes using quantitative PCR. Serum levels of 48 cytokines and growth factors were determined using Bio-Plex Pro Human Cytokine Assay 21-plex and 27-plex magnetic bead suspension panels. y g y Methods A double-blind, placebo-controlled randomized study was conducted enrolling patients undergoing colorectal cancer surgery. Placebo or a formulation of L. acidophilus, L. plantarum, B. lactis and S. boulardii was administered starting 1  day before operation and continuing for 15  days post operation. Patients were followed-up for 30 days with the development of postoperative complications as the primary outcome. PAXGene tubes and serum were collected on postoperative day 4 for measurement of gene expression and serum cytokines (ClinicalTrials.gov NCT02313519). y g Results Administration of P signifi cantly decreased the rate of all postoperative major complications (28.6% vs. 48.8% of placebo, P = 0.010, odds ratio: 0.42). Major benefi t was found in the reduction of the postoperative pneumonia rate (2.4% vs. 11.3%, P = 0.029), of wound infections (7.1% vs. 20.0%, P = 0.020), of anastomotic leakage (1.2% vs. 8.8%, P = 0.031) and of the need for mechanical ventilation (20.2% vs. 35.0%, P = 0.037). The time until hospital discharge was shortened as Results Activity and protein concentration of soluble form of CD73 and messenger RNA level of CD73 all decreased along with the disease severity (P ≤0.01 for all). The activity of soluble form of CD73 at admission predicted the development of severe pancreatitis in diff erent groups of the patients. P387 Intraabdominal hypertension in burn patients Compliance alterations during the study CRS (ml/cmH2O) CCW (ml/cmH2O) CL (ml/cmH2O) Minutes A B P value A B P value A B P value 0 35 ± 8 31 ± 4 NS 58 ± 20 46.1 ± 11 NS 99 ± 22 98 ± 14 NS 20 14 ± 4** 12 ± 3** NS 20 ± 6** 17.7 ± 3** NS 52 ± 21** 35 ± 11** NS 40 13 ± 4** 12 ± 1** NS 19 ± 6** 16.8 ± 3** NS 47 ± 23** 36 ± 13** NS 60a 12 ± 3** 11 ± 2** NS 20 ± 5** 18.4 ± 4** NS 41 ± 20 28 ± 7** NS 80 13 ± 3** 10 ± 1** NS 20 ± 6 18.2 ± 44 NS 41 ± 21** 24 ± 6** <0.05 100 12 ± 3** 10 ± 2** NS 21 ± 8** 19.9 ± 5** NS 37 ± 20** 19 ± 4** <0.05 120 12 ± 3** 10 ± 1** NS 20 ± 6** 21.9 ± 7** NS 37 ± 20** 17 ± 3** <0.05 140 12 ± 3** 9 ± 1** NS 21 ± 7** 21.8 ± 6** NS 37 ± 20** 17 ± 5** <0.05 160 12 ± 3** 9 ± 1** NS 21 ± 7** 22.3 ± 7** NS 37 ± 19** 16 ± 4** <0.05 180 33 ± 7** 16 ± 5** <0.001 50 ± 18 77.0 ± 18** <0.05 81 ± 20* 21 ± 7** <0.001 aSepsis induction. Comparison with baseline: *P <0.05, ** P<0.01. Table 1 (abstract P388). Compliance alterations during the study S137 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 2 (abstract P388). 1. Salmi M, Jalkanen S. Semin Immunopathol. 2014;36:163-76. 2. Maksimow M, et al. Crit Care Med. 2014; 42:2556 P387 Intraabdominal hypertension in burn patients Respiratory system resistance alterations during the study RRSisp (cmH2O/l/minute) RRSexp (cmH2O/l/minute) Minutes A B P value A B P value 0 8.1 ± 0.8 8.3 ± 0.7 NS 13.6 ± 4.1 15.5 ± 3.7 NS 20 7.8 ± 0.6 8.1 ± 0.7 NS 17.1 ± 5.2** 19.3 ± 2.2** NS 40 7.8 ± 0.8 7.6 ± 0.9 NS 18.1 ± 5.4** 20.6 ± 3.1** NS 60a 7.6 ± 0.9 7.5 ± 1.1 NS 18.9 ± 4.4** 19.9 ± 4.7** NS 80 7.8 ± 1.2 8.2 ± 1.2 NS 18.6 ± 4.2** 21.1 ± 4.12** NS 100 7.8 ± 0.9 7.9 ± 0.9 NS 18.2 ± 4.5** 22.7 ± 4.5** NS 120 8 ± 0.6 9.1 ± 1.1 <0.05 18.6 ± 4.3** 20.9 ± 2.2** NS 140 7.7 ± 0.6 9.5 ± 1.2 <0.01 18.8 ± 3.5** 22.9 ± 3.2** <0.05 160 7.3 ± 0.7 9.6 ± 1.5* <0.01 17.7 ± 3.5** 22.2 ± 2.3** <0.01 180 8.1 ± 0.8 9.7 ± 1.2* <0.01 14.9 ± 3.3 18.9 ± 2.3* <0.05 aSepsis induction. Comparison with baseline: *P <0.05, **P <0.01. Table 2 (abstract P388). Respiratory system resistance alterations during the study P389 Severity markers in acute pancreatitis S Jalkanen University of Turku, Finland Critical Care 2015, 19(Suppl 1):P389 (doi: 10.1186/cc14469) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G T i1 EJ Gi ll B b li 2 P389 P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G Tsaousi1, EJ Giamarellos-Bourboulis2 1Aristotle University of Thessaloniki, Greece; 2 University of Athens Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P390 (doi: 10.1186/cc14470) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery K Kotzampassi1, G Stavrou1, G Damoraki2, M Georgitsi2, G Basdanis1, G Tsaousi1, EJ Giamarellos-Bourboulis2 1Aristotle University of Thessaloniki, Greece; 2 University of Athens Medical School, Athens, Greece Critical Care 2015, 19(Suppl 1):P390 (doi: 10.1186/cc14470) P390 Randomized, double-blind, placebo-controlled study of the effi cacy of four probiotics to modify the risk for postoperative complications in colorectal surgery Severity markers in acute pancreatitis Especially, activity of soluble form of CD73 was better than C-reactive protein or creatinine in predicting the severity of pancreatitis in the group of patients without any signs of organ failure at admission. In subgroup analyses of patients with severe pancreatitis and without organ dysfunction upon admission, IL- 8, hepatocyte growth factor and granulocyte colony-stimulating factor (G-CSF) levels predicted the development of severe pancreatitis, with G-CSF being the most accurate cytokine. Figure 1 (abstract P390). Figure 1 (abstract P390). Conclusion Activity of soluble form of CD73 and levels of certain cytokines at admission to the hospital have prognostic value in predicting the development of the severe form of acute pancreatitis. The possibility that combining them with other prognostic markers might improve prognostic accuracy requires further studies. References 2. Maksimow M, et al. Crit Care Med. 2014; 42:2556-64. 3. Nieminen A, et al. Crit Care. 2014 [Epub ahead of print]. Figure 1 (abstract P390). S138 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 well. Gene expression of SOCS3 was positively related with circulating IL-6 in the P group but not in the placebo group (Figure 1). Conclusion The studied P formulation signifi cantly decreased the risk of postoperative complications, namely mechanical ventilation, infections and anastomotic leakage. Modulation of the gene expression of SOCS3 is one suggested mechanism. thiamine 200  mg or placebo i.v. twice/day for 7  days. The primary outcome was lactate levels at 24 hours. Secondary outcomes included the SOFA score at 24 hours and mortality. Lactate levels at 24 hours were compared between groups using the Wilcoxon rank-sum test and categorical variables were compared using the Fisher’s exact test. Lactate values at 24 hours, for those who died before 24 hours, were imputed according to a predefi ned plan. We performed a preplanned analysis in those with baseline thiamine defi ciency (≤7 nmol/l). well. Gene expression of SOCS3 was positively related with circulating IL-6 in the P group but not in the placebo group (Figure 1).i thiamine 200  mg or placebo i.v. twice/day for 7  days. The primary outcome was lactate levels at 24 hours. Secondary outcomes included the SOFA score at 24 hours and mortality. Lactate levels at 24 hours were compared between groups using the Wilcoxon rank-sum test and categorical variables were compared using the Fisher’s exact test. Role of ultrasonography in detection of the localization of the nasoenteric tube R Dagli1, H Bayir2, Y Dadali1, T Tokmak1, Z Erbesler1 1Ahi Evran University Education and Research Hospital, Kirsehir, Turkey; 2Abant Izzet Baysal University, Medical School, Bolu, Turkey Critical Care 2015, 19(Suppl 1):P391 (doi: 10.1186/cc14471) Introduction In this study, we aimed to determine the success rate of nasoenteric tube (NET) insertion into the postpyloric area by ultrasonography (USG) and compare it with the commonly used method, direct abdominal radiography. g p y Methods Patients admitted to an adult ICU between April and July 2014 with an indication for NET insertion for enteral feeding were included in the study after informed consent was given from patients’ relatives. Nasoenteric feeding tubes were placed using the blind bedside method by a single anesthesiologist. Any motility stimulant agent was not used. The outside of the polyurethrane 8 F with unweighted NET (Bexen, Spain) and its guiding wire were lubricated with gel. The NET was inserted into the nostril after determination of the mouth– posterior ear–xiphoid distance and pushed on at least such a distance. Followed by auscultation of the gastric area and air infusion of 30 to 50 ml into the tube, the patient was positioned on their right side and the tube was advanced 20 to 30 cm more. Then the guiding wire inside the NET was removed. The patient was then brought to the supine position and NET was visualized by two radiologists simultaneously by M5 portable USG (Mindray, PRC), with a 3.5 MHz convex probe whether it passes through the postpyloric area or not. Localization of the tube was confi rmed with abdominal radiography in all patients. During the fi rst insertion of the NET, the ratios for inaccurate localization and correct placements through the postpyloric area were recorded and results were compared with abdominal radiography. Conclusion Thiamine defi ciency is prevalent in septic shock. Thiamine did not decrease overall median lactate levels at 24  hours. In the patients with thiamine defi ciency, there were statistically signifi cant lower lactate levels at 24  hours in the thiamine group and a large, although nonsignifi cant, diff erence in mortality. Unraveling the link between malnutrition and adverse clinical outcomes: association of acute and chronic malnutrition measures with blood biomarkers from diff erent pathophysiological systems S Felder, N Braun, A Kutz, M Batschwaroff , P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2015, 19(Suppl 1):P393 (doi: 10.1186/cc14473) Introduction Malnutrition is common in hospitalized medical patients and is associated with poor clinical outcomes. Whether malnutrition has a direct link to adverse outcomes or is rather a mirror of the severe patient condition remains debated. Our aim was to study the association of acute and chronic malnutrition status with blood biomarkers from diff erent pathophysiological concepts to better understand the underlying mechanisms of malnutrition. Results In this study, the bedside blind method was used for NET insertion into 34 patients. Eleven of the tubes were detected passing through the postpyloric area by USG. In one case the NET could not be seen in the postpyloric area by USG, but it was detected in the postpyloric area by control abdominal radiography. In 22 patients, NETs were detected in the stomach with control abdominal radiography. Success for NET placement with the bedside blind method and USG imaging was 35% versus 91.6%, respectively. Methods We prospectively followed consecutive adult medical inpatients hospitalized between February 2013 and October 2013 in a tertiary care Swiss hospital. Nutritional risk was assessed using the Nutritional Risk Screening (NRS 2002) score, which incorporates acute and chronic measures of malnutrition. Multiadjusted regression models were used to investigate the associations between acute and chronic nutritional risk and biomarkers mirroring infl ammation (CRP, PCT, proADM, leucocytes), stress (copeptin), renal dysfunction (creatinine, urea), nutritional status (vitamin D25, albumin, calcium, glucose), and hematological function (platelets, INR, Hb, RDW). Biomarker levels were transformed into deciles due to skewed distributions. Conclusion The success rate of the bedside blind method in the NET placement was low. It is clear that if any other placement techniques with high success rate will be applied, USG will be useful in a higher number of patients reducing the need for abdominal radiography. P392 Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial M Donnino1, LW Andersen1, M Chase1, KM Berg1, TA Giberson1, H Smithline2, M Tidswell2, R Wolfe1, M Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Critical Care 2015, 19(Suppl 1):P392 (doi: 10.1186/cc14472) Severity markers in acute pancreatitis Lactate values at 24 hours, for those who died before 24 hours, were imputed according to a predefi ned plan. We performed a preplanned analysis in those with baseline thiamine defi ciency (≤7 nmol/l). Conclusion The studied P formulation signifi cantly decreased the risk of postoperative complications, namely mechanical ventilation, infections and anastomotic leakage. Modulation of the gene expression of SOCS3 is one suggested mechanism. yi y ( ) Results We enrolled 88 patients; 43 received thiamine and 45 placebo. Baseline characteristics were similar between groups. We found no overall statistical signifi cant diff erence in 24-hour lactate levels between thiamine and placebo groups (2.5 (IQR: 1.5 to 3.4) vs. 2.6 (IQR: 1.6 to 5.1), P = 0.40). Fewer patients in the thiamine group had lactate levels >4 mmol/l at 24 hours (21% vs. 38%, P = 0.10) and this was statistically signifi cant if only evaluating survivors at 24 hours (7% vs. 33%, P = 0.03), although our preplanned analysis was to impute data. We found no diff erence in 24-hour SOFA score or mortality. A total of 28 (35%) patients were thiamine defi cient. Of the defi cient patients, those receiving thiamine had statistically signifi cant lower lactate levels at 24 hours (2.1 (IQR: 1.4 to 2.5) vs. 3.1 (IQR: 1.9 to 8.3), P = 0.03) and more patients in the placebo group had a lactate >4 mmol/l (38% vs. 7%, P = 0.07). Mortality in the thiamine and placebo groups was 13% and 46%, respectively (P = 0.10).i Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial We considered target calories to be 25 kcal/kg/day and target proteins to be 1.5 g/kg/day. Introduction The provision of nutrition in the critical care unit (CCU) has shifted from nutrition support to nutrition therapy, and the potential benefi ts derived from this in the recovery of the critically ill is being explored [1]. We audited the management of nutrition in the CCU in a South African Hospital against the American Society of Parenteral and Enteral Nutrition Guidelines. Furthermore, we reviewed the knowledge and confi dence of healthcare providers in the management of nutrition in the CCU. g y g p g g y Results Patients received more calories (78.3% vs. 59.1%) and more proteins (70.2% vs. 54.6%) post implementation. The mean percentage of patients in the post group who achieved >70% of required calories was 80.1% versus 30.9% in the pre group. The mean percentage of patients who achieved >70% of required proteins was 58.3% versus 32.1% in the pre group. p g p Conclusion The multipronged approach of the quality improvement methodology helped to increase the provision of calories and proteins in our population of critically ill surgical patients. However, there is still room for improvement in terms of achieving optimal enteral nutrition targets early in our population. There is also a need to look into sustaining such results. Methods Retrospective data collection of patients admitted to a four- bed CCU over a 4-month period in 2013. A survey was distributed to diff erent disciplines involved in patient nutrition in the CCU. Results Seventy-two patients were admitted to the CCU during this time period, and notes were able for 44. Three paediatric patients were excluded. Twenty-nine patients stayed for 2 or more days (the audit population). The median age of the audit population was 38, 19 were female. Sixteen were postoperative admissions. The median APACHE II score of the patients with suffi cient available data (n = 16) was 14 (range 6 to 34). The audit found that 21 of the patients had nutrition started in the CCU, with 15 having nutrition started within 48 hours. Only eight patients had a nutritional assessment done. A total of 45 responded to the survey: eight anaesthetists, 25 from surgical disciplines, seven CCU nurses, and fi ve dieticians. All agreed that nutrition should be started in the fi rst 48 hours, except from the surgeons only 14 (56%) agreed. Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial The average self-rating of knowledge of nutrition management in the CCU (1 = lowest, 5 = highest) was 2.1 with the dieticians and CCU nurses showing the highest confi dence with 3.4 and 2.6, respectively. The anaesthetists rated their knowledge at 1.9 and the surgeons rated themselves at 1.8. P397 Introduction Optimizing enteral nutrition early has been shown to be benefi cial in critically ill patients. However, underfeeding is still a common problem. The critically ill surgical patient often presents with additional challenges to optimal enteral feeding. The objective of this study was to improve enteral feeding practices in a surgical ICU. Quality improvement project to optimize enteral nutrition in a tertiary hospital’s surgical ICU g p g Conclusion Perioperative optimal nutrition support for at least 7 days could modulate the infl ammatory status and clinical outcome of severe malnourished surgical neoplasic patients. Could preoperative and postoperative optimal nutrition support modulate the infl ammatory response and clinical outcome of severe malnourished surgical patients with gastrointestinal neoplasia? p L Mirea, D Pavelescu, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P396 (doi: 10.1186/cc14476) p L Mirea, D Pavelescu, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2015, 19(Suppl 1):P396 (doi: 10.1186/cc14476) p L Mirea, D Pavelescu, I Grintescu Introduction Our aim was to assess whether perioperative and postoperative optimal 7-day nutrition support could modulate the infl ammatory status and clinical outcome of severe malnourished patients with surgery for gastrointestinal neoplasia. Methods A prospective randomized study of 64 patients with gastrointestinal neoplasia, severe malnourished BMI <18.5, albumin level <3 g/dl, BW loss >10%, NRS >3, scheduled for surgery, allocated into two groups. Group A: 32 patients, minimal enteral nutrition in the postoperative period according to tolerance, medium 500  kcal/day. Group B: 32 patients received optimal parenteral nutrition support (25 kcal/kg/day) 3 days before surgery and continued for at least 4 days postoperatively. We measured CRP, fi brinogen, IL-6, TNF, albumin level preoperative and at 96 hours, the incidence of complications, and the length of ICU stay.i Conclusion We found that there is poor management of nutrition in the CCU. This is paired with limited knowledge and low confi dence in management amongst the attending staff . Evidence would suggest that the development and dissemination of clear hospital guidelines could improve rates of correct management [2]. However, the lack of uniform guidance based on strong evidence from the leading global authorities on nutrition suggests that, in order to improve implementation of adequate nutrition, more research is urgently required. References 1. McClave S, et al. JPEN J Parenter Enteral Nutr. 2009;33:277-316. 2. Martin CM, et al. CMAJ. 2004;170:197-204. Results There was a signifi cant decrease in the values of CRP, IL-6, TNF, albumin at 96  hours in group B. No diff erence in fi brinogen. A signifi cantly lower rate of complications and a shorter time of ICU stay were observed in group B. See Figures 1 and 2 (overleaf). Thiamine as a metabolic resuscitator in septic shock: a randomized, double-blind, placebo-controlled, pilot trial Results A total of 529 patients (mean age 72 years, 57.1% male) were included. Overall, there was a signifi cant association of NRS and most biomarkers of infl ammation, stress, renal function, nutrition and the hematological system (coeffi cient and 95% CI): CRP 0.021, P = 0.0021, PCT 0.28, P = 0.003, proADM 0.4, P <0.001, copeptin 0.44, P <0.001, urea 0.28, P = 0.002, vitamin D25 –0.23, P = 0.012, albumin –0.6, P <0.001, hemoglobin –0.5, P <0.001, RDW 0.46, P <0.001. These associations remained robust after adjustment for sociodemographics (model 1), comorbidities (model 2) and main medical diagnosis (model 3). Subgroup analysis suggested that mainly the acute part of malnutrition and not chronic malnutrition was associated with an increase in biomarker levels. g H Smithline2, M Tidswell2, R Wolfe1, M Cocchi1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Baystate Medical Center, Springfi eld, MA, USA Introduction The objective was to determine whether the administration of thiamine mitigates elevated lactate levels in patients with septic shock. Thiamine is essential for aerobic metabolism and we have found that thiamine levels are low and inversely correlated with lactate levels in patients with sepsis. Conclusion Acute malnutrition was associated with a pronounced infl ammatory response and an increase in biomarkers from diff erent pathophysiological systems which may partly explain the link between malnutrition and adverse medical outcomes. However, interventional trials are needed to prove causal relationships. Methods We performed a randomized, double-blind, placebo- controlled, two-center trial from January 2010 to October 2014. We enrolled patients with septic shock, elevated lactate (≥3 mmol/l) and no obvious competing cause of lactate elevation. Patients received S139 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P394 Evaluation of the provision of nutrition in a South African provincial hospital S Kudsk-Iversen, R Matos-Puig, R Naidoo, S Moorad Stanger Provincial Hospital, Stanger, South Africa Critical Care 2015, 19(Suppl 1):P394 (doi: 10.1186/cc14474) interventions were conducted with lectures to physicians and nurses. Visual aids in the form of screensavers at each bedside computer served as reminders to the team to optimize feeding. A subsequent audit was then conducted to determine the improvement in achieving the desired outcomes, namely the amount of calories and proteins received as well as the proportion of patients who achieved >70% of their target calories and proteins. 1. McClave S, et al. JPEN J Parenter Enteral Nutr. 2009;33:277-316. 2. Martin CM, et al. CMAJ. 2004;170:197-204. P395 P395 Quality improvement project to optimize enteral nutrition in a tertiary hospital’s surgical ICU J Li, LY Koh, JH Yang, C Khoo, T Ter, BH Tan National University Health System, Singapore Critical Care 2015, 19(Suppl 1):P395 (doi: 10.1186/cc14475) Does discontinuation of the use of hydroxyethyl starches in the critically ill cardiac surgery patient have an impact on caloric intake? E De Waele, K De Bondt, S Mattens, J Czapla, J Nijs, M La Meir, D Nguyen, PM Honoré, H Spapen Universitair Ziekenhuis Brussel, Brussels, Belgium Critical Care 2015, 19(Suppl 1):P397 (doi: 10.1186/cc14477) Methods The Clinical Practice Improvement Programme is a local quality improvement initiative involving a multidisciplinary team aimed at identifying and improving defi ciencies in the process of delivery of care. A team led by an intensivist, consisting of doctors, surgeons, nurses and a pharmacist, was formed to improve enteral feeding practices in a surgical ICU. The quality improvement methodology was employed. An audit was carried out to determine the problem of underfeeding in the unit. Root cause analyses were conducted and team members identifi ed key barriers to optimal feeding and areas for improvement. Protocols were developed to standardize and encourage early enteral feeding as well as to reduce the time feeds are interrupted for patients who were going for surgeries or for various other reasons. Educational Introduction After research revealed unwanted eff ects of the use of starches in critically ill patients, its use in the immediate postoperative period of cardiac surgery patients came to an abrupt ending. However, they constitute an important source of non-intended calories, providing 4 calories per gram. We investigated whether this phenomenon (involuntary) attributed to an increase in caloric debt for this critically ill patient population. Methods We retrospectively searched a database of 417 elective cardiac surgery patients, representing 5,004 observation-days. Caloric S140 Critical Care 2015, Volume 19 Suppl 1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P396). Results 1. Figure 2 (abstract P396). Results 2. Figure 1 (abstract P396). Results 1. Figure 1 (abstract P396) Results 1 Figure 1 (abstract P396). Results 1. Figure 2 (abstract P396). Results 2. Figure 2 (abstract P396). Results 2. by aggressive nutrition therapy, in the general population of an ICU. Cancer patients are more prone to be at nutritional risk due to the disease and treatment complications. Our aim was to characterize NUTRIC score behavior in the population of patients admitted to an oncologic ICU. intake was evaluated in the group of patients before and after the cessation of starch use. Results Patient characteristics and caloric needs were comparable: 2,054 ± 395 kcal/day and 2,056 ± 347 kcal/day. Does discontinuation of the use of hydroxyethyl starches in the critically ill cardiac surgery patient have an impact on caloric intake? The 140 patients who in the immediate postoperative period had volume resuscitation without the use of starches had a mean non-intended fl uid caloric intake of 69 (± 36.3) kcal/day. The group of 277 patients who received starches in the postoperative period had a mean non-intended fl uid caloric intake of 105 (± 100.2) kcal/day. g Methods Between January and June 2014 we applied the NUTRIC score to all patients, age >18 years, without cerebral death criteria and with a length of stay (LOS) >72  hours. Data were collected and analyzed using SPSS v20.0. To evaluate the impact on mortality we used logistic regression. y Conclusion Withdrawal of the use of starches resulted in a 34% decrease of non-intended caloric intake by fl uids, contributing to caloric debt. Whether outcome is infl uenced and/or whether these fi ndings are clinically relevant needs further research. g Results Sixty-nine patients were included, 23 women (33.3%) and 46 men (66.7%). Most patients were aged between 50 and 75 years (72.5%) and had normal range weight 58% (n = 40). The mean LOS was 11.56 (minimum: 3 to maximum: 69). The most common motive for admission was sepsis (7.7%, n = 26). APACHE II score was above 15 in 77% of the patients (n = 53) and SOFA score was superior to 6 in 56.5% (n = 30). The NUTRIC score was low risk in 42% (n = 29) of the patients and high in 58% (n = 40). Twenty-eight-day mortality was 26.1% (n = 18). A high NUTRIC score corresponded to a 22-fold increased odds of dying in the fi rst 28 days (P <0.001). Both APACHE II and SOFA were mortality predictors alone, with an increase of 1 point in APACHE score corresponding to an increase of 14% (P = 0.002) and an increase of Early calorie-dense immune nutrition in haemodynamically compromised cardiac patients f Early calorie-dense immune nutrition in haemodynamically compromised cardiac patients S Efremov, V Lomivorotov Research Institute of Circulation Pathology, Novosibirsk, Russia Critical Care 2015, 19(Suppl 1):P399 (doi: 10.1186/cc14479) Introduction The aims of present study were to test the hypothesis that early enteral nutrition (EN) with calorie-dense food supplemented with glutamine improves recovery of nutritional status in critically ill cardiac patients and to evaluate their resting energy expenditure (REE). Results Six out of nine patients had no glycogen present in the muscle (score = 0). The other three patients had glycogen scores between 5 and 15 which are well below scores of healthy individuals (reference 50 to 70). As a comparison we collected post-competition levels in competitive athletes, which decrease their glycogen stores (score 15 to 25) but are well above those of most critically ill patients we have studied. p g gy p Methods A prospective randomised study of 40 adult cardiac patients undergoing elective cardiopulmonary bypass surgery no more than 24  hours before eligibility assessment, complicated with acute heart failure syndrome. Patients were randomised to receive either standard isocaloric isonitrogenic early EN (standard group, n  = 20) or immunomodulating early EN (immune group, n  = 20). The daily energy target was set using REE measured by indirect calorimetry (CCM Express; Medgraphics, St. Paul, MN, USA). Serum prealbumin, transferrin, C-reactive protein, blood lactate and clinical characteristics were analysed. Conclusion This is the fi rst time that muscle glycogen stores have been evaluated in critical illness. Our data show severe glycogen depletion in ICU patients which probably leads to muscle catabolism necessary for gluconeogenesis, eventually resulting in cachexia. This fi nding poses severe metabolic challenges for ICU patients in which interfering with recovery can contribute to poor survival. In light of our fi ndings, re-evaluation of nutritional protocols and potential anabolic/anti- catabolic therapy to decrease muscle catabolism may improve survival. Diff erent therapeutics that may prevent hypermetabolism (such as beta-blockers) should be re-evaluated along with anabolic agents (that is, oxandrolone) which could counteract the severe catabolic response in critical illness. Monitoring of muscle glycogen repletion could signal the transition from the catabolic to anabolic phase. y Results The actual REE was an average of 6.8 and 7.5  kcal/kg/day higher than the REE calculated using the Harris–Benedict equation and empiric approach (25 kcal/kg/day), respectively (Figure 1). Early calorie-dense immune nutrition in haemodynamically compromised cardiac patients f Early EN with immune formula was associated with higher levels of prealbumin concentration on the 14th day (0.13 ± 0.01 g/l and 0.21 ± 0.1 g/l; P = 0.04) and transferrin on the 3rd, 5th, 7th, and 14th day (P <0.05) after surgery. Conclusion Haemodynamically compromised cardiac patients have increased REE, which in the absence of indirect calorimetry should be set at 30 kcal/kg/day. Early EN using a calorie-dense immune formula leads to better recovery of nutritional status as assessed by serum protein levels. Plasma glutamine after acute or elective admission on the ICU H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) NUTRIC score in oncologic patients NUTRIC score in oncologic patients A Patrão, L Bei, F Coelho A Patrão, L Bei, F Coelho Instituto Português de Oncologia – Porto, Portugal g g , g Critical Care 2015, 19(Suppl 1):P398 (doi: 10.1186/cc14478) Introduction The NUTRIC score is a tool designed to quantify the risk of critically ill patients developing adverse events that may be modifi ed S141 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 1 point in SOFA corresponding to an increase in the odds of being dead at 28 days (P = 0.002). Body mass index, age, number of comorbidities, and days in the ICU did not correlate with mortality. Conclusion The NUTRIC score is a good tool in cancer patients to predict 28-day mortality. Nevertheless, the only compounds of the NUTRIC score that correlated independently with mortality were APACHE II and SOFA scores. Further investigation towards the inclusion of other categories such as tumor staging and the type of tumor could be useful to develop a specifi c prognostic tool for this population. 1 point in SOFA corresponding to an increase in the odds of being dead at 28 days (P = 0.002). Body mass index, age, number of comorbidities, and days in the ICU did not correlate with mortality. Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) Measurement of skeletal muscle glycogen status in critically ill patients: a new approach in critical care monitoring I San Millan, J Hill, P Wischmeyer University of Colorado, Aurora, CO, USA Critical Care 2015, 19(Suppl 1):P400 (doi: 10.1186/cc14480) Conclusion The NUTRIC score is a good tool in cancer patients to predict 28-day mortality. Nevertheless, the only compounds of the NUTRIC score that correlated independently with mortality were APACHE II and SOFA scores. Further investigation towards the inclusion of other categories such as tumor staging and the type of tumor could be useful to develop a specifi c prognostic tool for this population. Introduction Critically ill patients experience hypermetabolism in- creas ing substrate utilization, especially glucose oxidation. Glycogen is the main source of glucose in the body, being 85% and 15% stored in skeletal muscle and liver respectively. Since glycogen stores are limited we evaluated the hypothesis that critical illness could be associated with glycogen depletion leading to skeletal muscle catabolism for gluconeogenesis and eventually resulting in cachexia, an important determinant of future ICU survival and ICU-acquired weakness. Methods Nine critically ill patients (58.75  ± 25 to 75  years old) with an ICU stay from 1  day to 5  weeks were evaluated for skeletal muscle glycogen content using a rapid, non-invasive high-frequency ultrasound methodology (MuscleSound®, Denver, CO, USA). Scans were obtained from the rectus femoris and vastus lateralis muscles. Glycogen content was measured with a score from 0 to 100 according to the MuscleSound® scale. Patients had a variety of primary diagnoses including septic shock (n  = 3), hemorrhagic shock/abdominal hypertension (n = 1), hypovolemic shock/post major oncologic surgery (n = 1), trauma (n = 3), and burn injury (n = 1). Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis Methods Sepsis was induced by E. coli LPS. GLN was infused: i.v. through the femoral vein (0.5 g/kg); enterally (E) through jejunostomy (0.5 g/kg); and i.v. + E. Blood was drawn continuously from the femoral artery and the portal vein for GLN plasma levels in systemic-S and portal-P circulation. P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) p Results In healthy swine, GLN levels remained stable, both in S and P; i.v. infusion, and even more i.v. + E signifi cantly increased GLN in the S circulation (P <0.001), whereas E infusion failed to do so (P = 0.4). On the contrary, GLN P levels were signifi cantly increased after i.v. + E infusion, as well as after E infusion (P <0.001) and to a lesser extent, after i.v. (P <0.001). In sepsis, both S and P GLN levels decreased signifi cantly. Introduction Intravenous fi sh oil (FO) lipid emulsions (LEs) are rich in ω-3 polyunsaturated fatty acids, which exhibit anti-infl ammatory and immunomodulatory eff ects. We previously demonstrated that FO- containing emulsions may be able to decrease mortality and ventilation days in the critically ill. Over the last year, several additional randomized controlled trials (RCTs) of FO-based LEs have been published. Therefore, the purpose of this meta-analysis was to update our systematic review aimed to elucidate the effi cacy of FO-based LEs on clinical outcomes in the critically ill. Figure 1 (abstract P404). Figure 1 (abstract P404). y Methods We searched computerized databases from 1980 to 2014. Overall mortality was the primary outcome and secondary outcomes were infections, ICU and hospital length of stay (LOS), and mechanical ventilation (MV) days. We included RCTs conducted in critically ill adult patients that evaluated FO-based LEs in parenteral nutrition (PN) or enterally fed patients. We analyzed data using RevMan 5.1 (Cochrane IMS, Oxford, UK) with a random eff ects model. f Results A total of 10 RCTs (n  = 733), including four trials published over the last year, met inclusion criteria. Plasma glutamine after acute or elective admission on the IC H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) Figure 1 (abstract P399). Indirect calorimetry measured signifi cantly higher resting energy expenditures. Introduction Low plasma glutamine concentrations are associated with unfavourable outcome at acute ICU admission. We questioned whether there is a diff erence in plasma glutamine level after acute or elective ICU admission. Methods We performed a single-centre prospective observational study in a 22-bed mixed ICU. Exclusion criteria were age <18 years and total parental nutrition at admission. Patients were divided into two groups: elective surgery and acute admissions. Blood samples were taken at ICU admission and daily at 6.00 a.m. Glutamine levels were measured using the Bioprofi le 100 plus analyser (Nova Biomedical UK, Cheshire, UK). A Mann–Whitney U test was used to detect diff erences between groups and a Bonferroni method to correct for multiple comparisons. Results We included 88 patients after elective surgery (76 cardiac and 12 general surgery) and 90 patients after acute admission (27 sepsis, 17 acute surgery, two trauma and 44 medical). Baseline characteristics are presented in Table 1. Plasma glutamine levels at admission were signifi cantly lower in acute patients compared with elective surgery, 0.25  mmol/l (IQR 0.09 to 0.37) versus 0.43 mol/l (IQR 0.33 to 0.55) Figure 1 (abstract P399). Indirect calorimetry measured signifi cantly higher resting energy expenditures. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S142 Table 1 (abstract P402) Elective Acute Age (years) 68 ± 10 59 ± 17* APACHE IV 46 ± 14 67 ± 29* LOS ICU 2 (2 to 2) 4 (2 to 7)* Survival (n) 88 85* Mean ± SD or median (IQR). *P <0.01. 0.17, heterogeneity I2 = 0%), and hospital LOS (WMD –4.06; 95% CI, –10.14 to 2.03; P = 0.19, I2 = 89%, P <0.00001), without eff ect on ICU LOS. See Figure 1. Conclusion FO-based LEs may be associated with a reduction in infections, as well as clinically important reductions in duration of ventilation, and hospital LOS. Nevertheless, according to current literature there is inadequate evidence to give a fi nal recommendation on the routine use of FO-containing emulsions in PN and/or as a pharmaconutrient strategy in enterally fed critically ill patients. Further large-scale RCTs which should aim to consolidate potential positive treatment eff ects are warranted. (P <0.001). There appeared to be a signifi cant correlation between the APACHE IV score and glutamine levels (R = 0.52, P <0.001). P403 P403 Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis P Langlois1, R Dhaliwal2, M Lemieux2, D Heyland3, W Manzanares4 1Hospital Fleurimont, Sherbrooke, QC, Canada; 2Kingston General Hospital, Kingston, ON, Canada; 3Queen´s University, Kingston, ON, Canada; 4University Hospital, Montevideo, Uruguay Critical Care 2015, 19(Suppl 1):P403 (doi: 10.1186/cc14483) P404 Glutamine administration in sepsis: enteral, parenteral or both? Experimental study in swine G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) Glutamine administration in sepsis: enteral, parenteral or both? Experimental study in swine G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) G Stavrou1, E Filidou2, K Arvanitidis2, K Fotiadis1, V Grosomanidis1, A Ioannidis1, G Tsaousi1, A Michalopoulos1, G Kolios2, K Kotzampassi1 1Aristotle University of Thessaloniki, Greece; 2Democritus University of Thrace, Alexandroupolis, Greece Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) yp Conclusion Plasma glutamine levels were signifi cantly lower after acute admission compared with elective surgery. In both groups a considerable amount of patients had decreased glutamine levels, but this was not independently associated with the type of admission. In contrast to previous studies we found that glutamine levels were determined by severity of illness and the presence of an infection. p Critical Care 2015, 19(Suppl 1):P404 (doi: 10.1186/cc14484) Introduction Glutamine (GLN) is recommended in the critically ill for i.v. administration but enteral use is not quite clarifi ed. We decided to measure GLN plasma levels in healthy and septic swine after GLN given by i.v., enteral or both routes, over a 3-hour period. Plasma glutamine after acute or elective admission on the IC H Buter, M Koopmans MCL, Leeuwarden, the Netherlands Critical Care 2015, 19(Suppl 1):P402 (doi: 10.1186/cc14482) Moreover, in a backward linear regression analysis this correlation was independently associated with APACHE IV scores and the presence of infection, but not with the type of admission.i P406 P406 Reliability of a new French-language triage algorithm for out-of- hours primary care calls: the SALOMON rule E Brasseur1, A Ghuysen1, AF Donneau2, V D’Orio1 1C.H.U. of Liège, Belgium; 2University of Liège, Belgium Critical Care 2015, 19(Suppl 1):P406 (doi: 10.1186/cc14486) Figure 2 (abstract P404). Introduction Because of increased workloads associated with primary care physician (PCP) shortage, many western countries have been facing the diffi cult challenge of optimizing their out-of-hours primary care services. PCPs initially gathered in small rotation groups, and then further collaborations led to larger-scale cooperatives. In such models, implementation of patient call triage is mandatory to increase the effi ciency, quality and safety of care [1]. Organization models diff er, from the PCP performing all patient calls to nurses and nurse assistants answering calls and performing triage, but no validated triage algorithm has been reported to date. We developed a specifi c French- language triage algorithm called SALOMON (Système Algorithmique Liégeois d’Orientation pour la Médecine Omnipraticienne Nocturne) in order to guide nurse triage PCP out-of-hours calls. The present study tested this algorithm reliability. As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P  = 0.08). See Figures 1 and 2. As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P  = 0.08). See Figures 1 and 2. Conclusion In our experimental early sepsis model, a combination of E and i.v. GLN seems to be the most appropriate; this results in high GLN levels for the functional needs, including those of the gut mucosa. Reference 1. Crit Care. 2014;18:R76. P405 Methods The SALOMON algorithm is based on 53 common presentation fl owcharts using specifi c discriminators to triage calls into four categories according to the level of care required: emergency medical services, nonemergent visit to local emergency department, PCP home visit or PCP delayed consultation. Using an appropriate statistical test, we assessed the accuracy of presentation fl owchart and triage category selections attributed to 130 clinical scenarios, by 10 diff erent nurses, in comparison with references established by a local team of experts, at two diff erent time periods: immediately after training (T0) and 3 to 6 months after algorithm practice (T1).l Prehospital factors associated with an ICU admission from the emergency department Reference Reference 1. Huibers L, et al. Scand J Prim Health Care. 2011;29:198-209. 1. Huibers L, et al. Scand J Prim Health Care. 2011;29:198-209. Prehospital factors associated with an ICU admission from the emergency department TA Williams1, J Finn1, D Fatovich2, D Brink3, KM Ho2, H Tohira4 1Curtin University, Bentley, Australia; 2Royal Perth Hospital, Perth, Australia; 3St John Ambulance – WA, Belmont, Australia; 4 Curtin University Health Sciences, Bentley, Australia Critical Care 2015, 19(Suppl 1):P405 (doi: 10.1186/cc14485) Introduction This study aimed to describe the patient characteristics and prehospital factors associated with an ICU admission from the ED. There is a paucity of information about the early recognition of critical illness by paramedics; especially in the Australian prehospital setting. Methods A retrospective cohort study, July 2012 to June 2014, conducted in the Perth metropolitan area, which is served by a single ambulance service. Adult patients were included if transported to a public hospital ED that used the ED information system (EDIS) (seven of eight EDs) and were admitted to the ICU from the ED (ED-ICU group). Patients aged <16  years, those from rural areas or transfers were excluded. We used existing ambulance clinical data linked to EDIS data. Prehospital cohort characteristics are described using univariate statistical techniques. Logistic regression was conducted with admission to the ICU from the ED (critical illness surrogate) as the outcome variable. Variables included in regression models were age, sex, paramedic-identifi ed urgency, that is the time patients should be seen by a doctor based on the Australasian Triage Scale, paramedic- identifi ed patient problem and the time taken from the ambulance service receiving the call to hospital arrival. Physiological variables: systolic blood pressure (SBP), heart rate (HR), respiratory rate (RR), temperature, oxygen saturation, and GCS were included in the logistic models. Results Overall selection of fl owcharts was accurate for 94.1% of scenarios at T0 and 98.7% at T1. Triage category selection was correct for 93.3% of scenarios at T0 and 98.4% at T1. Both fl owchart selection and triage category were correct in of 89.5% case in T0 and at 97.5% T1. When an incorrect fl owchart was used, triage category remained accurate in 64.9% and 70.5% respectively. Both fl owchart and triage selection accuracy improved signifi cantly from T0 to T1 (P <0.0001). y gi y Conclusion The results of the present study revealed that using the SALOMON algorithm is reliable for out-of-hours PCP call triage by nurses. Validity of this rule may be further evaluated through its actual implementation in real-life conditions. Reference Intravenous fi sh oil lipid emulsions in ICU patients: an updated systematic review and meta-analysis There was considerable heterogeneity in interventions tested in these trials. No eff ect on overall mortality was found. When the results of fi ve RCTs that reported infections were aggregated, FO-containing emulsions signifi cantly reduced infections (RR 0.64; 95% CI, 0.44 to 0.92; P = 0.02, heterogeneity I2  = 0%). Furthermore, FO-based LEs were associated with a trend toward a reduction in MV days (WMD, –1.41; 95% CI, –3.43 to 0.61; P = Figure 1 (abstract P404). Figure 1 (abstract P403). Eff ects of parenteral fi sh oil lipid emulsions on infections. S143 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 As previously, i.v. (P = 0.001) and even more i.v. + E (P <0.001) infusion signifi cantly increased S GLN levels, while E infusion failed to have any eff ect. In the P vein, both i.v. (P = 0.02) and i.v. + E (P <0.001) GLN increased signifi cantly, whereas the E had no eff ect (P  = 0.08). See Figures 1 and 2. C l i I i l l i d l bi i f Figure 2 (abstract P404). (all P <0.001) except the time taken from receiving the call to hospital arrival (P = 0.48). Figure 2 (abstract P404). Conclusion Three-quarters of the ED-ICU patients were transported to the ED with high urgency. Currently no prehospital severity of illness or early warning system (EWS) is used in our ambulance service. Given the small proportion of ED-ICU patients who presented with abnormal observations, it is unlikely that introducing an EWS would alter practice or patient outcome. P407 Adequacy of trained assistance, emergency equipment and drugs at emergency calls in the ICU and in remote areas of the hospital I Kolic1, S Unell2, A Watts2, A Barry2, J Short2 1South London Healthcare NHS Trust, London, UK; 2Lewisham and Greenwich NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P407 (doi: 10.1186/cc14487) Results Of the 142,448 eligible patients transported by ambulance, 1,076 (0.75%) were admitted to the ICU from the ED: the ED-ICU group was younger (mean 53 vs. 61 years, P <0.001). Seventeen percent of ICU patients were transported as Urgency 1 (resuscitation/immediate) and 58% as Urgency 2 (within 10 minutes) while 70% of non-ICU patients were transported as Urgency 3 to 5 (P <0.001). Thirteen percent of ICU patients had a SBP <90 mmHg, 15% had a HR ≥130 and 19% had a RR >30. Drug overdose (21%) and respiratory conditions (18%) were the most common ICU conditions identifi ed by paramedics for the ED-ICU group. All variables entered into the logistic models were signifi cant Introduction We aimed to identify the adequacy of assistance provided and to assess correct anaesthetic equipment and drug availability at emergency calls made in the ICU and in remote areas of the hospital. Emergency calls often involve managing critically ill patients with the highest mortality results. The importance of a clinical team with the necessary competencies and the right level of resources are paramount. Methods We undertook a prospective survey of all adult patients with emergency calls put out to the anaesthetic team in a London district S144 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 general hospital over a 6-week period. We performed a snapshot audit of equipment in resuscitation trolleys across each ward and in the radiology department. We compared the data collected on available equipment with the standard set by the Resuscitation Council (UK) Recommended Minimum Equipment Checklist [1]. The survey addressed the availability, clinical competency and appropriate duration of stay of the anaesthetic assistant at the emergency calls. Further qualitative data were collected on the availability of required emergency drugs. general hospital over a 6-week period. We performed a snapshot audit of equipment in resuscitation trolleys across each ward and in the radiology department. Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P408 (doi: 10.1186/cc14488) g g p J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK Critical Care 2015 19(Suppl 1):P408 (doi: 10 1186/cc14488) J Bannard-Smith, S Abbas, S Ingleby, C Fullwood, S Jones, J Eddleston Manchester Royal Infi rmary, Manchester, UK f Results The intervention signifi cantly increased satisfaction amongst staff regarding: identifi cation of the team leader and other key staff members at the response; and time out eff ectiveness in reducing repetition and improving staff understanding of the patient’s status and medical issues. We found no signifi cant change in staff perceptions regarding the clarity of the ongoing treatment plan at the end of the MET call response. Introduction There is widespread concern regarding excess mortality for patients admitted to hospital out of hours. We introduced an electronic track and trigger system (Patientrack) with automated alerts in a large university teaching hospital between 2010 and 2012. The system computes the patient’s early warning score and alerts medical staff via a pager. It is operational 24 hours a day, 7 days a week and could be an eff ective tool to reduce variations in mortality throughout the working week. Conclusion Utilising a low-cost intervention in a regional setting, we were successful in improving staff perceptions of role allocation and communication within our MET call responses. The intervention also led to signifi cantly increased overall satisfaction with the MET call system. Through our surveys we have identifi ed other facets of the MET call response that also require attention. Given our encouraging results we are designing a follow-up intervention incorporating structured multidisciplinary training in MET call scenarios. g Methods We extracted hospital outcome data for all admissions during the fi nancial years between 2007 and 2014. We identifi ed variables that predicted mortality and incorporated them into a multivariate logistic regression model to assess risk of death for admissions in hours (9:00 am to 5:00 pm, Monday to Friday) versus out of hours (all other times). P409 Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) P409 Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) Reference 1. Resuscitation Council (UK). Recommended minimum equipment for in hospital adult resuscitation (October 2004). https://www.resus.org.uk/pages/ eqipIHAR.html. 1. Resuscitation Council (UK). Recommended minimum equipment for in hospital adult resuscitation (October 2004). https://www.resus.org.uk/pages/ eqipIHAR.html. 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Critical Care 2015, 19(Suppl 1):P409 (doi: 10.1186/cc14489) Introduction Medical emergency team (MET) calls are quickly becoming an integral part of the response to a deteriorating patient in Australia. Conceptually the MET call response incorporates a structured approach, but in practice this can quickly disintegrate. This collapse of method can leave patients without clear treatment plans and staff disenfranchised. We sought to improve the process of the MET call response at our regional hospital by introducing targeted interventions focused on teamwork, communication, leadership and role allocation. Introduction Medical emergency team (MET) calls are quickly becoming an integral part of the response to a deteriorating patient in Australia. Conceptually the MET call response incorporates a structured approach, but in practice this can quickly disintegrate. This collapse of method can leave patients without clear treatment plans and staff disenfranchised. We sought to improve the process of the MET call response at our regional hospital by introducing targeted interventions focused on teamwork, communication, leadership and role allocation. Methods We invited junior doctors and nurses to complete a survey designed by a multidisciplinary MET Call Working Group; 138 staff (40% of population) completed the survey. Based on analysis of responses, a focused three-pronged intervention was formulated and implemented hospital wide. The arms of the intervention were: identifi cation of the name and role of each staff member using highly visible labels; role allocation according to policy written through a multidisciplinary working group; and a time out during the response allowing a structured synopsis of the patient’s current status to be communicated to the team. The intervention was preceded by extensive staff education, and 175 staff (50%) completed the survey 6 months later to assess its success. Results The intervention signifi cantly increased satisfaction amongst staff regarding: identifi cation of the team leader and other key staff members at the response; and time out eff ectiveness in reducing repetition and improving staff understanding of the patient’s status and medical issues. We found no signifi cant change in staff perceptions regarding the clarity of the ongoing treatment plan at the end of the MET call response. Conclusion Emergency calls require standards to be met involving the competency of responding team members and adequate resources. This leads us to question whether guidelines should exist regarding the clinical competency and timeliness of the assistant available to the physician at emergency calls. 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia Methods We invited junior doctors and nurses to complete a survey designed by a multidisciplinary MET Call Working Group; 138 staff (40% of population) completed the survey. Based on analysis of responses, a focused three-pronged intervention was formulated and implemented hospital wide. The arms of the intervention were: identifi cation of the name and role of each staff member using highly visible labels; role allocation according to policy written through a multidisciplinary working group; and a time out during the response allowing a structured synopsis of the patient’s current status to be communicated to the team. The intervention was preceded by extensive staff education, and 175 staff (50%) completed the survey 6 months later to assess its success.i Revitalising the medical emergency team call KP Verma1, S Jasiowski2, K Jones2 1Melbourne Health, Melbourne, Australia; 2Ballarat Health Services, Ballarat, Australia C l C (S l ) P (d / ) P407 We compared the data collected on available equipment with the standard set by the Resuscitation Council (UK) Recommended Minimum Equipment Checklist [1]. The survey addressed the availability, clinical competency and appropriate duration of stay of the anaesthetic assistant at the emergency calls. Further qualitative data were collected on the availability of required emergency drugs. sex, unplanned admission and admission from supportive care. Risk of death for out-of-hours admissions was not signifi cantly diff erent to in- hours for any year (1.01 (0.92 to 1.11), P = 0.784). There was a signifi cant fall in risk of death over the 7-year period compared with baseline values in 2007/08 (Table 1). Conclusion In our cohort there was no evidence of increased mortality for patients admitted out of hours compared with in hours. This remained true after adjustment for age, sex, emergency admissions and admission source. Our data demonstrated an overall fall in risk of death over the study period. The introduction of Patientrack could have contributed to this reduction in mortality. Results During the study period 44 emergency calls were attended. Twenty-three (52%) of these calls were in the accident and emergency department, and four (9%) in the ICU. Survey results demonstrated two cases where no anaesthetic assistant arrived at the emergency call put out to them. In cases where timely assistance was available, the assistant did not have the adequate clinical and anaesthetic skills required by the attending physician. In 6% of cases where skilled assistance was required (n = 2), it was felt that the assistant did not stay for the clinically required length of time. Emergency drugs required were found to not be available in 11% of cases (n = 5) and in 17% of cases (n = 6) the necessary emergency equipment was not available. Data were collected on equipment from 17 resuscitation trolleys. The inadequacies identifi ed were the oxygen cylinders were fi lled less than 75% full in 41% cases (n = 7) and end-tidal capnography was identifi ed to be absent. P409 Use of an electronic early warning score and mortality for patients admitted out of hours to a large teaching hospital P410 S Results Data were available for 1,180,268 hospital admissions, of which 7,264 (0.6%) died. Predictors for hospital mortality included: age, male P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) Among true cardiac arrest events, 92.6% was due to pulseless electrical activity/asystole and 7.7% was due to ventricular fi brillation (VF)/pulseless ventricular tachycardia (VT); both of these did not have any diff erence on the initial outcome. But having an initial rhythm of VF/pulseless VT had 90% more chance for discharge from the hospital, with P  = 0.04. Although arrival time of the CBT did not have any infl uence on the fi nal outcome, duration of resuscitation ≤20 minutes had an odds ratio of 10.6 with P <0.001 favoring return of spontaneous circulation over death after controlling for age. Of the 203 patients who had true cardiac arrest events, 43 (21.2%) were discharged from the hospital. Good neurological outcome at discharge was seen among 22 (10.8%) of the patients based on Cerebral Performance Category Score. Conclusion Our experience shows that out of every 1,000 patients admitted to our hospital, about fi ve sustained cardiac arrest, of whom only 11.3% survived to hospital discharge with good neurological recovery. Variation in the eff ectiveness of the cardiopulmonary resuscitation quality in comparison with world data could be due to the inherent diff erence in the severity of the primary illness in the patients and diversity in the reported data. P411 Evaluation of emergency call Code Blue over a 5-year period N Bakan, G Karaoren, S Tomrk, S Keskin Istanbul Umraniye Training and Research Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P411 (doi: 10.1186/cc14491) Introduction Code systems are the emergency call and management systems for rapid response in healthcare institutions. The main aim of these systems is to provide common institutional understanding of what is necessary to be done immediately at the time of an event. Code Blue (CB), which is used throughout the world and was described in the 2008 service quality standards of Turkey, defi nes the necessary emergency intervention in cases of respiratory or cardiac arrest. This study aimed to evaluate the clinical and application data of patients for whom a CB call was made between 2009 and 2013. Methods After approval of local ethics committee, retrospective examination was made of CB forms. P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. Methods All Code Blue activations from 1 January 2012 to 31 December 2012 were analyzed retrospectively. Data were gathered from the Code Blue form and fi ner details of individual patients were linked through their medical records. Code Blue was activated only for events that happened outside the medical and surgical ICUs. Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. Introduction Numerous American and European studies have associated survival rates of in-hospital cardiac arrest (IHCA) with diff erent quality markers. There has been a paucity of studies that explain IHCA in Asian populations. This study was conducted to assess the characteristics and survival among patients suff ering from IHCA. f Methods All Code Blue activations from 1 January 2012 to 31 December 2012 were analyzed retrospectively. Data were gathered from the Code Blue form and fi ner details of individual patients were linked through their medical records. Code Blue was activated only for events that happened outside the medical and surgical ICUs. Conclusion The MET has been successfully implemented, with demand for its services having increased by 32.7% in 1 year. The unadjusted immediate mortality rate of patients for whom a MET/cardiac arrest call is activated is 3.64%. Response time had no infl uence on mortality, most probably due to the rapid response time. Immediate mortality was low, probably as a result of early adequate intervention. Further evaluation of overall hospital mortality is warranted for future studies. pp g Results A total of 260 Code Blue activations were made, out of which there were 203 true cardiac arrest events among 40,168 in-patients; the cumulative incidence of the same was 0.51%. Mean (SD) duration of arrival of the Code Blue Team (CBT) to the scene was 64.5 (27.7) seconds. Cardiovascular illness was the predominant baseline morbidity but none of the baseline illness showed increased risk of mortality in this group. P410 Successful implementation of a medical emergency team: 2-year experience in a teaching hospital A Tridente, J Elmore, R Varia, T Mahambrey St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P410 (doi: 10.1186/cc14490) Table 1 (abstract P408). Overall risk of death ratios compared to 2007/08 Year ROD 95% CI 2008/09 1.01 0.73 to 1.5 2009/10 1.03 0.74 to 1.5 2010/11 0.46* 0.33 to 0.7 2011/12 0.31* 0.22 to 0.4 2012/13 0.27* 0.2 to 0.39 2013/14 0.26* 0.2 to 0.37 *P <0.001. Table 1 (abstract P408). Overall risk of death ratios compared to 2007/08 Introduction A medical emergency team (MET) was introduced in our institution in January 2012 to provide timely response to the needs of acutely ill inpatients and cardiac arrest calls. The MET assesses the patient and prescribes a management plan for the responsible team to follow; promptly stabilising and transferring patients to a place of safety where required. We aimed at evaluating the eff ects of introducing the MET on clinically relevant processes and outcomes. Methods Prospective data were analysed using STATA 10.1. The primary outcome measure was immediate mortality (defi ned as mortality at S145 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The time taken to reach patients conformed with the global standard mean 2 to 3  minutes [1]. The rates of erroneous CB and time to reach patients reduced each year due to more staff experienced and knowledgeable in CB and the structuring of the emergency clinic. Conclusion The time taken to reach patients conformed with the global standard mean 2 to 3  minutes [1]. The rates of erroneous CB and time to reach patients reduced each year due to more staff experienced and knowledgeable in CB and the structuring of the emergency clinic. conclusion of the MET intervention); the secondary outcome measures were admission to critical care after a MET call and cardiac arrest. conclusion of the MET intervention); the secondary outcome measures were admission to critical care after a MET call and cardiac arrest. Results A total of 5,763 MET calls were made between 9 January 2012 and 4 March 2014, of which 5,310 (92.1%) were MET calls, 349 (6.1%) cardiac arrest calls, 36 (0.6%) false alarms and 68 (1.2%) unclassifi ed. The number of calls increased by 32.7% from 2,255 in 2012 to 2,993 in 2013, with all month-specifi c comparisons showing signifi cant increases in MET activity (ranging from 0.5% to 103.6% increases). P410 Successful implementation of a medical emergency team: 2-year experience in a teaching hospital A Tridente, J Elmore, R Varia, T Mahambrey St Helens and Knowsley, Liverpool, UK Critical Care 2015, 19(Suppl 1):P410 (doi: 10.1186/cc14490) MET activity displayed cyclical yearly changes, with the winter months and the month of August (junior doctors’ changeover period) being particularly busy. Median response time (interquartile range) was 1 (1 to 2) minutes, with 99.1% calls attended to within 3 minutes. There were 210 (3.64%) immediate deaths (with no signifi cant diff erences between years), 112 (1.9%) patient transfers to critical care, 233 (4%) patients were transferred to other locations (other than critical care) while 4,697 (81.5%) patients remained on the ward of origin. In 408 cases (7.1%) a do-not-resuscitate order was instituted. On multiple logistic regression analyses, when the type of call was taken into consideration, the response time had no infl uence on primary (mortality OR = 0.83, 95% CI = 0.63 to 1.09, P = 0.18) and secondary outcomes (admission to critical care OR = 0.85, 95% CI = 0.62 to 1.17, P = 0.33; subsequent cardiac arrest OR = 0.57, 95% CI = 0.27 to 1.2, P = 0.14). References 1. Abella BS, et al. JAMA. 2005;293:305-10. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. 1. Abella BS, et al. JAMA. 2005;293:305-10. 1. Abella BS, et al. JAMA. 2005;293:305 10. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. 2. Merchant RM, et al. Crit Care Med. 2011;39:2401-6. Results From CB calls for a total of 1,195 patients over the 5-year period, 1,035 (86.6%) were evaluated. The rate of erroneous CB was 36.9%. Patients comprised 413 (39.9%) females and 622 (60.1%) males with a mean age of 59.73 ± 23.13 years (range, 0.1 to 102 years). The distribution of total cases over the 5 years (2009 to 2013) was 15.5%, 25.2%, 26.5%, 19% and 13.8% respectively. Distribution according to clinic was emergency internal (37.5%), internal (16.5%) and emergency surgical (9.5%). Clinical diagnosis was cardiac 28.8%, neurological 15.6% and end-stage cancer 13.5%. A total 19.9% of the patients were those discharged from intensive care. The total survival rate was 59.6%. The duration of CPR in survivors was statistically longer than in nonsurvivors (P <0.01). There was no statistically signifi cant relationship between the duration of CPR and age (P >0.05). The overall mean time taken to reach the patient was 102.71 ± 22.47 seconds, which reduced to 93.64  ± 19.91  seconds in 2013. The APACHE II–PRISM scores and mortality rates were low in the cases of erroneous CB (P <0.05). P412 Attention Code Blue: a comprehension of in-hospital cardiac arrest from a multispeciality hospital in South India M Hisham, MN Sivakumar, T Sureshkumar, R Senthil Kumar, A Satheesh Kovai Medical Center and Hospital, Coimbatore, India Critical Care 2015, 19(Suppl 1):P412 (doi: 10.1186/cc14492) The age and gender of the patient, diagnosis, department to which admitted, time of CB call, reason for CB, whether or not CB was appropriate, whether or not CPR was applied, duration of CPR if applied, APACHE II and PRISM scores and predicted mortality were recorded from the hospital automated record system and the CB form. Patients who refused treatment or who could not reach the necessary parameters for the calculation of APACHE II and PRISM scores were excluded. References Reference 1. Nolan JP, et al. European Resuscitation Council guidelines for resuscitation 2010 section 1. Executive summary. Resuscitation. 2010;81:1219-76. P412 Are we failing to teach cardiopulmonary resuscitation (CPR) in schools? A pilot study to assess CPR and automated external defi brillator training in London schools J Salciccioli, D Marshall, M Sykes, A Wood, S Joppa, M Sinha, PB Lim Imperial College London, UK Critical Care 2015, 19(Suppl 1):P413 (doi: 10.1186/cc14493) Are we failing to teach cardiopulmonary resuscitation (CPR) in schools? A pilot study to assess CPR and automated external defi brillator training in London schools J Salciccioli, D Marshall, M Sykes, A Wood, S Joppa, M Sinha, PB Lim Imperial College London, UK Critical Care 2015, 19(Suppl 1):P413 (doi: 10.1186/cc14493) TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest Methods We conducted a registered audit of CPR and AED training in London schools. Secondary schools were identifi ed via web links for each of the London Borough Councils. Telephone interviews with school staff familiar with CPR and AED training practices were conducted prospectively using a standardized web-based survey. All survey response data were captured electronically. We defi ned universal training as any programme which delivers CPR and AED training to all students in the school. We used simple descriptive statistics to summarise the results. J Callerova1, R Skulec2, J Knor3, V Cerny3 1Emergency Medical Service of Central Bohemian Region, Beroun, Czech Republic; 2Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015, 19(Suppl 1):P416 (doi: 10.1186/cc14496) J Callerova1, R Skulec2, J Knor3, V Cerny3 1Emergency Medical Service of Central Bohemian Region, Beroun, Czech Republic; 2Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic Critical Care 2015 19(Suppl 1):P416 (doi: 10 1186/cc14496) Introduction Implementation of point-of-care ultrasound examination into cardiopulmonary resuscitation (CPR) may increase diagnostic accuracy for determining the cause of cardiac arrest. However, current protocols either do not refl ect all causes detectable by ultrasound or are too complicated for prehospital use. Thus, we decided to construct and validate a new protocol TRACE (ThoRacic and Abdominal sonography in Cardiac arrEst) for ultrasound examination during out-of-hospital cardiac arrest (OHCA). Results A total of 51 schools completed the survey covering an estimated student population of 54,037. There were four (8%) schools that provide universal training programmes and an additional 23 (45%) off er optional training programmes for students. There were 16 (31%) schools which have an AED available on the school premises. The most common reasons for not having a universal CPR training programme is the requirement for additional class time (15/51; 29%) and that funding is unavailable for such a programme (12/51; 24%). There were three students who died from sudden cardiac arrest over the period of the past 10 years. Methods We designed a new protocol for ultrasound examination during OHCA to increase the success rate for the establishment of OHCA cause. Body position during transport in a refractory cardiac arrest porcine model p Methods Data were prospectively collected from January 2011 to January 2012 from 336 female medical and pharmacy students undergoing CPR training at the Lithuanian University of Health Sciences. During the training process, the instructors performed a simple 5-second intervention (Andrew’s manoeuvre) with all of the rescuers in the study group. The instructor pushed 10 times on the shoulders of each trainee while she performed CCs to achieve the maximal required compression depth. Immediately after training, the participants were asked to perform a 6-minute BLS test on a manikin that was connected to a PC with SkillReporter™ System software (Laerdal, Norway); the quality of the participants’ CPR skills was then evaluated. Body position during transport in a refractory cardiac arrest porcine model J Belohlavek1, M Mlcek2, M Huptych3, T Boucek1, T Belza2, P Krupickova2, O Kittnar2 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) Introduction Cardiac arrest patients are not transported only supine. The eff ect of body position on resuscitability and cerebral perfusion in a 30° and 60° incline is not known.i Results The CC depth in the study group increased by 6.4 mm (P <0.001) compared with the control group (52.9 vs. 46.6 mm). A regression analysis showed that Andrew’s manoeuvre increased the depth of the CCs among women by 14.87 × (1 – 0.01 × weight) mm.i Methods Twenty-fi ve female pigs were subjected to a simulated cardiac arrest (3  minutes no fl ow, 5  minutes mechanical CPR). Next, animals were randomly assigned to one of the three groups: GROUP 60 (n  = 8), 60° incline for 3  minutes to simulate transport in space restricted elevator; GROUP 30 (n  = 8), 30° incline for 8  minutes to simulate staircase transport; and GROUP 0, with no incline. During subsequent standard CPR including rescue ECMO, resuscitability and cerebral perfusion were assessed. Conclusion Andrew’s manoeuvre during CPR training signifi cantly improved the performance of the female rescuers and helped them achieve the CC depth required by 2010 resuscitation guidelines. It is most eff ective among the women with the lowest body weight. Are we failing to teach cardiopulmonary resuscitation (CPR) in schools? A pilot study to assess CPR and automated external defi brillator training in London schools Introduction Mortality from cardiac arrest remains high [1]. Bystander cardiopulmonary resuscitation (CPR) and the use of automated external defi brillators (AED) are two of the most important factors favouring survival [2]. CPR/AED training in schools is a recommended intervention for signifi cantly improving training rates across a large population [3]. The current practice for CPR/AED training in London schools is unknown. The primary aim of this study was to assess current S146 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 practices relating to CPR and AED training in London secondary schools. 1. Krikscionaitiene A, et al. Magical manoeuvre: a 5-s instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth. Eur J Emerg Med. 2014;21:424-8. TRACE: a new protocol for ultrasound examination during out-of- hospital cardiac arrest The subcostal view was performed during planned rhythm check to assess the presence of cardiac tamponade and size of the right and left ventricle and inferior caval vein. Thereafter, during ongoing cardiac compressions, Morrison’s pouch and right pleural space were investigated to exclude intraperitoneal and inrapleural free fl uid. The same procedure was applied on the left side of the body. Finally, the anterior thoracic view was done to exclude pneumothorax. Working diagnosis was compared with the fi nal in-hospital diagnosis or autopsy. Conclusion CPR and AED training rates in London secondary schools are low. The majority of schools do not have an AED available on premises. The most common reason for not providing CPR training is the requirement for additional class time. These data highlight an opportunity to vastly improve CPR training rates in a large population. Future studies should assess programmes which are cost-eff ective and which do not require signifi cant amounts of additional class time. References 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87. 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. 1. Berdowski J, et al. Resuscitation. 2010;81:1479-87. Results We examined 40 consecutive OHCA patients. Correct cause of OHCA during CPR was recognised in 38 patients (95%). Leading causes were acute coronary syndrome (55.0%), pulmonary embolism (15.0%) and complication of chronic heart failure (10.0%). Incorrect recognition was performed in one patient with respiratory cause, originally considered as pulmonary embolism, and in another with pulmonary embolism, considered as respiratory cause. One rhythm check was suffi cient to perform TRACE in 31 patients, in the other two interruptions of cardiac compressions were required. Return of spontaneous circulation was achieved in 15 (37.5%) patients, favourable neurological outcome at hospital discharge in eight (20%) patients. Specifi c therapy to aff ect the cause of OHCA was applied during OHCA in 12 (30%) patients. 2. Go AS, et al. Circulation. 2014;129:e28. 3. Cave DM, et al. Circulation. 2011;123:691-706. Magical manoeuvre: a 5-second instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth Magical manoeuvre: a 5-second instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth A Krikscionaitiene, A Pranskunas, M Dambrauskiene, N Jasinskas, Z Dambrauskas, E Vaitkaitiene, J Vencloviene, D Vaitkaitis Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P415 (doi: 10.1186/cc14495) A Krikscionaitiene, A Pranskunas, M Dambrauskiene, N Jasinskas, Z Dambrauskas, E Vaitkaitiene, J Vencloviene, D Vaitkaitis Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2015, 19(Suppl 1):P415 (doi: 10.1186/cc14495) g Conclusion Implementation of the TRACE protocol to the CPR process was feasible, required minimal interruption of cardiac compressions and resulted in a high recognition rate for the cause of OHCA. Introduction Adequate chest compression (CC) depth is crucial for resuscitation outcomes. Lightweight rescuers, particularly women, are often unable to achieve the required 5 to 6  cm CC depth. This nonrandomised cohort study investigated new strategies to improve CC performance. P417 Body position during transport in a refractory cardiac arrest porcine model J Belohlavek1, M Mlcek2, M Huptych3, T Boucek1, T Belza2, P Krupickova2, O Kittnar2 1General University Hospital, Prague, Czech Republic; 2Charles University in Prague, Czech Republic; 3Czech Technical University in Prague, Czech Republic Critical Care 2015, 19(Suppl 1):P417 (doi: 10.1186/cc14497) Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Ch 1 ZM Li 1 JH T 1 S S h 1 M H i Bi I k d 1 B L 2 Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Chua1, ZM Lin1, JH Tan1, S Surentheran1, M Haris Bin Iskander1, B Leong2 1National University of Singapore, Singapore; 2National University Hospital, Singapore Critical Care 2015 19(Suppl 1):P418 (doi: 10 1186/cc14498) Introduction Despite several large studies concerning out-of-hospital cardiac arrests in recent years, it is not clear whether their in-hospital counterparts have benefi ted from advances in resuscitation as well as post-resuscitation care. Introduction Despite several large studies concerning out-of-hospital cardiac arrests in recent years, it is not clear whether their in-hospital counterparts have benefi ted from advances in resuscitation as well as post-resuscitation care. p Methods We identifi ed all cases of in-hospital cardiac arrest (IHCA) occurring in the National University Hospital in Singapore from 1 June 2008 to 31 May 2009. Patients for which IHCA occurred but where no resuscitation was attempted were excluded. Key outcomes were classifi ed as primary (survival to discharge) and secondary (return of spontaneous circulation). Additionally, various arrest characteristics were analysed to identify predictive factors for survival to discharge with level of signifi cant set at P <0.05. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. Figure 2 (abstract P419). Comparison complications of CPR between the OHCA group and the IHCA group. gi Results Among 353 unique cases of IHCA analysed, 63 patients (17.8%) had a shockable rhythm (ventricular fi brillation and pulseless ventricular tachycardia) of which 17 (27.0%) survived to discharge. While 290 (82.2%) patients presented with nonshockable rhythm (asystole or pulseless electrical activity), only 32 patients (11%) survived to discharge. For patients who survived to discharge, univariate analysis showed that event location (P = 0.016), nationality (P = 0.035), paying class (P = 0.038), use of ECG monitoring (P = 0.048), initial cardiac rhythm (P = 0.000) and presence of a house offi cer (P = 0.005) were statistically signifi cant. Multivariate analysis showed that patients with shockable rhythms were 2.52 times more likely to survive but other factors were not signifi cant. For patients who attained ROSC, univariate analysis showed that time of day (P = 0.006), event location (P = 0.000), and number of adrenaline doses administered (P = 0.000) were statistically signifi cant. Comparison of complications secondary to cardiopulmonary resuscitation between out-of-hospital cardiac arrest and in-hospital cardiac arrest M Seung Y Park M Seung, Y Park Yonsei University Severance Hospital/Yonsei University College of Medicine, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P419 (doi: 10.1186/cc14499) M Seung, Y Park Yonsei University Severance Hospital/Yonsei University College of Medicine, Seoul, South Korea Critical Care 2015, 19(Suppl 1):P419 (doi: 10.1186/cc14499) Introduction Chest compression during cardiopulmonary resuscitation (CPR) could bring out unintended complications which are mainly composed of chest injuries. The aim of this study was to assess whether there was a signifi cant diff erence in the complications of CPR between out-of-hospital cardiac arrest (OHCA) and in-hospital cardiac arrest (IHCA) survivors using multidetector computed tomography (MDCT). g p g p y Methods We performed a retrospective cohort study in the emergency departments of two academic tertiary care centres from January 2009 to May 2014. We enrolled both OHCA and IHCA patients who underwent successful CPR. The enrolled patients had undergone a chest CT within 48 hours after ROSC. We evaluated the MDCT fi ndings of the CPR-related chest injuries and compared complications between OHCA and IHCA patients.i Figure 1 (abstract P417). Figure 1 (abstract P417). (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1. (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1. p Results A total of 148 patients were fi nally enrolled in this study, OHCA were 89 (60.1%) and IHCA were 59 (39.8%). The mean CPR time, both in-hospital and total, was longer in OHCA survivors. Rib fractures were Conclusion Positional changes during simulated refractory cardiac arrest in this experimental model signifi cantly aff ect resuscitability and brain perfusion. Animals subjected to shorter time in a more inclined (GROUP 60) position were more easily resuscitated; however, cerebral blood fl ow was better preserved in GROUP 30. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. Figure 1 (abstract P419). Patient demographics and clinical fi ndings. P418 Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Chua1, ZM Lin1, JH Tan1, S Surentheran1, M Haris Bin Iskander1, B Leong2 1National University of Singapore, Singapore; 2National University Hospital, Singapore Critical Care 2015, 19(Suppl 1):P418 (doi: 10.1186/cc14498) Body position during transport in a refractory cardiac arrest porcine model Reference Results Attainment of ROSC (3, 5, 5 in respective groups, P = 0.021), time to ROSC (15:24 (13:26; 16:02) vs. 19:19 (18:28; 19:37) vs. 9:10 minutes (8:28; 9:41), respectively, P = 0.005) signifi cantly diff ered. Changes in carotid blood fl ow according to the respective periods of the protocol 1. Krikscionaitiene A, et al. Magical manoeuvre: a 5-s instructor’s intervention helps lightweight female rescuers achieve the required chest compression depth. Eur J Emerg Med. 2014;21:424-8. S147 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (baseline, cardiac arrest, initial supine CPR and 30° vs. 60° CPR) are depicted in Figure 1 Figure 1 (abstract P417). Figure 1 (abstract P417). Impact of intra-arrest fl uid loading with diff erent doses of crystalloid infusion on hemodynamics in experimental cardiac arrest R Skulec1, A Truhlar1, R Parizkova1, Z Turek1, D Astapenko1, J Dudakova2, V Cerny3 R Skulec1, A Truhlar1, R Parizkova1, Z Turek1, D Astapenko1, J Dudakova2, V Cerny3 y 1Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 2Emergency Medical Service of Central Bohemian Region, Kladno, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic y 1Charles University in Prague, University Hospital Hradec Kralove, Czech Republic; 2Emergency Medical Service of Central Bohemian Region, Kladno, Czech Republic; 3J.E. Purkinje University, Masaryk Hospital, Usti nad Labem, Czech Republic l l d Critical Care 2015, 19(Suppl 1):P420 (doi: 10.1186/cc14500) Introduction Fluid loading during cardiopulmonary resuscitation for nonhypovolemic cardiac arrest remains controversial. Thus, we conducted an experimental study comparing the impact of two diff erent doses of balanced crystalloid infusion on hemodynamics in a porcine model of ventricular fi brillation.i Conclusion Although our study has a number of methodological limitations, these results about incidence in cardiac arrest survivors corroborate previous retrospective reports. It is possible that every cardiac arrest survivor has had to live a NDE, regardless of brain mechanisms associated with experience, but only some patients remember it. If some chronic medications, such as benzodiazepine, may decrease memorization, the role of the elements of the clinical context about NDE during resuscitation is speculative References i Methods Ventricular fi brillation was induced for 15  minutes in 19 anesthetized domestic pigs. Before induction, the animals were randomized to receive either 1,000  ml (34  ± 3  ml/kg, group A, n  = 7) or 500  ml (16  ± 2  ml/kg, group B, n  = 7) of balanced crystalloid solution or to undergo no fl uid loading during CPR (group C, n = 5). After spontaneous circulation (ROSC) was restored, the animals were observed for 90 minutes. 1. Van Lommel P, van Wees R, Meyers V, Elff erich I. Near-death experience in survivors of cardiac arrest: a prospective study in the Netherlands. Lancet. 2001;358:2039-45. Results In all groups, signifi cant increase of intracranial pressure followed by its decrease after ROSC was observed. While in groups B (from 12 ± 2 to 18 ± 2 mmHg, P <0.05) and C (from 13 ± 1 to 18 ± 3 mmHg, P <0.05) it was comparable (P >0.05), the rise of intracranial pressure in group A was signifi cantly higher (from 12  ± 3 to 23  ± 3 mmHg, P <0.05). f References 1. Kim EY, Yang HJ, Sung YM, et al. Multidetector CT fi ndings of skeletal chest injuries secondary to cardiopulmonary resuscitation. Resuscitation. 2011;82:1285-8. Kim EY, Yang HJ, Sung YM, et al. Multidetector CT fi ndings of skeleta Methods In a retrospective study from 2005 to 2012, 295 consecutive cardiac patients who were successfully resuscitated after cardiac arrest were enrolled. In total, 204 (69%) were alive during the research period (mean delay: 55 months). A total of 118 (40%), over 18 years, able to answer a short standardized interview were included in the study when they accepted to participate. Demographic, medical, pharmacological and psychological data were recorded and we used the Greyson NDEs scale to identify and characterize NDEs. Descriptive and unifactorial analysis was performed using the Jacknife method and Wald test according to low event frequency. 2. Kim MJ, Park YS, Kim SW, et al. Chest injury following cardiopulmonary resuscitation: a prospective computed tomography evaluation. Resuscitation. 2013;84:361-4. g y Results From our 118 reports, 20 described a core experience and 18 (15.3%) met the criteria for NDEs (Greyson NDEs total score >6/32 (7 to 19)). Only one patient recounted a negative experience. Regarding the risk factors for NDEs, using univariate analysis, we found for demographic data: woman (CI: 1.11 (1.10 to 1.12), P  <0.0001), age under 60 (CI: 1.23 (1.21 to 1.24), P <0.0001), prior knowledge of NDEs (CI: 1.97 (1.95 to 1.99)) and previous NDE (CI: 5.82 (4.19 to 8.08)). According to the history of previous disease, we found an increased risk for pulmonary disease (CI: 1.75 (1.73 to 1.77)), rheumatic disease (CI: 3.79 (3.75 to 3.84)), endocrine disease (CI: 1.45 (1.43 to 1.46)), and a decrease for cardiac disease (CI: 0.65 (0.64 to 0.66)), psychiatric disease (CI: 0.71 (0.69 to 0.72)) and digestive tract disease (CI: 0.71 (0.69 to 0.72)). For previous pharmacological treatment we found a decrease of risk for all classes and particularly when two drugs were simultaneously given (CI: 0.37 (0.36 to 0.38)). Impact of intra-arrest fl uid loading with diff erent doses of crystalloid infusion on hemodynamics in experimental cardiac arrest Whereas coronary perfusion pressure was lower in group A than in control group C during volume loading, fl uid infusion induced its mild increase in group B (group A: 12.1  ± 2.4, group B: 16.0 ± 2.6, group C: 13.6 ± 2.8 mmHg, P = 0.043). Decrease of cerebral perfusion pressure was equal in all groups. Cardiac index 10 minutes after ROSC signifi cantly diff ered among all groups (group A: 8.9 ± 2.2, group B: 7.1 ± 1.3, group C: 4.9 ± 1.9 l/minute/m2, P = 0.007) and the dose of crystalloid infusion during cardiac arrest positively correlated with cardiac index increase (r = 0.815, P <0.001).i 2. Parnia S, Spearpoint K, de Vos G, Fenwick P, Goldberg D, Yang J, et al. AWARE – AWAreness during REsuscitation – a prospective study. Resuscitation. 2014;85:1799-805. 3. Greyson B. The near-death experience scale. Construction, reliability, and validity. J Nerv Ment Dis. 1983;171:369-75. 3. Greyson B. The near-death experience scale. Construction, reliability, and validity. J Nerv Ment Dis. 1983;171:369-75. Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Centre Hospitalier Universitaire, Reims Cedex, France Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Near-death experiences in survivors of cardiac arrest: a study about demographic, medical, pharmacological and psychological context F Lallier, G Velly, A Leon Centre Hospitalier Universitaire, Reims Cedex, France Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) p Critical Care 2015, 19(Suppl 1):P421 (doi: 10.1186/cc14501) Introduction Near-death experiences (NDEs) are increasingly being reported as a clear reality of clinical signifi cance. Previous studies, essentially, have been trying to estimate their incidence in various populations, notably after cardiac arrest resuscitation, and to understand the implication of resuscitation characteristics [1,2]. Using the Greyson NDE scale [3], the present retrospective study aimed at exploring cardiac arrest survivors and the correlations between NDE and physiological, medical, psychological and pharmacological context. i Conclusion Frequency of rib fractures and multiple rib fractures were higher in OHCA survivors. Further investigation is needed into the relation between the location of CPR and the CPR-related injuries, eff orts to reduce the complications after CPR. Predictors of return of spontaneous circulation and survival in in- hospital cardiac arrest: a retrospective study in a single institution JL Ch 1 ZM Li 1 JH T 1 S S h 1 M H i Bi I k d 1 B L 2 Multivariate analysis showed that an arrest occurring in the ICU setting was 2.9 times more likely to attain ROSC (95% CI: 1.02 to 5.59, P = 0.044). Conclusion The results of this study have described some key predictive factors regarding positive outcomes in IHCA in Singapore. These are vital in understanding important features regarding IHCAs and will aid in developing policies to help improve care and survival in this group of patients. S148 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P421 detected more in OHCA survivors. Frequency of multiple rib fractures was higher in OHCA survivors. Frequency of sternum fractures was higher in OHCA survivors, showing no signifi cant diff erence. In lung injuries, lung contusion and pneumothorax account for the large part, and OHCA survivors had higher incidence in both complications but statistically insignifi cant. Major complications occurred in eight cases in OHCA survivors and three cases in IHCA survivors during the study period. After adjusting for the time factor in multiple logistic regression analysis, rib fractures and multiple rib fractures became statistically signifi cant in OHCA survivors. See Figures 1 and 2. Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest Eighty-three percent (n = 45) of survivors admitted to the ICU went to the cardiac cathlab before ICU admission compared with 53% (n  = 39) of nonsurvivors. Forty-three percent of survivors had PPCI compared with 26% of nonsurvivors. Eighty- one percent (n  = 44) of survivors received therapeutic hypothermia compared with 62% (n = 48) of nonsurvivors. Introduction Mild hypothermia and fever control have been shown to improve neurological outcomes post cardiac arrest. Common methods to induce hypothermia include body surface cooling and intravascular cooling; however, a new approach using a catheter placed into the esophagus has recently become available.i p g y Methods We report the fi rst three cases of temperature control using an esophageal cooling device (ECD). The ECD was placed orally in a similar fashion to orogastric tubes. Temperature reduction was achieved by connecting the ECD to a commercially available heat exchange unit (Blanketrol II or III). Conclusion Over 3 consecutive years our annual case mix, ICU and hospital mortalities for OOHCA patients admitted to the ICU have remained stable, while our annual pre-ICU cathlab and PPCI utilisation have risen consistently in both survivors and nonsurvivors. ICU survivors were more likely to have had a shockable rhythm, been to the cathlab, and received PPCI and TH, but all may simply refl ect selection bias. Any benefi t these conferred to cardiac patients may have been off set by our liberal ICU admission policy to OOHCAs with nonshockable rhythms. Access to 24–7 PPCI in this group may determine survival and we suggest that OOHCA patients should be taken directly to regional heart attack centres. Results The fi rst patient, a 59-year-old male (73 kg), was admitted after successful resuscitation from a protracted out-of hospital cardiac arrest. His initial temperature was 35°C, which is within our current institutional protocol of 34 to 36°C. Several hours after arrival, his temperature slowly increased to 35.8°C despite application of a cooling blanket and ice packs to the groin and axilla. The ECD was inserted and a reduction of temperature to 34.8°C was achieved within 3  hours. The patient expired on day 3. The second patient, a 54-year-old female (95 kg), was admitted after resuscitation from an out-of-hospital PEA arrest. Despite initiating our cooling protocol with surface-cooling blankets and cold intravenous saline, she mounted a fever peaking at 38.3°C shortly after admission. Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, V Achan, M Davies, M Peck Frimley Park Hospital, Frimley, UK y p y Critical Care 2015, 19(Suppl 1):P422 (doi: 10.1186/cc14502) y p y Critical Care 2015, 19(Suppl 1):P422 (doi: 10.1186/cc14502) Conclusion Fluid loading during CPR had signifi cant impact on hemodynamics in our experimental model. While a high dose led to unintentional increase of intracranial pressure and decrease of coronary perfusion pressure, a low dose did not aff ect intracranial pressure and was associated with mild increase of coronary perfusion pressure during cardiac arrest. Introduction Our 700-bed hospital has a 24–7 cathlab service that routinely investigates patients with indications prior to ICU admission following out-of-hospital cardiac arrest (OOHCA). Our aim was to compare survivors and nonsurvivors and evaluate utilisation and S149 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 nonshockable rhythms. However, access to 24–7 PPCI may determine survival and we suggest that all OHCA patients should be taken directly to regional heart attack centres. nonshockable rhythms. However, access to 24–7 PPCI may determine survival and we suggest that all OHCA patients should be taken directly to regional heart attack centres. prognostic impact of angiography and primary percutaneous coronary intervention (PPCI) in this patient group. Methods A retrospective analysis using Trust electronic databases (Symphony, WardWatcher, PICIS, PRISM) of all OOHCA patients admitted to our ICU over 3 consecutive years between 1 November 2011 and 31 October 2014. P424 Targeted temperature management after cardiac arrest and fever control with an esophageal cooling device A Hegazy, D Lapierre, E Althenayan University of Western Ontario, London, ON, Canada Critical Care 2015, 19(Suppl 1):P424 (doi: 10.1186/cc14504) Results A total of 351 patients presented to our emergency department (ED) following OOHCA in this period, and of these 50% died in the ED, 37% were admitted to the ICU and 13% were admitted elsewhere. Of the 129 patients admitted to the ICU, median age was 66 (range 18 to 93), 71% were male, 68% had a shockable presenting rhythm, median ICU LOS was 3.75 (range 1 to 34 days) and ICU and hospital mortalities were 50% and 60% respectively. Eighty-nine percent (n = 48) of OOHCA survivors admitted to the ICU had a shockable rhythm compared with 55% (n = 41) of nonsurvivors. P425 P425 Is selective nasopharyngeal brain cooling detrimental to neuroprotection? M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P425 (doi: 10.1186/cc14505) P423 Utilisation and prognostic impact of angiography and primary percutaneous coronary intervention prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, M Davies, V Achan, M Peck Frimley Park Hospital, Frimley, UK Critical Care 2015, 19(Suppl 1):P423 (doi: 10.1186/cc14503) Utilisation and prognostic impact of angiography and primary percutaneous coronary intervention prior to intensive care admission for patients following out-of-hospital cardiac arrest L Eveson, S Shrestha, M Davies, V Achan, M Peck Frimley Park Hospital, Frimley, UK Critical Care 2015, 19(Suppl 1):P423 (doi: 10.1186/cc14503) Introduction Our 700-bed hospital has a 24–7 cathlab service that routinely investigates patients with indications prior to ICU admission following out-of-hospital cardiac arrest (OHCA). Our aim was to compare ICU survivors and nonsurvivors and evaluate the utilisation and prognostic impact of angiography and primary percutaneous coronary intervention (PPCI) in this patient group. f Conclusion The ECD is a novel technology that can be used for temperature management post cardiac arrest and for fever control in critically ill patients. Despite patients mounting a febrile response, temperature control was achieved and maintained successfully. The device was reported as being easy to use, by both physicians and nurses. y p g p Methods A retrospective analysis using Trust electronic databases (Symphony, WardWatcher, PICIS, PRISM) of all OHCA patients admitted to our ICU over 3 consecutive years between 1 November 2011 and 31 October 2014. Results A total of 351 patients presented to our hospital following OHCA in this period, and of these 50% died in the ED, 37% were admitted to the ICU and 13% elsewhere. Of the 129 patients admitted to the ICU, median age was 66 (range 18 to 93), 71% were male, 68% had a shockable presenting rhythm, median ICNARC score was 31 (range 10 to 66), median ICU LOS was 3.75 (range 1 to 34 days) and ICU and hospital mortality were 50% and 60% respectively. ICU survivors were more likely to have had a shockable rhythm (89 vs. 55%), been to the cathlab (83 vs. 53%), received PPCI (43 vs. 26%) and TH (82 vs. 62%) and had lower median ICNARC scores (26 vs. 35) than nonsurvivors. Over the 3 consecutive years, pre-ICU cathlab and PPCI utilisation increased annually in ICU survivors (73 vs. 86 vs. 89% and 45 vs. 54 vs. 59% respectively) and nonsurvivors (40 vs. 38 vs. 50% and 20 vs. 27 vs. 31% respectively). However, our annual ICU and hospital mortality remained unchanged (46 vs. 51 vs. 51% and 60 vs. 57 vs. 62% respectively). Utilisation and prognostic impact of cathlab investigation prior to intensive care admission for patients following out-of-hospital cardiac arrest After ECD insertion and confi rming the external heat exchanger connection, her temperature gradually dropped to 35.7°C over a period of 4 hours. She subsequently made a favorable neurological recovery and was discharged home at day 23. The third patient, a 47-year-old male patient (86 kg) presented with an ongoing fever secondary to necrotizing pneumonia in the postoperative period after coronary artery bypass grafting. His fever was unresponsive to empiric antibiotic therapy, antipyretics, cooling blankets, and ice packs. ECD insertion resulted in a decrease in temperature from 39.5°C to 36.5°C in less than 5 hours. The patient eventually made a full recovery and was discharged home after 59 days. In all three patients, placement of the device occurred in less than 3  minutes and ease of use was reported as excellent by nursing staff and physicians. P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) Introduction Uncontrolled shivering may have negative consequences by increasing metabolic demand and subsequently neutralize the benefi ts of therapeutic normothermia [1]. Previous anti-shivering protocols that utilize the least sedation have been described in therapeutic temperature modulation (TTM) [2]. Our aim is to describe and evaluate an anti-shivering protocol that emphasizes the least sedating regimen with the least number of pharmacologic agents for patients undergoing therapeutic normothermia. brain temperature, intracranial pressure (ICP), core temperatures and vital signs were continuously recorded. Cooling was terminated once the brain reached 32°C and the animals were allowed to passively rewarm. Results In the SNBC group, the brain target temperature was reached in 54 ± 11 minutes. The mean ICP dropped precipitously to a nadir of –15 mmHg. TCD showed signifi cant vasospasm in the MCA, compared with the internal carotid artery (ICA), during the entire cooling phase (Table 1). Upon termination of cooling, the brain temperature spontaneously rewarmed to core temperature in 13  ± 4  minutes. Rewarming was associated with hyperemia and elevation of ICP. In group 2, there was no cerebral vasospasm or hyperemia during cooling and rewarming respectively.i Methods This retrospective chart review includes patients with neurologic injury who underwent TTM from March 2013 to November 2014 and were evaluated for the following outcomes: percentage of total patient hours in each score of the Bedside Shivering Assessment Scale (BSAS) at 72  hours, 168  hours, and total duration of TTM; percentage of total patient days in each tier of the anti-shivering protocol at 72  hours, 168  hours, and total duration of TTM; de- escalation of anti-shivering agents with or without the necessary need for re-escalation; ICU and hospital length of stay (LOS); rescue agents utilized; and hospital mortality. Conclusion SNBC is associated with signifi cant vasospasm of the MCA. In addition, spontaneous and rapid rewarming of the brain, vasodilation, rapid reperfusion, and rebound elevation of ICP, all occurring minutes after termination of SNBC, are likely to be detrimental to an already ischemic brain. y Results Evaluation of 47 patients who underwent TTM resulted in a total of 505 patient-days of TTM with 6,967 BSAS hours. Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) 2 yg Methods A prospective, observational study during therapeutic hypothermia (24 hours –33°C) in 82 post-CA patients monitored with near-infrared spectroscopy. Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) y Results Forty-three patients (52%) survived in CPC 1 to 2 until 180 days post CA. The mean MAP range associated with maximal survival was 76 to 86 mmHg (OR = 2.63, 95% CI (1.01; 6.88), P = 0.04). The mean SVO2 range associated with maximal survival was 67 to 72% (OR  = 8.23, 95% CI (2.07; 32.68), P  = 0.001). In two separate multivariate models, a mean MAP (OR = 3.88, 95% CI (1.22; 12.33), P = 0.02) and a mean SVO2 (OR = 8.79, 95% CI (1.69; 18.36), P = 0.01) in the optimal range persisted as independently associated with increased survival after correction for presence of early bystander CPR and presenting shockable rhythm. Based on more than 1,625,000 data points, we found a strong linear relation between SVO2 (range 40 to 90%) and average cerebral saturation (R2 = 0.86) and between MAP and average cerebral saturation for MAPs between 40 and 87 mmHg (R2 = 0.70). Based on our hemodynamic model, the optimal MAP and SVO2 were determined to be 87 mmHg and 72%. Introduction Therapeutic hypothermia (TH) is the international standard of care for all comatose patients after cardiac arrest, but criticism focuses on poor outcomes. We sought to develop criteria to identify American urban patients more likely to benefi t from TH. Introduction Therapeutic hypothermia (TH) is the international standard of care for all comatose patients after cardiac arrest, but criticism focuses on poor outcomes. We sought to develop criteria to identify American urban patients more likely to benefi t from TH. Methods A retrospective chart review of 107 consecutive adults under- going TH in downtown New Orleans from 2010 to 2014 yielded records for 99 patients with all 44 survivors or families contacted up to 4 years. Results Sixty-nine males and 38 females with a mean age of 60.2 years showed 63 dead (58%) and 44 survivors (42%). Presenting cardiac rhythm was divided into shockable (pulseless ventricular tachycardia, ventricular fi brillation) and nonshockable (pulseless electrical activity, asystole). Presenting in shockable rhythms with ROSC <20  minutes were 21 patients with 15 (71%) survivors (P = 0.001). P427 Pharmacologic evaluation of shivering management in neurologically injured patients utilizing therapeutic normothermia T Lam, B Heather, J Jancik Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2015, 19(Suppl 1):P427 (doi: 10.1186/cc14507) Overall, patients spent 85.5% of total hours at BSAS 0, 11.4% of total hours at BSAS 1, 2.5% of total hours at BSAS 2, and 0.6% of total hours at BSAS 3. Patients were in tier 0 of the anti-shivering protocol 33.1% of the time, in tier 1 of the anti-shivering protocol 20.6% of the time, in tier 2 of the anti-shivering protocol 43.6% of the time, and in tier 3 of the anti- shivering protocol 2.8% of the total duration of TTM. There were 487 rescue doses of fentanyl and 243 rescue doses of meperidine that were required for shivering. Patients had a mean ICU LOS of 19 days, mean hospital LOS of 21 days, and a mortality rate of 23.4%.fi P426 Conclusion This study demonstrates a high level of effi cacy of our protocol and the feasibility of de-escalation to limit the number of pharmacologic interventions. With our patient population spending a large percentage of time without shivering, it would suggest that this protocol could be revised further by utilizing rescue agents more frequently in order to prevent escalation of therapy to the next tier. References Hemodynamic targets during therapeutic hypothermia after cardiac arrest: a prospective observational study K Ameloot, I Meex, C Genbrugge, W Boer, F Jans, B Ferdinande, W Mullens, M Dupont, C Dedeyne, J Dens ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P426 (doi: 10.1186/cc14506) 1. Badjiatia N, et al. Crit Care Med. 2009;37:S250-7. 2. Choi HA, et al. Neurocrit Care. 2011;14:389-94. 1. Badjiatia N, et al. Crit Care Med. 2009;37:S250-7. 2. Choi HA, et al. Neurocrit Care. 2011;14:389-94. Introduction In analogy with sepsis, current postcardiac arrest (post- CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVO2 above 70%. This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival. Introduction In analogy with sepsis, current postcardiac arrest (post- CA) guidelines recommend to target mean arterial pressure (MAP) above 65 mmHg and SVO2 above 70%. This is unsupported by mortality or cerebral perfusion data. The aim of this study was to explore the associations between MAP, SVO2, cerebral oxygenation and survival. Methods A prospective, observational study during therapeutic hypothermia (24 hours –33°C) in 82 post-CA patients monitored with near-infrared spectroscopy. P428 Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) Is selective nasopharyngeal brain cooling detrimental to neuroprotection? M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA M Kumar, L Johnson, A Goldberg, M Kashiouris, L Keenan, A Rabinstein Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P425 (doi: 10.1186/cc14505) Introduction Clinical outcomes vary depending on the method used to induce therapeutic hypothermia following stroke or cardiac arrest. In swine, we tested the hypothesis that selective nasopharyngeal brain cooling (SNBC), in contrast to systemic hypothermia, adversely impacts cerebral perfusion. Methods In two groups of animals (34 to 35  kg), blood fl ow in the right middle cerebral artery (MCA) was measured using transcranial Doppler (TCD). In group 1, SNBC was initiated using perfl uorohexane aerosol (1  ml/kg/minute) and oxygen (1  l/kg/minute) through a nasopharyngeal catheter atomizer. In group 2, the animals were body surface cooled using water-circulating blankets (4°C). In both groups, Conclusion ICU survivors were more likely to have had a shockable rhythm, been to the cathlab, received PPCI and TH and been less sick than nonsurvivors, but these may simply refl ect selection and other biases. Any benefi t these factors did confer to cardiac patients may have been off set by our liberal ICU admission policy to OHCAs with S150 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 brain temperature, intracranial pressure (ICP), core temperatures and vital signs were continuously recorded. Cooling was terminated once the brain reached 32°C and the animals were allowed to passively rewarm. Results In the SNBC group, the brain target temperature was reached in 54 ± 11 minutes. The mean ICP dropped precipitously to a nadir of –15 mmHg. TCD showed signifi cant vasospasm in the MCA, compared with the internal carotid artery (ICA), during the entire cooling phase (Table 1). Upon termination of cooling, the brain temperature spontaneously rewarmed to core temperature in 13  ± 4  minutes. Rewarming was associated with hyperemia and elevation of ICP. In group 2, there was no cerebral vasospasm or hyperemia during cooling and rewarming respectively. Conclusion SNBC is associated with signifi cant vasospasm of the MCA. In addition, spontaneous and rapid rewarming of the brain, vasodilation, rapid reperfusion, and rebound elevation of ICP, all occurring minutes after termination of SNBC, are likely to be detrimental to an already ischemic brain. Is selective nasopharyngeal brain cooling detrimental to neuroprotection? P426 Hemodynamic targets during therapeutic hypothermia after cardiac arrest: a prospective observational study K Ameloot, I Meex, C Genbrugge, W Boer, F Jans, B Ferdinande, W Mullens, M Dupont, C Dedeyne, J Dens ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P426 (doi: 10.1186/cc14506) Introduction In analogy with sepsis current postcardiac arrest (post- Table 1 (abstract P425). Cerebral vasospasm during SNBC MCA fl ow ICA fl ow velocity velocity Lindegaard Pulsatility ICP Intervention (cm/second) (cm/second) ratio ratio (mmHg) Baseline 62 41 1.51 0.77 13 SNBC cooling 128 52 2.46 0.48 –15 SNBC rewarming 96 44 1.74 1.12 21 Table 1 (abstract P425). Cerebral vasospasm during SNBC Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest p g C Endisch, C Storm, CJ Ploner, C Leithner C Endisch, C Storm, CJ Ploner, C Leithner Methods With IRB approval, we prospectively measured rSO2 during ALS in consecutive OHCA patients. One sensor of the Equanox™ 7600 (NONIN) was applied on the patient’s forehead’s right side when the medical emergency team arrived in an OHCA. ROSC was defi ned as ROSC >20 minutes. Charité Universitätsmedizin Berlin, Germany Charité Universitätsmedizin Berlin, Germany Critical Care 2015, 19(Suppl 1):P431 (doi: 10.1186/cc14511) y Critical Care 2015, 19(Suppl 1):P431 (doi: 10.1186/cc14511) Introduction Electrical median nerve stimulation elicits a burst of high-frequency oscillations (HFOs) superimposing onto the cortical short-latency potential. Digital fi ltering of somatosensory evoked potentials (SSEPs) enables non-invasive analysis of these HFOs. The late HFO component following the cortical N2O peak is ascribed to spiking activity of cortical neurons. Results We included 88 prehospital cardiac arrest patients between December 2011 and October 2014 with eight (9%) patients with CPC 1 or 2. Twenty-seven patients of the nonsurvivors had ROSC >20 minutes and one patient had CPC 3 at hospital discharge. We observed no signifi cant diff erence between both groups in age (P  = 0.161), time between emergency call and start of ALS (P  = 0.788) and duration of basic life support performed by bystanders, general practitioners or paramedics (P  = 0.649). The initial rhythm was asystole in one (12.5%) survivor and in 50 (62.5%) nonsurvivors (P = 0.009), ventricular fi brillation in six (75%) survivors and 13 (16%) nonsurvivors (P = 0.001), and pulseless electrical activity in one (12.5%) survivor and 17 (21%) nonsurvivors (P = 1.00). The cardiac arrest was witnessed in all survivors (100%) and in 49 (61%) nonsurvivors (P = 0.046). First measured rSO2 was 38% (27 to 67) in the survivor group compared with 22% (8 to 32) in the nonsurvivor group (P = 0.004). Also a signifi cant diff erence was found in mean rSO2 until 1  minute before ROSC between survivors and nonsurvivors, respectively 46% (40 to 74) and 34% (22 to 42). We observed no signifi cant diff erence in increase of rSO2 until 1 minute before ROSC between survivors 12.5% (5 to 21) and nonsurvivors 11% (5 to 18) (P = 0.719). Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) Time >20 minutes until ROSC in shockable rhythms had fi ve patients with three survivors (78%, P  = 0.001). Presenting in nonshockable rhythms with ROSC <20  minutes were 54 patients with 18 survivors (33%, P  = 0.001). ROSC >20 minutes in nonshockable rhythms had 19 patients with two survivors (8%, P = 0.001). Survivors of shockable rhythms showed 19 (100%) living post TH. Fifteen survivors (79%, n = 19, P = 0.001) had CPC Conclusion The optimal SVO2 (72%) and MAP (87  mmHg) derived from our hemodynamic model matched with the observed SVO2 (67 to 72%) and MAP (76 to 86 mmHg) associated with maximal survival. Prospective interventional studies to reach or maintain these targets are needed to confi rm these fi ndings. S151 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of this study were to investigate the association between hemoglobin, cerebral oxygenation (SctO2) and outcome in post-CA patients. score 1 or 2 with four survivors (21%, n = 19) having a CPC score of 3. A total of 25 survived nonshockable rhythm. Acute survival of patients with nonshockable rhythm showed 18 expired <72 hours (72%, n = 25) with long-term survival of four patients (5%, n = 74) and CPC scores of 1 or 2 (P = 0.001). Interestingly, patients with time to ROSC <20 minutes exhibiting more than one loss of sustained ROSC showed 100% mortality (P = 0.001). Patients presenting with shockable >20 minutes ROSC had overall survival of 70% (P = 0.001), but those undergoing >3 cardiac rhythm changes had 100% mortality (P = 0.001). yg 2 Methods A prospective observational study in 82 post-CA patients during hypothermia in the fi rst 24 hours of ICU stay. Hemoglobin was determined hourly together with a corresponding SctO2 by NIRS and SVO2 in patients with a pulmonary artery catheter (n = 62). 2 Results Based on 2,099 paired data, we found a strong linear relationship between hemoglobin and average SctO2 (SctO2 = 0.70 × hemoglobin + 56 (R2 = 20.84, P = 10–6)). Given the previously suggested SctO2 target between 66 and 68%, hemoglobin levels below 10  g/dl generally resulted in suboptimal brain oxygenation. Forty-three patients (52%) had a good neurological outcome (CPC 1 to 3) at 180 days post CA. Somatosensory evoked high-frequency oscillations and prognostication after cardiac arrest y Methods We retrospectively studied late HFO components of median nerve SSEPs obtained 24  hours to 4  days after cardiac arrest in patients treated with mild hypothermia (33°C for 24 hours). Cortical average recordings were digitally fi ltered at 450 to 750 Hz and noise- corrected maximum peak-to-peak amplitudes of the cortical late HFO bursts determined. Outcome upon ICU discharge was dichotomized according to the Cerebral Performance Category (CPC) scale. CPC 1 to 3 was classifi ed as good outcome, CPC 4 to 5 (unresponsive wakefulness syndrome and death) as poor outcome. Results Of 307 consecutive patients, 153 (50%) achieved good outcome (CPC 1 to 3) and 154 (50%) had poor outcome. Late HFO bursts were present in 102 (33%) recordings. Among patients with late HFO amplitudes above 0.1 μV, 26 had CPC 1 to 3, none had CPC 4 and eight died. Case review indicated causes of death other than hypoxic encephalopathy in all patients who died despite HFO amplitudes above 0.1 μV. Conclusion We found cortical late HFO bursts, obtained by digital fi ltering of standard SSEP recordings, in a signifi cant proportion of patients after cardiac arrest treated with mild hypothermia. Our data indicate that the presence of late HFO bursts with amplitudes above 0.1 μV may confi rm absence of severe hypoxic encephalopathy early after cardiac arrest with high specifi city. Conclusion We observed a signifi cant diff erence in fi rst measured rSO2 and mean rSO2 until 1 minute before ROSC between patients with good neurological outcome (CPC 1 or 2) at hospital discharge and patients with worse neurological outcome or nonsurvivors (CPC 3 or 4 or 5). However, no signifi cant diff erence was observed in the increase between both groups. Larger studies are necessary to confi rm these results. P432 P432 One-size-fi ts-all or patient-tailored hemodynamic targets in post-cardiac arrest patients: an observational near-infrared spectroscopy study on cerebral autoregulation C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) Predictors of survival of therapeutic hypothermia based on analysis of a consecutive American inner-city population over 4 years BR Roberts, HT Toca, JM Martinez Louisiana State University Health Sciences Center – New Orleans, LA, USA Critical Care 2015, 19(Suppl 1):P428 (doi: 10.1186/cc14508) There was a signifi cant association between average hemoglobin above 12.3 g/dl and good neurological outcome (OR = 2.88, 95% CI = 1.02; 8.16, P = 0.04). In a multivariate model, this association persisted after correction for comorbidities and age by the modifi ed Charlson score (OR = 2.99, 95% CI = 1.05; 8.53, P = 0.03). This association was entirely driven by results obtained in patients with an average SVO2 below 70% (OR = 17.55, 95% CI = 1.67; 184.41, P = 0.01). Conclusion Patients presenting with shockable rhythms undergoing TH had overall acute survival of 70% followed by long-term survival of 100% after 4 years. In contrast, patients presenting with nonshockable rhythm had long-term survival of 5%. TH is not recommended. Diff erence in cerebral saturation during cardiopulmonary resuscitation between survivors with favorable neurological outcome and compromised neurological outcome at hospital discharge C Genbrugge1, W Boer1, I Meex2, F Jans1, C Deyne1, J Dens1 1ZOL, Genk, Belgium; 2Hasselt University, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P429 (doi: 10.1186/cc14509) Diff erence in cerebral saturation during cardiopulmonary resuscitation between survivors with favorable neurological outcome and compromised neurological outcome at hospital discharge C Genbrugge , W Boer , I Meex , F Jans , C Deyne , J Dens 1ZOL, Genk, Belgium; 2Hasselt University, Hasselt, Belgium Critical Care 2015, 19(Suppl 1):P429 (doi: 10.1186/cc14509) Conclusion There is a steep linear relationship between hemoglobin and SctO2 in post-CA patients with hemoglobin levels below 10 g/dl generally resulting in cerebral desaturation. Average hemoglobin below 12.3 g/ dl was independently associated with worse neurological outcome 180  days post CA. An interventional trial is necessary to investigate whether maintaining higher hemoglobin would improve outcome. Introduction During out-of-hospital cardiac arrest (OHCA) monitoring possibilities are limited. Recently, the role of cerebral oximetry, using near-infrared spectroscopy, during ALS was investigated. In this study we determined whether the magnitude of increase in cerebral saturation (rSO2) or mean rSO2 during prehospital ALS was associated with good neurological outcome at hospital discharge (Cerebral Performance Category (CPC) 1 or 2). P431 Response of regional oxygen saturation technologies during hypoxia Results Thirty-four simplifi ed EEG samples were analysed. According to standard EEG, 11 patients showed a DS pattern, three had CI, six showed BS, four showed PEDs and 10 had an SE. Neurophysiologists interpreted all samples with a high accuracy. Mean sensitivity was 82.12% and mean specifi city was 91.88%. Only one SE was missed by one neurophysiologist. Unfortunately, only one PED was confi rmed by both neurophysiologists. Interobserver reliability was high (κ  = 0.843). High correlations were found for the comparison of full and simplifi ed EEG for both neurophysiologists (r = 0.809). Further, the two inexperienced physicians identifi ed SE with a sensitivity of 85% and specifi city of 98%. Introduction The purpose of this study was to determine the rate and magnitude of response to hypoxia for three diff erent regional oxygen saturation (rSO2) devices. rSO2 technologies are focused on absolute accuracy without consideration of response characteristics. Current rSO2 technologies assume that the oxygen saturation is a fi xed ratio of arterial and venous blood. Cerebral arteries have an oxygenation- related vasoactivity that may change the arterial/venous ratio during hypoxia. Thus, absolute rSO2 accuracy may be less important compared with sensitivity to changes in cerebral rSO2. pi y Conclusion Simplifi ed EEG monitoring, using BIS, resulted in high accuracy of a simple classifi cation system in post-CA patients. Not only neurophysiologists, but also treating physicians were capable to identify SE, which may play an important role in the early detection of SE. We suggest using BIS as a screening tool in post-CA patients to save valuable time in the detection of SE, without replacing the need for full EEG monitoring for confi rmation. 2 Methods Ten subjects completed the study and are included in the analysis. One INVOS sensor (SAFB-SM) was placed on the left side and one Equanox (8000CA) or Foresight (1 July 2007 or 1 July 2005) sensor (alternated between subjects) was placed on the right side of the forehead for bilateral monitoring. Desaturation was induced by adjusting the inspiratory gas mixture of O2/N2. Desaturation was titrated from room air to achieve a plateau of 70% arterial oxygen saturation (SpO2). Resaturation was induced by rapid change in FiO2 to 1.0. After 5  minutes of SpO2 100%, the process was repeated by desaturation to SpO2 70% and rapid return to SpO2 100%. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients neurologic outcome in postcardiac arrest patients I Meex, K Ameloot, C Genbrugge, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P430 (doi: 10.1186/cc14510) neurologic outcome in postcardiac arrest patients I Meex, K Ameloot, C Genbrugge, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P430 (doi: 10.1186/cc14510) p py y g C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) C Genbrugge, K Ameloot, I Meex, W Boer, F Jans, W Mullens, M Dupont, B Ferdinande, J Dens, C Dedeyne ZOL Genk, Belgium Critical Care 2015, 19(Suppl 1):P432 (doi: 10.1186/cc14512) Introduction The safety of a restrictive transfusion threshold of 7 g/dl applied in all critically ill patients can be questioned in postcardiac arrest (post-CA) patients since these are phenotypically clearly distinct. The aims Introduction A subgroup of post-CA patients with disturbed cerebral autoregulation might benefi t from higher mean arterial pressures S152 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 in seconds and the magnitude of absolute change may result in better resolution to detect physiological changes. Clinical studies are required to elucidate the clinical relevance of the diff erences. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. Methods A prospective observational study in 51 post-CA patients monitored with near-infrared spectroscopy. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. (MAPs). We aimed to (1) investigate whether patients with disturbed autoregulation have a worse prognosis, (2) phenotype these patients, (3) defi ne an individual optimal MAP and (4) investigate whether time under this individual optimal MAP is associated with outcome. p Methods A prospective observational study in 51 post-CA patients monitored with near-infrared spectroscopy. Diff erences in cerebral saturation measured during prehospital advanced life support, between patients with presumed cardiac origin and noncardiac origin of cardiac arrest C Genbrugge1, W Boer1, K Anseeuw2, I Meex3, F Jans1, J Dens1, C De Deyne1 2 g p Results The rate of rSO2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was signifi cantly faster for INVOS (42 ± 16) compared with Foresight (57 ± 20) (P <0.05). There was signifi cant diff erence in total rSO2 change between INVOS (23 ± 4%) and Equanox (15 ± 4%) during desaturation and resaturation (P <0.005) and between INVOS compared with Foresight (20 ± 5%) (P <0.05). Conclusion All rSO2 devices followed the SpO2 slope during desaturation as expected. The diff erences between the devices in terms of total rSO2 change reached statistical signifi cance. There were also signifi cant diff erences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change 2 Results The rate of rSO2 change during desaturation was similar for all devices with an average slope factor of 0.17 for Foresight, 0.16 for Equanox and 0.21 for INVOS. The rate of rSO2 change in seconds during resaturation from SaO2 70% to SaO2 100% was signifi cantly faster for INVOS (42 ± 16) compared with Foresight (57 ± 20) (P <0.05). There was signifi cant diff erence in total rSO2 change between INVOS (23 ± 4%) and Equanox (15 ± 4%) during desaturation and resaturation (P <0.005) and between INVOS compared with Foresight (20 ± 5%) (P <0.05). Introduction During out-of hospital cardiac arrest (OHCA) cerebral saturation may provide relevant information on cerebral oxygenation. In this study we examined the time course in cerebral saturation (rSO2) during prehospital ALS comparing patients with a presumed cardiac origin (survivor = Sc, nonsurvivor = NSc) of arrest and noncardiac origin (survivor = Snc, nonsurvivor = NSnc) of arrest. Conclusion All rSO2 devices followed the SpO2 slope during desaturation as expected. The diff erences between the devices in terms of total rSO2 change reached statistical signifi cance. There were also signifi cant diff erences in the rate of rSO2 change in seconds between INVOS and Foresight during resaturation. The rate of absolute change Methods With IRB approval, we prospectively measured rSO2 from the start of ALS in consecutive OHCA patients. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients P434 P434 Use of bispectral index EEG monitoring for a fast and reliable detection of epileptic activity in postcardiac arrest patients J Haesen1, L Desteghe1, I Meex2, C Genbrugge2, J Demeestere2, J Dens2, L Ernon2, C De Deyne2 1Universiteit Hasselt, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium Critical Care 2015, 19(Suppl 1):P434 (doi: 10.1186/cc14514) p py Results (1)  In multivariate analysis, patients with preserved auto- regulation (33.65%) had a signifi cant higher 180-day survival rate (OR = 4.62, 95% CI (1.06; 20.06), P = 0.04). (2) Phenotypically, a higher proportion of patients with disturbed autoregulation had pre- CA hypertension (31  ± 47 vs. 65  ± 49%, P  = 0.02) suggesting that right shifting of autoregulation is caused by chronic adaptation of cerebral blood fl ow to higher blood pressures. Based on an index of autoregulation (COX), the average COX-predicted optimal MAP was 85 mmHg in patients with preserved and 100 mmHg in patients with disturbed autoregulation. (3)  An individual optimal MAP could be determined in 33/51 patients. (4) The time under the individual optimal MAP was negatively associated with survival (OR = 0.97, 95% CI (0.96; 0.99), P = 0.02). The time under previously proposed fi xed targets (65, 70, 75, 80 mmHg) was not associated with a diff erential survival rate. Conclusion Cerebral autoregulation was shown to be disturbed in 35% of post-CA patients of which a majority had pre-CA hypertension. Disturbed cerebral autoregulation within the fi rst 24 hours after CA is associated with a worse outcome. In contrast to uniform MAP goals, the time spent under a patient-tailored optimal MAP, based on an index of autoregulation, was negatively associated with survival. Results (1)  In multivariate analysis, patients with preserved auto- regulation (33.65%) had a signifi cant higher 180-day survival rate (OR = 4.62, 95% CI (1.06; 20.06), P = 0.04). (2) Phenotypically, a higher proportion of patients with disturbed autoregulation had pre- CA hypertension (31  ± 47 vs. 65  ± 49%, P  = 0.02) suggesting that right shifting of autoregulation is caused by chronic adaptation of cerebral blood fl ow to higher blood pressures. Based on an index of autoregulation (COX), the average COX-predicted optimal MAP was 85 mmHg in patients with preserved and 100 mmHg in patients with disturbed autoregulation. (3)  An individual optimal MAP could be determined in 33/51 patients. Response of regional oxygen saturation technologies during hypoxia Cerebral and pulse oximetry data were recorded during the study and the time of each FiO2 change and plateau was recorded. rSO2 levels at 10, 20, 40, 60 and 80% of the total SpO2 response were calculated for each device to assess the rate of rSO2 change. The rate of rSO2 change in seconds and total rSO2 change were compared. Association between hemoglobin, cerebral oxygenation and neurologic outcome in postcardiac arrest patients (4) The time under the individual optimal MAP was negatively associated with survival (OR = 0.97, 95% CI (0.96; 0.99), P = 0.02). The time under previously proposed fi xed targets (65, 70, 75, 80 mmHg) was not associated with a diff erential survival rate. Introduction Assessment of prognosis in postcardiac arrest (post- CA) patients became very challenging since the introduction of therapeutic hypothermia (TH). Continuous EEG monitoring has been proposed to improve prognostication; however, its use is limited due to diffi culties in ready interpretation. Thus emerges the need for a simple EEG montage. The bispectral index (BIS) monitor is a simplifi ed EEG system, mainly calculating an index ranging from 0 (isoelectric EEG) to 100 (full consciousness) to provide information on hypnotic depth of anaesthesia. The aim of the study was to validate the accuracy of simplifi ed EEG monitoring in a CA setting. pi g g Methods BIS monitoring (BIS VISTATM) was applied to collect frontotemporal data in TH-treated CA patients. A standard 19-channel EEG was performed after return to normothermia. Afterwards, small EEG frames coincident with the time of full EEG registration were extracted from the BIS monitor. We asked two neurophysiologists to indicate the presence of status epilepticus (SE), cerebral inactivity (CI), burst suppression (BS), periodic epileptiformic discharges (PEDs) or a diff use slowing pattern (DS). In addition, these samples were analysed by two inexperienced physicians, who were asked to indicate the presence of SE. P436 Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) C Storm, CJ Ploner, C Leithner Introduction Bilaterally absent cortical somatosensory evoked potentials (SSEPs) predict poor outcome after cardiac arrest. A threshold for the amplitude of early cortical SSEPs above which patients may survive with good outcome has not been determined. Thus, tolerable noise levels for the interpretation of cortical SSEPs are poorly defi ned. Furthermore, it has not been systematically investigated whether high amplitudes of cortical SSEPs may exclude severe hypoxic encephalopathy incompatible with re-awakening. Conclusion In patients with cardiac arrest and targeted temperature management at 33°C, an NSE serum concentration of >90 μg/l strongly indicates poor outcome. NSE producing tumors, acute brain diseases, severe hematologic diseases, use of an intra-aortic balloon pump and blood transfusions need to be considered as potential confounders. An NSE serum concentration of <17  μg/l largely excludes hypoxic encephalopathy incompatible with re-awakening. p p y p g Methods We prospectively studied SSEPs after median nerve stimulation obtained 24 hours to 4 days after cardiac arrest in patients treated with targeted temperature management at 33°C for 24 hours. Amplitudes of cortical SSEPs were determined, if at least two peripheral, spinal and cortical recordings per side were available, spinal potentials were bilaterally reproducible and cortical noise level was below 0.25 μV. Cortical SSEP amplitude was defi ned as largest amplitude of a reproducible cortical SSEP of four cortical recordings (two per side) within 50 milliseconds after stimulation. Outcome was assessed upon ICU discharge using the Cerebral Performance Category (CPC) scale. CPC 1 to 3 was defi ned as good outcome, CPC 4 to 5 as poor outcome. Results Of 318 consecutive patients examined, 293 had complete SSEP recordings with reproducible spinal potentials and cortical noise levels below 0.25 μV. Of those, 137 (47%) had good outcome and 156 (53%) had poor outcome. The lowest amplitude of the early cortical SSEPs in a survivor with good outcome was 0.62 μV. All 79 patients with amplitudes below this threshold had poor outcome. None of 27 patients who survived with CPC 4 (unresponsive wakefulness syndrome) had cortical SSEP amplitudes above 2.5 μV. Twenty-four patients with amplitudes above this limit died. Detailed case review indicated a cause of death other than hypoxic encephalopathy in these patients. P436 l Results Of 601 included patients, 309 (51%) had good outcome. An NSE serum concentration threshold of 90  μg/l predicted poor outcome with a positive predictive value of 0.98 and a sensitivity of 0.51. Three patients survived with good outcome despite an NSE serum concentration >90 μg/l. In two of these patients NSE elevations had been documented prior to cardiac arrest. One patient had a neuroendocrine tumor of the pancreas, the other patient suff ered from encephalitis of unknown etiology and an osteomyelofi brosis. Potential confounders in the third patient were an ovarian carcinoma, the use of an intra-aortic balloon pump and blood transfusions shortly after cardiac arrest. Only 16 of 205 patients with NSE <17  μg/l had poor outcome, the majority of these patients died from causes other than hypoxic encephalopathy. Diff erences in cerebral saturation measured during prehospital advanced life support, between patients with presumed cardiac origin and noncardiac origin of cardiac arrest One sensor (Equanox™ 7600; Nonin) was applied on the patient’s forehead’s right side when S153 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 the medical emergency team arrived at the OHCA setting. ROSC was defi ned as ROSC >20 minutes. Retrospectively, included patients were divided into two groups with respect to their presumed origin of arrest. Results Between December 2011 and October 2014, 113 OHCA patients were included. We observed a signifi cant diff erence in asystole and VF as initial rhythm between NSc and NSnc, respectively (P = 0.035 and P = 0.001). In both groups of NS, duration of ALS was signifi cant longer compared with the two S groups (P  = 0.001 in both comparisons). We observed no signifi cant diff erence in fi rst measured rSO2, mean rSO2 until 1 minute before ROSC and increase in rSO2 until 1 minute before ROSC (respectively P = 0.123, P = 0.501, P = 0.265) between Sc and Snc. However, when we compare the nonsurvivors of cardiac with noncardiac origin, we observed a signifi cant diff erence in mean rSO2 until 1 minute before ROSC, 35% (27 to 44) in the NSc group and 27 (21 to 34) in the NSnc group (P = 0.026). First measured rSO2 was 24.5% (13 to 34) in the NSc group and 14 (4 to 28) in the NSnc group (P = 0.069) trending to be signifi cantly diff erent. No signifi cant diff erence was observed in increase until 1 minute before ROSC between both groups of NS (P = 0.920). Signifi cant diff erences was observed in mean rSO2 until 1 minute before ROSC and increase in rSO2 between Snc and NSnc (P = 0.033; P <0.001) and between Sc and NSc (P = 0.001; P <0.001).i Prognostic value of neuron-specifi c enolase after cardiac arrest and targeted temperature management Prognostic value of neuron-specifi c enolase after cardiac arrest and targeted temperature management g p g K Streitberger, C Leithner, CJ Ploner, C Storm Charité Universitätsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P437 (doi: 10.1186/cc14517) K Streitberger, C Leithner, CJ Ploner, C Storm Charité Universitätsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P437 (doi: 10.1186/cc14517) Introduction The serum concentration of neuron-specifi c enolase (NSE) has been established as a highly specifi c predictor of poor outcome after cardiac arrest. Recent studies have indicated that patients treated with targeted temperature management at 33°C for 24 hours may have good outcome despite NSE serum concentrations considerably higher than the cutoff established for normothermic patients. The threshold above which survival with good outcome becomes very unlikely, its positive predictive value and sensitivity for prediction of poor outcome have not been established in this patient group. Furthermore, a threshold below which hypoxic encephalopathy may be largely excluded has not been determined. Methods From 2006 through 2014 we prospectively included in- hospital and out-of-hospital cardiac arrest patients treated with targeted temperature management at 33°C for 24  hours. The NSE serum concentration was determined 3 days after cardiac arrest and the outcome was assessed according to the Cerebral Performance Category (CPC) upon ICU discharge. CPC 1 to 3 was defi ned as good outcome and CPC 4 to 5 as poor outcome. Individual case review was performed in patients with good outcome despite very high NSE serum concentration and in patients with poor outcome despite very low NSE serum concentration. Conclusion We can conclude that NSc have a signifi cant higher mean rSO2 and trend to have a signifi cant diff erence in fi rst measured rSO2 compared with NSnc. However, no signifi cant diff erence was observed between Sc and Snc. P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522 Results One hundred and ninety-three patients (110 males, 83 females, mean age 48.2  ± 18.3  years) were studied. The mean time interval between collapse and onset of resuscitation was 2.3  ± 2.1  minutes. A total of 65.3% arrests were witnessed. Sustained ROSC occurred in 36.8% patients and the SOHD was 24.9%. The initial rhythm recorded during CPR was asystole in 133 patients, pulseless electrical activity in 21 patients and ventricular fi brillation/tachycardia (VF/VT) in 39 patients. SOHD for these rhythms was 8.3%, 33.3% and 76.9%, respectively. On univariate analysis, type of rhythm, witnessed arrests and time to resuscitation were associated with sustained ROSC and SOHD. On multivariate analysis, only type of rhythm, VF/VT (P = 0.000) and PEA (P  = 0.017), were signifi cantly associated with SOHD, while witnessed arrest and time to resuscitation were not. Introduction The association between blood alcohol level (BAL) on mortality and neurologic outcome in patients with polytrauma and head injury is not clear and the data in the literature are sometimes confl icting. Some studies suggest a possible neuroprotective eff ect of alcohol and increased survival while others show the opposite. The rationale for this study was to investigate whether BAL has any impact on presentation, neurologic outcome and survival in patients with combination of polytrauma and head injury. y j y Methods This is a retrospective study of 43 polytraumatized patients with head injury who were intubated and treated by the prehospital unit and transported to the trauma center. Patients were grouped according to their BAL into BAL+ (>0.5  mg/l) and BAL– (≤0.5  mg/l). Inclusion criteria were age ≥18, Injury Severity Score ≥16 and head Abbreviated Injury Scale (AIS) ≥3. Physiological parameters and outcome with respect to survival to hospital discharge (STHD) and functional outcomes were analyzed. Severity of injuries was measured using the Trauma–Injury Severity Score (TRISS) and head injury using AIS. Functional outcome was measured using the Glasgow Outcome Scale (GOS), Cerebral Performance Category (CPC) and Glasgow Coma Conclusion Sustained ROSC occurred in 36.8% patients and the SOHD was 24.9%. Outcome of cardiopulmonary resuscitation in cancer patients in an Indian tertiary cancer center Conclusion Ten percent of patients resuscitated after CA and admitted to the ICU become OD, consisting of up to 31% of the total number of OD in our center. Patients resuscitated after CA who suff er severe irreversible brain damage or are brain dead can thus substantially expand the donor pool. This justifi es extensive resuscitation eff orts, if not to save lives, then to save organs. This might be reassuring for families, staff and the community. Introduction Cardiopulmonary resuscitation (CPR) after cardiac arrest in cancer patients is often discouraged as it is associated with poor outcome. In our 700-bed tertiary cancer hospital in Mumbai, India, the ICU runs an in-hospital cardiac arrest team (CAT). We reviewed our data to determine outcome from CPR, identify factors associated with improved outcomes and justify the presence of a CAT in our cancer hospital. p Methods All in-hospital patients from November 2012 to November 2014 (2-year period) with unanticipated cardiorespiratory arrests were included. Data were recorded prospectively using the Utstein template. Only patients with cardiac arrest rhythms were included. Patients with anticipated progression towards arrest, those with seizures, hypotension without dysarrythmias or other medical emergencies were excluded. The outcomes studied were return of spontaneous circulation (ROSC) and survival on hospital discharge (SOHD). Binary logistic regression analysis was performed to determine factors associated with ROSC and SOHD. P436 Amplitudes of cortical somatosensory evoked potentials and outcome prediction after cardiac arrest C Storm, CJ Ploner, C Leithner Charite Universitaetsmedizin, Berlin, Germany Critical Care 2015, 19(Suppl 1):P436 (doi: 10.1186/cc14516) P438 38 Resuscitated cardiac arrest in patients admitted with acute heart failure: analysis of a large prospective AHEAD network registry J Parenica1, J Belohlavek2, J Spinar1, G Dostalova2, S Havranek2, A Linhart2 R Miklik1, L Dusek3, J Jarkovsky3 1University Hospital Brno, Czech Republic; 2General University Hospital, Prague, Czech Republic; 3Masaryk University, Brno, Czech Republic Critical Care 2015, 19(Suppl 1):P438 (doi: 10.1186/cc14518) Introduction Heart failure is a frequent cause of cardiac arrest and vice versa, cardiac arrest frequently complicates acute heart failure episodes. We aimed to characterize the infl uence of cardiac arrest on outcome of acute heart failure patients admitted to hospital. Methods The AHEAD registry includes patients hospitalized for acute heart failure from 10 PCI and fi ve non-PCI centers in the Czech Republic. The data were collected from September 2006 to October 2012. Conclusion Our data indicate that the prognostic value of SSEP after cardiac arrest extends beyond a mere absent–present dichotomy. Bilaterally absent as well as very low amplitude SSEPs predict poor outcome with high positive predictive value. SSEPs should not be used for prognostication, if noise in cortical recordings could mask critically low amplitudes. High amplitudes of early cortical SSEPs strongly argue against severe hypoxic encephalopathy incompatible with re-awakening. Results In the respective period, a total of 6,242 patients were enrolled into the registry. Resuscitated cardiac arrest occurred in 313 patients prehospitally and in 484 after admission; the remaining 5,445 patients were not resuscitated during their index hospitalization. Patients resuscitated after admission in comparison with prehospitally resuscitated were older, had lower left ventricle ejection fraction, more S154 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P438). Overall mortality (including hospitalization). Total n = 6,242. P440 Outcome after CPR: when we cannot save lives, we can save organs C Caestecker, J Froyman, P Lormans, W Stockman AZ Delta, Roeselare, Belgium Critical Care 2015, 19(Suppl 1):P440 (doi: 10.1186/cc14520) Introduction Patients resuscitated after cardiac arrest (CA) who suff er bad neurologic outcome or become brain dead might become organ donors (OD) when well managed and identifi ed. We report on the cohort of patients resuscitated after in-hospital or out-of-hospital CA becoming OD in a tertiary community hospital with an intensive donor identifi cation program. i g Methods Data from our 28-bed mixed medical/surgical adult ICU in a 900-bed tertiary hospital were analyzed from 2010 to 2014. y y Results Our ICU admitted 2,320 patients/year. Overall ICU mortality in this period was 7.4%. A summary of the results is presented in Table 1. Of the 219 patients admitted after CA, 21 (10%) became OD. This resulted in 55 successfully transplanted organs (28 kidneys, 17 livers, seven lung pairs, three hearts). Of note, good outcome (CPC 1 and 2) was achieved in 55 patients (25%). frequently suff ered cardiogenic shock, had more organ dysfunctions and died more frequently, respectively, with hospital mortality of 79.5% versus 29.1%, P <0.001; see also Figure 1. Table 1 (abstract P440) Organ Donors ICU admission Year donors after CA DCD/DBD after CA 2010 11 3 0/3 40 2011 9 5 3/2 41 2012 18 4 3/1 47 2013 17 5 4/1 53 2014 13 4 1/3 38 Total 68 21 (31%) 11/10 219 Conclusion In patients hospitalized for acute heart failure, both prehospital and postadmission resuscitated cardiac arrest is a severe complication associated with signifi cantly morbidity and mortality. Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Endothelial dysfunction in acute brain injury and the development of cerebral ischemia S Van Ierssel1, VM Conraads1, EM Van Craenenbroeck1, Y Liu2, AI Maas1, PM Parizel1, VY Hoymans1, CJ Vrints1, PG Jorens1 1University Hospital Antwerp, Edegem, Belgium; 2The Third Xiangya Hospital, Changsha, China Critical Care 2015, 19(Suppl 1):P443 (doi: 10.1186/cc14523) Introduction Since most patients with acute brain injury are treated head-up at 30 to 45°, there can be a height diff erence of up to 15 cm between the heart and the ear canal. This causes a diff erence between mathematical CPP and true CPP of up to 11 mmHg depending on the zero reference level used and the body length of the patient (Figure 1). We conducted a survey to analyze the current practice on CPP targets and zero reference levels in diff erent ICUs. Introduction Cerebral ischemia (CeI) is a major complicating event after acute brain injury (ABI) in which endothelial dysfunction is a key player. p y Methods We studied cellular markers of endothelial dysfunction and peripheral reactive hyperemia index (RHI) in 26 patients with ABI at admission and after 6 and 12 days, and compared these with healthy volunteers (n = 15). CeI was determined clinically or using computer tomography.i f Methods Neuro-ICU physicians were invited to participate in a survey on ICP and CPP targets and the level of measurement. Results The results of 30 centers are summarized in Table 1. Most centers (83.3%) use head-up elevation of 30 to 45° and consider an ICP of 20 mmHg as the threshold to start treating ICP (80%). More variation is noted in minimal and maximal CPP threshold. All centers measure ICP at the ear canal. Twenty-seven centers (90%) measure MAP at the heart, three centers measure MAP at the ear canal. These three centers use >50, 60 and 65 mmHg as minimal CPP and raise the bed to 30 to 45°. The two centers using minimal CPP >60 mmHg do not apply an upper limit. g p y Results In patients with ABI, RHI at admission was signifi cantly reduced compared with healthy subjects (P = 0.003), coinciding with a decrease in circulating endothelial progenitor cells (EPC) (P = 0.002) (Table 1). The RHI recovered in eight patients without development of CeI (Figure 1, black), but failed to fully recover by day 12 in three out of four patients that developed CeI (Figure 1, red). P442 Eff ect of alcohol in blood on neurological outcome and survival of patients with combination of polytrauma and head injury S Pristovnik1, M Strnad2, V Vujanoviæ1, T Pelcl1, V Borovnik-Lesjak1 1Health Center Dr. Adolf Drolc, Maribor, Slovenia; 2Medical Faculty Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P442 (doi: 10.1186/cc14522) A reduced response time, witnessed arrest and type of rhythm are associated with ROSC and improved SOHD. The type of rhythm was independently associated with SOHD, with VF/VT and PEA having improved survival while asystolic patients had a poor outcome. These considerations justify the presence of a CAT in our cancer hospital. S155 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P443). Evolution in time of markers of endothelial dysfunction after acute brain injury Scale (GCS) at discharge. Diff erences among groups were analyzed using Student’s t test, the Mann–Whitney U test and the chi-square test. Results BAL did not have a signifi cant eff ect on presenting physiological parameters. There was a signifi cant diff erence in the age of the groups, showing that patients in the BAL+ group were younger (32.6 vs. 56.8 years; 95% confi dence level; P = 0.000). BAL had a lasting eff ect on functional measures of neurological outcome which showed better results in the BAL+ group; it had signifi cantly better GOS (4.7 vs. 3.9; 95% confi dence level; P = 0.027), and GCS at discharge was on the border of statistical signifi cance (15 vs. 14; 95% confi dence level; P = 0.05). Other variables (TRISS, AIS, STHD, CPC) did not show statistically signifi cant diff erences among groups. gif g g p Conclusion Presence of alcohol in the blood had a positive eff ect on neurological outcome but there was no signifi cant diff erence in survival. However, the positive results in neurologic outcome in the BAL+ group may also be due to the fact that this group was younger. The small number of patients in the study is another limiting factor of the interpretation. Therefore, the neuroprotective role of alcohol needs further clarifi cation. Conclusion Patients with ABI exhibit impaired microvascular endo thelial function measured as RHI and a decreased circulating level of EPC. P444 P444 P444 Survey on ICP, target CPP and MAP measurement level in patients with severe acute brain injury in diff erent ICUs M Van Laer1, K Deschilder2, W Stockman2 1UZ Brussel, Brussels, Belgium; 2AZ Delta, Roeselare, Belgium Critical Care 2015, 19(Suppl 1):P444 (doi: 10.1186/cc14524) P445 with traumatic brain injuries defi ned as a Glasgow Coma Score <8 upon admission. Patients included were 18 to 59  years of age and were mechanically ventilated with intracranial pressure monitoring for greater than 72 hours. The primary outcome evaluated was the number of patients treated for confi rmed infections. Secondary outcomes included the antibiotic length of therapy (LOT), antibiotic days of therapy (DOT), number of cultures, and ICU and hospital length of stay (LOS). DOT was defi ned as the sum of days on which each antibacterial drug was administered. P445 Comparison of 15oxygen positron emission tomography and near-infrared spectroscopy for measurement of cerebral physiology J Simpson1, N Sudhan1, H Hare2, J Donnelly1, X Liu1, F Aigbirhio1, T Fryer1, G Stocks-Gee1, P Smielewski1, D Bulte2, J Coles1 1University of Cambridge, UK; 2University of Oxford, UK Critical Care 2015, 19(Suppl 1):P445 (doi: 10.1186/cc14525) Introduction The gold standard technique for imaging cerebral blood fl ow (CBF) and metabolism is 15oxygen positron emission tomography (15O PET). Continuous near-infrared spectroscopy (NIRS) has been used to assess adequacy of cerebral oxygenation following stroke, traumatic brain injury and subarachnoid haemorrhage [1], and measurements have been compared with jugular oxygen saturation. In this study we compared NIRS with 15O PET within healthy volunteers. g Results A total of 23 patients treated with normothermia and 119 patients in the control group were evaluated between January 2009 and September 2014. The number of patients treated for confi rmed infections was similar between groups (normothermia: 73.9%, control: 80%, P  = 0.803). Empiric antibiotic therapy was more commonly utilized in the normothermia group at 34% versus 20.5% (P = 0.173). Antibiotic LOT and DOT were 13.8 versus 10.8 days (P = 0.157) and 18.3 versus 16.2 days (P = 0.575) in the normothermia versus control groups, respectively. Total culture rate was lower in the normothermia group with 13.2 versus 8.78 (P = 0.0002) cultures per patient. No signifi cant diff erence in hospital LOS (normothermia: 23 days, control: 18 days, P = 0.158) or ICU LOS (normothermia: 17 days, control: 15 days, P = 0.185) was demonstrated.i y Methods Fifteen healthy subjects were recruited (12 male, average age 38 years); PET precluded females of reproductive age. Steady-state 15O PET with arterial sampling was performed to measure CBF, cerebral metabolic rate of oxygen (CMRO2), oxygen extraction ratio (OEF) and cerebral blood volume (CBV) [2]. g References 1. Zweifel C, et al. Stroke. 2010;41:1963-8. 1. Zweifel C, et al. Stroke. 2010;41:1963-8. 2. Coles JP, et al. JCBFM. 2004;24:202-11. 3. Culver JP, et al. JCBFM. 2003;23:911-24. f Results Sixty-four treatments (32 HTS, 32 Man) were studied among 26 patients (19 TBI, seven SAH; age 42  ± 17  years, time from injury to treatment 2.6 ± 1.9 days). Man and HTS eff ectively decreased ICP (coeffi cient = –2.5 mmHg, 95% CI = –3.2 to –1.8 mmHg and –2.9 (–3.8 to –2.0) respectively; both P < 0.0001). No signifi cant diff erence was found in CMD lactate, pyruvate, glucose and PbtO2 after HTS or Man treatment. CMD glutamate decreased signifi cantly after Man (–0.73 ( –1.41 to –0.052); P <0.05), but not after HTS. Eff ect of osmotherapy with mannitol or hypertonic saline on cerebral oxygenation and metabolism in patients with intracranial hypertension after severe brain injury yp j T Suys, H Quintard, C Patet, M Oddo Introduction Osmotherapy with mannitol (Man) or hypertonic saline (HTS) is currently used to treat elevated intracranial pressure after severe acute brain injury (sABI); however, their eff ect on cerebral oxygenation and metabolism has not been extensively evaluated. Methods A retrospective analysis of a cohort of patients with sABI after traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) monitored with cerebral microdialysis (CMD), brain oxygen (PbtO2) and ICP, who were treated with Man (20%, 0.5 g/kg) or HTS (7.5%, 100 ml) for ICP >25 mmHg. Osmotherapy was administered over 20 minutes and each patient’s individual response to intervention was analyzed up to 120 minutes following infusion. Only episodes where no other hypertonic solute was administered within 2  hours before or after treatment were selected. Variables analyzed included CMD lactate, pyruvate, glucose, glutamate, lactate/pyruvate ratio, and main brain physiologic variables ICP, PbtO2, CPP. Analysis was conducted using mixed-eff ects multilevel regression. Introduction Osmotherapy with mannitol (Man) or hypertonic saline (HTS) is currently used to treat elevated intracranial pressure after severe acute brain injury (sABI); however, their eff ect on cerebral oxygenation and metabolism has not been extensively evaluated. Conclusion We found no relationship between NIRS and baseline physiology as determined by 15O PET. Further studies should assess the dynamic response of NIRS to a measured change in physiology in patients. Further confi nes of NIRS include its limited and focal brain coverage. Conclusion We found no relationship between NIRS and baseline physiology as determined by 15O PET. Further studies should assess the dynamic response of NIRS to a measured change in physiology in patients. Further confi nes of NIRS include its limited and focal brain coverage. P445 Simultaneously, NIRS data were collected using a Hamamatsu NIRO 200 with sensors on either side of the forehead. NIRS OEF was calculated from tissue oxygen saturation, SaO2 and an assumed arterial/venous blood volume ratio of 30/70 [3]. Results The frontal region 15O PET CBF, CMRO2, OEF and CBV were mean (SD) 44.9 (10) ml/100 ml/minute, 158.7 (24.7) μmol/100 ml/minute, 45.8 (7.3)%, and 2.8 (0.8)  ml respectively, and there was no relationship between NIRS and 15O PET (Figure 1). Conclusion Rates of confi rmed infections and number of antibiotic days were similar between the normothermia and control groups, suggesting that normothermia does not increase infection risk. However, the number of cultures obtained in the control group was signifi cantly greater than the normothermia group with a trend toward increased empiric antibiotic use. Figure 1 (abstract P445). Linear correlation between NIRS and PET OEF. Reference Figure 1 (abstract P445). Linear correlation between NIRS and PET OEF. Reference 1. Greer DM, et al. Stroke. 2008;39:3029-35. 1. Greer DM, et al. Stroke. 2008;39:3029-35. Endothelial dysfunction in acute brain injury and the development of cerebral ischemia Despite recovery Figure 1 (abstract P443). y Conclusion Considering the infl uence of position on CPP, the variations among centers and the narrow range of CPP thresholds, future studies and guidelines should describe where MAP is measured. Alternatively, we propose a new formula for CPP: true CPP = MAP(heart) – ICP(ear) – height diff erence (heart to ear canal (cm)) × 0.73. Conclusion Considering the infl uence of position on CPP, the variations among centers and the narrow range of CPP thresholds, future studies and guidelines should describe where MAP is measured. Alternatively, we propose a new formula for CPP: true CPP = MAP(heart) – ICP(ear) – height diff erence (heart to ear canal (cm)) × 0.73. Figure 1 (abstract P443). Figure 1 (abstract P444). Table 1 (abstract P444) ICP threshold (mmHg); n (%) >15; 2 (6.7%) >18; 1 (3.3%) >20; 24 (80%) >25; 3 (10%) Minimal CPP (mmHg); n (%) >50; 6 (20%) >55; 4 (13.3%) >60; 16 (53.3%) >65 to 70; 4 (13.3%) Maximal CPP (mmHg); n (%) <70 to 75; 8 (26.7%) <80; 8 (26.7%) <85; 2 (6.7%) No limit; 12 (40%) Figure 1 (abstract P444). Figure 1 (abstract P444). S156 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P445 Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate C Patet1, H Quintard1, T Suys1, L Pellerin2, P Magistretti3, M Oddo1 1CHUV – Lausanne University Hospital, Lausanne, Switzerland; 2University of Lausanne, Switzerland; 3Brain Mind Institute, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P448 (doi: 10.1186/cc14528) Introduction In patients with acute brain injury (ABI), increased cerebral energy demand is frequent, potentially leading to cerebral glucose depletion (GD) and poor outcome. In this setting, lactate may act as supplemental fuel. We examined dynamics of cerebral lactate supply during prolonged GD in ABI. Results ICP registrations were made parallel with all TCD measurements in 51 patients. Intraparenchymal ICP monitoring was inserted within the fi rst 3 hours after trauma and there was no complication (infections, intracranial hemorrhage, or technical failure) related to invasive ICP monitoring. Increased ICP was noted upon transducer insertion in 38 children with male prevalence (10 girls, 28 boys). Median GCS was 6, indicating the magnitude of injury in this group of patients. The overall results of the 38 patients showed a strong correlation between the ICP and PI and during outbursts of ICP with a correlation coeffi cient of r = 0.89 (ICP >20 mmHg) and r = 0.90 (ICP <20 mmHg). The relation between ICP and PI was estimated by the linear regression equation: ICP = 22,299 × PI – 10,705 (ICP >20 mmHg) and ICP = 38,592 × PI – 16,972 (ICP <20 mmHg). The CPP and PI were correlated signifi cantly during the changes in intracranial pressure. However, a better correlation was found when ICP >20 mmHg and PPC <50 mmHg (PI was 2.4 ± 0.89 when CPP was 35.96 ± 4.48 with a correlation coeffi cient of Pearson r = 0.80) than when ICP <20 mmHg and CPP >50 mmHg (PI was 0.78 ± 0.14 when CPP was 57.11 ± 9.62 with a correlation coeffi cient of Pearson r =0.76). y Methods We retrospectively analyzed severe ABI (18 TBI, eight SAH) monitored with brain oxygen and cerebral microdialysis (CMD) to measure hourly levels of cerebral extracellular glucose, lactate, pyruvate and lactate/pyruvate ratio (LPR). Variations of CMD variables were analyzed as a function of GD (defi ned as spontaneous decreases of CMD glucose from normal to low (<1.0 mM), at least 2 hours) and increased cerebral energy demand (LPR >25). 1. Paolin A, Manuali A, Di Paola F, Boccaletto F, Caputo P, Zanata R, et al. Reliability in diagnosis of brain death. Intensive Care Med. 1995;21:657-62. 2. Gan TJ, Glass PS, Windsor A, Payne F, Rosow C, Sebel P, et al. Bispectral index monitoring allows faster emergence and improved recovery from propofol, alfentanil, and nitrous oxide anesthesia. Anesthesiology. 1997;87:808-15. Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury g Conclusion The BIS is a noninvasive, simple, and easy to interpret method, showing a perfect correlation with the other diagnostic methods. BIS can be used in severely comatose patients as an assessment of brain death. j y H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate Results During GD (60 episodes; 26 patients), we found an increase of CMD lactate (4 ± 2.3 vs. 5.4 ± 2.9 mM) and LPR (27 ± 6 vs. 35 ± 9; all P <0.005) while brain oxygen and blood lactate remained normal. Dynamics of lactate and glucose supply were studied by analyzing the relationship between blood and CMD samples. No correlation between blood and brain lactate was found when brain glucose and LPR were normal (r = –0.12, P = 0.48; Figure 1), while this correlation became linear during GD, progressively rising to r = 0.53 (P <0.0001) when energy demand increased, suggesting increased cerebral lactate availability. The correlation between blood and brain glucose changed in the opposite direction, decreasing from r = 0.78 to 0.37 (P <0.0001) during GD and at LPR >25. Conclusion The absolute value of the PI is a reliable noninvasive indicator of ICP in pediatric severe TBI. A strong correlation between PI and ICP was demonstrated. Therefore, PI may be of guiding value in the invasive ICP placement decision in the neurointensive care patient when ICP monitoring is not systematically performed. In particular, ICP monitoring remains as grade C in the latest guidelines of management of STBI in children published in 2012. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. P449 Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) Figure 1 (abstract P448). Correlations between blood and brain lactate. Figure 1 (abstract P448). Correlations between blood and brain lactate. P450 Bispectral index as a predictor of unsalvageable traumatic brain injury Introduction The aim was to evaluate the accuracy of bispectral index (BIS) monitoring for the diagnosis of brain death in severely comatose patients. We aimed to determine the utility of the BIS as a tool for clinical evaluation of the moment of brain death. Methods A prospective observational study with waiver of consent was conducted in the trauma ICU for 2 years from October 2012 to September 2014. Monitoring of BIS occurred during patient stay in the ICU. Population: 62 severely comatose patients (Glasgow Coma Score less than 6) admitted to the ICU mainly because of intracerebral hemorrhage, head injury, or postanoxic coma. BIS was recorded continuously during the hospitalization in the ICU. Where necessary, clinical brain death was confi rmed by EEG or brain stem test. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Conclusion Energy dysfunction is associated with increased supply of nonhypoxic cerebral lactate. Our data suggest lactate may act as alternative substrate after ABI when availability of cerebral glucose is reduced. Results Twenty-nine patients were already clinically brain dead at the time of admission, and their individual BIS values were 0. Twenty-four patients were not clinically brain dead at the time of admission, and their individual BIS values were between 20 and 30. These patients became brain dead, and their individual BIS values dropped to 0 in a few hours to a few days. Seventeen patients who did not become brain dead during their hospitalization in the ICU had persistent electrocerebral activity on EEG, and their average BIS values remained above 31. P449 Correlation between intracranial pressure and pulsatility index measured by transcranial Doppler in children with severe trauma brain injury H Bouguetof, M Negadi, K El Halimi, D Boumendil, Z Chentouf Mentouri University Ahmed Benbella Oran 1, Oran, Algeria Critical Care 2015, 19(Suppl 1):P449 (doi: 10.1186/cc14529) P448 Methods A prospective and descriptive study conducted in our PICU from June 2011 to December 2013. We investigated 51 children with severe trauma brain injury (TBI). The TCD measurements were routinely performed bilaterally on the middle cerebral artery parallel to the ICP registration. The ICP and CPP data were correlated to PI and the correlation coeffi cient calculated. To control the linear correlation, the residuals were tested for normal distribution around the regression line. P448 Neuroenergetic response to prolonged cerebral glucose depletion after severe brain injury and the role of lactate C Patet1, H Quintard1, T Suys1, L Pellerin2, P Magistretti3, M Oddo1 1CHUV – Lausanne University Hospital, Lausanne, Switzerland; 2University of Lausanne, Switzerland; 3Brain Mind Institute, Lausanne, Switzerland Critical Care 2015, 19(Suppl 1):P448 (doi: 10.1186/cc14528) Evaluation of infection risk and antibiotic exposure in traumatic brain injury patients treated with therapeutic normothermia h k , , Hennepin County Medical Center, Minneapolis, MN, USA Hennepin County Medical Center, Minneapolis, MN, USA p y p Critical Care 2015, 19(Suppl 1):P446 (doi: 10.1186/cc14526) Introduction The purpose of this study is to assess the rate of confi rmed infections, antibiotic exposure, and monitoring practices with normothermia protocol utilization for traumatic brain injury patients. Treatment and prevention of fever is a focus of therapy for patients with severe neurological injury as fever has been identifi ed as an independent risk factor for morbidity and mortality [1]. Conclusion Osmotherapy with Man and HTS treatment had no eff ect on cerebral oxygenation and metabolism. Man, but not HTS, favorably reduced brain glutamate. These fi ndings support further investigation to test the value of alternative osmotic agents (such as hypertonic lactate) that may reduce ICP and at the same time improve cerebral metabolism after sABI. Methods The retrospective chart review analyzed outcomes of maintaining normothermia at 36.5°C versus a similar population without temperature control as a standard of care in patients admitted Acknowledgements Supported by grants from the Swiss National Science Foundation and the Novartis Foundation for Biomedical Research. S157 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P448 References Introduction This study was designed to see whether there is a correlation between the transcranial Doppler (TCD) parameters and the CCP and intracranial pressure (ICP) monitoring during the cerebral hemodynamic changes and to evaluate ICP indirectly by TCD. S158 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 PbtO2 decreased and the microdialysis showed continuous signs of ischemia and cellular degradation. P451 Neuromonitoring of patients with severe traumatic brain injury at the bedside M Aries1, JG Regtien1, M Czosnyka2, J Donnelly2, P Smielewski2 1UMCG, Groningen, the Netherlands; 2Addenbrookes Hospital, University of Cambridge, UK Critical Care 2015, 19(Suppl 1):P451 (doi: 10.1186/cc14531) Conclusion This experimental study documents that during protracted pronounced hemorrhagic shock, cerebral energy metabolism was severely compromised and exhibited a biochemical pattern typical of ischemia and cellular degradation. After retransfusion this pattern continued. From intravenous microdialysis in the sagittal sinus, it is possible to achieve semiquantitative information of the intracerebral redox state. Accordingly, it might be possible to monitor the global cerebral energy state continuously with a strictly extracerebral technique. This technique might be valuable in various severe conditions during critical care when cerebral energy metabolism may be compromised; for example, resuscitation after cardiac standstill, open heart surgery, multitrauma and so forth. Interestingly the study also showed that after reinfusion of blood other parts of the body recovered, evaluated by microdialysis, but the brain showed signs of damage, making the brain the limiting organ in hemorrhagic shock. Introduction Measurement of intracranial pressure (ICP) and arterial blood pressure is used to derive cerebral perfusion pressure (CPP) and to guide targeted therapy of severe traumatic brain injury (TBI) necessitating ICU admission. In this review we discuss the evidence for ICP monitoring, CPP calculation, and ICP/CPP-guided therapy after severe TBI. Despite its widespread use, there is currently no class I evidence that ICP/CPP-guided therapy improves outcomes. Similarly, no class I evidence can currently advise the ideal CPP. y Methods A review of current literature with special focus on autoregulation (PRx)-guided CPP treatment in TBI patients.i Results Optimal CPP is probably patient, time, and pathology specifi c and related to cerebral autoregulation status. P453 P453 Amantadine sulfate treatment in cases with brain injury in the ICU: a retrospective clinical trial S Goksu Tomruk, N Bakan, G Karaören, A Salvarcý Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P453 (doi: 10.1186/cc14533) P452 y Results The patients’ diagnoses included two post-CPR, fi ve intracranial and one subdural hematoma, one CVA, one postoperative aneurysm, one subarachnoid hemorrhage and one brain contusion. Table  1 (overleaf) presents the fi ndings. The AS therapy was initiated between days 3 and 33 of admission in all patients other than Patients 2 and 8. A dramatic improvement was observed in a patient with both GCS and JFK- CRS score of 5 when AS therapy was initiated in month 5 and the patient was discharged for home care. In Patient 9, AS therapy was withdrawn on day 5 due to persistent thrombocytopenia (TP) despite exclusion of other reasons; subsequently, improvement was observed in TP. The complications were relatively less severe with average acceptability. p p y References 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2. Jaeger M, et al. Crit Care Med. 2010;38:1343-7. 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2. Jaeger M, et al. Crit Care Med. 2010;38:1343-7. 1. Aries MJ, et al. Crit Care Med. 2012;40:2456-63. 2 Jaeger M et al Crit Care Med 2010;38:1343 7 Amantadine sulfate treatment in cases with brain injury in the ICU: a retrospective clinical trial S Goksu Tomruk, N Bakan, G Karaören, A Salvarcý Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P453 (doi: 10.1186/cc14533) Introduction Improvement of recovery is a challenging process in cases with varying degrees of severe brain injury (BI) requiring intensive care. Amantadine sulfate (AS) is recommended for use in cases with brain injury. The Coma Remission Scale (JFK-CRS) consists of auditory–visual– motor–mouth–tongue functions, communication and awareness scales; provides a score between 0 and 23; and shows numeric recovery from coma. The aim of this study was to evaluate outcomes and eff ects of AS used for neurorecovery on the Glasgow Coma Scale (GCS) and JFK-CRS in our ICUs during the last 5 months. Conclusion The management of TBI is likely to become increasingly based on a more comprehensive monitoring and management approach rather than relying on absolute numbers of ICP and CPP in isolation. This will allow individual optimization of perfusion and therefore of oxygen and energy substrate delivery. We await further robust, high-quality evidence to support the benefi ts of using more sophisticated monitoring tools like the autoregulation- guided CPP concept during the ICU management of TBI. For the near future, more important is a greater understanding of the underlying pathophysiology. Methods After approval of the Ethics Committee, we recruited 12 patients with brain injury resulted from trauma or hemorrhage who had initial GCS of ≤9 and received AS (500 mg, twice daily over 10 days) during the recovery period. In all subjects, age, gender, diagnosis, initial APACHE II score, time of initiation of AS therapy, JFK-CRS and GCS scores, aspartate aminotransferase, alanine aminotransferase, BUN, creatinine, platelet count, electrocardiography fi ndings, electrolyte values and arterial blood gas values on days 1, 6, 10 and 14 were recorded.i Technique for continuous bedside monitoring of the global cerebral energy state y R Jakobsen1, A Granfeldt2, TH Nielsen1, P Toft1, CH Nordström1 1Odense University Hospital, Odense, Denmark; 2Regional Hospital of Randers, Denmark Critical Care 2015, 19(Suppl 1):P452 (doi: 10.1186/cc14532) Introduction In the present experimental study we explore whether cerebral venous lactate/pyruvate ratio (LP ratio) measured by intra- vascular microdialysis during induced hemorrhagic shock may be used as a surrogate marker for compromised cerebral oxidative metabolism. Methods Six female pigs were anesthetized and vital parameters was recorded. Microdialysis catheters were placed in cerebral hemisphere parenchyma, the superior sagittal sinus and femoral artery. Brain tissue oxygenation (PbtO2) and intracranial pressure (ICP) was recorded. Hemorrhagic shock was achieved by bleeding the animals to a mean arterial pressure (MAP) of approximately 35 mmHg. Animals were kept at a MAP of about 30 to 40 mmHg for 90 minutes. The animals were resuscitated with reinfusion of shed blood followed by 3 hours of observation. Conclusion We suggest that an AS dose of 1,000 mg/day (over 10 days) seems to improve neurorecovery in BI patients with good tolerability. Prospective controlled studies with large, homogeneous BI populations will better defi ne the role of AS for recovery and complications. P454 References The fact that optimal CPP and autoregulation status varies between individual patients and over time makes it an attractive bedside tool to serve as a (simplifi ed) model to investigate the use of autoregulation (PRx) status to fi ne tune or feedback clinical treatments in individual sedated TBI patients (optimal CPP concept) [1]. Evidence is emerging for the role of other monitors (representing (local) metabolism, oxygen supply/use, perfusion, neuronal functioning) that enable the intensivist to employ an individualized multimodality monitoring approach in TBI [2]. P453 Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) P454 Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Prognostic value of ubiquitin carboxy-terminal hydrolase L1 in patients with moderate or severe traumatic brain injury: a systematic review M Shemilt1, JF Laforest1, F Lauzier1, A Boutin1, D Fergusson2, R Zarychanski3, L Moore1, L McIntyre2, L Nadeau1 1CHU de Québec, QC, Canada; 2Ottawa Health Research Institute, Ottawa, ON, Canada; 3University of Manitoba, Winnipeg, MB, Canada Critical Care 2015, 19(Suppl 1):P454 (doi: 10.1186/cc14534) Results In the cerebral hemisphere, hemorrhagic shock caused a marked increase in the LP ratio, while a signifi cant but minor increase was observed in the sagittal sinus. The LP ratio increased and continued doing so to a very high level. In the femoral artery, the shock period was associated with a slight increase of the LP ratio. The increase in the LP ratio in the sagittal sinus was markedly and signifi cantly higher than in the arterial blood. Further, the dynamic changes in the LP ratio in the sagittal sinus followed that of the parenchyma, not the arterial blood. After infusion of blood ICP increased, cerebral perfusion pressure and Introduction Traumatic brain injury (TBI) prognostication is a developing fi eld striving to identify indicators, notably neurochemical S159 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P453). Findings of 12 patients receiving amantadine sulfate Table 1 (abstract P453). P454 The main purpose of this study was to determine the impact of CB2R inhibition on leukocyte activation within the microcirculation following endotoxin challenge in an experimental stroke model. Methods This was a prospective, randomized animal study. Five experimental groups (male C57BL/6 mice, age: 6 to 8  weeks) were subjected to the following treatments: control; endotoxemia (LPS 5 mg/kg, i.v.); transient cerebral hypoxia–ischemia (HI) + endotoxemia; HI + endotoxemia + CB2R antagonist (AM630 2.5 mg/kg, i.v.). HI was induced by unilateral carotid artery occlusion, followed by 50-minute exposure to a low oxygen atmosphere (8% O2). The CB2R antagonist was given 15 minutes prior to LPS administration. Intravital microscopy was carried out 2  hours after LPS administration. Brains were then extracted and stained with tetrazolium chloride to calculate the infarct volume. The primary outcome measurement in this study regarding the immune response after stroke was the quantifi cation of leukocyte adhesion following endotoxin challenge in submucosal venules of the gut – an important organ in the development of multiorgan failure in endotoxemia and sepsis. Methods The MEDLINE, Embase, The Cochrane Library and BIOSIS electronic databases, conference abstracts and existing narrative reviews were searched from their inception to July 2013. Cohort studies including patients with moderate or severe TBI having evaluated the prognostic value of UCHL-1 according to mortality or the Glasgow Outcome Scale (GOS) were considered. Data concerning patients, outcomes, study methods, and laboratory methods were abstracted. Pooled results were planned to be presented using mean diff erences and analyzed using random eff ect models, as well as sensitivity analyses to explain potential heterogeneity.i Results Our search strategy yielded 2,257 articles, of which fi ve studies corresponded to our inclusion criteria (n  = 730). All studies were performed by the same group of researchers. Five studies reported mortality (n = 515), two studies reported GOS (n = 58). Results from all included studies observed that UCHL-1 was a signifi cant predictor of mortality. However, only two studies represented a unique study population, thus precluding a meta-analysis. Results Compared with endotoxemic animals without CNS injury, mice subjected to HI displayed reduced leukocyte activation in intestinal submucosal venules indicative of CIDS. Administration of the CB2R antagonist in animals with CIDS challenged with endotoxin restored peripheral leukocyte recruitment without a detrimental impact on infarct size. P454 Findings of 12 patients receiving amantadine sulfate Initiation of Patient number Sex/age (years) APACHE II GCS A/B/C AS therapy (day) CRS score D/E/F/G Complications Outcome 1 M/14 24 3/5/7 6 0/4/5/6 H, J Still in ICU 2 F/78 33 8/8/10 84 0/0/0/0 K Ex 3 F/75 35 5/6/4 4 1/1/0/Ex K, L Ex 4 M/24 29 3/3/3 6 0/0/Ex K, L Ex 5 M/27 27 4/4/15 6 1/13/18/22 L, M, J Discharged 6 F/70 45 3/3/3 20 0/0/0/0 – Ex 7 F/72 22 5/5/12 3 4/7/11/17 K, L, I Discharged 8 F/53 27 3/5/10 150 5/7/10/14 K Discharged 9 F/38 34 4/4/4 33 0/0/1/1 H, J Still in ICU 10 M/50 26 4/6/13 8 0/7/18/19 L, J Discharged 11 M/63 24 8/10/12 14 6/8/8/12 – Discharged 12 M/50 31 4/6/11 6 2/5/7/9 L, J Still in ICU F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, convulsion. F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, l i F, female; M, male. GCS, Glasgow Coma Scale: A, at admission to ICU; B, at initiation of AS therapy; C, outcome value. CRS: D, value on day 1; E, value on day 6; F, value on day 10; G, value on day 14. Complications observed: H, low platelet; J, ALT increase by twofold; K, BUN increase by twofold; L, AST increase by twofold; M, convulsion. biomarkers, of long-term outcomes. Among these, ubiquitin carboxy- terminal hydrolase L1 (UCH-L1) is currently being investigated to defi ne its potential prognostic value. The objective of this systematic review is to determine the ability of UCH-L1 to predict prognosis following a moderate or severe TBI. CIDS. P455 Cannabinoid 2 receptor antagonism reverses central nervous system injury-induced immune defi ciency syndrome IB Burkovskiy, J Zhou, G Robertson, C Lehmann Dalhousie University, Halifax, NS, Canada Critical Care 2015, 19(Suppl 1):P455 (doi: 10.1186/cc14535) P454 Conclusion In this systematic review, we observed that all published studies on UCHL-1 were conducted by the same group of investigators and presented results from an intersecting cohort of patients. Due to the paucity of data, we could not perform a pooled analysis and conclude on the association of this biomarker with long-term prognosis. Assays using UCHL-1 were only recently developed and further studies done by diff erent research teams will be needed to determine the reproducibility and validity of UCH-L1 as a potential prognostic tool. Conclusion CB2R-related modulation of leukocyte activation is involved in the impaired immune response following CNS injury. Future studies will explore the CB2R pathway in order to develop novel therapies to improve the immune response in CIDS. P456 Implementation process of a large multicenter study in trauma AF Turgeon, TBI-Prognosis Team in collaboration with the CCCTG CHU de Québec, QC, Canada Critical Care 2015, 19(Suppl 1):P456 (doi: 10.1186/cc14536) Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Results Overall, 33.4  weeks (95% CI  = 24.8 to 42.1) were required on average from the start-up package mailing to centers and the start of the screening process. Data sharing and fi nancial agreement were mainly responsible for this delay with an average of 28.6 weeks (95% CI = 20.4 to 36.7) needed to complete the agreement with the coordinating center. REB approval was obtained on average 17.5 weeks (95% CI = 13.8 to 21.2) from the shipping of the study package to the participating centers. Eighty percent of the REBs had members with prior experience in multicenter clinical research, and more than half with specifi c clinical expertise in critical care medicine or neurology/ neurosurgery. A standardized electronic REB submission process was used in most REBs (60%). All centers had experience in implementing multicenter clinical studies. PIs had experience conducting from zero to 40 prior clinical studies and from 3 to 24  years of research experience with protected research time ranging from 5 to 75%. RCs had managed from zero to more than 50 clinical studies. Most centers (87%) organized specifi c presentation of the study to the critical care medical staff (93%), while some (60%) presented the study to other medical teams. Agreements from other departments such as radiology (87% of centers), electrophysiology (80%) and clinical imaging (73%) were requested in most centers. p L Tsentsiper, E Kondratyeva, S Kondratyev, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P458 (doi: 10.1186/cc14538) p L Tsentsiper, E Kondratyeva, S Kondratyev, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint-Petersburg, Russia Critical Care 2015, 19(Suppl 1):P458 (doi: 10.1186/cc14538) Introduction Currently, the most common way to predict the outcome of acute brain damage is to study the level of protein S-100 in the serum. This method lacks specifi city as the concentration of protein S-100 signifi cantly increases with age, more for men than women, and there are no data on prognostically signifi cant changes in the level of S-100 after removal of the tumor and cerebral hemorrhages. Endothelins, vasopressin, some cytokines, excess sodium or calcium in serum, activation of the sympathoadrenal system, and tachycardia are the stimulants of brain natriuretic peptide production. Levels of N-terminal pro-brain natriuretic peptide in brain injury patients The rise of the natriuretic peptide level in cases of acute brain damage has a functionally adaptive nature, based on vasodilation, diuretic action peptide and ability to reduce sympathoadrenal system activity. Thus, we can suppose that the more severe the damage, the higher the stimulation of natriuretic peptide. In this study we investigate the level of N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with severe brain damage and fi nd correlation between the level of peptide and outcome. Endothelins, vasopressin, some cytokines, excess sodium or calcium in serum, activation of the sympathoadrenal system, and tachycardia are the stimulants of brain natriuretic peptide production. The rise of the natriuretic peptide level in cases of acute brain damage has a functionally adaptive nature, based on vasodilation, diuretic action peptide and ability to reduce sympathoadrenal system activity. Thus, we can suppose that the more severe the damage, the higher the stimulation of natriuretic peptide. In this study we investigate the level of N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with severe brain damage and fi nd correlation between the level of peptide and outcome. Conclusion The implementation of a large Canadian multicenter and multifaceted clinical study in the trauma population involved a signifi cant amount of time and energy from both the coordinating and the participating sites. The variable experience of participating sites and teams, as well as the involvement of diff erent medical disciplines, may have had an impact on study implementation time. Delays for REB approval but also for data sharing and fi nancial agreement must be taken into consideration in the timeline for implementing large multicenter clinical studies in trauma. Methods We studied 110 patients having brain injuries of various origins. All patients were divided into four groups. All patients were 20 to 72 years old, 58 men and 52 women. Group 1 (n = 17) – acute TBI, group 2 (n = 29) – patients operated on for the brain tumor, group 3 (n = 36) – hemorrhagic stroke, group 4 (n = 28) – vegetative state. We measured the level of brain natriuretic peptide on days 1 to 3, and then every 7 days for 21 days. Results All patients with severe acute brain damage (groups 1, 2, 3) had a level of NT-proBNP higher than normal (normal 0 to 125 pg/ml). Signifi cant diff erence in values between the groups was not observed. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P458 the ongoing Pan-Canadian TBI-Prognosis Study. Descriptive statistics were used. the ongoing Pan-Canadian TBI-Prognosis Study. Descriptive statistics were used. Levels of N-terminal pro-brain natriuretic peptide in brain injury patients Level of NT-proBNP above 700  pg/ml and/or the absence of its reduction to normal dynamic indicators was marked by an unfavorable outcome of the disease – severe disability (n = 25) or death (n = 18). For patients from group 4 regardless of their age, sex, severity of condition and treatment results in a level of NT-proBNP below 250 pg/ml. P457 Optic nerve sheath diameter by bedside ultrasound is a reliable screening test for cerebral edema in the comatose ICU patient A Mohamed, N Alharbi, N Salahuddin, I Hussain, O Solaiman King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2015, 19(Suppl 1):P457 (doi: 10.1186/cc14537) Conclusion In cases of acute severe brain damage the level of NT- proBNP signifi cantly increased. Correlation between the level of NT-proBNP and etiology of acute brain damage was not observed. If the level of NT-proBNP is above 700 pg/ml and/or in the absence of its reduction to normal, then poor outcome of the disease – severe disability or death  – can be predicted. Level of NT-proBNP cannot be used as an indicator for the severity of the status for patients in a vegetative state in contrast to patients with acute brain damage. Introduction ICU patients may remain comatose after resolution of critical illness. Frequently this is due to delayed sedative clearance but may also result from increases in intracranial pressure (ICP) and cerebral edema. We proposed that measurement of the optic nerve sheath diameter (ONSD) is a rapid, bedside screening test that can be used to quickly identify patients with cerebral edema and increased ICP. y y Methods This was a prospective, observational study carried on consecutive patients admitted to a multidisciplinary medical and surgical ICU. Stable patients with unexplained coma and scheduled for brain imaging were included. Patients with obvious ocular trauma or on sedative, narcotic infusions were excluded. ONSD was measured using a 7.5 to 10 MHz linear array ultrasound transducer probe placed on the closed eye in the transverse axis. The ONSD was measured at a predefi ned point 3 mm posterior to the globe. Both eyes were measured and the mean value used. The study protocol was approved by the Hospital Research Ethics Committee (RAC Prop No. 2141 103). Statistical analysis was carried out using SPSS version 20.0. Cannabinoid 2 receptor antagonism reverses central nervous system injury-induced immune defi ciency syndrome IB Burkovskiy, J Zhou, G Robertson, C Lehmann Dalhousie University, Halifax, NS, Canada Critical Care 2015, 19(Suppl 1):P455 (doi: 10.1186/cc14535) Introduction Our purpose was to evaluate the time from shipping of the study start-up package to study screening, as well as conditions that may impact this process, in the context of a large-scale multifaceted and multicenter clinical study in trauma. Introduction Our purpose was to evaluate the time from shipping of the study start-up package to study screening, as well as conditions that may impact this process, in the context of a large-scale multifaceted and multicenter clinical study in trauma. Introduction Central nervous system (CNS) injury, such as stroke, is known to increase susceptibility to infections that adversely aff ect clinical outcome. This impaired immune response to infection is termed CNS injury-induced immune defi ciency syndrome (CIDS). Activation of the cannabinoid 2 receptor (CB2R) suppresses immune function suggesting that antagonism of this receptor may overcome Methods We designed a survey questionnaire based on four domains: REB characteristics and process, centers’ characteristics, experience of the study and clinical teams, and center-specifi c implementation approaches. The questionnaire was self-administered to all lead research coordinators of the 17 level 1 participating trauma centers in S160 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P459 C b Cerebral oximetry monitoring in pediatric seizure patients in the emergency department T Abramo1, B Schnieder1, E Storm1, N Hobart-Porter1, Z Hu1, N Todd1, L Crawley1, M Meredith2, S Godbold1 1University of Arkansas School of Medicine, Little Rock, AR, USA; 2University of Tennessee School of Medicine, Memphis, TN, USA Critical Care 2015, 19(Suppl 1):P459 (doi: 10.1186/cc14539) Introduction During ictal/post-ictal events, altered cerebral physiology occurs: increased neuronal activity causes signifi cant increase in cerebral metabolism with changes in ipsilateral cerebral blood fl ow. Standard PED seizure monitoring is by O2SAT and ETCO2 which yield no direct data about regional cerebral oxygenation/physiology (rSO2). Signifi cant abnormal hemispheric cerebral physiology resulting in neurological injury can occur without knowing because the current monitoring system could not detect the abnormal hemispheric abnormality. Cerebral oximetry can provide a rapid, non-invasive detection of each hemisphere’s cerebral physiologic changes during ictal/post-ictal phases. The aim was to study left and right rSO2 values in patients in the pre and post seizure periods and in nonseizing controls. Methods An observational study of seizing and nonseizing patient’s left and right rSO2 readings compared with nonseizure patients. Results ONSD was measured in 43 patients; mean age was 62  ± 19.2 years, 48% (n = 20) were female. Admitting diagnosis was sepsis in 24% (n = 10), intracranial vascular event in 21% (n = 9), cardiac arrest in 12% (n = 5), hepatic encephalopathy in 7% (n = 3), malignancy with metastases in 7% (n = 3) and other causes in 28% (n = 12). The ONSD measured correlated highly between eyes, Spearman’s ρ  = 0.799, P ≤0.001. The area under the ROC curve for detecting cerebral edema was 0.812 (95% CI  = 0.667 to 0.957). Using a 0.58  cm cutoff ONSD diameter, the sensitivity was 86%, specifi city 74%, negative predictive value 96% and the positive likelihood ratio = 3.3. Conclusion This study suggests that bedside measurement of ONSD by ultrasound performs well as an initial screening test for cerebral edema. The identifi ed cutoff value of 0.58 cm can be used to detect cerebral edema with reasonable accuracy. S161 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P459). Results No diff erence for ictal left and right rSO2 readings across ages. See Figure 1. of our institute. In post-anoxic comatose patients we recorded EEG during hypothermia to assure burst suppression. P460 Neurophysiological tests in the neuro ICU P460 Neurophysiological tests in the neuro ICU A Marudi1, Y Valzani2, F Valzania1, M Carpentiero1, C Parenti1, S Scacchetti1, S Baroni1, E Bertellini1 1Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy; 2University of Modena and Reggio Emilia, Modena, Italy Critical Care 2015, 19(Suppl 1):P460 (doi: 10.1186/cc14540) P459 C b In post-traumatic brain-injured patients with a persistent comatose state we use EEG to detect nonconvulsive states which potentially can increase secondary brain injury if untreated. In malignant epilepticus status we use EEG to monitor the eff ect of therapy and to modify it. In patients who present profound weakness of legs and hands we performed EMG to distinguish primary peripheral myopolyneuropathy (Guillian Barrè, miastenia gravis) from secondary illness acquired in the ICU (critical polyneuropathy, critical myopathy) [3]. Conclusion We have demonstrated abnormal hemispheric cerebral physiology during focal or generalized ictal activity. In patients with generalized seizures, the left and right rSO2 values were signifi cantly decreased. In patients with focal seizures, the ipsilateral rSO2 values were signifi cantly diff erent from the contralateral rSO2 readings and correlated to the hemisphere experiencing the focal seizure. In certain patients, during the ictal phase their rSO2 readings rose and stayed or rose then dropped. Overall, cerebral oximetry has shown great monitoring potential for actively seizing patients in the emergency department. Conclusion NPT can improve management of patients admitted to the neuro ICU. The data provided can modify therapeutic strategies and improve outcome in these settings of patients. R f References 1. Guerit JM. Curr Opin Crit Care. 2010;16:98-104. 2. Bednarik J, et al. Intensive Care Med. 2003;29:1505-14. 3. Pohhmann-Eden B, et al. Intensive Care Med. 1997;23:301-8. References 1. Guerit JM. Curr Opin Crit Care. 2010;16:98-104. 2. Bednarik J, et al. Intensive Care Med. 2003;29:1505-14. 3. Pohhmann-Eden B, et al. Intensive Care Med. 1997;23:301-8. References 1. Guerit JM. Curr Opin Crit Care. 2010;16:98-104. 2. Bednarik J, et al. Intensive Care Med. 2003;29:1505-14. 3. Pohhmann-Eden B, et al. Intensive Care Med. 1997;23:301-8. Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery Patients were randomized into two groups: MAP group, in which MAP S162 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 was maintained above 70 mmHg or within 20% from baseline (n = 78); or CPP group, in which CPP was maintained above 60 mmHg or within 20% from baseline (n = 54). ICP, MAP and CPP were assessed every hour of anesthesia. Time of recovery of consciousness, incidence of POD and length of stay in the ICU and in the hospital were also evaluated. ischemia-operated animals with normothermia (normothermia  + ischemia group); sham-operated animals with hyperthermia (hyperthermia  + sham group); and ischemia-operated animals with hyperthermia (hyperthermia  + ischemia group). Hyperthermia (39.5 ± 0.2°C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30  minutes before and during ischemia–reperfusion.i y Results Initial ICP was 14 ± 3 mmHg in the MAP group and 15 ± 2 mmHg in the CPP group. During the anesthesia it was stable without any signifi cant change. Decreasing of MAP after induction of anesthesia was similar in two groups and it was stable during the anesthesia. The frequency of use of vasopressors and infusion rate was higher in the CPP group. Time of recovery of consciousness in the MAP group was higher (28 ± 7 minutes vs. 18 ± 5 minutes (P <0.05)). The incidence of postoperative delirium was higher in the MAP group (18% vs. 11% in the CPP group (P <0.05)). There were no signifi cant diff erences between two groups in other complications. Total length of stay in the ICU and in the hospital was higher in the MAP group (6 ± 2 days vs. 4 ± 2 (P <0.05) and 15 ± 3 days vs. 12 ± 2 in the N group (P <0.05)). Results In the normothermia + ischemia group, a signifi cant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5  days after ischemia–reperfusion. In the hyperthermia  + ischemia group, neuronal death in the SP of the CA1 occurred at 1  day post ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2  days post ischemia. In addition, we examined activation of astrocytes and microglia using immunohistochemistry for anti-glial fi brillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). Hydroquinone shows neuroprotective potential in an experimental ischemic stroke model via attenuation of blood–brain barrier disruption CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P464 (doi: 10.1186/cc14544) JH Cho, CW Park, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P462 (doi: 10.1186/cc14542) Introduction Therapeutic exercise is an integral component of rehabilitation for patients with stroke. Despite the high prevalence of cerebral ischemia in the older population, the mechanisms linking restorative exercise to memory recovery from ischemic stroke have not been completely understood in aged animals. In this study, we investigated eff ects of long-term exercise on neuronal death and memory recovery in the aged gerbil hippocampus after transient cerebral ischemia. We also investigated changes in gliosis, ischemia- induced myelin repair, microvessels, neurogenesis, and growth factor immunoreactivity in the hippocampus to study possible mechanisms of restorative exercise in memory recovery. Introduction Hydroquinone (HQ), a major benzene metabolite, occurs naturally in various plants and food, and is also manufactured for commercial use. Although many studies have demonstrated the various biological eff ects of HQ, the neuroprotective eff ects of HQ following ischemic stroke have not been investigated. Therefore, in this study, we fi rst examined the neuroprotective eff ects of HQ against ischemic damage in a focal cerebral ischemia rat model. Methods It was proven that pre and post treatment with 100  mg/ kg HQ protects from ischemia-induced cerebral damage, which was confi rmed by evaluation of neurological defi cit, positron-emission tomography and 2,3,5-triphenyltetrazoliumchloride staining. Methods The gerbils were divided into four groups (n = 12 in each group): the sham-operated group (Sham), 4-week sedentary group following ischemia (SD4), 1-week treadmill group following ischemia (TR1) and 4-week treadmill group following ischemia (TR4). Treadmill exercise was stared at 5 days after ischemia/reperfusion (I/R) and lasted for 1 or 4 weeks, and the animals were sacrifi ced 31 days after ischemia. Results In this study, 4 weeks of treadmill exercise facilitated memory recovery despite neuronal damage in the CA1 region after I/R. On the other hand, the long-term treadmill exercise alleviated the increased gliosis in the CA1 region, and increased the myelin repairing and microvessels in the CA1 region and DG, and enhanced the ischemia- induced cell proliferation, neuroblast diff erentiation, neuronal maturation of the newly generated cells, and BDNF expression in the ischemic DG of the aged gerbil. Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery Introduction Neurophysiological tests (NPTs) are important prog- nostic and diagnostic tools for patients admitted to the modern neuro ICU (NICU) [1]. Electroencephalography (EEG), somasensorial evoked potentials (SSEP), auditory brainstem response (ABR) and electromyography (EMG) complete clinical examination and radio- logical fi ndings in patients suff ering from post-traumatic brain injury, post-anoxic brain injury, refractory male epiletticus status, and neuromuscular illness. We evaluate the spread of NPTs in our NICU. Methods We collected data from patients admitted to our NICU from January 2014 to November 2014. We recorded the admission diagnosis and the NPT applied. y j I Zabolotskikh, N Trembach I Zabolotskikh, N Trembach Kuban State Medical University, Krasnodar, Russia Kuban State Medical University, Krasnodar, Russia Critical Care 2015, 19(Suppl 1):P461 (doi: 10.1186/cc14541) y Critical Care 2015, 19(Suppl 1):P461 (doi: 10.1186/cc14541) Introduction Increased intracranial pressure (ICP) adversely aff ects anesthesia due to a disturbed cerebral blood fl ow. In older patients this disturbance may increase the incidence of postoperative delirium (POD) and may lead to a poor outcome [1]. The standard hemodynamic protocol involves maintaining the mean arterial blood pressure (MAP), but in patients with intracranial hypertension it may not be enough to maintain adequate cerebral perfusion. The purpose of this study was to evaluate the protocol of maintaining cerebral perfusion pressure (CPP) in the prevention of postoperative delirium in older patients in abdominal surgery. Methods We collected data from patients admitted to our NICU from January 2014 to November 2014. We recorded the admission diagnosis and the NPT applied. Results From January 2014 to November 2014 we performed 521 EEG, 45 SSEP/ABR and 10 EMG. In post-anoxic and post-traumatic brain- injured comatose patients we performed EEG, SSEP and ABR 24 to 48 hours after the admission to predict later prognosis and expected neurological defi cit [2]. In the presence of a benign pattern no further evaluation was performed; in the presence of a malignant pattern the NPTs were repeated every 48 to 72 hours according to the protocol Methods A total of 132 ASA 3 patients, undergoing major abdominal surgery (duration 5.2 (4.3 to 6.5) hours) with ICP >12 mmHg evaluated by a venous ophthalmodynamometry [2], were included in our research. Goal-directed cerebral hemodynamic strategy decreases the incidence of postoperative delirium in patients with intracranial hypertension in major abdominal surgery GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia  + ischemia group than those in the normothermia  + ischemia group.i y g p Conclusion A goal-directed hemodynamic protocol of maintaining CPP can decrease the incidence of POD in older patients with intracranial hypertension after major abdominal surgery compared with a protocol of maintaining MAP. Conclusion Based on our fi ndings, we suggest that experimentally hyperthermic precondition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus. Hydroquinone shows neuroprotective potential in an experimental ischemic stroke model via attenuation of blood–brain barrier disruption y y Results In addition, pre and post treatment with 100  mg/kg HQ signifi cantly attenuated ischemia-induced Evans blue dye extra- vasation, and signifi cantly increased the immunoreactivities and protein levels of SMI-71 and glucose transporter-1 (GLUT-1), which were well known as useful makers of endothelial cells, in ischemic cortex compared with a vehicle-treated group.l Conclusion Briefl y, these results indicate that pre and post treatment with HQ can protect from ischemic damage induced by transient focal cerebral ischemia, and the neuroprotective eff ects of HQ may be closely associated with the prevention of BBB disruption via increase of SMI-71 and GLUT-1 expressions. P462 P464 Eff ects of long-term exercise on memory recovery in the aged gerbil hippocampus after transient cerebral ischemia CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P464 (doi: 10.1186/cc14544) References 1. Zabolotskikh I, et al. Eur J Anesth. 2013;30:114-5. 1. Zabolotskikh I, et al. Eur J Anesth. 2013;30:114-5. 2. Firsching R, et al. J Neurosurg. 2011;115:371-4. P463 Impact of hyperthermia before and during ischemia reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia CW Park, JH Cho, TG Ohk, MC Shin, MH Won Kangwon National Univeristy, Chuncheon-si, Gangwondo, South Korea Critical Care 2015, 19(Suppl 1):P463 (doi: 10.1186/cc14543) Conclusion These results suggest that, in the aged gerbil, long-term treadmill exercise after ischemic stroke could restore the impaired short-term memory function through the cumulative eff ects of multiple neurorestorative processes. Prognostic value of blood lactate and glucose levels after aneurysmal subarachnoid hemorrhage y g S Dijkland1, K Van Donkelaar2, W Van den Bergh2, J Bakker1, D Dippel1, M Nijsten2, M Van der Jagt1 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) j g 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) 1Erasmus MC – University Medical Center, Rotterdam, the Netherlands; 2University Medical Center Groningen, the Netherlands Critical Care 2015, 19(Suppl 1):P466 (doi: 10.1186/cc14546) Results A total of 307 patients were included in the validation cohort. The observed 60-day case fatality rate was 30.6%. Discrimination was good, and was considerably better for the model with WFNS grade at treatment decision (AUC = 0.89) compared with the model with WFNS grade on admission (AUC  = 0.82). Calibration was poor, with mean predicted probabilities of 17.0% for the model with WFNS grade on admission and 17.7% for the model with WFNS grade at the time of treatment decision. Introduction In critically ill patients, blood lactate on admission is associated with outcome, but in patients with aneurysmal sub- arachnoid hemorrhage (SAH) this has not been investigated. We studied the association of early circulating lactate and glucose with unfavorable disease course. The prognostic role of both lactate and glucose was studied, hypothesizing that both may be increased due to sympathetic activation after SAH [1]. y p Methods In this retrospective cohort study we included consecutive patients with aneurysmal SAH admitted to the ICUs of two university hospitals in the Netherlands between November 2006 and December 2011. Exclusion criteria were: nonaneurysmal SAH, ICU admission >24 hours after ictus, death ≤48 hours after admission and no lactate measurement <24 hours after admission. Maximum blood lactate and glucose levels within the fi rst 24 hours after SAH were used for analyses. The outcomes were DCI, defi ned as a new hypodensity on brain CT due to DCI, and poor outcome, defi ned as a modifi ed Rankin Scale of 4, 5 or death 3 to 6 months after the ictus. We performed proportional hazard analyses to assess the associations of lactate and glucose with DCI, and logistic regression was used to assess the associations with poor outcome. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion Maximum lactate in the acute phase after aneurysmal SAH is associated with both DCI-related infarction and poor outcome. Once glucose was considered, early lactate remained independently associated with poor outcome, while glucose, instead of lactate, was associated with DCI. These routinely available laboratory measurements may improve identifi cation of patients at risk for complications or poor outcome after SAH. Confi rmation of the pathophysiological signifi cance of lactate and glucose in prospective research is warranted. Reference (PaO2) is associated with decreased long-term survival in patients with spontaneous intracerebral hemorrhage (ICH) treated in the ICU. (PaO2) is associated with decreased long-term survival in patients with spontaneous intracerebral hemorrhage (ICH) treated in the ICU. Methods Data on primary admissions for adult patients (>18  years) treated for ICH in Finnish ICUs between 2003 and 2012 were collected from a nationwide ICU database. Patients were divided into three groups according to the PaO2 value associated with the lowest measured PaO2/ FIO2 ratio during the fi rst 24 hours after ICU admission. High arterial oxygen tension was defi ned as PaO2 >19.9 kPa; intermediate as PaO2 13 to 19.9 kPa; and low as PaO2 <13 kPa. The primary outcome was 6-month mortality. 1. Kaukonen K, et al. Crit Care Med. 2014;42:1379-85. Results Of the 3,033 patients, 63% (n = 1,923) had low PaO2, 29% (n = 892) intermediate PaO2, and 7% (n = 218) high PaO2. Forty-nine percent of the patients died during the 6-month follow-up. Of these, 75% died before discharge from hospital. Univariate analysis showed that 6-month mortality was higher in the high PaO2 group (61%) compared with the intermediate and low PaO2 groups (52% and 46% respectively, P  <0.001). Multivariate analysis, however, showed no statistically signifi cant correlation between high PaO2 and mortality (with the low PaO2 group as the reference category, odds ratio for death (OR) for high PaO2 = 1.10, 95% confi dence interval (CI) = 0.76 to 1.58 and for intermediate PaO2 = 0.96, 95% CI = 0.78 to 1.17). P467 Prediction of 60-day case fatality after aneurysmal subarachnoid hemorrhage: external validation of a prediction model S Dijkland, B Roozenbeek, P Brouwer, H Lingsma, D Dippel, L Vergouw, M Vergouwen, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P467 (doi: 10.1186/cc14547) Introduction Aneurysmal subarachnoid hemorrhage (SAH) is a devastating disease with substantial morbidity and mortality. Prognostic modeling is an important instrument to identify high- risk patients in both clinical practice and research settings. Recently, a prognostic model to predict 60-day case fatality after aneurysmal SAH was developed with data from the International Subarachnoid Aneurysm Trial (ISAT) [1]. Our aim was to externally validate this model in a retrospective cohort of consecutive SAH patients. 2 Conclusion High PaO2 is not predictive of 6-month mortality in patients treated for spontaneous ICH in the ICU. Therefore, targeting higher PaO2 values appears to be a safe approach in order to avoid hypoxemia. References 1. Rincon F, Kang J, Maltenfort M, et al. Association between hyperoxia and mortality after stroke. Crit Care Med. 2014;42:387-96. 1. Rincon F, Kang J, Maltenfort M, et al. Association between hyperoxia and mortality after stroke. Crit Care Med. 2014;42:387-96. p p Methods We included consecutive aneurysmal SAH patients admitted to one university hospital between October 2007 and October 2011. Exclusion criteria were: age <18  years, hospital admission >28  days after SAH, nonaneurysmal SAH, explicit objection by the patient to view the medical data and missing data on 60-day case fatality. The model’s predictors were age, maximum lumen size of the aneurysm, Fisher grade and World Federation of Neurological Surgeons (WFNS) grade. Two versions of the model were validated: one with WFNS grade scored on admission and the other with WFNS grade assessed at the time of treatment decision, as a proxy to WFNS grade at randomization used in the ISAT. The outcome was 60-day case fatality. Model performance was assessed by studying discrimination, expressed by the area under the receiver operating characteristic curve (AUC), and calibration. 2. de Jonge E, Peelen L, Keijzers PJ, et al. Association between administered oxygen, arterial partial oxygen pressure and mortality in mechanically ventilated intensive care unit patients. Crit Care. 2008;12:R156. P467 P467 Prediction of 60-day case fatality after aneurysmal subarachnoid hemorrhage: external validation of a prediction model S Dijkland, B Roozenbeek, P Brouwer, H Lingsma, D Dippel, L Vergouw, M Vergouwen, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P467 (doi: 10.1186/cc14547) Reference 1. Risselada R, et al. Eur J Epidemiol. 2010;25:261-6. P468 Prognostic value of blood lactate and glucose levels after aneurysmal subarachnoid hemorrhage Multivariable analyses were adjusted for established predictors for DCI and poor outcome.i Conclusion Our results indicate that the ISAT prediction model is generalizable, since the model showed adequate performance in an independent, unselected cohort of aneurysmal SAH patients. The model discriminated well between patients who died and those who survived the fi rst 60 days after SAH. Additionally, use of WFNS grade at the time of treatment decision of the ruptured aneurysm improved model performance. However, since predicted probabilities were lower than observed probabilities, the ISAT prediction model needs to be adapted before use in clinical practice. Association between high arterial oxygen survival after intracerebral hemorrhage Introduction Hyperthermia can exacerbate the brain damage pro- duced by ischemia. In the present study, we investigated eff ects of hyperthermia before and during ischemia–reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofl uorescence staining. Methods The animals were randomly assigned to four groups: sham- operated animals with normothermia (normothermia + sham group); M Fallenius1, R Raj1, M Reinikainen2, S Bendel3, MB Skrifvars1 1Helsinki University Hospital, Helsinki, Finland; 2North Karelia Central Hospital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2015, 19(Suppl 1):P465 (doi: 10.1186/cc14545) M Fallenius1, R Raj1, M Reinikainen2, S Bendel3, MB Skrifvars1 1Helsinki University Hospital, Helsinki, Finland; 2North Karelia Central Hospital, Joensuu, Finland; 3Kuopio University Hospital, Kuopio, Finland Critical Care 2015, 19(Suppl 1):P465 (doi: 10.1186/cc14545) Introduction Liberal use of oxygen after brain insults remains controversial [1,2]. We studied whether high arterial oxygen tension S163 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Cortical spreading depolarizations in patients with intracerebral hemorrhage: preliminary datal Results Two hundred and eighty-fi ve patients were included in the analyses. DCI occurred in 84 patients (29%) and 106 patients (39%) had poor outcome. Lactate was independently associated with DCI (adjusted HR = 1.16, 95% CI = 1.04 to 1.30) and poor outcome (adjusted OR = 1.53, 95% CI = 1.25 to 1.94). Maximum lactate and glucose were strongly related (Spearman’s ρ  = 0.55, P  <0.001). In multivariable analyses including both lactate and glucose as independent variables, only lactate was independently related to poor outcome (OR = 1.42, 95% CI = 1.11 to 1.81), and only glucose was independently associated with DCI (HR = 1.10, 95% CI = 1.02 to 1.19). AJ Schiefecker1, R Beer1, M Kofl er1, B Pfausler1, I Unterberger1, P Lackner1, G Broessner1, P Rhomberg1, F Sohm1, M Mulino1, C Thome1, M Fabricius2, E Schmutzhard1, R Helbok1 1Medical University of Innsbruck, Austria; 2Rigshospitalet, Copenhagen, Denmark Critical Care 2015, 19(Suppl 1):P468 (doi: 10.1186/cc14548) Introduction Perihematomal edema (PHE) expansion contributes to increased morbidity and mortality after spontaneous intracerebral S164 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 positivity on admission is an independent prognostic predictor in acute ischemic stroke. Reference Figure 1 (abstract P469). hemorrhage (ICH). Pathophysiology of PHE is incompletely understood. Recently, the role of cortical spreading depolarizations (CSDs) in secondary brain injury was established in subarachnoid and traumatic brain injury patients. However, the value of CSDs after ICH is not known. Methods Patients with ICH fulfi lling the inclusion criteria were prospectively enrolled in the observational COSBID study (Co-operative Study on Brain Injury Depolarisations). g.BSamp (g.tec, Austria) connected to PowerLab and LabChart software (Adinstruments) was used for electrocorticography (EcoG). Electrocardiogram patches at the patient’s shoulder and bed served as groundings, and a surface reference electrode was glued on the mastoid. The duration of EcoG depressions was defi ned as the time between depression onset and start of EcoG recovery in the integral of power calculations (0.5 to 45 Hz; 60 seconds time constant decay). Brain tissue oxygen tension (PbtO2), cerebral blood fl ow (CBF), cerebral metabolism and intracranial pressure were monitored in the PHE region. Data are presented as median and interquartile range. q g Results Eighteen patients with ICH (ICH volume: 54 (33 to 69)  ml) were analyzed. Hematoma evacuation was performed in 17 patients, one subject underwent craniectomy only. Troponin level as a predictor of prognosis in patients with acute ischemic stroke H Bayir1, R Dagli2, H Kaymaz2, I Yildiz1, H Kocoglu1 1Abant Izzet Baysal University, Medical School, Bolu, Turkey; 2Ahi Evran University Education and Research Hospital, Kirsehir, Turkey Critical Care 2015, 19(Suppl 1):P469 (doi: 10.1186/cc14549) H Bayir1, R Dagli2, H Kaymaz2, I Yildiz1, H Kocoglu1 1Abant Izzet Baysal University, Medical School, Bolu, Turkey; 2Ahi Evran University Education and Research Hospital, Kirsehir, Turkey Critical Care 2015, 19(Suppl 1):P469 (doi: 10.1186/cc14549) Methods The data from 1 million National Health Insurance bene fi ciaries were utilized. All adult benefi ciaries were followed from 1 January 2005 until 31  December 2012 to identify those who developed ischemic stroke. Meanwhile, each identifi ed patient with burn injury was matched with 100 unexposed patients based on the high-dimensional propensity score. Cox regression models were applied to compare the hazards of ischemic stroke in the matched cohorts. Introduction The aim of this study was to identify the association between troponin level and the outcome in patients with acute ischemic stroke. Introduction The aim of this study was to identify the association between troponin level and the outcome in patients with acute ischemic stroke. Methods We retrospectively investigated 152 patients admitted to our reanimation ICU for cerebrovascular accident between 1 January 2013 and 31 December 2013. Inclusion criteria were as follows: patients with acute ischemic stroke, measurement of serum troponin level and electrocardiography performed within 24  hours of admission. Not included were patients with intracerebral hemorrhage, no brain imaging or electrocardiography, previous myocardial infarction, stable or unstable angina pectoris before admission, previous coronary angioplasty or coronary bypass surgery. Figure 1 (abstract P470). Patient fl owchart of enrollment. g p y y yp g y Results Of 152 patients, 51 patients were excluded from the study because of the exclusion criteria. The serum troponin level was elevated in 81 patients. The patients were divided into two groups; patients in group 1 (n = 81) with serum troponin level >0.01, and those in group 2 (n = 20) with serum troponin level ≤0.01. For 1-month follow-up results of patients, death had occurred in 50.6% (n = 41) of patients in group 1 and in 25% (n = 5) of patients in group 2. There was a signifi cant positive correlation between the increase in troponin level and death within 1 month (r = 0.205; P = 0.040). P469 Introduction The results of studies attempting to assess the risks of ischemic stroke in patients with burn injury have been confl icting. We investigated the risks of ischemic stroke in hospitalized burn injury patients in Taiwan to evaluate whether the risk is higher compared with the general population.i P470 P470 Increased risk of ischemic stroke in patients with burn injury: a nationwide cohort study in Taiwan TY Hung1, YC Su2 1Zhongxing Branch of Taipei City Hospital, Taipei, Taiwan; 2Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan Critical Care 2015, 19(Suppl 1):P470 (doi: 10.1186/cc14550) Cortical spreading depolarizations in patients with intracerebral hemorrhage: preliminary datal Patients were 60 (55 to 67) years old and 38% female. Monitoring time per patient was 10 (6 to 14) days. A total of 129 CSDs with 16 (10 to 29) minutes of EcoG depression were observed. Eighty-four percent (n  = 15) of patients showed expansion of PHE by 25 (10 to 50) ml within 3 to 6 days after bleeding. Neuromonitoring probes were 35 (23 to 58) mm distant from the EcoG strip. CSDs occurred in 73% (n  = 11) of patients with PHE expansion. The interval between CSDs was 98  minutes (25 to 308). CSDs were associated with a signifi cant decrease of PbtO2 (–4 mmHg (–3; –7); duration 10 (5 to 23) minutes) in 68% (52/77), CBF changes in 95% (19/20) and metabolic derangement in 80% (80/100) of CSDs. PHE expansion was observed in all patients with spreading convulsions (n = 2) and patients with repetitive CSDs occurring as clusters (n = 3). positivity on admission is an independent prognostic predictor in acute ischemic stroke. positivity on admission is an independent prognostic predictor in acute ischemic stroke. Reference 1. Whiteley W, Chong WL, Sengupta A, Sandercock P. Blood markers for the prognosis of ischemic stroke: a systematic review. Stroke. 2009;40:e380-9. P470 Increased risk of ischemic stroke in patients with burn injury: a nationwide cohort study in Taiwan TY Hung1 YC Su2 Conclusion CSDs are common in ICH patients and associated with perihematomal PbtO2 decreases and metabolic derangement. Especially, clusters of CSDs might be associated with detrimental metabolic changes of the perihematomal brain tissue. TY Hung , YC Su 1Zhongxing Branch of Taipei City Hospital, Taipei, Taiwan; 2Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan Critical Care 2015, 19(Suppl 1):P470 (doi: 10.1186/cc14550) TY Hung , YC Su 1Zhongxing Branch of Taipei City Hospital, Taipei, Taiwan; 2Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Chiayi, Taiwan Critical Care 2015, 19(Suppl 1):P470 (doi: 10.1186/cc14550) Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patientsf R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil fi y Results Ninety-nine of 204 included patients developed ICU-AW. Patients with ICU-AW had higher APACHE IV and SOFA scores, a longer duration of mechanical ventilation, longer ICU stay, and died more often on the ICU compared with ICU patients without ICU-AW. Maximal levels of IL-1β, IL-6, IL-8, IL-10, TNFα, IFNγ, fractalkine and sICAM-1 were higher in patients who developed ICU-AW compared with patients who did not develop ICU-AW (univariable analysis). PC 1 to 4 derived from maximal levels explained >69% of the total variance in the data. Multivariable logistic regression models showed that PC 1 (mainly loaded by IL-6, IL-8 and IL-10) and PC 4 (mainly loaded by sP-selectin) were signifi cantly higher in patients with ICU-AW compared with patients without ICU-AW (OR of 1.27 (95% CI = 1.02 to 1.60) and 1.55 (1.06 to 2.27) respectively). Introduction Coronary artery bypass grafting (CABG) surgery usually improves myocardial contractility, reducing cardiovascular events. However, it is a high-risk procedure associated with signifi cant neurological complications, including stroke, delirium and cognitive impairment. The pathophysiology of these complications is not very well known, and may include low fl ow state after surgery, low cardiac output, embolism and reperfusion lesion. The aim of this study is to prospectively evaluate the cerebral hemodynamics through transcranial color and spectral Doppler sonography in high-risk patients undergoing cardiac surgery with a pump. y Methods This was a prospective, single-center study, performed at the Heart Institute from University of São Paulo. From May to November 2014 we included 35 patients in the study, aged older than 18 years old, submitted to CABG with a pump, with EuroSCORE higher than 6 or left ventricular ejection fraction lower than 40%. Transcranial color and spectral Doppler sonography was performed 48 hours before surgery (T0), 7 days (T1) and 6 months after surgery (T2). We used a probe of 2.5 to 2  MHz (Doppler Box DWL/Compumedics, Singen, Germany). All recordings were taken with the patient in a supine position. P471f Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patients R Juliana, G Ferreira, L Camara, S Zeff erino, D Azevedo, R Groehs, M Lima, R Nogueira, E Bor-Seng-Shu, E Osawa, J Jardim, J Almeida, F Galas, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P471 (doi: 10.1186/cc14551) P472 ncreased early systemic infl ammation in patients with ICU-acquired weakness k h d ll h k E Witteveen, L Wieske, C Verhamme, T Van der Poll, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P472 (doi: 10.1186/cc14552) Introduction Infl ammation may be important in the pathogenesis of ICU-acquired weakness (ICU-AW) since SIRS, sepsis and multiple organ failure are the main risk factors. Local infl ammation has been found in muscle and nerve tissue of patients with ICU-AW, but little is known about the association with systemic infl ammation. We hypothesized that systemic infl ammation is increased in patients who develop ICU- AW compared with patients who do not develop ICU-AW. Figure 2 (abstract P470). Burn injury patients have higher adjusted hazard ratio of ischemic stroke. Results A total of 743,237 patients were enrolled. After matching, 1,763 burn injury patients and 176,300 unexposed patients were selected. The adjusted hazard ratio of ischemic stroke was signifi cantly increased in burn injury patients (1.84; 95% CI, 1.43 to 2.36). Such phenomenon remained signifi cantly after 12 months (1.54; 95% CI, 1.11 to 2.13). See Figures 1 and 2. p p p Methods Newly admitted ICU patients ≥48  hours on mechanical ventilation were included. Daily plasma samples were collected from leftover plasma. Muscle strength was evaluated as soon as patients were awake and attentive. ICU-AW was defi ned by a mean Medical Research Council score <4. IL-1β, IL-6, IL-8, IL-10, IL-13, TNFα, IFNγ, fractalkine, GM-CSF, sICAM-1, sE-selectin and sP-selectin were measured in plasma samples of days 0, 2 and 4 using cytometric bead arrays and FACS. Diff erences of maximum levels between patients with and without ICU- AW were calculated using Mann–Whitney U tests. Principal component (PC) analysis was used to avoid multicollinearity and to reduce the set of mediators into a smaller set of PCs. To investigate whether diff erent infl ammatory profi les are associated with development of ICU-AW, we used multivariable logistic regression models of selected PCs, corrected for a priori selected variables, being age, gender, BMI, sepsis, SOFA score, APACHE IV score, immune insuffi ciency and corticosteroids. Conclusion The risk of ischemic stroke is higher in patients hospitalized with burn injury than in the general population, and the eff ects may be extended longer than expected previously. f 1. Cho SJ, Minn YK, Kwon KH. Stroke after burn. Cerebrovasc Dis. 2007;24:261-3. Troponin level as a predictor of prognosis in patients with acute ischemic stroke The best cutoff point revealed by the ROC curve of troponin was 0.291 mg/l; at which the sensitivity was 73% and the specifi city was 79% when used for prediction of death within 1 month (area = 0.319, CI = 0.214 to 0.423, P = 0.021; Figure 1). Conclusion These results suggest that increased serum troponin g Conclusion These results suggest that increased serum troponin level at admission is associated with higher mortality rate. Troponin Figure 1 (abstract P470). Patient fl owchart of enrollment. S165 Critical Care 2015, Volume 19 Suppl 1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 2 (abstract P470). Burn injury patients have higher adjusted hazard ratio of ischemic stroke. at T0, 0.85 ± 0.24 at T1 and 0.91 ± 0.25 at T2, P = 0.146). There was a signifi cant diff erence in the levels of hemoglobin (13.19 ± g/dl 1.97 at T0 and 9.64 ± 1.48 g/dl at T1, P = 0.002). However, this diff erence was not maintained at T2 (12.7 ± 2.02 g/dl at T2, P = 0.252). There were no diff erences regarding PETCO2 at the time points. f g g 2 p Conclusion After cardiac surgery with a pump in high-risk patients, improvement of cerebral hemodynamic occurs, perhaps due to the optimization of cardiovascular function. These fi ndings must be better investigated. Eff ect of coronary artery bypass grafting surgery with a pump on cerebral blood fl ow in high-risk patientsf We measured the middle cerebral artery mean fl ow velocity and pulsatility index. The end-expiratory pressure of CO2 (PETCO2) was measured with infrared capnography attached to a face mask. Blood pressure, hematocrit and axillary temperature was also recorded. Conclusion Development of ICU-AW is associated with increased systemic infl ammation in the fi rst days after ICU admission. li Acknowledgement This research was supported by the Center for Translational Molecular Medicine (MARS). P473f P472 P472 Increased early systemic infl ammation in patients with ICU-acquired weakness E Witteveen, L Wieske, C Verhamme, T Van der Poll, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P472 (doi: 10.1186/cc14552) Predictive value for weakness and 1-year mortality of screening electrophysiology tests in the ICU g Results A total of 115 ICU patients were included. The average age was 61 years and 67% of the patients were male. ICU admission diagnoses were 53% acute surgery, 14% elective surgery and 33% were admitted for medical nonsurgical reasons. Inter-rater reliability of the DEMMI was high: intraclass correlation coeffi cient (ICC) ranging from >0.91 (range 0.85 to 0.94) at ICU admission, >0.98 (range 0.96 to 0.99) at the MICU and >0.97 (range 0.96 to 0.98) at the general ward. Internal consistency reliabilities (Cronbach α) of the DEMMI were 0.84, 0.87 and 0.98 at the ICU, MICU and hospital ward respectively. Validity coeffi cients (Spearman’s rank correlations) with BI, Katz-ADL and MMT were 0.63, –0.45 and 0.62. Introduction Muscle weakness in long-stay ICU patients contributes to 1-year mortality [1]. Whether electrophysiological screening is an alternative diagnostic tool also in unconscious/uncooperative patients remains unknown. The aims of this study were to determine the diagnostic properties of abnormal compound muscle action potential (CMAP), sensory nerve action potential (SNAP) and spontaneous electrical activity (SEA) for Medical Research Council (MRC)-sum score defi ned weakness and their predictive value for 1-year mortality. i p y y Methods Data were prospectively collected during the EPaNIC trial (ClinicalTrials.gov NCT00512122) [2] from October 2008 to November 2010. From day 8 onwards, nerve conduction studies and electromyography were performed weekly in 642 long-stay and 88 randomly selected short-stay patients and muscle strength was assessed in cooperative patients using the MRC-sum score. The electrophysiologist was blinded for the clinical assessments of the physiotherapists and vice versa. The two primary outcomes were: sensitivity, specifi city, positive and negative predictive values of abnormal CMAP, SNAP and SEA for weakness (MRC-sum score <48); and the predictive value for 1-year mortality of abnormal fi ndings on fi rst electrophysiological screening. This association was assessed by univariate and multivariate analyses correcting for weakness and other risk factors, including baseline risk factors, comorbidities, illness severity and ICU exposures. Conclusion The DEMMI is a reliable, responsive and feasible measure ment instrument for the assessment of mobility in critically ill ICU patients. R f 1. Stevens RD. Intensive Care Med. 2007;33:1876-91. 2. de Morton NA, et al. Health Qual Life Outcomes. 2008;6:63. 2. de Morton NA, et al. Health Qual Life Outcomes. 2008;6:63. Eff ect of prolonged critical care admissions on upper and lower limb muscle architecture g pp Methods Following ethical approval, patients who were intubated and ventilated in one of two critical care departments were assented to take part in the study by their next of kin. B-mode ultrasound scans of the right biceps, vastus lateralis and the medial head of gastrocnemius were performed on days 1, 5 and 10. Scans were not performed in patients once they were free of sedation. Muscle thickness (MT) was measured in all three muscles, with pennation angle (PA) being measured in the lower limb muscles. Fascicle length (FL) was derived from PA and MT. Results Twenty patients were recruited, of which 15 were scanned on day 5, and eight were scanned on day 10. In the biceps, there were no alterations in MT over 5 or 10 days. MT of the vastus lateralis signifi cantly decreased on day 5 (1.77 ± 0.06 mm muscle loss, P = 0.03) and day 10 (5.58 ± 0.09 mm muscle loss, P = 0.01). There was also a signifi cant loss in PA over 5 days (1.48 ± 0.63°, P = 0.01) and 10 days (2.96 ± 0.72°, P = 0.01). However, FL was unchanged over time. There was a signifi cant relationship between size of PA and percentage loss of PA and FL in over 5 days. Loss of MT and PA (MT: 3.21 ± 2.08 mm lost, PA: 2.19 ± 1.64°) was observed in the medial gastrocnemius over 10 days, but did not approach signifi cance. Large fascicles on day 1 were associated with greater percentage loss of FL on day 5 (P = 0.012). Methods Following ethical approval, patients who were intubated and ventilated in one of two critical care departments were assented to take part in the study by their next of kin. B-mode ultrasound scans of the right biceps, vastus lateralis and the medial head of gastrocnemius were performed on days 1, 5 and 10. Scans were not performed in patients once they were free of sedation. Muscle thickness (MT) was measured in all three muscles, with pennation angle (PA) being measured in the lower limb muscles. Fascicle length (FL) was derived from PA and MT. Conclusion The diagnostic properties of electrophysiological screening vary over time. Abnormal CMAP documented early during critical illness carries information about longer-term outcome, which should be further investigated mechanistically. Acknowledgement HVM and GH contributed equally. Acknowledgement HVM and GH contributed equally. Eff ect of prolonged critical care admissions on upper and lower limb muscle architecture References 1. Hermans G, et al. Am J Respir Crit Care Med. 2014;190:410-20. 2. Casaer M, et al. N Eng J Med. 2011;365:506-17. 1. Hermans G, et al. Am J Respir Crit Care Med. 2014;190:410-20. 2. Casaer M, et al. N Eng J Med. 2011;365:506-17. g Results Twenty patients were recruited, of which 15 were scanned on day 5, and eight were scanned on day 10. In the biceps, there were no alterations in MT over 5 or 10 days. MT of the vastus lateralis signifi cantly decreased on day 5 (1.77 ± 0.06 mm muscle loss, P = 0.03) and day 10 (5.58 ± 0.09 mm muscle loss, P = 0.01). There was also a signifi cant loss in PA over 5 days (1.48 ± 0.63°, P = 0.01) and 10 days (2.96 ± 0.72°, P = 0.01). However, FL was unchanged over time. There was a signifi cant relationship between size of PA and percentage loss of PA and FL in over 5 days. Loss of MT and PA (MT: 3.21 ± 2.08 mm lost, PA: 2.19 ± 1.64°) was observed in the medial gastrocnemius over 10 days, but did not approach signifi cance. Large fascicles on day 1 were associated with greater percentage loss of FL on day 5 (P = 0.012). P475 P474 y Methods The inter-rater reliability and validity were determined in a prospective observational study. Patients were included and assessed by two independent raters until hospital discharge. Reliability was expressed using the intraclass correlations (ICC). To evaluate the validity, the DEMMI scores were compared with the Barthel Index (BI), Katz-ADL and manual muscle testing (MMT). Predictive value for weakness and 1-year mortality of screening electrophysiology tests in the ICU H Van Mechelen1, G Hermans1, F Bruyninckx2, T Vanhullebusch1, B Clerckx1, P Meersseman2, Y Debaveye1, MP Casaer1, A Wilmer2, PJ Wouters1, I Vanhorebeek1, R Gosselink1, G Van den Berghe1 1KU Leuven, Belgium; 2UZ Leuven, Belgium Critical Care 2015, 19(Suppl 1):P474 (doi: 10.1186/cc14554) Psychometric properties of the de Morton Mobility Index in ICU patients J Sommers1, T Vredeveld2, J Horn1, RH Engelbert2, R Lindeboom1, M Vd Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2Amsterdam School of Health Professions (ASHP), University of Applied Sciences, Amsterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P475 (doi: 10.1186/cc14555) Conclusion In the lower limb, we have shown that MT and PA alterations occur in the fi rst 10 days. Patients with larger PA and FL appear to lose a greater percentage of angle and fascicle length in the fi rst 5 days. In contrast, we have demonstrated a sparing eff ect on the muscles of the upper limb compared with the lower limb. These fi ndings may have implications for rehabilitation and interventions to preserve muscle mass. Introduction Many ICU patients develop ICU-acquired muscle weak- ness (ICU-AW) due to inactivity and critical illness. ICU-AW is associated with short-term and long-term physical impairments and impaired functional status [1]. The de Morton Mobility Index (DEMMI) was developed to measure changes in mobility across clinical settings and proved to be reliable, feasible and sensitive to small but clinically relevant changes in functioning [2]. Our aim was to evaluate the psychometric properties of the DEMMI in ICU patients. Eff ect of prolonged critical care admissions on upper and lower limb muscle architecture P Turton1, R Hay1, JK Taylor2, J Little2, J McPhee3, I Welters4 1Royal Liverpool and Broadgreen University Hospitals, Liverpool, UK; 2Warrington and Halton Hospitals NHS Trust, Warrington, UK 3Manchester Metropolitan University, Manchester, UK; 4University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P473 (doi: 10.1186/cc14553) Results The mean age of patients was 64 years; most patients were male (74%). Middle cerebral artery mean fl ow velocity increased signifi cantly after cardiac surgery. It was 53.89 ± 17.23 m/second at T0, 61.48 ± 15.18 m/second at T1 and 59.27 ± 16.12 m/second at T2 (P = 0.029). The pulsatility index was similar at all time points (0.88 ± 0.25 Introduction Muscle wasting is a common consequence of long- term stay in the critical care environment, which may slow down the rehabilitation of survivors. Previous ultrasound studies have S166 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 (35.6% vs. 15.2%, P <0.001). This association remained signifi cant after correction for weakness and other risk factors (OR: 2.463 (95% CI: 1.113 to 5.452), P = 0.026). Also among conscious/cooperative patients without weakness, reduced CMAP was independently associated with a higher likelihood of death within 1 year (HR: 2.818 (95% CI: 1.074 to 7.391), P = 0.035). (35.6% vs. 15.2%, P <0.001). This association remained signifi cant after correction for weakness and other risk factors (OR: 2.463 (95% CI: 1.113 to 5.452), P = 0.026). Also among conscious/cooperative patients without weakness, reduced CMAP was independently associated with a higher likelihood of death within 1 year (HR: 2.818 (95% CI: 1.074 to 7.391), P = 0.035). demonstrated a loss of cross-sectional area of lower limb muscles during a 10-day intensive care stay. In this study, we have looked at how markers of muscle architecture (muscle thickness, pennation angle and fascicle length) change in the lower limb, as well as looking at changes in muscle thickness in the upper limb. demonstrated a loss of cross-sectional area of lower limb muscles during a 10-day intensive care stay. In this study, we have looked at how markers of muscle architecture (muscle thickness, pennation angle and fascicle length) change in the lower limb, as well as looking at changes in muscle thickness in the upper limb. Prevalence of psychiatric disorders in trauma patients: results from a major trauma unit M Adlam, A Feehan, V Metaxa Methods This was a prospective observational study of trauma patients from two critical care trauma centers. We excluded patients who had ICU stay <48  hours and those with severe traumatic brain injury (TBI) (GCS ≤8). Patients were followed until ICU discharge, resolution of delirium, death or ICU length of stay >28 days. Delirium was assessed daily using the Confusion Assessment Method for the ICU until the end of the follow-up period. Demographic and admission data, daily consumption of medications, and environmental factors (that is, presence of clock, TV/radio, and so forth) were collected daily. Univariate analysis was performed using Cox regression analysis to identify risk factors for delirium. The independent eff ect of modifi able risk factors was assessed using multivariate Cox regression analysis adjusting for severity of illness and nonmodifi able risk factors. King’s College Hospital, London, UK g g p Critical Care 2015, 19(Suppl 1):P477 (doi: 10.1186/cc14557) Introduction Mental illness has been recognised as a potential risk factor both for intentional and unintentional injury. About 50% of patients presenting with self-infl icting injuries in emergency departments had previous known psychiatric disorder (PD) [1,2], whereas individuals with mental illness were admitted for unintentional injury twice as often as those without [3]. We aimed to assess the prevalence of PD in trauma patients being admitted to a major trauma ICU and compare it with the nontrauma population. Methods We retrospectively reviewed all admissions from January 2010 to December 2013 in a tertiary, mixed ICU that serves a London major trauma centre (MTC) hospital. Data obtained were age, APACHE II score, reason for admission, length of stay (LOS), mortality and a diagnosis of depression, bipolar, self-harm, psychosis, schizophrenia and suicide attempt. j g yi Results We enrolled 150 trauma patients resulting mostly from falls (40%) and motor vehicle accidents (28.7%) over 14 months. Patients with TBI accounted for 56.7% while polytrauma patients without TBI accounted for 43.3%. Mean ICU length of stay was 8.1 ± 7.1 days, 69.3% required mechanical ventilation, 14.7% required a tracheostomy. Delirium developed in 58 patients (38.7%) (mean age 62.9 ± 15.7, mean APACHE score 15.4 ± 6.1, mean ISS score 23.4 ± 9.1). Univariate analysis revealed that delirium was signifi cantly associated with the following nonmodifi able risk factors: age (per 10-year range), APACHE II score (per 10-point increase), need of mechanical ventilation, presence of TBI and pre-existing diabetes. j References 1. Schecter WP, et al. Arch Surg. 2005;140:90. 2. Colman I, et al. Acad Emerg Med. 2004;11:136. 3 W JJ t l J T 2011 61 1299 2. Colman I, et al. Acad Emerg Med. 2004;11:136. 3. Wan JJ, et al. J Trauma. 2011;61:1299. Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study Conclusion BNP and eGFR in ICU admission was associated with delirium. PCT and D-dimer in ICU admission was not associated with delirium. BNP and eGFR might evaluate a predictor of delirium in ICU. References 1. Ely EW, et al. JAMA. 2004;291:1753-62. 1. Ely EW, et al. JAMA. 2004;291:1753-62. Introduction Delirium is associated with signifi cant morbidity and mortality in critically ill medical and surgical patients. However, patients suff ering from trauma are generally excluded from these studies. Our objectives were to assess the incidence of delirium and identify modifi able risk factors associated with delirium among critically ill trauma patients. y 2. van den Boogaard M, et al. Crit Care. 2011;15:R297. B-type natriuretic peptide and estimated glomerular fi ltration rate at ICU admission as a predictor of delirium Introduction Delirium in the ICU is a predictor of mortality and cognitive impairment at hospital discharge. Although several pathways for delirium have been described, it is very diffi cult to predict the occurrence of delirium. In this study, we examined plasma biomarkers in delirious and nondelirious patients at admission and whether the biomarkers can predict onset of delirium. y Results A total of 730 patients were electrophysiologically screened, of which 432 were tested for weakness. On day 8, only normal CMAP excluded weakness with a high negative predictive value (80.5%). By day 15, abnormal SNAP and the presence of SEA revealed a high positive predictive value (91.7% and 80.0%, respectively). On day 8, only a reduced CMAP was associated with higher 1-year mortality S167 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We targeted 103 ICU patients in Okayama University Hospital between April 2013 and February 2014. Delirium was diagnosed using the Confusion Assessment Method  – ICU. On admission, blood was obtained for biomarker analysis. Patients with severe head injury and under 16 years old were excluded. P <0.05 was considered statistically signifi cant. Conclusion Trauma patients with PD have increased mortality and LOS. MTCs provide a unique opportunity to identify mental illness during hospitalisation through screening and intervention programmes. Integration of mental health services into ICU care should be examined, as it might provide an effi cient and cost-eff ective way of decreasing the risk of reinjury. gi Results Thirty-seven delirious and 66 nondelirious patients were included. We found that delirious patients tented to have higher B-type natriuretic peptide (BNP) levels and to have lower estimated glomerular fi ltration rate (eGFR) (BNP: delirious patients 188.6 pg/ml, nondelirious patients 78.2  pg/ml (P  = 0.001); eGFR: delirious patients 58.6  ml/ minute/1.73  m2, nondelirious patients 81.3  ml/minute/1.73  m2 (P  = 0.020)). Procalcitonin (PCT) and D-dimer were almost the same between delirious and nondelirious patients (PCT: delirious patients 0.202 ng/ml, nondelirious patients 0.150 ng/ml (P = 0.613); D-dimer: delirious patients 5.25 ng/ml, nondelirious patients 5.35 ng/ml (P = 0.714)). P478i Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study MA Duceppe1, A Elliott1, M Para1, MC Poirier1, M Delisle1, AJ Frenette2, D Deckelbaum1, T Razek1, M Desjardins2, JC Bertrand2, F Bernard2, P Rico2, L Burry3, D Williamson2, MM Perreault1 1The Montreal General Hospital, Montreal, QC, Canada; 2Hôpital du Sacré- Coeur de Montreal, QC, Canada; 3Mount Sinai Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P478 (doi: 10.1186/cc14558) Modifi able risk factors for delirium in critically ill trauma patients: a multicenter prospective study MA Duceppe1, A Elliott1, M Para1, MC Poirier1, M Delisle1, AJ Frenette2, D Deckelbaum1, T Razek1, M Desjardins2, JC Bertrand2, F Bernard2, P Rico2, L Burry3, D Williamson2, MM Perreault1 1The Montreal General Hospital, Montreal, QC, Canada; 2Hôpital du Sacré- Coeur de Montreal, QC, Canada; 3Mount Sinai Hospital, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P478 (doi: 10.1186/cc14558) Prevalence of psychiatric disorders in trauma patients: results from a major trauma unit In a multivariate analysis when adjusting for the nonmodifi able risk factors, opioids (adjusted HR = 0.37, 95% CI (0.14 to 1.0)), presence of a TV/radio in the room (adjusted HR = 0.28, 95% CI (0.12 to 0.67)), and number of hours mobilized (adjusted HR = 0.77, 95% CI (0.68 to 0.88)) had a protective eff ect on delirium; whereas use of physical restraints (adjusted HR = 2.20, 95% CI (1.11 to 4.35)) and active infection (adjusted HR = 2.08, 95% CI (1.16 to 3.71)) remained strongly associated with delirium. Results Of 978 trauma patients admitted to the ICU, 68 (7%) had a known PD. Their diagnoses are shown in Figure 1. Median APACHE II score and unadjusted mortality were 13 and 18% respectively in the PD group (15 and 12% in the entire cohort, P >0.05). Patients suff ering from more than one diagnosis or self-harm alone had increased median LOS (6 vs. 4 days in the entire cohort, P >0.05). Figure 1 (abstract P477). Conclusion Considering the long-term consequences of delirium, steps should be implemented to prevent its development in trauma and include optimizing opioids and mobilizing patients while limiting use of physical restraints. References References 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. 2. Pisani MA, et al. Am J Respir Crit Care Med. 2009;180:1092-7. 3. van den Boogaard M, et al. Intensive Care Med. 2014;40:361-9. 4. Ely EW, et al. JAMA. 2001;286:2703-10. 1. Page VJ, et al. Routine Crit Care. 2009;13:R16. Propofol sedation reduces contraction and motion of diaphragm in humans: preliminary results p y G Ranieri, M Luigi, F Belsito, M Rocco, RA De Blasi Sant’Andrea, Rome, Italy Results The PRE-DELIRIC scores predicted a mean rate of delirium of 39%. PRE-DELIRIC risk scores ranged from 4 to 93% (Figure 1). Six (15%) patients developed delirium in the fi rst 24 hours following extubation. Fifteen (37%) of patients were predicted 20% or less probability of delirium. Twelve (29%) patients developed delirium at any point during their ICU stay. This resulted in 36 total delirium bed-days. Introduction Among drugs used for sedation, propofol has a primary role [1]. Despite propofol being described to exert a relaxant eff ect on skeletal muscle, no data showing its action on diaphragm are reported. The aim of this observational study on humans is to apply ultrasound to assess propofol’s eff ect on diaphragmatic contraction and motion during endoscopic procedures. y y Conclusion Our observation that <30% of patients experienced delirium is less than the reported prevalence in similar settings and our own audits. This study demonstrates that there is some agreement between recorded rates of delirium and predicted rates using PRE- DELIRIC. We suggest that PRE-DELIRIC can be used in quality/audit work on UK ICUs in order to assess attempts to improve the management of delirium. Further work is required to assess the utility of PRE-DELIRIC as a risk assessment tool in individual patients. Methods We investigated seven consecutive patients undergoing gastroscopy or colonoscopy in the endoscopy unit of our hospital. Patients received propofol at a dose able to induce and maintain sedation to level 6 of the Ramsay Sedation Scale during the procedure. Measurements were obtained on right side of the thorax in millimeters; diaphragmatic motion (DM) and diaphragmatic motion at maximal inspiration (DM forced) were measured in M-Mode with a 3.5  MHz array convex probe placed on the midclavicular line using the liver acoustic window. Thickness at end inspiration (TEI) and thickness at end expiration (TEE) were measured in M-Mode with a 10 MHz vascular probe. The thickening fraction (TF) was calculated: (TEI – TEE) / TEE [2]. Time points of measurements were taken when the patient arrived in the surgery room (Baseline), 1 minute after level 6 of the Ramsey Sedation Scale was obtained (Sedation) and 5 minutes after the patient had a recovery to level 1 on the Ramsey Sedation Scale (Awakening). P479 Evaluation of the PRE-DELIRIC delirium prediction tool on a general ICU J Hanison, S Umar, K Acharya, D Conway Manchester Royal Infi rmary, Manchester, UK Critical Care 2015, 19(Suppl 1):P479 (doi: 10.1186/cc14559) Introduction Delirium is a frequently occurring complication of critical care, occurring in approximately 45% of unplanned UK ICU admissions Introduction Delirium is a frequently occurring complication of critical care, occurring in approximately 45% of unplanned UK ICU admissions Figure 1 (abstract P477). Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S168 Figure 1 (abstract P479). Range of PRE-DELIRIC scores. Methods A literature study via MEDLINE and Embase search with keywords ‘PRIS’, ‘lactic acid’, ‘propofol’ and ‘sedation’. All cases in English, French and Spanish were indentifi ed. Exclusion criteria were onset >48 hours, unclear description of time pattern and dose. p p Results Twenty-two cases of early PRIS were found. These concerned 10 pediatric versus 12 adult patients. Eleven were identifi ed in the ICU versus 11 in the operating room. The survival rate of early PRIS was 95.5%, and morbidity was restricted to four patients. In the adult subgroup, the mean propofol dose was 4.9  mg/kg/hour. Triggering factors such as use of catecholamines and corticosteroids were found in 36.4% and 45% of patients. In total, 3/22 cases matched Bray’s defi nition of PRIS. In 14/22 cases, lactic acidosis was interpreted as onset of PRIS. Conclusion Compared with a review by Fudickar [1], we found signifi cant diff erences in critical dose, risk factors, symptomatology and morbidity/mortality between PRIS and early PRIS cases. As criticisms are off ered, a question is whether these cases really are the onset of the fatal syndrome PRIS. Therefore, we completed diff erential diagnosis of lactic acidosis and found that not all possible causes (for example, hyperglycemia, ketonemia, pharmacologic confounders as biguanides, epinephrine) were ruled out in most cases. This is important since PRIS is an exclusion diagnosis. The existence of early PRIS should indeed be questioned and investigated by large, multicenter observational trials. Reference [1]. The presence of delirium in critical care is an independent risk factor for mortality; for every day of delirium, there is an additional 10% relative risk of death at 1 year [2]. A delirium prediction tool PRE- DELIRIC has been recently developed and calibrated in a multinational project [3]. This study aimed to determine the utility of PRE-DELIRIC on our ICU. q Reference 1. Fudickar A. Propofol infusion syndrome in anaesthesia and intensive care medicine. Curr Opin Anaesthesiol. 2006;19:404-10. Methods This study prospectively investigated 41 patients. Medical and surgical general ICU patients were included after 24 hours of sedation and mechanical ventilation. The researchers calculated PRE-DELIRIC scores for each patient. PRE-DELIRIC involves recording 10 variables, submitted into an online algorithm that estimates the percentage risk of delirium. We diagnosed delirium with the CAM-ICU which was performed 12 hourly [4]. P484 Prolonged dexmedetomidine infusion and drug withdrawal in critically ill children A Haenecour, A Goodwin, W Seto, C Urbain, P Laussen, C Balit The Hospital for Sick Children, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P484 (doi: 10.1186/cc14564) Prolonged dexmedetomidine infusion and drug withdrawal in critically ill children A Haenecour, A Goodwin, W Seto, C Urbain, P Laussen, C Balit The Hospital for Sick Children, Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P484 (doi: 10.1186/cc14564) Results One hundred and twenty-six of 174 respondents indicated that they practice in the ICU setting. Sixty-six per cent were specialists and mainly anaesthesiologists (42%), whilst 32% were critical care subspecialists. The respondents indicated that on average 30 ± 20% of their patients experience delirium. Eighty per cent of the respondents indicated that delirium impacts signifi cantly negatively on patient outcomes whilst 1% indicated that there was no such association. Delirium screening is achieved mainly by clinical assessment (77%). Twenty-four per cent utilise an objective tool to screen for delirium and amongst them the CAM-ICU is utilised by 80%. Amongst delirious patients the sedative of choice is dexmedetomidine in the majority. However, 20% prescribe midazolam as a fi rst choice in this setting. Conclusion The fi ndings are comparable with reports of similar surveys conducted in other regions. The delirium screening method is inadequate as the vast majority do not utilise an objective method. Introduction We investigated the incidence, symptoms and risk factors for withdrawal associated with prolonged dexmedetomidine use. Dexmedetomidine is an α2-adrenergic receptor agonist, with anxiolytic, analgesic and sedative properties. Intended for short-term use, there is increasing literature describing prolonged use for sedation. However, this raises the potential of withdrawal syndrome and there is no recommendation for the discontinuation of dexmedetomidine. Other goals included determining the hemodynamic eff ects of discontinuation of dexmedetomidine and role of clonidine in patients with prolonged dexmedetomidine use. g Methods A retrospective review of patients admitted to the critical care unit who had exposure to dexmedetomidine for longer than 48  hours, between 1 January 2014 and 15 July 2014. Data included patient demographics, dexmedetomidine exposure (bolus dose, total cumulative dose, duration), other sedative exposure, withdrawal symptoms measured by WAT-1 score, nursing subjective assessment and treatment given for withdrawal. Each potential withdrawal episode was reviewed by two reviewers. Hemodynamic parameters were analyzed to assess hemodynamic changes associated with discontinuation of dexmedetomidine. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. i Results During propofol administration TEI reduced 19% whereas after awakening it increased 14.5% but did not reach baseline. Conversely TEE did not change during the study. During propofol sedation, TF decreased 34% and returned to baseline after recovery. DM showed 29% reduction during propofol administration whereas the forced diaphragmatic motion tested when patients were conscious (forced DM) did not evidence any change. y g Conclusion In this observational study, ultrasound assessed that propofol causes a reduction of diaphragmatic contraction and motion during endoscopic procedures. P484 Descriptive statistics were used with t test and chi-square test. Median and interquartile range (IQR) are reported. 8 Delirium knowledge and assessment by ICU practitioners in South Africa: results of a national survey S Chetty, F Paruk University of the Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P482 (doi: 10.1186/cc14562) Delirium knowledge and assessment by ICU practitioners in South Africa: results of a national survey S Chetty, F Paruk University of the Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P482 (doi: 10.1186/cc14562) p g p Conclusion These results are consistent with the hypothesis of 2 days reduction of the median weaning time in the loxapine group, but the diff erence was not statistically signifi cant. Loxapine reduces the need for resuming sedation during weaning from MV. Given the quality of the data and methodology, these results may be useful in future meta-analyses. Introduction Delirium recognition in critically ill patients is considered to be important taking into account the poor outcomes associated with its occurrence. The purpose of this study was to evaluate knowledge pertaining to delirium as well as the implementation of screening practices. This study constituted a component of a survey that explored current sedation-related practices in South African ICUs. Methods Following approval from the University Human Research ethics committee, a validated questionnaire was distributed electronically to physician members of various medical databases in South Africa as South Africa does not have a formal registry of critical care practitioners. g References g y Results The trial was discontinued after 101 patients had been randomized because of insuffi cient enrollment. Fifteen patients withdrew consent, leaving 86 patients for analysis. Forty-seven patients were assigned to the loxapine group and 39 to the placebo group. Median time to successful extubation was 3.2  days in the loxapine group and 5 days in the placebo group (RR = 1.2, 95% CI = 0.75 to 1.88; P = 0.45). During the fi rst 24 hours, sedation was more frequently resumed in the placebo group (44% vs. 17%, P = 0.01). One patient had a transient seizure in the loxapine group. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. 1. Singh H, et al. Cochrane Database Syst Rev. 2008;4:CD006268. 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. g , y ; 2. Matamis D, et al. Intensive Care Med. 2013;39:801-10. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. Results The trial was discontinued after 101 patients had been randomized because of insuffi cient enrollment. Fifteen patients withdrew consent, leaving 86 patients for analysis. Forty-seven patients were assigned to the loxapine group and 39 to the placebo group. Median time to successful extubation was 3.2  days in the loxapine group and 5 days in the placebo group (RR = 1.2, 95% CI = 0.75 to 1.88; P = 0.45). During the fi rst 24 hours, sedation was more frequently resumed in the placebo group (44% vs. 17%, P = 0.01). One patient had a transient seizure in the loxapine group. Data analyzed are reported in Table  1 and expressed as mean (SD). *ANOVA was used to compare data for repeated measurements. Post hoc statistical comparison with Bonferroni’s test was used to identify signifi cant variations. than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. The primary endpoint was the time between the fi rst administration of loxapine or placebo and successful extubation (no reintubation in the following 48 hours). Three hundred patients were necessary to have 90% power to detect a 2-day reduction of weaning time in the loxapine group with a one-sided type I error rate of 5%. than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. Short-term propofol infusion syndrome (PRIS): fact or fi ction? A systematic review on early PRIS in intensive care and anesthesia J Vandenbrande University Hospitals Leuven, Belgium University Hospitals Leuven, Belgium y Critical Care 2015, 19(Suppl 1):P480 (doi: 10.1186/cc14560) Table 1 (abstract P481) Variable Baseline Sedation Awakening P value Thickness end inspiration (mm) 3.25 (0.21) a2.66 (0.20) b,c3.22 (0.30) <0.001* Thickness end expiration (mm) 2.11 (0.20) d2.00 (0.15) 2.07 (0.15) 0.112* Thickening fraction 0.54 (0.07) e0.36 (0.10) f,g0.49 (0.11) <0.001* Diaphragmatic motion (mm) 18.66 (2.23) h13.27 (4.93) 15.31 (0.40) 0.055 Diaphragmatic motion forced 54.61 (19.34) – 56.34 (13.75) 0.298 (mm) aP <0.001 versus baseline. bP = 0.043 versus baseline. cP <0.001 versus sedation. dP = 0.030 versus baseline. eP =0.012 versus baseline. fP =0.353 versus baseline. gP = 0.041 versus sedation. hP = 0.022 versus baseline. Introduction Propofol infusion syndrome (PRIS) is a rare propofol complication, leading to cardiac failure. It was fi rst described in critically ill children and in adults with traumatic brain injury. Pathophysiology is unknown although common factors are the prolonged (>48 hours) use of high-dose (>5 mg/kg/hour) propofol combined with elevated levels of catecholamines and corticosteroids. Recently, case reports of early- onset PRIS during anesthesia and in the early postoperative setting were published. In many of these, lactic acidosis is interpreted as onset of PRIS. Criticism off ers that it might concern a poor diff erential diagnostic approach or an observational bias. Also, lactic acidosis is not an obligate PRIS symptom and incidence of lactic acidosis during propofol sedation is unknown. To gain insight into the incidence and characteristics of early PRIS, we performed a systematic review on early PRIS cases. S169 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 than 48 hours who were potential candidates for weaning from the ventilator and who exhibited agitation defi ned by a Richmond Agitation Sedation Scale (RASS) >2 after sedation withdrawal were randomly assigned to receive either loxapine or placebo. All participants were masked to group of allocation. After randomization, patients received 150 mg loxapine or placebo by nasogastric tube. RASS was monitored every 4 hours. A second dose of loxapine or placebo was administered if agitation persisted or worsened. In case of severe agitation, usual sedation (benzodiazepines and morphinic agents) was immediately resumed. Extubation was contemplated when patients were conscious and calm. Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients Introduction Recently, several animal studies observed that dexmedetomidine (DEX), a new sedative and α2-adrenoceptor agonist, inhibited the infl ammatory responses [1-3]. Moreover, DEX was reported to have anti-infl ammatory eff ects in patients [4,5]. However, these studies were the small-sized studies and there are few studies about the eff ects of long-term administration of DEX in severe septic patients. The present study evaluated the eff ects of long-term administration of DEX on infl ammatory responses and severity in severe septic patients. We hypothesize that the administration of DEX has benefi cial eff ects for severe septic patients. y y y Methods We investigate the role of DEX as a sedative agent used during recovery from deep sedation and weaning from extracorporeal support in patients on vv-ECMO. From May 2014 to October 2014 we prospectively enrolled seven patients aff ected by ARDS of diff erent etiologies treated with vv-ECMO. The mean age was 53.7 ± 7.9 years and the mean ICU stay was 21.4 ± 11.5 days. Initially, all patients were sedated with association of opioids and GABA receptor agonists, following the internal protocol. At the time of weaning from ECMO, ruled out cardiovascular instability, we started the administration of DEX (0.7 μg/kg/hour, without initial bolus) with progressive decrease of the dose of other sedative drugs. if Methods In 66 patients (M/F 44/22, mean age 66 years) with severe sepsis, who were administered propofol (0.5 to 4.0  mg/kg/hour) only for sedation, 42 patients (M/F 28/14, mean age 67  years) were administered DEX (0.2 to 0.7  μg/kg/hour) for more than 24  hours in addition to propofol (DEX group). Twenty-four patients were not administered DEX (Control group). Primary outcome were changes in infl ammatory responses at 48 hours after the administration of DEX or none, and secondary outcomes were changes in APACHE II and SOFA scores at 48 hours after the administration of DEX or none. Results The mean duration of DEX infusion was 6.1 ± 4.8 days. Except for one patient, who received DEX as a single drug after suspension of other sedatives, a low-dose infusion of another sedative (<50% compared with initial dose) was maintained. Three patients presented adverse events: two bradycardia and one hypotension. P486 P486 Short-term sedation of mechanically ventilated ICU patients with propofol, benzodiazepines, or dexmedetomidine: systematic review and meta-analysis on awakening and recovery times A Feuersenger1, L Pradelli2, A Aliano2, JF Baron3, M Westphal1 1Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany; 2AdRes, Torino, Italy; 3Fresenius Kabi ELAMA, Paris, France Critical Care 2015, 19(Suppl 1):P486 (doi: 10.1186/cc14566) Conclusion In the present study, the long-term administration of DEX has benefi cial eff ects of infl ammatory responses and severity for severe septic patients. Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) In four patients DEX was discontinued after recovery of respiratory function; in two patients deeper sedation for ventilatory dyssynchrony was needed so other sedative drugs were started. Only in one patient was the drug suspended for extreme bradycardia, resolved after suspension. Results The administration of DEX occurred for a mean 130 hours (24 to 433  hours) in the DEX group. White blood cell counts, C-reactive protein (CRP) and procalcitonin (PCT) in both groups signifi cantly decreased after the administration of DEX or none. However, CRP and PCT in the DEX group were signifi cantly lower than those in the control group: CRP 7.7 (5.0) versus 13.6 (7.9)  mg/dl; P  <0.05, PCT 7.6 (11.7) versus 18.6 (11.6) ng/ml; P <0.05, mean (SD). APACHE II and SOFA scores in both groups decreased after the administration of DEX or none, but APACHE II and SOFA scores in the DEX group were lower than those in the control group: APACHE II 10.8 (4.8) versus 15.2 (5.1); P <0.05, SOFA 3.6 (2.0) versus 5.8 (2.9); P <0.05, mean (SD). p y p Conclusion In our study, DEX allowed the reduction of doses of other sedative drugs during weaning from vv-ECMO; this may lead to a cooperative sedation, promoting spontaneous breathing. Side eff ects described and the cost–benefi t ratio must still be verifi ed extensively in patients during weaning from ECMO. P487f Eff ects of administration of dexmedetomidine on infl ammatory responses and severity in severe septic patients T Taniguchi, M Okajima, K Sato, T Noda Kanazawa University, Kanazawa, Japan Critical Care 2015, 19(Suppl 1):P487 (doi: 10.1186/cc14567) P485 Weaning from extracorporeal membrane oxygenation: experience with dexmedetomidine in seven adult ARDS patients M Cozzolino, A Franci, A Peris, L Tadini Buoninsegni, B Loriga A.O.U. Careggi, Firenze, Italy Critical Care 2015, 19(Suppl 1):P485 (doi: 10.1186/cc14565) Introduction Sedation in the ICU is a basic therapeutic procedure to increase tolerance of invasive treatments and reduce discomfort. Extracorporeal membrane oxygenation (ECMO) is a highly invasive treatment and prolonged sedation may be required. Patients undergoing ECMO represent a challenge with respect to sedation. Initially, deep sedation may be required to optimize ventilation and circuit–patient fl ows and to minimize oxygen consumption. The other critical phase is represented by weaning from ECMO support. Optimal sedation is not clearly defi ned, moreover there are no data on sedation practices with dexmedetomidine (DEX) in adult patients undergoing ECMO. In contrast to other sedatives, DEX has analgosedative eff ects without respiratory depression, and could be useful to facilitate spontaneous respiratory activity during recovery from sedation. i Conclusion In conclusion, Pr is associated with shorter awakening and recovery times after STS than BDZ, while no diff erence could be shown when Pr was compared with Dx. P487f Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 to help tolerate intubation and reduce pain and anxiety. Propofol (Pr) is a widely used option, but other viable alternatives for short-term sedation (STS; that is, <24 hours) include benzodiazepines (BDZ) and dexmedetomidine (Dx). We aimed at pooling all available evidence on the comparative eff ects of Pr in terms of awakening and recovery times after STS in mechanically ventilated ICU patients. with withdrawal were cumulative dose (P = 0.01) and duration of use of dexmedetomidine (P = 0.02). Duration of opioids exposure prior to dexmedetomidine wean was also a risk factor for withdrawal (P = 0.01). Use of clonidine as a transition from dexmedetomidine did not protect against withdrawal (P = 0.59). g Conclusion This study showed that withdrawal syndrome is associated with prolonged infusion of dexmedetomidine. Patients with higher cumulative doses and longer duration of exposure were more at risk. Our results suggested that clonidine use is not protective for withdrawal from dexmedetomidine. y Methods We planned a systematic literature review searching Medline and Scopus and performed a meta-analysis on direct comparisons reporting on weaning time (Tw), duration of mechanical ventilation (Tmv), time to extubation (Tex) and length of stay in the ICU (Ticu). The primary analysis considered only data from RCTs, while in a secondary analysis observational studies were also included.i y Results The literature search identifi ed 15 relevant RCTs, of which 11 versus BDZ, and a further fi ve observational studies, of which one versus BDZ. When compared with BDZ, Pr associated with signifi cantly reduced Tw (–1.6 hours, 95% CI: –2.5 to –0.8), Tmv (–2.0 hours, 95% CI: –3.7 to –0.2), and Ticu (–5.0 hours, 95% CI: –8.5 to –1.4); no statistically signifi cant diff erence resulted when comparing Pr and Dx. When nonrandomized evidence was included, results did not change signifi cantly. Loxapine to control agitation during weaning from mechanical ventilation: a randomized controlled trial S Gaudry1, B Sztrymf2, R Sonneville3, B Megarbanne4, C Clec’h5, J Ricard1, D Hajage3, D Dreyfuss1 1Hôpital Louis Mourier, Colombes, France; 2Hôpital Béclère, Clamart, France; 3Hôpital Bichat, Paris, France; 4Hôpital Lariboisière, Paris, France; 5Hôpital Avicennes, Bobigny, France Critical Care 2015, 19(Suppl 1):P483 (doi: 10.1186/cc14563) Results A total of 53 patients accounted for 69 unique dexmedetomidine treatment courses. Median age at the time of dexmedetomidine infusion was 5  months (range 1  day to 3  years). Dexmedetomidine dose ranged from 0.1 to 2  μg/kg/hour with a median cumulative dose of 87  μg/kg (IQR 53, 156). Median duration of exposure to dexmedetomidine was 124  hours (IQR 76, 178) with a maximum duration of 466 hours. We identifi ed 24 separate episodes of withdrawal (incidence 35%). Most common symptoms were agitation (100%), fever (67%), vomiting/retching (46%), loose stools (29%) and decreased sleep (20%). Statistical analysis showed that factors signifi cantly associated Introduction Weaning from prolonged mechanical ventilation (MV) in the ICU may be impeded by the occurrence of agitation. Loxapine had the ability to control agitation without aff ecting the effi cacy of spontaneous ventilation in an observational study, justifying the implementation of a randomized controlled trial. Methods We conducted a multicenter, placebo-controlled, parallel group, randomized trial at fi ve French ICUs between November 2011 and November 2013. Patients (aged >18  years) under MV for more S170 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P490 Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) Results A total of 77 children with a median age of 15 (4 to 84) months, weight of 10 (5.7 to 20) kg and length of ICU stay of 8 (5 to 14) days received DEX, with a mortality rate of 9%. Indications were: weaning from mechanical ventilation (32.5%), neurosurgical postoperative (NCI) and upper airway surgery (VAS) (24.7%), non-invasive ventilation (13%), refractory tachycardia (6.5%) and other indications (23.3%). There was no diff erence between the initial and maximum doses and infusion length. There was a signifi cant decrease in MAP and HR after 6 hours infusion of DEX in the total group; however, no signifi cant diff erence occurred between groups when analyzing MAP and HR 24 hours after the start of infusion (P = 0.798 and 0.379, one-way ANOVA, respectively). In six patients (8%) DEX was suspended for possible side eff ects. Introduction To determine which of three methods of pain manage- ment provided the best pain control in severe ASA III older patients in the surgical ICU (SICU). As technology improves, more older patients benefi t from surgery and need SICU care. Older surgery patients frequently present two medical problems. First is unspecifi c symptoms and decreased pain sensation resulting in delayed diagnosis. Second, they usually are not given enough perioperative pain relief. Optimal pain management results in perioperative stable hemodynamic status and decreased morbidity, mortality, length of stay and medical costs. Methods A retrospective cohort study chart review of 1,872 all-cause patients in a 16-bed SICU during April 2011 to September 2012. Un- consciousness, uncooperative, ASA <III and <65-year-old patients were excluded. The primary point was to compare eff ectiveness of three diff erent methods of pain management: P.R.N. i.v. Demerol/NSAID (D/N), i.v. patient-controlled analgesia (PCA) and patient-controlled epidural analgesia (PCEA), in three diff erent conditions: rest, movement and coughing, with visual analogue scales (VAS 0 to 100). Secondary point was patient satisfaction. Introduction To determine which of three methods of pain manage- ment provided the best pain control in severe ASA III older patients in the surgical ICU (SICU). As technology improves, more older patients benefi t from surgery and need SICU care. Older surgery patients frequently present two medical problems. P490 Best pain management for critical older patients in the surgical ICU W Huang Mackay Memorial Hospital, Taipei, Taiwan Critical Care 2015, 19(Suppl 1):P490 (doi: 10.1186/cc14570) First is unspecifi c symptoms and decreased pain sensation resulting in delayed diagnosis. Second, they usually are not given enough perioperative pain relief. Optimal pain management results in perioperative stable hemodynamic status and decreased morbidity, mortality, length of stay and medical costs. Methods A retrospective cohort study chart review of 1,872 all-cause patients in a 16-bed SICU during April 2011 to September 2012. Un- consciousness, uncooperative, ASA <III and <65-year-old patients were excluded. The primary point was to compare eff ectiveness of three diff erent methods of pain management: P.R.N. i.v. Demerol/NSAID (D/N), i.v. patient-controlled analgesia (PCA) and patient-controlled epidural analgesia (PCEA), in three diff erent conditions: rest, movement and coughing, with visual analogue scales (VAS 0 to 100). Secondary point was patient satisfaction. f Conclusion Increased DEX indications have been observed in the pediatric population. In this study DEX was demonstrated to be a safe and tolerable drug with few side eff ects, especially related to the cardiovascular system. References 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 1. Pichot C, Ghignone M, Quintin L. Dexmedetomidine and clonidine: from second- to fi rst-line sedative agents in the critical care setting? J Intensive Care Med. 2012;27:219-37. 2. Afonso J RF. Dexmedetomidina: Papel Atual em Anestesia e Cuidados Intensivos. Rev Bras Anestesiol. 2012;62:118-33. 3. Panzer O, Moitra V, et al. Pharmacology of sedative-analgesic agents: dexmedetomidine, remifentanil, ketamine, volatile anesthetics, and the role of peripheral mu antagonists. Crit Care Clin. 2009;25:451-69. 4. Hsu CD CJ. Pharmacologic agents for pediatric procedural sedation outside of the operating room. UpToDate2014. http://www.uptodate.com/contents/ pharmacologic-agents-for-pediatric-procedural-sedation. 5. Tobias JD. Dexmedetomidine: applications in pediatric critical care and pediatric anesthesiology. Pediatr Crit Care Med. 2007;8:115-31. 2. Afonso J RF. Dexmedetomidina: Papel Atual em Anestesia e Cuidados Intensivos. Rev Bras Anestesiol. 2012;62:118-33. p p Results A total of 1,292 patients were excluded. VAS results are presented in Table  1 as mean  ± SD. At rest, the PCEA group is signifi cantly better than the other two groups. When at movement, there is no diff erence between the PCEA group and the D/N group but both are better than the PCA group. While coughing, the PCEA group is worse than the D/N group, although there is no diff erence between the PCEA group and the PCA group. The PCEA group gets the best grades in patient satisfaction. 3. Panzer O, Moitra V, et al. Pharmacology of sedative-analgesic agents: dexmedetomidine, remifentanil, ketamine, volatile anesthetics, and the role of peripheral mu antagonists. Crit Care Clin. 2009;25:451-69. 4. Hsu CD CJ. Pharmacologic agents for pediatric procedural sedation outside of the operating room. UpToDate2014. http://www.uptodate.com/contents/ pharmacologic-agents-for-pediatric-procedural-sedation. 5. Tobias JD. Dexmedetomidine: applications in pediatric critical care and pediatric anesthesiology. Pediatr Crit Care Med. 2007;8:115-31. Psychometric comparison of three behavioral scales for the assessment of pain in critically ill patients unable to self-report p y p G Papakitsos1, A Kapsali1, T Papakitsou2 p , p , p 1GHA, Arta, Greece; 2General Hospital Messologi, Greece p g Critical Care 2015, 19(Suppl 1):P489 (doi: 10.1186/cc14569) Introduction Pain assessment is associated with important out omes in ICU patients but remains challenging, particularly in non communicative patients. Use of a reliable tool is paramount to allow any implementation of sedation/analgesia protocols in a multidisciplinary team. This study compared psychometric properties (inter-rater agreement primarily; validity, responsiveness and feasibility secondarily) of three pain scales: Behavioural Pain Scale (BPS/BPS-NI, that is BPS for non-intubated patients), Critical Care Pain Observation Tool (CPOT) and Non-Verbal Pain Scale (NVPS), the pain tool routinely used in this 16-bed medical ICU. Conclusion PCEA provided better pain control at rest than the other two methods, whereas P.R.N. Demerol/NSAID and PCEA were somewhat better than PCA when patients were moving. While coughing, P.R.N. Demerol/NSAID provided the best pain control. However, patient satisfaction was signifi cantly better with PCEA. p p References References 1. Taniguchi T, et al. Crit Care Med. 2004;32:1322-6. 2. Qiano H, et al. Crit Care. 2009;13:R136. 3. Wu Y, et al. Mediators Infl amm. 2013;2013:562154. 4. Pandharipande PP, et al. Crit Care. 2010;14:R38. 5. Ji F, et al. Circulation. 2013;127:1576-84. 1. Taniguchi T, et al. Crit Care Med. 2004;32:1322-6. 2. Qiano H, et al. Crit Care. 2009;13:R136.l Introduction Sedation in the ICU is crucial in reduction of patients’ discomfort, in particular in patients undergoing mechanical ventilation Introduction Sedation in the ICU is crucial in reduction of patients’ discomfort, in particular in patients undergoing mechanical ventilation S171 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P488 FRAP values. We found that FRAP values were inversely correlated with APACHE II score (r = –0.266, P <0.01) suggesting that, in trauma patients, increased antioxidant response, as measured by the FRAP assay, could be a pathophysiological response to stress. Albumin and uric acid concentrations reproduced the FRAP trend with severity. Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, P <0.002) and head trauma (β = –0.219, P <0.045). These results accen- tuate the infl uence of trauma location and severity in TAC changes. The TAC response in ED patients reinforces the need for adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. P488 Characteristics of the use of dexmedetomidine in critically ill children: a Brazilian study PL Lago, C Andreolio, J Piva, E Baldasso Hospital de Clinicas de Porto Alegre, Brazil Critical Care 2015, 19(Suppl 1):P488 (doi: 10.1186/cc14568 Table 1 (abstract P490) Demerol/NSAID PCA PCEA (n = 449) (n = 62) (n = 69) P value VAS (rest) 21 ± 14 * 19 ± 9* 12 ± 10 <0.001 VAS (movement) 35 ± 20 41 ± 12* 33 ± 11 0.02 VAS (coughing) 40 ± 24* 58 ± 14 51 ± 14 <0.001 *Signifi cant diff erence. P488 Characteristics of the use of dexmedetomidine in critically ill children: a Brazilian study Conclusion FRAP values in trauma ED patients are independently infl uenced by age (β = 0.271, P <0.021), APACHE II score (β = –0.356, P <0.002) and head trauma (β = –0.219, P <0.045). These results accen- tuate the infl uence of trauma location and severity in TAC changes. The TAC response in ED patients reinforces the need for adequate tailoring of treatments aimed at their recovery, such as antioxidant therapies. Introduction To describe the main indications, doses, infusion length and side eff ects of dexmedetomidine (DEX) administered to children and adolescents admitted to the pediatric ICU (PICU). p Methods A retrospective observational study including children (<18 years) admitted to a Brazilian PICU who received DEX between November 2011 and June 2014. Demographic data, indications, initial dose, maximum dose and time of infusion of DEX, side eff ects and impact on heart rate (HR) and mean arterial pressure (MAP) 6 and 24 hours after the start of infusion. P491 P491 Subanesthetic xenon increases erythropoietin levels in humans and remains traceable in the fi rst 24 hours after exposure: a randomized controlled trial J Ney, C Stoppe, M Brenke, A Goetzenich, S Kraemer, G Schaelte, A Fahlenkamp, R Rossaint, M Coburn RWTH Aachen University, Aachen, Germany Critical Care 2015, 19(Suppl 1):P491 (doi: 10.1186/cc14571) P491 Subanesthetic xenon increases erythropoietin levels in humans and remains traceable in the fi rst 24 hours after exposure: a randomized controlled trial J Ney, C Stoppe, M Brenke, A Goetzenich, S Kraemer, G Schaelte, A Fahlenkamp, R Rossaint, M Coburn RWTH Aachen University, Aachen, Germany Critical Care 2015, 19(Suppl 1):P491 (doi: 10.1186/cc14571) Methods In a prospective observational study of ED polytraumatized patients (n = 23, mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score of 11 ± 6) we measured (in the fi rst 24 hours) plasma TAC by the ferric reducing activity/antioxidant power (FRAP). For control subjects, we used age-matched and gender-matched volunteers (n = 32). We also evaluated the contribution of antioxidant molecules (uric acid, bilirubin, and albumin) to these values.f J Ney, C Stoppe, M Brenke, A Goetzenich, S Kraemer, G Schaelte, A Fahlenkamp, R Rossaint, M Coburn RWTH Aachen University, Aachen, Germany Critical Care 2015, 19(Suppl 1):P491 (doi: 10.1186/cc14571) Introduction The noble gas xenon was recently amended to the list of prohibited substances by the World Anti-Doping Agency as Introduction The noble gas xenon was recently amended to the list of prohibited substances by the World Anti-Doping Agency as Results Polytraumatized patients show diff erences in TAC with reference to control subjects. ED polytraumatized patients show high Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S172 Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. system (Pall Medical), a new device for sedation in ICU patients [2]. The system delivers volatile anesthetics in mechanically ventilated patients. An open reservoir scavenger and a dedicated gas fi lter avoid residual volatile anesthetic halogenate escaping into the room air. g p g Methods Ten mechanically ventilated patients electively admitted for ICU postoperative monitoring were sedated with sevofl urane delivered with the MIRUS™ system. Two patients were excluded from the analysis because inclusion criteria had been lost during the study period. Analgesia was obtained with morphine sulfate: bolus 0.1 mg/ kg i.v. at the end of surgery and 0.2 to 0.4 mg/kg/24 hours. i References Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. 1. Reade MC, et al. N Engl J Med. 2014;370:444. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. 2. Bomberg H, et al. Anaesthesia. 2014;69:1241. it is supposed to trigger the production of HIF-1α and subsequently erythropoietin. Subsequently, researchers and clinicians started a scientifi c discussion about the potential clinical benefi t in support of humans exposed to high demand, such as critically ill patients. The objective of this study was therefore to evaluate the eff ect of xenon on serum levels of erythropoietin in healthy volunteers. Bispectral index-guided anesthesia on time to tracheal extubation after onpump cardiac surgery Bispectral index-guided anesthesia on time to tracheal extubation after onpump cardiac surgery E Kaval, P Zeyneloglu, A Camkiran, A Sezgin, A Pirat, G Arslan Baskent University Faculty of Medicine, Ankara, Turkey Critical Care 2015, 19(Suppl 1):P493 (doi: 10.1186/cc14573) y y Methods This is a monocenter, randomized, blinded, crossover trial, which was registered at ClinicalTrials.gov (NCT01285271). Healthy study test persons were spontaneously breathing randomly 1  hour of xenon 30% (Xe/O2 30%/65%) or control gas (N2/O2 30%/65%). The primary outcome parameter was the erythropoietin level 24  hours after exposure. Secondary outcome parameters are xenon’s elimination kinetics measured in blood and exhalation samples. Introduction Electroencephalographic-based cerebral monitors such as the bispectral index (BIS) have been used for titration of both inhalational and intravenous anesthetics during general anesthesia. Titration of anesthetics using these monitors may facilitate an earlier recovery from general anesthesia and less consumption of anesthetics. The primary aim of this study was to investigate whether BIS-guided anesthesia would reduce time to tracheal extubation when compared with minimum alveolar concentration (MAC)-guided anesthesia in patients undergoing onpump cardiac surgery. Results The application of xenon increases erythropoietin levels with a maximum 24  hours after exposure (1.32 (0.99 to 1.66) P  = 0.033) compared with the baseline values and compared with control values (0.87 (0.68 to 1.05) P = 0.012, Figure 1). Xenon was gas chromatographically traceable in blood and exhalation probes up to 24 hours after exposure. Methods Fifty patients undergoing elective coronary artery bypass grafting surgery from a single tertiary referral university hospital were randomized to BIS-guided anesthesia (Group BIS, n  = 25) and MAC-guided anesthesia (Group MAC, n = 25). The inspired desfl urane concentration was titrated to maintain a BIS value of 40 to 60 in Group BIS and an age adjusted minimum alveolar concentration of 0.7 to 1 was used in Group MAC. Time to tracheal extubation across the two groups was the primary outcome measure. Secondary outcomes included intraoperative desfl urane consumption, postoperative complications, and lengths of stay in the ICU and hospital. p Conclusion One hour of a subanesthetic level of xenon increases erythropoietin levels in healthy study test persons and remains gas chromatographically traceable in blood and exhalation probes 24 hours after exposure. These fi ndings may stimulate larger studies to confi rm these results and to open new avenues for the therapeutic use of xenon in critically ill patients. P491 The primary endpoint was to achieve predefi nite levels of sedation (Riker scale 4). Secondary endpoints were the assessment of hemodynamic stability (MAP and HR), blood lactates, any type of side eff ects, and sevofl urane consumption. Data were collected at the following times: admission to the ICU (T1), 1 hour after initiation of sedation (T2), and 1 hour after sedation withdrawal (T3). Results were expressed as median (IQR) or mean (SD), where appropriate. Figure 1 (abstract P491). Erythropoietin levels after 30% xenon exposure. ( ), pp p Results The local ethical board approved the protocol. Median duration of sedation was 4 (5.5 to 2) hours. Predefi nite levels of sedation were achieved in all patients with a median MAC of sevofl urane of 0.5 (0.5 to 0.3)% and with a median gas consumption of 9.9 (14.3 to 5.3) ml/hour. MAP and HR values at T1 were 86.5 (97 to 80.8) mmHg and 81.5 (103.8 to 65) bpm, respectively; at T2, 74.5 (89 to 69.5) mmHg and 74 (82 to 58.3) bpm, respectively; and at T3 92.5 (101 to 76.8) mmHg and 74 (88.5 to 66.3) bpm, respectively. Lactates were always normal. Mechanical ventilation was interrupted 5.4 (3.1) minutes after withdrawal of sevofl urane and respiratory parameters always were within normal values. Finally, no side eff ects were registered at any phase of the study. Conclusion This pilot study shows that MIRUS™ is eff ective and safe in delivering sevofl urane for sedation at a predefi nite target level in postsurgical patients, without side eff ects. Further data with a larger number of patients and for a longer duration of sedation are required to confi rm these positive, preliminary observations. References P492 MIRUS™, a new system for sedation with halogenates in the ICU: a preliminary study of feasibility in postsurgical patients P Mancinelli, S Romagnoli, C Chelazzi, G Zagli, E Bonicolini, A Belardinelli, AR De Gaudio Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Critical Care 2015, 19(Suppl 1):P492 (doi: 10.1186/cc14572) Results Demographic features, logistic EuroSCOREs, duration of cardiopulmonary bypass and surgery were similar in both groups. Mean desfl urane consumption was signifi cantly lower in Group BIS (11.9 ± 1.7 ml/hour) compared with Group MAC (13.4 ± 3.0 ml/hour) (P = 0.031). Time to tracheal extubation was not signifi cantly diff erent between the groups (13.3 ± 9.6 hours vs. 17.0 ± 22.4 hours) (P = 0.68). Incidences of postoperative complications were similar and lengths of stay in the ICU and hospital were 2.4 ± 0.7 days versus 3.2 ± 2.7 days Introduction Sedation is standard practice in the ICU [1]. The aim of this study was to investigate the effi ciency and safety of the MIRUS™ Introduction Sedation is standard practice in the ICU [1]. The aim of this study was to investigate the effi ciency and safety of the MIRUS™ S173 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods The study was approved by the appropriate institutional review board. After written informed consent, 63 ASA I to III patients undergoing elective hip or knee replacement surgery under general anaesthesia were included. Patients were randomly organised into three groups (20 to 22 each). Anaesthesia was induced with intravenous anaesthetics. After tracheal intubation MIRUS™ automatically adjusted the end-tidal VA concentration to 1.0 MAC. Patients were ventilated with the Puritan Bennett 840 ICU ventilator. After 1 hour of anaesthesia with 1.0 MAC the ventilator mode was switched from SIMV VC+ (totally controlled ventilation, passive patient, with a tidal volume of 8 ml/IBW) to proportional assist ventilation with 50% support (active patient). At the end of surgery the MIRUS™ system was stopped (MAC set to 0.0) and recovery times were measured. and 5.3 ± 1.2 days versus 6.5 ± 3.1 days in Group BIS and Group MAC respectively (P >0.05 for all). and 5.3 ± 1.2 days versus 6.5 ± 3.1 days in Group BIS and Group MAC respectively (P >0.05 for all). Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) A Koroša1, A Markota2, F Svenšek2, A Sinkovič2 1University of Maribor, Slovenia; 2University Medical Centre Maribor, Slovenia Critical Care 2015, 19(Suppl 1):P494 (doi: 10.1186/cc14574) y Results Patients were comparable in age, height, weight and operation time. In 60/63 patients a MAC of 1.0 was reached by MIRUS™. Therefore, ISO 11.2 ± 3.3 ml/hour, SEVO 24.3 ± 4.8 ml/hour or DES 41.7 ± 7.9 ml/ hour (mean ± SD; t test: P <0.001) were used during passive ventilation. During patients’ active ventilation, mean VA consumptions of ISO 9.6 ± 5.1 ml/hour, SEVO 19.4 ± 9.6 ml/hour or DES 35.5 ± 23.0 ml/hour were detected (NS between passive and active patients). ISO was the cheapest VA (€2.70  ± 3.10/hour passive patient, €1.90  ± 2.30 active patient), followed by SEVO (€8.40 ± 3.70 passive patient and €6.8 ± 3.8 active patient) and DES (€9.6 ± 4.1 passive patient and €8.6 ± 6.5 active patient). Recovery times were signifi cantly shorter after SEVO and DES compared with ISO (minutes:seconds; ISO 9:31 ± 6:04, SEVO 6:19 ± 2:56, DES 5:27 ± 1:59). Introduction The aim of this study is to present our experience with sevofl urane in the ICU, outline which patients were sedated with sevofl urane and present the safety profi le. Sevofl urane has some potential advantages over intravenous sedation: rapid elimination and few interactions. The optimal role of sevofl urane in ICU is not known. Methods We performed a retrospective study on adult patients who were sedated with sevofl urane in the medical ICU. The decision to use sevofl urane was left to the attending physician. Institutional ethics committee approval was obtained. The target mean alveolar concentration in all patients was 0.5 to 1%. The AnaConDa® device (Sedana Medical, Uppsala, Sweden) was used along with the Anastasia® (Sedana Medical) gas monitor. Data were obtained from patients’ medical records. P495 Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia V Vinnikov, D Drees, J Herzog-Niescery, P Gude, H Vogelsang, B Cevik, T Weber, M Bellgardt St. Josef-Hospital, Ruhr-Universität, Bochum, Germany Critical Care 2015, 19(Suppl 1):P495 (doi: 10.1186/cc14575) Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery JL Iribarren, JJ Jimenez, N Perez, M Brouard, R Perez, O Gonzalez, A Arbesu, R Martinez, ML Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P496 (doi: 10.1186/cc14576) Interaction between etomidate and beta tumoral necrosis factor on hemodynamic response after cardiac surgery JL Iribarren, JJ Jimenez, N Perez, M Brouard, R Perez, O Gonzalez, A Arbesu, R Martinez, ML Mora Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2015, 19(Suppl 1):P496 (doi: 10.1186/cc14576) Introduction The use of etomidate is a risk factor for relative adrenal insuffi ciency in patients undergoing cardiopulmonary bypass (CPB) [1]. The objective was to determine the possible interaction between Conclusion We identifi ed sevofl urane as an appropriate sedation agent in a diverse group of patients. Sevofl urane advantages over intravenous sedation could be more pronounced in some patient groups (for example, successful resuscitation after cardiac arrest). The safety profi le of sevofl urane sedation was comparable with intravenous sedation [1]. Reference Figure 1 (abstract P496). 1. Mesnil et al. Intensive Care Med. 2011;37:933-41. 1. Mesnil et al. Intensive Care Med. 2011;37:933-41. Reference 1. Bomberg H, et al. Anaesthesia. 2014;69:1241-50. 1. Bomberg H, et al. Anaesthesia. 2014;69:1241-50. P496 Use of sevofl urane in the medical ICU: 2-year experience, patient and safety profi le Introduction The aim of this study is to present our experience with sevofl urane in the ICU, outline which patients were sedated with sevofl urane and present the safety profi le. Sevofl urane has some potential advantages over intravenous sedation: rapid elimination and few interactions. The optimal role of sevofl urane in ICU is not known. l Methods We performed a retrospective study on adult patients who were sedated with sevofl urane in the medical ICU. The decision to use sevofl urane was left to the attending physician. Institutional ethics committee approval was obtained. The target mean alveolar concentration in all patients was 0.5 to 1%. The AnaConDa® device (Sedana Medical, Uppsala, Sweden) was used along with the Anastasia® (Sedana Medical) gas monitor. Data were obtained from patients’ medical records. Conclusion This study showed that MIRUS™ could automatically control end-tidal VA concentrations in ventilated and spontaneously breathing patients. Using ISO reduces costs. Further studies must be taken to analyse feasibility, costs and recovery times of ISO, SEVO and DES used for sedation in an ICU setting. f Results We included 61 adult patients who were admitted from April 2012 to November 2014. Mean age was 62.6 ± 14.9 years, 39 (63.9%) were male. ICU mortality was 41%, hospital mortality was 43%. Mean duration of sevofl urane use was 3.56 ± 2.31 days. Admission diagnoses were: successful resuscitation after cardiac arrest (44.2%), sepsis (37.7%), cardiogenic shock (4.9%), pancreatitis (3.3%) and liver failure, acute exacerbation of COPD, asthma, tetanus and intracerebral hemorrhage (1.6% each). During treatment with sevofl urane, no patients developed malignant hyperthermia, new hyperkalemia or QT prolongation. In three (4.9%) patients, worsening liver function tests prompted sevofl urane discontinuation. Ischemic hepatitis was considered an alternative in all three patients. Seven (11.4%) patients developed renal failure while receiving sevofl urane. Sevofl urane was continued in all patients and renal failure was attributed to alternative diagnoses. No self-extubations were recorded. In seven (11.4%) patients, sevofl urane was discontinued because of worsening ventilation. In six (9.8%) patients, unexpected awakening occurred. Eight patients (13.1%) had symptoms of delirium after sevofl urane inhalations were concluded.il Reference P492 Conclusion Intraoperative use of BIS monitoring in patients undergoing onpump cardiac surgery reduced desfl urane requirement but BIS- guided anesthesia did not facilitate time to extubation and lengths of stay in the ICU and hospital. P494 Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia l V Vinnikov, D Drees, J Herzog-Niescery, P Gude, H Vogelsang, B Cevik, T Weber, M Bellgardt St. Josef-Hospital, Ruhr-Universität, Bochum, Germany Critical Care 2015, 19(Suppl 1):P495 (doi: 10.1186/cc14575) Introduction The new MIRUS™ system as well as the established AnaConDa® system uses a refl ector to conserve volatile anaesthetics (VA) [1]. Both systems act with commercially available ICU ventilators. In contrast to AnaConDa®, MIRUS™ includes an automated control of end-tidal VA concentrations. In this study we compared feasibility, costs and recovery times after anaesthesia with isofl urane (ISO), sevofl urane (SEVO) or desfl urane (DES) in ventilated and spontaneously breathing patients. S174 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of more signifi cant impact than medication errors. We assessed the incidence and characteristics of operation-related MEs to improve patient safety in such patients. etomidate and beta tumoral necrosis factor (TNFβ) polymorphism on hemodynamics after CPB. Methods A prospective cohort study on CPB patients who received etomidate or not during anesthetic induction during 2008 to 2011. Demographic and postoperative variables were collected. We tested the Hardy–Weinberg equilibrium in order to avoid selection bias. V18 SPSS was used. y Methods The Japan Adverse Event (JET) study was a prospective cohort study which had evaluated AEs and MEs at two tertiary care hospitals. We included all adult patients aged ≥15 years old who had operations over a 2-month period. The primary outcome of this study was the operation-related MEs, defi ned as any deviation from appropriate process of an operation or perioperative care. Trained nurses placed at each participating hospital reviewed all charts daily on weekdays, along with laboratories, incident reports, and prescription queries to collect any potential event. They also collected the characteristics of the patients in the cohort. Some operation-related MEs are associated with operation-related AEs, which are operation-related preventable AEs. After those suspected events were collected, physician reviewers independently evaluated them and classifi ed them as operation- related MEs, AEs, or rule violations. Physician reviewers assessed and rated operation-related AEs according to the symptom and the severity of injury. Results We studied 433 patients undergoing CPB, 285 (65.8%) men and 148 (34.2%) women, 66 ± 6 years, EuroSCORE I 5.3 ± 4%. TNFβ was in Hardy–Weinberg equilibrium (χ2: 0.6; P = 0.42). Visualising patients’ dynamics in the ICU and predicting mortality in real-time using big data MK Komorowski, AF Faisal MK Komorowski, AF Faisal Imperial College London, UK Critical Care 2015, 19(Suppl 1):P499 (doi: 10.1186/cc14579) , Imperial College London, UK Critical Care 2015, 19(Suppl 1):P499 (doi: 10.1186/cc14579) Introduction As informatisation of hospitals continues to spread, increasing amounts of healthcare related data are being collected, and the ICU is no exception. Large datasets are being made available to the scientifi c community, and off er the potential to answer clinical questions and to develop the next generation of clinical tools. A demonstration of such a tool is presented here, built using data from the Multiparameter Intelligent Monitoring in Intensive Care II (MIMIC-II) open database. Methods All of the adult patients who died during their stay in the ICU were included, as well as a matched cohort of patients who survived for more than 28 days after discharge. Data regarding their vital signs, laboratory tests and demographics were collected. Using Matlab, a graphical method involving principal component analysis was developed. The expected mortality was computed using the k-nearest neighbours’ method and compared with several classifi cation algorithms (logistic regression, random forest, support vector machine, Gaussian mixture models). g Conclusion The fi ndings are comparable with reports of sedation surveys conducted in other countries. There is an evidence–practice gap that needs to be addressed. Sedation practices in South African ICUs: results of a national survey F Paruk, S Chetty University of Witwatersrand, Johannesburg, South Africa Critical Care 2015, 19(Suppl 1):P497 (doi: 10.1186/cc14577) Introduction There has been a paradigm shift in the approach to sedation of critically ill patients. The purpose of this study was to evaluate current sedation-related practices in South African ICUs. Conclusion Ninety-three percent of operation-related MEs resulted in operation-related AEs and 21% of them resulted in life-threatening events. Prevention of operation-related MEs should improve the mortality of surgical patients. p Methods A validated questionnaire was distributed electronically to physician members of various medical databases in South Africa as South Africa does not have a formal registry of critical care practitioners. Results One hundred and twenty-six of 174 respondents indicated that they practice in the ICU setting. Sixty-six per cent were specialists and mainly anaesthesiologists (42%), whilst 32% were critical care subspecialists. The public and private-sector representation was 64% and 46% respectively. A written sedation guideline is implemented by 42%. Forty-three per cent utilise a sedation scale, with the Ramsey Sedation Scale being the commonest in use. However, 38% of sedation scale users do so infrequently. Daily interruption of sedation is practiced by 75%. Light sedation is targeted by 42% and 14% do not follow any sedation targets. Upon admission and on subsequent days, sedation targets are achieved most of the time by 48% and 69% of the respondents respectively. Whilst a wide variety of sedatives are prescribed, midazolam constitutes the most commonly prescribed agent. Dexmedetomidine is the agent of choice for postcardiac surgery patients with cardiovascular comorbidities, delirious patients, during weaning and for non-invasive ventilation. Propofol is the agent of choice amongst neurological patients. The respondents indicated that there is a need for local sedation guidelines.i Automated control of end-tidal volatile anaesthetic concentration using the MIRUS™ system: a comparison of isofl urane, sevofl urane and desfl urane in anaesthesia A total of 254 (58.7%) patients received etomidate, 152 out of them required vasoactive drugs. Homozygous G was defi ned as unfavorable TNFβ versus the A allele [2]. Using the general linear model after adjusting for sex and amines dose at 4  hours, an independent association was observed between the systemic vascular resistance index (SVRI) at 4 hours and the use of etomidate (F: 18; P <0.001): 1,849 (95% CI: 1,673 to 2,024) versus 2,493 (95% CI: 2,258 to 2,729) dinas.seg/cm5.m2, the presence of homozygous G (F: 6.5; P  = 0.01), and also showed a signifi cant etomidate–homozygous G interaction (F: 22.8: P <0.001): 1,687 (95% CI: 1,350 to 2,023) versus 3,041 (95% CI: 2,589 to 3,492) dinas seg/cm5. m2 (Figure 1). Results This study included 389 patients with 6,624 patient-days. The median age of patients was 69 years and 224 (58%) were male. Among these 389 patients, 31 patients had 46 operation-related MEs during their hospital stay and the incidence of operation-related MEs was 12 per 100 patients. Operation-related AEs occurred in 29 patients with 43 events. The most frequent symptoms for operation-related MEs were skin (26%), bleeding (21%), and central nervous system (14%). Among 46 operation-related MEs, 43 (93%) were not intercepted, and they resulted in operation-related AEs that were considered as preventable operation-related AEs. Nine of preventable operation-related AEs (21%) were fatal or life-threatening: fi ve were nerve injury during operation and stroke after neurosurgical operation, and one biliary peritonitis after gastrectomy and cholecystectomy, and tension pneumothorax after lung lobectomy, and two unexpected massive bleeding due to vessels injury. Conclusion Etomidate use is associated with lower postoperative SVRI which is increased in the presence of G homozygosity for TNFβ polymorphism. P498 Epidemiology of operation-related medical errors in inpatients in Japan: the JET study p y p References 1. Iribarren JL, et al. J Cardiothorac Surg. 2010;5:26. 1. Iribarren JL, et al. J Cardiothorac Surg. 2010;5:26. 2. Iribarren et al. Interact Cardiovasc Thorac Surg. 2008;7:1071-4. 2. Iribarren et al. Interact Cardiovasc Thorac Surg. 2008;7:1071-4. Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study y M Soares1, JM Kahn2, FA Bozza1, T Lisboa3, LP Azevedo4, W Viana5, L Brauer6, PE Brasil1, DC Angus2, JI Salluh1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Sta. Casa de Porto Alegre, Brazil; 4Hospital Sírio Libanês, São Paulo, Brazil; 5Hospital Copa DOr, Rio de Janeiro, Brazil; 6Hospital Sao Luiz Itaim, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P500 (doi: 10.1186/cc14580) Figure 1 (abstract P501). HEWS prior to critical event. Methods We conducted a prospectively identifi ed, retrospectively gathered cohort study at two hospitals of consecutively admitted medical and surgical patients over a 6-month period. One hospital had a rapid response team (RRT) and used HEWS with a trigger of 5 while the other was undergoing implementation of the HEWS without a RRT. HEWS was calculated for each patient on the fi rst day of admission and for the 3 days prior to inpatient arrest or death. A study investigator reviewed all events for accuracy. Our outcome of interest was a composite of inpatient cardiac arrest and hospital mortality. Introduction The aim was to investigate the impact of organizational factors on patient outcomes in a large sample of Brazilian ICUs. Introduction The aim was to investigate the impact of organizational factors on patient outcomes in a large sample of Brazilian ICUs. Methods A retrospective cohort study of 59,483 patients admitted to 78 ICUs in 51 hospitals during 2013. We retrieved demographic, clinical and outcome data from an electronic ICU quality registry (Epimed Monitor System). We surveyed ICUs using a standardized questionnaire regarding hospital and ICU structure, organization, staffi ng patterns, process of care, and family care policies. We used multilevel logistic regression analysis to identify characteristics associated with hospital mortality. y Results There were 7,138 patients admitted over 6 months. We found 0.5% of patients suff ered an inpatient arrest and 3.6% of patients died. Moreover, 66% of patients who died or arrested were admitted to the hospital without a RRT. Patients who arrested or died had more comorbidities defi ned by the Charlson Comorbidity Index (CCI) of 6.0 and 8.2 respectively compared with the general population, which had a CCI of 5.2. Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Results A total of 6,084 patients were included in the analyses, adding up to more than 12 million data points. Using this multidimensional dataset, a 3D representation of the clusters of survivors and nonsurvivors was built, showing how their trajectories diverge through time. Patterns in the evolution of individuals or subgroups of patients can be identifi ed using this approach. For example, the evolution of a new patient can be visualised, progressing through the clusters as his severity changes. His expected mortality can be predicted at any point in time, with an AUC ROC constantly above 0.85. Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) p y Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) p y Y Ohta1, M Sakuma1, D Bates2, T Morimoto1 1Hyogo College of Medicine, Hyogo, Japan; 2Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Critical Care 2015, 19(Suppl 1):P498 (doi: 10.1186/cc14578) Introduction Operation therapy is more invasive than medication therapy and then operation-related medical errors (MEs) might be Introduction Operation therapy is more invasive than medication therapy and then operation-related medical errors (MEs) might be S175 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P501). HEWS prior to critical event. Conclusion Machine learning tools off er an appealing mathematical framework for modelling complex medical situations. This proof of concept demonstrates that the application of computational sciences to high-quality data such as the MIMIC-II database has the potential to lead to the development of meaningful tools which will ultimately be capable of assisting physicians in making the right decision at the right time for an individual patient. P500 P500 Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study M Soares1, JM Kahn2, FA Bozza1, T Lisboa3, LP Azevedo4, W Viana5, L Brauer6, PE Brasil1, DC Angus2, JI Salluh1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Sta. Casa de Porto Alegre, Brazil; 4Hospital Sírio Libanês, São Paulo, Brazil; 5Hospital Copa DOr, Rio de Janeiro, Brazil; 6Hospital Sao Luiz Itaim, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P500 (doi: 10.1186/cc14580) Epidemiology of operation-related medical errors in inpatients in Japan: the JET study Only tight cooperation between clinicians and data scientists can help close the gap that currently separates these two worlds, for the ultimate benefi t of patients. Organizational factors and patient outcomes in Brazilian ICUs: the ORCHESTRA study The median and mean HEWS at time of admission was 1 and 1.7 for the general population, 2 and 2.4 for patients who suff ered an inpatient arrest and fi nally 3 and 3.8 for those who died. There was a rise in median HEWS from 2 to 5 in the 24 hours prior to in patient arrest or death. See Figure 1. y Results ICUs were mostly medical or medical–surgical (62.79%) and located in private hospitals (67.86%). Approximately half (40.51%) had critical care training programs. Median physician and nurse staff – bed ratios were 0.15 (IQR, 0.12 to 0.19) and 0.71 (0.61 to 0.84); board- certifi ed intensivists were present 24/7 in 16 (21%) of ICUs. Routine clinical rounds occurred in 67 (86%) and daily clinical checklists were used in 36 (46%) ICUs. Most frequently implemented protocols focused on sepsis management and VAP and CLABSI prevention. Median number of patients per center was 898 (IQR 585 to 1,715) and there were 67% medical admissions; 18% patients received mechanical ventilation (MV). Median SAPS 3 score was 41 (33 to 52) points. ICU and hospital mortality rates were 9.6% and 14.3%, respectively. Adjusting for relevant patients’ characteristics (SAPS 3 score, diagnostic admission category, chronic health status, comorbidities, MV use), case-volume and type of ICU, the ICU size (OR = 1.50 (95% CI, 1.45 to 1.95), for 11 to 20 beds; OR = 2.02 (1.40 to 2.92), for >20 beds) and ≥2 clinical protocols (OR  = 0.65 (0.42 to 0.99)) were the organizational characteristics associated with mortality. g Conclusion We found that a 2.5-fold increase in HEWS occurred 24 hours prior to critical events. Similar to previous studies, a RRT in conjunction with HEWS is the best system to reduce unanticipated adverse events. An absolute HEWS of 5 and/or a rapidly increasing HEWS should trigger rapid assessment and treatment to reduce preventable inpatient deaths and arrests. Reference 1. McNeill, Bryden. Resuscitation. 2013;84:1652-67. 1. McNeill, Bryden. Resuscitation. 2013;84:1652-67. Conclusion In a large sample of Brazilian ICUs, the implementation of clinical protocols was associated with better outcomes. Conversely, mortality was higher in larger ICUs. Ethnicity and trial recruitment h y g g Acknowledgements Funded by IDOR, CNPq and FAPERJ. Endorsed by BRICNet. HL Cronshaw, SW Scott, S Bowrey, JP Thompsoni HL Cronshaw, SW Scott, S Bowrey, JP Thompson Leicester Royal Infi rmary, Leicester, UK , , y, Leicester Royal Infi rmary, Leicester, UK yi y Critical Care 2015, 19(Suppl 1):P502 (doi: 10.1186/cc14582 Introduction Surrogate consent and time-sensitive recruitment in critical care research is challenging, yet low enrolment numbers or omitting ethnic groups skew results and conclusions. Patients from diff erent ethnic groups may respond diff erently to therapeutics [1]. There are few data about the eff ect of ethnicity on recruitment into ICU trials. Our ICU recruits to national trials and serves an increasingly non- White British population (24% of population). We undertook this study to determine whether ethnicity aff ects ICU consent rates. Validation of an electronic early warning score using decision tree analysis: proposal M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) Introduction Decision tree analysis uses an algorithm to classify data items by recursively posing a series of questions about items within a dataset. Each question leads to another node and potentially more questions until a predefi ned end condition is reached or no more questions can be asked (Figure 1). We hypothesize that scores generated using the decision tree method will improve upon our existing Hamilton Early Warning Score (HEWS) for a composite endpoint of cardiac arrest, unplanned ICU admission or death. Introduction Timely availability of a kidney specialist poses a formidable challenge in ICUs located in tier II and tier III cities of the developing world. Renal replacement therapy (RRT) is often required in the ICU for acute renal failure patients but availability of a nephrologist/ specialist is scarce, leading to unnecessary and risky transfer to higher centers in metropolitans or even worse to death. We explored whether a remotely monitored ICU  – an electronic ICU (eICU)  – would help mitigate this demand–supply gap. Methods A database of 156,642 electronically captured vital signs from 6,757 consecutively admitted patients to eight medical and surgical wards will be used to train and test the decision tree early warning score. One-third of the data will be withheld from the algorithm for use as a testing set. The algorithm will look for signifi cant changes in vitals 72 hours prior to an outcome and develop the score based upon the resulting relative risk of the composite endpoint happening given a certain vital sign. The scores and predictions generated by the decision tree analysis will then be compared with that of the inception HEWS cohort. g y g Methods This retrospective study was conducted at four Critinext affi liates where the eICU was being used to provide 24 × 7 support on 89 ICU beds from a remote command center with intensivist and other requisite staff . The eICU had complete access to the patient’s real-time vitals, hemodynamic parameters, imaging, laboratory values, audiovisuals and appropriately engineered smart alerts. P501 Hamilton Early Warning Score: predict, prevent and protect B Tam, M Xu, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P501 (doi: 10.1186/cc14581) Introduction This study determined the pattern of decline prior to an inpatient arrest. We implemented the Hamilton Early Warning Score (HEWS) within our electronic vital signs documentation to track and trigger care for deteriorating patients. Other EWS have been described in the literature with varying success [1]. In contrast to previous observational studies, we chose to implement a score modifi ed from published EWS using the consensus opinion of a steering committee and evaluate the score in real time. f Methods We performed a retrospective review of screening logs from three national UK trials (PROMISE, BALTI-P, GAiNS) and one local trial (Nociceptin in Sepsis). We analysed consent rates of eligible patients by ethnicity, age, sex, interventional or observational trial, and ethnicity of the researcher seeking consent. We performed chi-squared analysis, and entered signifi cant values into a logistic regression model using SPSS v22. S176 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results The planned analysis for determining the discriminatory and predictive ability of the decision tree HEWS will be conducted with area under the receiver operating characteristic curves. We will test whether the current HEWS has the appropriate sensitivity and specifi city when compared with that of the decision tree score. The AUROC will be calculated for both the training set of data as well as the separate population of additional medical and surgical patients. The two scores will also be plotted along an effi ciency curve, comparing the percentage of vitals that precede a critical event with the percentage of vitals that produce a EWS value greater than or equal to a given EWS value. Results We identifi ed 332 eligible patients across all trials, of whom 37 (11%) were not White British (nWB). Analysis demonstrated consent/assent refusal being signifi cantly associated with: nWB (14, 38%, P <0.001), interventional trial (21, 25%, P = 0.003) and diff erent researcher–patient ethnicities (P <0.001). P504 Can an electronic ICU support timely renal replacement therapy in resource-limited areas of the developing world S Gupta1, A Kaushal1, S Dewan2, A Varma1 1Fortis Escorts Heart Institute, New Delhi, India; 2Fortis Memorial Research Institute, Gurgaon, India Critical Care 2015, 19(Suppl 1):P504 (doi: 10.1186/cc14584) P504 Can an electronic ICU support timely renal replacement therapy in resource-limited areas of the developing world S Gupta1, A Kaushal1, S Dewan2, A Varma1 1Fortis Escorts Heart Institute, New Delhi, India; 2Fortis Memorial Research Institute, Gurgaon, India Critical Care 2015, 19(Suppl 1):P504 (doi: 10.1186/cc14584) P504 P503 Validation of an electronic early warning score using decision tree analysis: proposal M Xu, B Tam, L Thabane, AE Fox-Robichaud McMaster University, Hamilton, ON, Canada Critical Care 2015, 19(Suppl 1):P503 (doi: 10.1186/cc14583) . Lilly CM, et al. Chest. 2014;145:500-7. 1. Bjornsson TD, et al. J Clin Pharmacol. 2003;43:943-67. P501 Logistic regression analysis confi rmed these as independent factors (nWB OR = 4.5, 95% CI = 2.1 to 9.8, P <0.001; interventional trial OR = 2.7, 95% CI = 1.4 to 5.2, P = 0.003; data points missing for researcher–patient ethnicity so variable excluded).f Conclusion This initial study suggests that ethnicity may aff ect assent/ consent to ICU research, with patients from diff erent ethnicities being four times less likely to be recruited. Whilst data are incomplete for researcher–patient ethnicity, our data suggest that this may be an important factor and may infl uence future consent processes. We believe that the role of ethnicity warrants further investigation, not only in clinical trials but also in areas such as organ donation. Reference Conclusion Decision tree analysis methodology with real-life vital signs can produce an EWS superior to previous observational studies. Using a decision tree, especially one that composites all vitals, may show that certain vitals are more predictive of critical events than others. Data will be used to further improve our current HEWS score. 1. Bjornsson TD, et al. J Clin Pharmacol. 2003;43:943-67. Validation of an electronic early warning score using decision tree analysis: proposal The eICU model was further extended in initiating and getting RRT done in patients whenever deemed necessary in times of unavailability of a specialist at the same site. Patient baseline demographics, including risk factors, severity score, all-cause mortality at 30 days, transfers to higher center for RRT and its prevention were recorded. Descriptive analysis was performed. Between-group comparison was performed by applying the chi-squared statistic, signifi cance was assumed at a value of P <0.05. Out of a total of 5,146 admissions, 752 inpatient fi les with acute kidney injury/acute renal failure were reviewed, January to July 2013 (n = 373) and July 2013 to January 2014 (n = 379) pre and post eICU implementation respectively.i Figure 1 (abstract P503). Sample heart rate decision tree. p p y Results While baseline demographics and the patient profi le in the two groups did not show statistically signifi cant diff erence, mean APACHE II score was 14.25 ± 1.94 and 14.65 ± 1.76 pre and post eICU respectively; there was a statistically signifi cant diff erence in all-cause mortality at 30 days which decreased from 31 (8.3%) to 16 (4.2%) pre and post eICU respectively, a reduction of >49% (P = 0.030) and transfer out for RRT came down by >77%, from 15 (4%) to two (0.5%) post eICU implementation (P <0.002). Conclusion Over the years there is now broad consensus over the benefi ts of eICU intervention in deprived areas [1]. There is now a need for a paradigm shift to elevate specialized care to improve outcomes. Our small study has clearly indicated the benefi ts in outcome and economics even while providing intervention in such remote areas. An eICU as a bridge to the demand–supply gap needs to be explored and utilized further to its full potential in the emerging world. Reference Reference Figure 1 (abstract P503). Sample heart rate decision tree. 1. Lilly CM, et al. Chest. 2014;145:500-7. S177 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Table 1 (abstract P506). Mean change in systolic and diastolic BP and HR in relation to ICU work experience Table 1 (abstract P506). Mean change in systolic and diastolic BP and HR in relation to ICU work experience P505 Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study M Soares1, DC Angus2, JI Salluh1, AB Cavalcanti3, F Colombari4, R Costa5, E Silva6, A Japiassu7, JM Kahn2, FA Bozza1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Hospital do Coracao, São Paulo, Brazil; 4Hospital Alemao Oswaldo Cruz, São Paulo, Brazil; 5Hospital Quinta DOr, Rio de Janeiro, Brazil; 6Hospital Israelita Albert Einstein, São Paulo, Brazil; 7Rede Amil de Hospitais, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P505 (doi: 10.1186/cc14585) Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study Group 1 Group 2 P value SBP (mmHg) 3.22 –0.40 0.053 DBP (mmHg) 2.08 –0.27 0.061 HR (bpm) 4.46 1.16 0.048 M Soares1, DC Angus2, JI Salluh1, AB Cavalcanti3, F Colombari4, R Costa5, E Silva6, A Japiassu7, JM Kahn2, FA Bozza1 1DOr Institute for Research and Education – IDOR, Rio De Janeiro, Brazil; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Hospital do Coracao, São Paulo, Brazil; 4Hospital Alemao Oswaldo Cruz, São Paulo, Brazil; 5Hospital Quinta DOr, Rio de Janeiro, Brazil; 6Hospital Israelita Albert Einstein, São Paulo, Brazil; 7Rede Amil de Hospitais, Rio de Janeiro, Brazil Critical Care 2015, 19(Suppl 1):P505 (doi: 10.1186/cc14585) Results The mean duration of working in an ICU was 7.3 ± 5.32 years (from 2 to 18 years). The nurses were grouped according to experience – Group A: 17 nurses with <10  years of experience (mean: 4.4  years), Group B: six nurses with >10 years (mean: 15.6 years). There were 640 BP–HR measurements. The mean systolic BP, diastolic BP and HR did not diff er between the two groups (systolic BP: 111.2 ± 10.9 vs. 113.7 ± 14.1 mmHg, P = 0.654; diastolic BP: 72.8 ± 8.2 vs. 71.9 ± 8.1 mmHg, P = 0.835; HR: 81.4 ± 7.2 vs. 78.1 ± 8.4 bpm, P = 0.365). Nevertheless, the mean change in BP and HR during the shift did diff er between the two groups, with the more experienced nurses showing a trivial reduction in systolic and diastolic BP and minor increase in HR whereas the less experienced ones showed slight increase in both BP and HR measurements (Table  1), reaching almost statistical signifi cance. For the less experienced nurses in Group 1, it was noted that the mean changes were bigger in night shifts although the limited number of measurements did not allow robust statistical analysis. Introduction The aim was to evaluate outcomes and resource use and to investigate the association between organizational factors and effi cient resource use in a large sample of Brazilian ICUs. Methods A retrospective cohort study in 59,483 patients (medical admissions: 39,734 (67%)) admitted to 78 ICUs (private hospitals, n = 67 (86%); medical or medical–surgical, n = 62 (79%)) during 2013. We retrieved demographic, clinical and outcome data from an electronic ICU quality registry (Epimed Monitor System). P506 Blood pressure and heart rate changes during shifts in ICU nurses in relation to their work experience A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) Prehospital transported patients: a resource for accessing prognostic risk factors p g C Bech1, M Brabrand2, A Lassen1 1Od U i it H it l Od C Bech1, M Brabrand2, A Lassen1 1Odense University Hospital, Odense, Denmark; 2Sydvestjysk Sygehus Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P507 (doi: 10.1186/cc14587) Conclusion We observed a great variability in outcome and resources in a large sample of Brazilian ICUs. Implementation of clinical protocols and nursing staffi ng patterns can be targets to improve the effi ciency in resource use in emerging countries such as Brazil. Critical Care 2015, 19(Suppl 1):P507 (doi: 10.1186/cc14587) Introduction The survival of patients transported by ambulance to the emergency department (ED) depends on clinical conditions, patient-related factors and organisational prehospital set up. Data and information concerning patients in the prehospital system could form a valuable resource for assessing potential risk factors associated with adverse outcomes and mortality. Our aim was to describe ambulance transports to the ED and identify prognostic factors accessible in the prehospital phase and associated with 7-day mortality.i Acknowledgements Funded by IDOR, CNPq and FAPERJ. Endorsed by BRICNet. Reference 1. Rothen et al. Intensive Care Med. 2007;33:1329-36. 1. Rothen et al. Intensive Care Med. 2007;33:1329-36. Outcomes and resource use in Brazilian ICUs: results from the ORCHESTRA study We surveyed ICUs using a standardized questionnaire regarding hospital and ICU structure, organization, staffi ng patterns, process of care and family care policies. Effi cient resource use was assessed by estimating standardized mortality rates (SMR) and standardized resource use (SRU) adjusted for the severity of illness according to the SAPS 3 score, as proposed by Rothen and colleagues [1]. y Conclusion The less experienced ICU nurses, with <10 years of ICU work experience, tended to increase their BP and HR levels during the shift, a fi nding probably heightened during night shifts. Further research, including not only cardiovascular parameters, is warranted to uncover the eff ects of shift-work pattern in ICU nurses, taking into account this specifi cally stressful work environment. g Results The median admissions per center was 898 (IQR 585 to 1,715) and SAPS 3 score was 41 (33 to 52) points. Median ICU length of stay was 2 (1 to 5) days, and ICU and hospital mortality rates were 9.6% and 14.3%, respectively. Estimated SMR and SRU were 0.97 (0.72 to 1.15) and 1.06 (0.89 to 1.37), respectively. There were 28 (36%) most effi cient (ICUs with both SMR and SRU <median), 28 (36%) least effi cient (ICUs with both SMR and SRU >median), 11 (14%) overachieving (ICUs with low SMR and high SRU) and 11 (14%) underachieving (ICUs with high SMR and low SRU) ICUs. Most effi cient ICUs were usually located in private and accredited hospitals, with step-down units and training programs in critical care. In univariate analyses comparing most and least effi cient ICUs, ≥2 clinical protocols (OR  = 7.22 (95% CI, 1.41 to 36.97)) and graduated nurse/bed ratio >0.25 (OR = 4.40 (1.04 to 18.60)) were associated with effi cient resource use. Daily checklists also tended to be associated with effi cient resource use (OR = 2.89 (0.95 to 8.72), P = 0.057). Blood pressure and heart rate changes during shifts in ICU nurses in relation to their work experience y y Methods We included all adult patients (≥18 years) with a fi rst-time ambulance transport to the ED at Odense University Hospital in the period 1 April 2012 to 30 September 2013. Ambulance personnel recorded vital signs and other clinical fi ndings on a structured form on paper during the ambulance transport. Each contact was linked to information from population-based healthcare registers in order to identify comorbid conditions and information on mortality. Demographic factors and fi rst registered vital sign were analysed by univariate logistic regression analysis, with 7-day mortality as outcome. Results In total, 18,572 fi rst-time ambulance contacts were identifi ed in the period of inclusion. Overall 7-day mortality was 4.3% (95% CI = 4.0 to 4.6). Univariate analysis showed increasing age, Charlson Comorbidity Index ≥2, vital parameters outside the normal reference range and summoned physician-assisted mobile emergency care units to be associated with 7-day mortality. Further analyses are currently being carried out. P508 Contribution of medical senior house offi cers to a medical referral in the emergency department i g Conclusion In 2011 the Royal College of Physicians emphasized the impact that the quality of the care provided within the fi rst 48 to 72 hours had on clinical outcomes. An evaluation of consultant input into acute admissions management revealed that hospitals in which two or more ward rounds of all acute medical unit patients were performed daily had a lower adjusted case fatality rate for patients with hospital stays over 7 days. Despite twice-daily consultant ward rounds of all new acute admissions and the addition of a third 4:00 pm round from Monday to Friday, only 62% of patients were seen by a consultant within 12  hours. With 67% of patients being admitted between the hours of 12:00 am and 12:00 pm, it is possible that the substitution of an evening round for one of the afternoon rounds would help increase the number of patients seen within the target time frame. This would require a change in the working pattern of the acute medicine consultants. f Introduction In Irish hospitals, the medical senior house offi cer (SHO) is the most junior fully qualifi ed doctor on the medical on-call team. After a patient has been seen by an emergency department doctor of any level, they are almost always referred directly to the medical SHO. This process has been shown to delay a patient’s ward admission by 3 hours 30 minutes [1]. We attempted to quantify the additional benefi t for the patient of being seen by the on-call medical SHO, in terms of patients discharged, new diagnoses reached, and new treatments initiated. g g Methods The emergency department notes and clinical charts of 182 patients were assessed. This constituted a random sample of patients referred by emergency department doctors to the medical team on call over a 2-month period (November to December 2011). Results Discharged: 3/182 (1.6%) of patients referred to the medical team were discharged directly by the medical SHO. Diagnosed: medical SHOs suggested a diagnosis which was diff erent from, or additional to, the ED doctor, in 52/182 cases (28.6%). However, the medical consultant only agreed with this diagnosis in 25 cases (13.7%). This means an incorrect new diagnosis was reached more often than not (14.9%). Treatment: the majority of cases (116/182 (63.7%)) saw no new treatment initiated by the medical SHO. P508 Contribution of medical senior house offi cers to a medical referral in the emergency department Of the rest, only 31 (17%) had a new treatment initiated by the medical SHO which was continued on by the medical consultant through the admission. P510 Interhospital critical care transfer delays result from organisational not geographical factors: secondary analysis of deteriorating ward patients in 49 UK hospitals DJ Wong1, SK Harris2 1King’s College Hospital, London, UK; 2University College London, UK Critical Care 2015, 19(Suppl 1):P510 (doi: 10.1186/cc14590) Conclusion Few direct discharges, new diagnoses, or key new treatments were initiated by the medical SHO in the emergency department. A paper from our hospital shows that more patients referred in by GPs to ED are admitted compared with those referred in to the acute medical assessment unit, with comparable disease severity (43% vs. 12.5) [2]. That paper highlighted the fact that the junior level of the medical NCHDs who see patients in the ED may contribute to their lack of discharging/decision-making zeal. Our survey further illustrated this feature. Our study provided no evidence that a formal medical assessment should delay a patient progressing to the medical ward. Additional genuine urgent OPD appointment slots could be another benefi cial measure. Introduction Critically ill patients may require interhospital transfer for specialist care or because of a lack of local ICU capacity. Harm is assumed from delays that result, but it is not clear whether these delays are due to transfer distances or defi ciencies in the organisation of care. Methods In total, 151 of 15,602 deteriorating ward patients in the (SPOT)light study [1] were transferred rather than admitted locally. We defi ned delay as the time from critical care assessment in the fi rst hospital to arrival in critical care in the second hospital. We used hospital postcodes to derive latitude and longitude, and calculated both geodesic (straight-line) distances (Figure 1) and road distances between the sites using R version 3.1.1 [2]. We compared daytime versus overnight (7:00  pm to 7:00  am) transfer durations assuming traffi c would contribute less to delay overnight. Mapping and visualisation was performed on Quantum GIS version 2.4 [3]. i References 1. Gilligan P, et al. The referral and complete evaluation time study. Eur J Emerg Med. 2010;17:349-53. 1. Gilligan P, et al. The referral and complete evaluation time study. Eur J Emerg Med. 2010;17:349-53. 2. Watts M, et al. Acute medical assessment units: an effi cient alternative to in-hospital acute medical care. Ir Med J. 2011;104:47-9. 2. Watts M, et al. Acute medical assessment units: an effi cient alternative to in-hospital acute medical care. Ir Med J. 2011;104:47-9. Results The median delay to admission was 22  hours (range 41 to 167  hours). The median geodesic distance was 18  km (range 1 to 141 km), and road distance was 24 km (range 2 to 180 km). Correlations between time delay and geodesic/road distances were weak (Figure 2, Blood pressure and heart rate changes during shifts in ICU nurses in relation to their work experience A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) A Ioannidis, E Terzenidou, D Gklava, I Politi, E Georgiadou, A Georgousi, V Aidonoudis, A Kalea, P Melitzana, N Gritsi-Gerogianni Thessaloniki General Hospital ‘Ippokratio’, Thessaloniki, Greece Critical Care 2015, 19(Suppl 1):P506 (doi: 10.1186/cc14586) Introduction The aim of the study was to assess the blood pressure (BP) and heart rate (HR) changes during shifts in ICU nurses in relation to their work experience. Our hypothesis was that less experienced nurses, in comparison with more experienced ones, would be subjected to more work stress and this could be demonstrated by higher changes in BP and HR during shifts. Methods We enrolled 23 nurses working in an 8-hour shift schedule at a general adult ICU. Demographic and clinical data were obtained by completing a short questionnaire. The nurses were invited to measure their BP and HR at the beginning, in the middle and at the end of their shift. An ESH/BSH-certifi ed automatic device was used for the BP and HR measurements. Conclusion We found that several prehospital-registered vital signs recorded by ambulance personnel at fi rst contact with the patient were prognostic factors of 7-day mortality. S178 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P508 of patients were fi rst seen by a consultant within 12 hours of admission, with a range from 23 minutes to 26 hours. When looking at patients admitted during the weekdays, 63% of them were seen within 12 hours; for those admitted at the weekend the fi gure was 57%. P508 Contribution of medical senior house offi cers to a medical referral in the emergency department GF Fitzpatrick University Hospital, Limerick, Ireland Critical Care 2015, 19(Suppl 1):P508 (doi: 10.1186/cc14588) Reference 1. Royal College of Physicians. Acute medical care: the right person in the right setting-fi rst time. Report of the Acute Medicine Task Force. London: RCP; 2007. Achieving a time to fi rst consultant review of under 12 hours for acutely ill medical patients B Greatorex, EC Colley B Greatorex, EC Colley Figure 1 (abstract P510). Map of transfers. y Great Western Hospital, Swindon, UK p , , Critical Care 2015, 19(Suppl 1):P509 (doi: 10.1186/cc14589) Critical Care 2015, 19(Suppl 1):P509 (doi: 10.1186/cc14589) Introduction In 2007, the Acute Medicine Task Force made recommendations about the operation and staffi ng of acute medical units (AMU). Consultant-led care was seen as critical to ensuring high standards of care for patients and maintaining effi cient patient fl ow [1]. It also recommended that during the hours when the AMU is staff ed by a consultant, all new patients should be seen within 6 to 8 hours. Patients admitted overnight should have a consultant review within 12 to 14 hours. Following the introduction of a 4:00 pm consultant ward round of newly admitted acute medical patients to the existing 8:00 am and 2:00 pm rounds, it was our intention to establish whether our trust was meeting those recommendations. g Methods We conducted a prospective survey of all new acute medical admissions over a 2-week period. Data collected included date and time of admission to the hospital, location on arrival, time of fi rst medical clerking, and time of fi rst consultant review. Results Data were collected for 420 admissions. Sixty-seven percent of patients were admitted to the hospital between 12:00 am and 12:00 pm with a peak occurring between 4:00 pm and 6:00 pm. Sixty-two percent Methods We conducted a prospective survey of all new acute medical admissions over a 2-week period. Data collected included date and time of admission to the hospital, location on arrival, time of fi rst medical clerking, and time of fi rst consultant review. i Results Data were collected for 420 admissions. Sixty-seven percent of patients were admitted to the hospital between 12:00 am and 12:00 pm with a peak occurring between 4:00 pm and 6:00 pm. Sixty-two percent Figure 1 (abstract P510). Map of transfers. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S179 R2 = 0.015 and 0.011, respectively). Transfer delays in the daytime and overnight were similar (Wilcoxon rank sum, P = 0.6). Conclusion Interhospital transfers are subject to clinically signifi cant delays, and substantial travel distances. Delays are only weakly correlated to distances travelled and may refl ect delays resulting from organisational ineffi ciencies. Achieving a time to fi rst consultant review of under 12 hours for acutely ill medical patients We infer that eff orts to improve the effi ciency of transfer should focus on local organisational issues. There was no diff erence in the duration taken for overnight versus daytime transfers. References 1. Harris SK, et al. Intensive Care Med. 2014;40 Suppl 1:127. 2. www.r-project.org. 3 qgis osgeo org Figure 2 (abstract P510). (a) Time duration versus geodesic distance (R2 = 0.015). (b) Time duration versus road distance (R2 = 0.011). in those patients who are discharged out of hours is nearly twice that of those discharged during the day [1]. These results have been replicated in our institution with a mortality of 8.7% (discharged 22:00 to 06:59) versus 4.8% (discharged 07:00 to 21:59). In the UK, NICE CG50 advised that transfer from critical care to the ward out of hours should be avoided and documented as an adverse event. We postulated that one important factor in our hospital is the decreased medical and nursing cover overnight and so looked at the delay from discharge to fi rst medical review and to outreach review. in those patients who are discharged out of hours is nearly twice that of those discharged during the day [1]. These results have been replicated in our institution with a mortality of 8.7% (discharged 22:00 to 06:59) versus 4.8% (discharged 07:00 to 21:59). In the UK, NICE CG50 advised that transfer from critical care to the ward out of hours should be avoided and documented as an adverse event. We postulated that one important factor in our hospital is the decreased medical and nursing cover overnight and so looked at the delay from discharge to fi rst medical review and to outreach review. Methods The case notes of 100 consecutive patients discharged to the ward between September 2013 and October 2013 were examined to identify the time of discharge from the ICU and the subsequent fi rst review by the receiving medical team and the Critical Care Outreach team. The grade of the doctor reviewing the patient was recorded. Results Of these 100 patients, 22 were discharged between 22:00 and 07:59. From the 100 case notes requested, only 50 were available for examination. Forty patients were discharged to the wards, with only 37 having further documented medical reviews in the notes. References 1. Gantner D, et al, Intensive Care Med. 2014;40:1528-35. 2. Flower M, et al, Crit Care. 2011;15:P464. R2 = 0.015 and 0.011, respectively). Transfer delays in the daytime and overnight were similar (Wilcoxon rank sum, P = 0.6).i P512 g Conclusion Interhospital transfers are subject to clinically signifi cant delays, and substantial travel distances. Delays are only weakly correlated to distances travelled and may refl ect delays resulting from organisational ineffi ciencies. We infer that eff orts to improve the effi ciency of transfer should focus on local organisational issues. There was no diff erence in the duration taken for overnight versus daytime transfers. R f Evaluation of patients with wild mushroom poisoning in the emergency department Introduction Wild mushroom poisoning (MP) is an important medical emergency that may have bad clinical outcome. We aimed to evaluate the clinical and laboratory features of patients with wild MP admitted to our emergency department in the Central Black Sea Region and to inform the emergency department physicians about early diagnosis and management of wild MP in the light of obtained data.i Achieving a time to fi rst consultant review of under 12 hours for acutely ill medical patients Only 62% of patients were reviewed by a consultant following intensive care, with over 20% of patients waiting more than 24 hours for any medical review. During this time 18% of patients received a review by the nurse- led outreach team. See Figure 1. Conclusion It is clear that a highly vulnerable group of patients who are recovering from critical illness [2] receive inadequate early follow-up within the hospital. We postulate that the delay in medical review and the lack of senior review may be caused by over 40% being discharged overnight and contribute to the increased mortality seen in our institution and the ANZICS study [1] with nighttime discharges. References P511 Delayed ICU discharges and medical follow-up: a cause of increased mortality? The BASIC Patient Safety course was only administered to staff from ICU1, and safety culture was assessed in both units before and after, using a survey based on the Hospital Survey on Patient Safety Culture [2]. Relative risk (95% CI) of improvement: baseline to follow-up in hospitals in patient safety domains, adjusted for duration of work in the unit (≤10  years vs. >10  years), was calculated. Responses were coded according to the Survey User’s Guide, and positive response percentages for each patient safety domain were compared with the 2012 Agency for Healthcare Research and Quality (AHRQ) ICU sample of 36,120 respondents. organizational culture to improve patient safety culture is considered important. We conducted a prospective, controlled study to assess the impact of a standardized patient safety course on an ICU’s patient safety culture, using a validated patient safety culture assessment tool. Methods Staff from two ICUs – ICU1 (tertiary referral hospital) and ICU2 (district hospital) – in Hong Kong were recruited to compare changes in the measured safety culture before and after a patient safety course. The BASIC Patient Safety course was only administered to staff from ICU1, and safety culture was assessed in both units before and after, using a survey based on the Hospital Survey on Patient Safety Culture [2]. Relative risk (95% CI) of improvement: baseline to follow-up in hospitals in patient safety domains, adjusted for duration of work in the unit (≤10  years vs. >10  years), was calculated. Responses were coded according to the Survey User’s Guide, and positive response percentages for each patient safety domain were compared with the 2012 Agency for Healthcare Research and Quality (AHRQ) ICU sample of 36,120 respondents. Conclusion Wild MP can cause bad clinical outcome. The public should be informed about the probable hazards of wild mushroom ingestion because collection and consumption of wild mushrooms from nature is common. Public health units should take protective precautions against wild MP. Education of health personals regarding MP will lead to successful results in patient management. p Results Preintervention and postintervention period response rates for ICU1 were 88.1% (37/42) and 79.3% (23/29); and for ICU2 63% (20/32) and 63% (15/24). P511 Delayed ICU discharges and medical follow-up: a cause of increased mortality? Methods This study was designed retrospectively by examining fi les of the patients with wild MP who were admitted to Ondokuz Mayis University Emergency Department, between January 2008 and December 2012. Patients older than 18 years were included in the study. Patients were evaluated according to gender, age, location, duration between mushroom intake and the start of clinical symptoms, time of application to hospital, clinical features and fi ndings and treatment method. The number of patients has been compared with the regional distribution of population, monthly temperature and average annual rainfall. y p , , Critical Care 2015, 19(Suppl 1):P511 (doi: 10.1186/cc14591) Introduction Discharge from intensive care is a potentially vulnerable time for patients who are recovering from critical illness. Recent data from the ANZICS group have highlighted that the mortality diff erence Figure 1 (abstract P511). S180 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results A total of 420 patients poisoned by wild mushrooms were studied. The male/female ratio was 1/1.5. The age of patients changed from 18 to 92 and mean age was 46 years. MP constituted 13.3% of all intoxication cases. The time when the fi rst symptom occurred after mushroom intake was a mean 2 (0.17 to 2.15) hours. Of the patients, 47.6% lived in villages, 38.6% in towns and 13.8% in city centers. Admissions were mostly made in autumn, with 57.6%. Eighty-six percent of intoxications happened because of wild mushrooms collected in nature. The most frequent symptoms were nausea (93.8%), and vomiting (87.1%). Increase in liver function tests in 47 patients was observed. Two of these patients died while 10 patients were transferred to further centers for liver transplantation. The remaining patients were discharged from the hospital. organizational culture to improve patient safety culture is considered important. We conducted a prospective, controlled study to assess the impact of a standardized patient safety course on an ICU’s patient safety culture, using a validated patient safety culture assessment tool. Methods Staff from two ICUs – ICU1 (tertiary referral hospital) and ICU2 (district hospital) – in Hong Kong were recruited to compare changes in the measured safety culture before and after a patient safety course. P513 Triage after drug overdose: eff ect of the introduction of a medical psychiatry unit on the allocation of ICU beds D Kleinloog, F Braam Houckgeest, D Sierink Tergooiziekenhuizen, Hilversum, the Netherlands Critical Care 2015, 19(Suppl 1):P513 (doi: 10.1186/cc14593) y y D Kleinloog, F Braam Houckgeest, D Sierink D Kleinloog, F Braam Houckgeest, D Sierink Tergooiziekenhuizen, Hilversum, the Netherlands g Critical Care 2015, 19(Suppl 1):P513 (doi: 10.1186/cc14593) Introduction Many patients with drug overdose are sedated, but do not have medical reasons to warrant ICU admission. Historically, monitoring behavior and suicide risk was done in the ICU, until the patient was awake enough for psychiatric consultation. Conclusion The study provides supportive evidence that a structured, reproducible short course on patient safety is associated with a general improvement in the ICU’s patient safety culture, measured with a validated safety culture assessment tool. Methods A medical psychiatry unit (MPU) was instituted as part of the Department of Clinical Psychiatry. For all patients with drug overdose in the emergency department, a risk assessment was made by the intensivist. Those without ICU indication (such as cardiac or respiratory monitoring) were admitted to the MPU. Alternatively, when awake enough, they were seen by the psychiatrist immediately. We performed an analysis of all patients with drug overdose, admitted to our ICU (before MPU n = 88, after MPU n = 191). We used the Welch t test for comparisons. P511 Delayed ICU discharges and medical follow-up: a cause of increased mortality? Post intervention, compared with ICU2, ICU1 showed signifi cantly improved perceptions of teamwork within the hospital unit, RR (95% CI for diff erence between ICUs) 1.55 (1.10 to 2.19, P = 0.01); and overall perception of safety, 1.94 (1.11 to 3.37, P = 0.02); but not increased frequency of reporting mistakes, 0.90 (0.33 to 2.49, P = 0.84). Overall, ICU1 demonstrated a greater improvement in positive responses in fi ve safety culture domains than staff from ICU2. Patient safety culture indices were generally poorer in the two ICUs than the average ICU in the AHRQ database. Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU Before MPU Since MPU (18 months) (51 months) Diff erence 95% CI P value Admissions per month 4.9 3.5 –1.4 –2.7 to –0.1 0.04 APACHE II score 8.4 12.5 +4.0 +1.8 to +6.2 0.0004 APACHE III score 30.9 40.3 + 9.4 +2.6 to +16.2 0.0069 Length of stay 0.8 1.3 + 0.5 –0.0 to +1.0 0.05 Conclusion Introduction of an MPU was associated with reduced numbers of patients with drug overdose admitted to the ICU. Those admitted to the ICU after the institution of the MPU were sicker, probably indicating more appropriate use of ICU beds. P514 Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Ling, G Joynt, A Lee, W Samy, H Fung, CD Gomersall The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) Introduction It is estimated that about one in 10 patients may be harmed by adverse events during their hospital stay [1]. Transforming Before MPU Since MPU (18 months) (51 months) Diff erence 95% CI P value Admissions per month 4.9 3.5 –1.4 –2.7 to –0.1 0.04 APACHE II score 8.4 12.5 +4.0 +1.8 to +6.2 0.0004 APACHE III score 30.9 40.3 + 9.4 +2.6 to +16.2 0.0069 Length of stay 0.8 1.3 + 0.5 –0.0 to +1.0 0.05 Methods Patients admitted to the ICU between 21 September 2014 and 2 October 2014 were included. If an investigation did not involve ionising radiation or was not requested by intensive care clinicians it was excluded. The indication for imaging was noted, and patient notes were analysed no less than 48 hours after the imaging was reported. Figure 1 (abstract P515). Conclusion Introduction of an MPU was associated with reduced numbers of patients with drug overdose admitted to the ICU. Those admitted to the ICU after the institution of the MPU were sicker, probably indicating more appropriate use of ICU beds. Audit of imaging documentation on an ICU l N Arora, SJ Glover p Results After institution of the MPU, there was a 28% reduction in the number of patients with drug overdose per month, admitted to the ICU. Also, patients admitted to the ICU were sicker and stayed longer (see Table 1). There were no patients admitted to the ICU after initial MPU admission. p pi Critical Care 2015, 19(Suppl 1):P515 (doi: 10.1186/cc14595) Introduction The Ionising Radiation (Medical Exposure) Regulations 2001 recommend to ‘ensure that a clinical evaluation of the outcome of each medical exposure is recorded’ [1]. This audit looked at whether ICU documentation of investigations involving ionising radiation could be improved. Anticipated benefi ts would be improved communication between the multidisciplinary team and better- informed decision-making. Table 1 (abstract P513). Patient numbers and disease severity before and after introduction of MPU References 1. de Vries EN, et al. Qual Saf Health Care. 2008;17;216-23. 2. Nieva VF, et al. Qual Saf Health Care. 2003;12 Suppl 2:ii17-23. P514 P514 Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Ling, G Joynt, A Lee, W Samy, H Fung, CD Gomersall The Chinese University of Hong Kong, Shatin, Hong Kong Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) P516 Barriers to the implementation of checklists in the ICU: a survey on the perceptions of 314 Brazilian critical care nurses and physicians J Salluh1, W Viana2, F Machado3, A Cavalvanti4, F Bozza1, M Soares1 1IDOR, Rio de Janeiro, Brazil; 2Hospital Copa D’OR, Rio de Janeiro, Brazil; 3UNIFESP, São Paulo, Brazil; 4Hcor, São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P516 (doi: 10.1186/cc14596) Introduction Checklists have been used increasingly in the ICU aiming at mitigating avoidable adverse events, but their content is variable and little is known about barriers to their full implementation. This survey reported practices on the use of checklists and perceptions regarding barriers on its implementation. Methods A web-based survey was conducted among a convenience sample of Brazilian ICU nurses and physicians between August and October 2014. Standard descriptive statistics were used. Results A total of 314 professionals, 104 nurses (33.1%) and 210 physicians (66.9%), responded. The majority (82.4%) had more than 5  years of experience in intensive care. Checklists were applied every day, including weekends, in only 49.8% (n  = 227). When we compared the barriers perceived by those working in smaller versus larger ICUs (<10 beds vs. >20 beds), the absence of a more comprehensive checklist content (93.6% vs. 81.9%, P = 0.026), the absence of specialized software or app (80.8% vs. 63.8%, P = 0.014), low availability of mobile devices (87.2% vs. 72.4%, P = 0.019), Internet unavailability (83.3% vs. 67.6%, P = 0.017), and the limited number of computers (88.5% vs. 76.2%, P = 0.0366) were the most often barriers to implementation. Checklists were applied with similar frequencies (<3 times a week, three to fi ve times a week, and every day, including on weekends, P  >0.05) regardless of the ICU size. When the type of tool used (paper vs. electronic) was considered, the main barrier highlighted was the lack of 100% Internet availability in the ICU (64.8% vs. 100%, P = 0.009). Users of paper form had higher demands for more comprehensive checklist content (84.3% vs. 63.6%, respectively, P = 0.037) and experienced more barriers to team adherence (98.1% vs. 86.4%, respectively, P = 0.034) as compared with those using specialized software. Conclusion Although checklists are recognized as valuable tools for the adherence to best practice in the ICU, it is diffi cult to ensure the uniformity of their daily use. Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Li G J t A L W S H F CD G ll y y The Chinese University of Hong Kong, Shatin, Hong Kong y g g g g Critical Care 2015, 19(Suppl 1):P514 (doi: 10.1186/cc14594) Introduction It is estimated that about one in 10 patients may be harmed by adverse events during their hospital stay [1]. Transforming Figure 1 (abstract P515). S181 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 to concentrate, multitasking and the need to frequently change priorities in patient care-related procedures. It is therefore a continuous challenge to communicate and implement plans in an early, effi cient and reliable way within the team and to constantly keep track about tasks that are completed, postponed or that are about to be forgotten. To facilitate every caregiver’s workfl ow and to early and effi ciently communicate, we implemented the After Round Comprehensive Plan Summary (ARCoPS) tool. to concentrate, multitasking and the need to frequently change priorities in patient care-related procedures. It is therefore a continuous challenge to communicate and implement plans in an early, effi cient and reliable way within the team and to constantly keep track about tasks that are completed, postponed or that are about to be forgotten. To facilitate every caregiver’s workfl ow and to early and effi ciently communicate, we implemented the After Round Comprehensive Plan Summary (ARCoPS) tool. Results As shown in Figure 1, imaging requests were generally poorly documented (61%). In total, 17/26 (65%) chest X-rays (CXRs) were documented. A total of 0/2 CT scans were documented, despite one showing acute changes. In total, 17/20 (85%) CXRs requested following procedures carried out on ITU (such as insertion of central venous catheters) were documented, and the three not documented had no signifi cant fi ndings. The six other CXRs were requested to investigate worsening respiratory function. None were documented. Five had signifi cant fi ndings. y Methods Most of our patient care-related therapeutic and diagnostic decisions are made during the morning round consisting of intensivists, nursing team, surgeons, respiratory therapists, dieticians, clinical pharmacists and physiotherapists. P516 Resource limitations in smaller ICUs and the absence of comprehensive digital tools, mobile devices and Internet availability preclude full compliance at the bedside. P518 P518 Multicenter Thai university-based surgical ICU study (Thai-SICU study): adverse events and outcome in the SICU S Kongsayreepong, K Chittawatanarat Siriraj Hospital, Mahidol University, Bangkok, Thailand Critical Care 2015, 19(Suppl 1):P518 (doi: 10.1186/cc14598) Introduction The aim of this Thai-SICU was to study the incidence and outcome of adverse events in nine SICU university-based hospitals in Thailand. Introduction The aim of this Thai-SICU was to study the incidence and outcome of adverse events in nine SICU university-based hospitals in Thailand. Methods This multicenter prospective observational study was done in >18 years old, admission >6 hours surgical patients admitted to the large, postgraduate medical training university-based SICU during April 2011 to January 2012. Patient data were divided into three main phases as admission, discharge data and daily CRFs during the ICU stay. The patients were followed until they were discharged from the ICU or up to 28 days of their ICU admission and up to 28 days following discharge from the ICU if they survived. Results Following a 19.7-month recruitment period, a total of 4,654 patients (17,579 ICU-days) were included in the analysis process. Admitted patients had the median age of 64 years. Most of the patients were admitted directly from the OR for postoperative monitoring with median APACHE II score 10, 23% were admitted with priority I who needed aggressive hemodynamic resuscitation and respiratory support. ICU mortality and 28-day mortality were 9.61% and 13.80%. Each day of ICU increment was associated with a 1.38-day increase of hospital stay (95% CI, 1.24 to 1.53; P <0.01). On the surveillance periods, the six most common adverse events were sepsis (19.5%), AKI (16.9%), new cardiac arrhythmias (6.17%), respiratory failure (5.83%), cardiac arrest (4.86%) and delirium (3.5%) respectively. The other events including reintubation within 72 hours, intraabdominal hypertension, acute MI, unplanned extubation, upper GI hemorrhage, pneumohemothorax, seizure, drug error and pulmonary aspiration were <3% each. The risk Prospective controlled study to compare the eff ects of a basic patient safety course on healthcare worker patient safety culture L Li G J t A L W S H F CD G ll Immediately thereafter, all plans for the next 24-hour period are again discussed, summarized, confi rmed, prioritized and organized by the multidisciplinary team under aspects of optimal patient care and workfl ow effi ciency by entering the plan data into a fl at screen visualized template connected to our intranet immediately accessible at every physician and nursing caregiver workstation. ii Conclusion Investigations following procedures were generally well documented, but investigations seeking pathology were not documented at all, regardless of the fi ndings. This may have infl uenced the management of the patient and compromised patient safety. As such, the audit was presented at a departmental meeting to emphasise the importance of imaging documentation. A place for investigations was added to the ICU patient list to improve communication between the team, and a second audit is planned to assess the impact of this. Reference Results Twelve months after implementation of the ARCoPS tool we conducted caregiver interviews and identifi ed the following eff ects: increase in treatment confi dence through standardized multidisciplinary decision-making; reduced loss of information through immediate plan summarization by the whole team; reduction of ambiguity and misunderstanding about care plans through early written documentation; higher level of security not to forget procedures or tasks; positive acceptance of the tool to fl exibly change priorities of care procedures; positive acceptance of the tool to mark accomplished tasks providing visual feedback about the care plan status; individual caregiver workfl ow economization through permanent treatment plan availability; and higher job satisfaction throughout the caregiver team. Conclusion ARCoPS tool utilization has become a daily routine in our ICU. It functions as an eff ective communication and workfl ow tool and has helped us to reduce patient care-related misunderstandings and delays. It also enhanced the economics of our work sequence, which also highly contributes to a better level of patient safety. Furthermore, it has markedly contributed to an improved level of quality of work for caregivers. 1. The ionising radiation (medical exposure) regulations 2000. London: Stationery Offi ce; 2000. 1. The ionising radiation (medical exposure) regulations 2000. London: Stationery Offi ce; 2000. P517 After Round Comprehensive Plan Summary tool: an effi ciency approach for the ICU R Marktanner, M Mostafa, A Shafei, H Hon, R Ashraf, P Sreedhara, N Syed, D Gharaibeh, A Taha Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2015, 19(Suppl 1):P517 (doi: 10.1186/cc14597) Introduction ICU workfl ow for physicians, nurses and other healthcare providers is often complicated by distractions, interruptions, diffi culties Introduction ICU workfl ow for physicians, nurses and other healthcare providers is often complicated by distractions, interruptions, diffi culties S182 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods We performed an observational study in a 17-bed, mixed clinical and surgical emergency department observation. Before the year 2012, the staff were composed of fi rst-year internal medicine and surgical residents. There were no senior physicians specifi cally assigned to this unit, and residents were supervised by the members of the emergency department team, who changed shifts on a daily basis. In February 2012, two senior physicians (one cardiologist and one intensive care doctor) were specifi cally assigned to supervise the internal medicine residents and provide horizontal care for medical patients during the day, from Monday to Friday. There was no change in assistance for surgical patients. The schedule for nights and weekends remained unchanged. Mortality and length of stay for medical and surgical patients were measured in 2011, 2012 and 2013.i of adverse events on 28-day mortality were signifi cant on cardiac arrest (RR = 9.45; P <0.01), ARDS (RR = 4.58; P <0.01), AKI (RR = 4.18; P <0.01), sepsis (RR = 3.62; P <0.01), iatrogenic pneumohemothorax (RR = 3.23; P <0.01), seizure (RR = 3.12; P <0.01), upper GI hemorrhage (RR = 2.97; P <0.01), cardiac arrhythmia (RR = 2.91; P <0.01), ALI (RR = 2.71; P <0.01), delirium (RR  = 2.13; P  <0.01), MI (RR  = 2.12; P  <0.01), unplanned extubation (RR = 2.06; P <0.01), abdominal hypertension (RR = 1.75; P <0.01) and reintubation within 72 hours (RR = 1.51; P = 0.02). Conclusion This is the largest systemic surveillance observation in the SICU. The study results are the reference for future research and also provide information for patient and relative advice when confronted with adverse events during SICU admission. P521 Results There were 6,203 surgical admissions during the 8-year study period. For both ICU and in-hospital mortality; the median LOS in days for survivors was 2.2 (IQR: 1.2 to 4.9) and that of nonsurvivors was 3.2 (IQR: 1.5 to 7.9). At 28 days, 1 year, and 2 years, the respective values were 2.2 (1.2 to 4.9) and 3.3 (1.5 to 6.7); 2.1 (1.2 to 4.7) and 3.1 (1.5 to 7.3); and 2.1 (1.2 to 4.6) and 3.0 (1.5 to 7.0), all P <0.0001. The greatest median LOS was found in neurosurgery and cardiothoracic surgery; 3.3 days (IQR: 1.7 to 9.5) and 3.1 days (IQR: 1.5 to 8.0) respectively. They corresponded to the specialities with the greatest percentage ICU (9.7% and 10.2%) and 2-year mortality (27.9%, and 35%). The greatest mortality and median LOS was found in ventriculostomy cases; 40.8% at 2 years and 10.6 days (IQR: 4.8 to 18.2). Retrospective observational cohort study of mortality and length of stay for surgical ICU admissions Introduction A signifi cant number require a postoperative ICU stay which may be prolonged [1]. Very limited data exist to characterise mortality and ICU length of stay (LOS) in diff erent surgical specialties [2,3]. We aim to describe this relationship in a large cohort of surgical patients. Conclusion Emergency department observation units are an alternative to alleviate emergency room overcrowding when there are no intensive care beds available. However, patients end up staying in those units for days and horizontal care by senior doctors may improve outcomes. p Methods We performed a retrospective observational cohort study of adult surgical admissions to the ICU of a large academic tertiary medical centre. The primary endpoint was 28-day mortality. We used simple descriptive statistics to characterise the population. The Wilcoxon rank- sum test or Kruskall–Wallis test was used, as appropriate, for tests of continuous data. Rejection for ICU admission: analysis and results of this modality of limitation of therapeutic eff ort Williams TA, et al. Br J Anaesth. 2010:104:459-64. P517 Results In the fi rst year after the implementation of the clinic intensive care team, mortality in internal medicine patients decreased from 47% to 34% in 2012 and 33% in 2013. Although other changes happened in this period (the number of beds decreased from 24 to 17, nurses and physical therapists were hired and trained specifi cally for this unit), we believe the horizontal staff was critical, because mortality between surgical patients remained almost unchanged in the same period of time (23% in 2011, 22% in 2012 and 23% in 2013), in spite of the structural improvements that equally aff ected those patients. Length of stay decreased from 6.35 days in 2011 to 3.43 in 2012 and 3.15 in 2013 in medical patients and from 3.9 days on average in 2011 to 3.2 in 2012 and 2.8 in 2013 in surgical patients. Rejection for ICU admission: analysis and results of this modality of limitation of therapeutic eff ort pf F Acosta, O Moreno, M Muñoz, R Fernandez, M Colmenero Hospital Universitario San Cecilio, Granada, Spain Critical Care 2015, 19(Suppl 1):P521 (doi: 10.1186/cc14601) Critical Care 2015, 19(Suppl 1):P521 (doi: 10.1186/cc14601) Introduction The aim was to know the frequency, criteria and implications of rejection for ICU admission to our ICU unit, a second- level hospital (18 beds). Introduction The aim was to know the frequency, criteria and implications of rejection for ICU admission to our ICU unit, a second- level hospital (18 beds). Methods An observational retrospective study in a time interval of 6  months (January to June 2013). We retrospectively registered all patients rejected for admission to our unit, analyzing the clinical rejection report used in our hospital. From this report we extracted diff erent variables: demographical (age, sex), provenance (emergency room, hospital), clinical (comorbidity, functional situation, diagnosis, reason of requesting admission), rejection motive (‘too good’, ‘too bad’, futility, lack of beds, patient rejection), whether it was defi nitive or conditional, whether the patient was admitted afterwards, and the state at hospital discharge. We realized a descriptive analysis (frequencies) and multivariant analysis of the factors related to futility rejection. Conclusion There is an association between postoperative ICU LOS and mortality that persists for at least 2 years after admission. Neurosurgery and cardiothoracic surgery patients appear to have a worse prognosis and also a more prolonged LOS. Our results may provide a more objective basis for clinical decisions, the use of limited resources, and inform on appropriate expectations of treatment. 1. Pearse RM, et al. Lancet. 2012:380:1059-65. y y j Results There were 165 rejections, which represents 25% of total ICU patients evaluated for admission. A total of 59.4% were male. Mean age was 69 ± 7 years (19 to 98). In total, 53.9% had more than two comorbidities (pluripathological) and 31.5% moderate to severe functional disability. The cause of rejection was in 55.2% of situations that the patient was ‘too good’, 37.6% related to qualitative futility, 4.8% was ‘too bad’ and in 1.2% a mix of lack of space (beds) and patient rejection. In the multivariant analysis the signifi cant variables related to futility rejection were age (by years) with an OR of 1.05 (1.02 to 1.08), severe functional disability, OR of 4.35 (2.09 to 9.06), and the hospital provenance with an OR of 2.82 (1.1 to 7.2). 2. Timmers TK, et al. Ann Surg. 2011:253:151-7. 3. References 1. Komindr A, et al. Int J Emerg Med. 2014;5:1-6. 1. Komindr A, et al. Int J Emerg Med. 2014;5:1-6. 2. Baugh C, et al. Health Care Manag Rev. 2011;36:28-37. 2. Baugh C, et al. Health Care Manag Rev. 2011;36:28-37. P519 Retrospective observational cohort study of mortality and length of stay for surgical ICU admissions M Hameed1, M Maruthappu2, D Marshall3, M Pimentel1, L Celi4, J Salciccioli3, J Shalhoub3 1University of Oxford, UK; 2National Institute for Health and Care Excellence, London, UK; 3Imperial College London, UK; 4Massachusetts Institute of Technology, Cambridge, MA, USA Critical Care 2015, 19(Suppl 1):P519 (doi: 10.1186/cc14599) Impact of age, physiological status and APACHE score on acceptance of patients to the ICU Introduction Evidence suggests that age, chronic health status and acute illness severity aff ect the decision-making of clinicians regarding admission to the ICU (ITU) [1-3]. This prospective service review assesses the impact of age, APACHE II score and WHO functional score towards admission acceptance or refusal to ITU in a tertiary-level facility. p Results Following implementation of the new transfer process, the average wait time decreased by 58 minutes, process time decreased by 9 minutes, and lead time decreased by 66.5 minutes. The handoff error rate decreased by 1.3 errors/patient and fi rst-time quality rate increased by 67%. Staff satisfaction improved from 48% to 73%. By elimination of the PACU stay, the costs involved in admission to the PACU were deferred. admission acceptance or refusal to ITU in a tertiary level facility. Methods Design: a planned prospective review of all referrals over a 14- day period. Data collection: review (LT, DP, SP) of case notes of patients referred to ITU with the following variables collected: age, sex, APACHE II scores, WHO functional status score, grade of referrer and source of referral. Data were collected on 37 patients: 22 accepted to ITU and 15 refused admission. Statistics: data were analyzed using GraphPad 6.05. Categorical variables were expressed as mean and standard error of mean. For unpaired variables, statistical signifi cance is determined using unpaired t test. P <0.05 is considered statistically signifi cant. Results The WHO functional status was the most signifi cant variable aff ecting admission (P <0.001). The APACHE score of patients admitted to ITU was signifi cantly lower than refused patients (P = 0.039). Patient age did not aff ect admission status (P = 0.15). See Table 1. Methods Design: a planned prospective review of all referrals over a 14- day period. Data collection: review (LT, DP, SP) of case notes of patients referred to ITU with the following variables collected: age, sex, APACHE II scores, WHO functional status score, grade of referrer and source of referral. Data were collected on 37 patients: 22 accepted to ITU and 15 refused admission. Statistics: data were analyzed using GraphPad 6.05. Categorical variables were expressed as mean and standard error of mean. For unpaired variables, statistical signifi cance is determined using unpaired t test. P <0.05 is considered statistically signifi cant.i Conclusion A single, multidisciplinary bedside handoff process between the OR and ICU leads to cost and time savings. g References References 1. Reignier J, et al. Crit Care Med. 2008;36:2076-83. 2. Garrouste-Orgeas M, et al. Crit Care Med. 2005;33:750-5. 3. Azoulay E, et al. Crit Care Med. 2001;29:2132-6. References 1. Reignier J, et al. Crit Care Med. 2008;36:2076-83. 2. Garrouste-Orgeas M, et al. Crit Care Med. 2005;33:750-5. 3. Azoulay E, et al. Crit Care Med. 2001;29:2132-6. Results Baseline consensus for the need and potential to improve was obtained (97.4% and 94.5%). Despite a large degree of heterogeneity of perceptions around current communication and rounding practices, it was possible to develop a set of interventions based on consensus that could be applied in this complex setting. These included a handoff bundle, an operational touch-base, a unit-level safety briefi ng, a unit- level safety check, a lean rounding bundle and board rounds. These core interventions were supported by several more detailed resources describing the evidence base around best handoff and rounding practices; and a feedback document that described all outputs and recommendations from the ICARUS project. A pilot PDSA cycle demonstrated a 55.3% and 76.3% improvement in key information transfer using a safety briefi ng and board round summary. Improved interprofessional communication, handover and ward rounds in critical care (ICARUS) Introduction There is growing evidence that optimising communication and patient assessment practices including ward rounds and handoff s can improve clinical, safety and operational outcomes, particularly in the critical care setting [1,2]. Here we describe the design and baseline phases of a 5-year project utilising improvement sciences to optimise the quality of interprofessional communication, handoff s and rounding in one of the largest critical care units in the UK. Conclusion This study was performed to assess decision-making for admission to the ITU in times of increased demand and limited availability. We want to validate our fi ndings from this short pilot study in a larger prospective study. Multivariate regression analysis will be used to identify signifi cant features that aff ect clinician decision- making in critical care admission. Methods We obtained institutional ethical and research approvals. We used mixed methods including interviews of opinion leaders and a representative cross-section of staff , roundtables, a survey targeting the whole critical care team (n = 546) and a Delphi exercise to generate a baseline consensus for the need to improve and a set of novel quality improvement interventions and tools. We tested two of these in a pilot plan–do–study–act (PDSA) cycle. Impact of age, physiological status and APACHE score on acceptance of patients to the ICU By elimination of redundant, nonvalue-added processes, less opportunity for medical errors occurred, with substantial improvements in fi rst-time quality. Such a process can be successfully attained while aff ecting staff satisfaction positively. g p y gi Results The WHO functional status was the most signifi cant variable aff ecting admission (P <0.001). The APACHE score of patients admitted to ITU was signifi cantly lower than refused patients (P = 0.039). Patient age did not aff ect admission status (P = 0.15). See Table 1. Table 1 (abstract P522) Accepted Refused P value Mean age 56.5 ± 4.4 65.3 ± 3.6 0.15 Sex 73% male 56% male N/A WHO 0.45 ± 0.2 2.21 ± 0.3 <0.001 APACHE 13.5 ± 1.6 18.8 ± 2.0 0.039 P524 Improved interprofessional communication, handover and ward rounds in critical care (ICARUS) P Hopkins, A Chan, S Rasoli, C Bell, K Peters, A Feehan, J Dawson King’s Health Partners AHSC, London, UK Critical Care 2015, 19(Suppl 1):P524 (doi: 10.1186/cc14604) Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness f SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) Introduction This Six Sigma project was initiated to evaluate and improve the transfer of care of patients from the OR to the ICU. Medical errors are responsible for billions of dollars in increased healthcare spending. Miscommunication among healthcare providers is a major contributor to these errors, with handoff s a particularly vulnerable period in the care process. At our institution, surgical patients with scheduled admissions to the ICU are fi rst recovered in the postanesthesia care unit (PACU). With this process, multiple, unstructured, and individual handoff s occur in parallel between providers, which may lead to communication errors, diff erential information sharing, content variability, care delays, and ineffi ciency. Conclusion Despite wide heterogeneity in baseline beliefs, opinions and perceptions around inter-professional communication, rounding and handoff s, we were able to develop a novel set of feasible quality improvement interventions, targeting these areas, that can be applied in a large complex critical care setting. Furthermore, they can be driven by improvement science methodology and tested for eff ectiveness using qualitative and quantitative measures. We now plan to use these interventions to deliver quality improvements in communication practices in parallel with the planning and implementation phases of a new critical care facility and electronic clinical information system. R f Implementation of a horizontal intensive care team can impact effi ciency in an emergency observation unit in Brazil S Ribeiro, C Petrini, L Marino, R Brandao-Neto Hospital das Clinicas School of Medicine, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P520 (doi: 10.1186/cc14600) fi y g y S Ribeiro, C Petrini, L Marino, R Brandao-Neto Hospital das Clinicas School of Medicine, University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P520 (doi: 10.1186/cc14600) Introduction Emergency department observation units are often used to monitor critical patients in a situation of constant emergency department overcrowding and lack of intensive care beds. However, those units are often understaff ed and might not have enough personnel and equipment to provide the same quality of care as a conventional ICU. Conclusion Rejection for admission to ICU units is a frequent medical activity in our day-to-day job. The type of patient most rejected is cardiologic, mostly evaluated for thoracic pain probably ischemic but with low risk. In second place we found patients for which we decide rejection based on subjective qualitative futility, related mostly to age, prior functional disability and provenance. S183 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P522 were conducted, which began by defi ning the current process via direct observation and value stream mapping of orthopedic and neurosurgical patients. A new process was then introduced, including direct transfer of the patient to the ICU and a single, structured, bedside report between all care providers. A standardized handoff tool was implemented. We used process times, wait times and information content as process measures and handoff errors as outcome measures. A 10-point satisfaction score was also measured. were conducted, which began by defi ning the current process via direct observation and value stream mapping of orthopedic and neurosurgical patients. A new process was then introduced, including direct transfer of the patient to the ICU and a single, structured, bedside report between all care providers. A standardized handoff tool was implemented. We used process times, wait times and information content as process measures and handoff errors as outcome measures. A 10-point satisfaction score was also measured. P523 P523 Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) P523 Lean Six Sigma handoff process between operating room and pediatric ICU: improvement in patient safety, effi ciency and eff ectiveness SJ Gleich, ME Nemergut, AA Stans, DT Haile, SA Feigal, AL Heinrich, CL Bosley, JW Ward, S Tripathi Mayo Clinic, Rochester, MN, USA Critical Care 2015, 19(Suppl 1):P523 (doi: 10.1186/cc14603) P526 Out-of-hours discharge from critical care: does it matter? M Ahmed, G Lumley, S Nourse, A Hurding, A Thomas, M Healy The Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P526 (doi: 10.1186/cc14606) Introduction The aim of this audit was to assess the clinical impact of out-of-hours (OOH) discharge from the adult critical care unit (ACCU) in a tertiary care hospital. Discharging patients from critical care OOH has been associated with both higher mortality and increased rate of readmissions [1,2]. As a result, national guidance stipulates that OOH discharges from critical care should be avoided [3]. Results A total of 2,248 emergency admissions were analysed. The majority of patients were admitted within 1 week of hospital admission (n = 1,897). They were younger and had lower APACHE II scores (15 vs. 19; P <0.001). APACHE II scores were the same in all other groups (groups 2 to 5). Patients admitted to the ICU 3 weeks following hospital admission had signifi cantly higher hospital mortality (up to 50%; P <0.001) and ICU length of stay (12 ± 15 vs. 8 ± 10 days; P <0.001). ICU mortality remained the same in all groups (20 to 28%). ICNARC and SOFA scores were equal between the groups. The post-ICU lengths of stay were signifi cantly longer in groups 3 to 5. In-hospital CPR prior to admission to ICU was lower in patients from groups 4 and 5, probably signifying appropriate DNAR decisions made on the ward. Methods A retrospective snapshot analysis of patients was conducted at least 48  hours after discharge from the ACCU in October 2014. Patients discharged OOH (20:00 to 07:59) were compared with those discharged in hours (IH; 08:00 to 19:59). Analysis included: patient demographics, APACHE II score, handover procedure prior to discharge, follow-up by the receiving team, appropriate and timely drug prescribing, recognising and acting upon clinical deterioration and readmission rates. Conclusion Prolonged pre-ICU hospital admission is associated with higher hospital but not ICU mortality. Commonly measured scores do not refl ect this higher mortality in patients admitted after prolonged stay in hospital. Further research into parameters that may refl ect changes in physiological reserves may strengthen these scores for such patients. Results A total of 161 patients were discharged from the ACCU in October 2014. Of these, 46% of the patients were discharged OOH. P526 Forty-one (of 74) OOH and 19 (of 87) IH discharges were sampled for further analysis. The baseline demographics and APACHE II score were similar between both groups. The majority of discharges were delayed (>4 hours, 90% IH vs. 93% OOH). More patients had nursing handover completed if discharged IH (88% IH vs. 61% OOH) and doctors’ summaries were less frequently completed OOH (83% OOH vs. 94.4% IH). A management plan for the ward was outlined in 94% Pre-ICU length of hospital stay is a predictor of hospital but not ICU mortality y K Flavin, D Hall, G Marshall, P Zolfaghari g Royal London Hospital, London, UK Royal London Hospital, London, UK Critical Care 2015, 19(Suppl 1):P527 (doi: 10.1186/cc14607) Introduction Prolonged hospital stay prior to admission to the ICU was previously shown to be independently associated with poorer outcome [1,2]. This is probably due to slow deterioration of physiological function in hospital and infl uenced by processes leading to critical care admission [2]. We investigated whether commonly measured severity scoring systems (Acute Physiology and Chronic Health Evaluation (APACHE) II, Intensive Care National Audit and Research Centre (ICNARC) and Sequential Organ Failure Assessment (SOFA) scores) are signifi cantly diff erent in patients admitted with prolonged pre-ICU hospital length of stay, and describe mortality and hospital length of stay. Conclusion There is a higher prevalence of adverse events in readmitted patients with similarity in the type of adverse eff ects despite readmission. g References 1. Priestap F, et al. Crit Care Med. 2006;34:2946-51. p 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 3 Acutely ill patients in hospital NICE; 2007 2. Hanane T, et al. Crit Care Med. 2008;36:2232-7. 3. Acutely ill patients in hospital. NICE; 2007 gii Results The readmission rate was 15% (n = 392), particularly among males (55% (n  = 214)). A 14.3% adverse event rate was observed among the readmitted patients (9.5% among those who were not readmitted). The readmitted patients were older (median (md): 68.0 (95% CI: 65.7 to 67.3) vs. md: 71.0 (95% CI: 67.1 to 71.3); P <0.05) and remained hospitalized for a longer period of time (md: 5.0 (95% CI: 13.2 to 20.7) vs. md: 11.0 (95% CI: 17.0 to 33.2); P <0.05), but not necessarily in the CICU (P = 106).The most prevalent adverse events in readmitted patients were pressure ulcers (n = 16 (4.1%)), drug administration error (n = 13 (3.3%)) and enteral feeding tube (n = 10 (2.6%)). Meanwhile, among the nonreadmitted, phlebitis due to peripheral vein access and pressure ulcers (n = 42 (1.9%)), drug administration error (n = 41 (1.9%)) and enteral feeding tube (n = 31 (1.4%)). There was a tendency (P = 0.71) that readmitted patients were presented with higher prevalence of pressure ulcers (n = 16 (4.1%) vs. n = 42 (1.9%)). 3. Acutely ill patients in hospital. NICE; 2007 Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) Introduction Security policies for the patient are of interest to any health professional. The rates of adverse events in hospitals reach values ranging between 3.7 and 16.6%, being the highest range (40 to 70%) considered preventable or avoidable [1]. The objective of this study is to assess the prevalence and types of adverse eff ects in intensive care patients according to readmission. g p Conclusion This audit compares with current evidence suggesting harm from OOH discharge despite most discharges being delayed. Discharging patients is a complex hospital process, but focus needs to be on discharging patients IH. Therefore, areas for improvement include targeting forward fl ow of patients throughout the hospital, completion of handover documents IH and publication of guidance for receiving teams. Methods During 4 years the data from 2,582 patients admitted to the coronary ICU (CICU) were analyzed in the coronary care unit of the city of Presidente Prudente, Brazil. We analyzed the rate of readmissions, length of stay and the mainly detected adverse events. We considered signifi cant P <0.05 two-tailed and confi dence intervals at 95% (CI). p Reference 1. Forster AJ, et al. CMAJ. 2004;170:345-9. 1. Forster AJ, et al. CMAJ. 2004;170:345-9. y Methods A retrospective analysis of prospectively collected data of all emergency admissions in the ICNARC database of a 44-bed adult critical care unit within a major trauma centre over a 2-year period. Demographic data, APACHE II score, SOFA score, ICNARC model score, mortality, and length of hospital stay prior to and after ICU admission were collected. Five groups of patients were defi ned as follows: those admitted to ICU within 1 week of hospitalization (group 1), within 8 to 14 days (group 2), within 15 to 21 days (group 3), within 22 to 28 days (group 4), and more than 28 days (group 5). Chi-squared and ANOVA tests were performed using the SOFA statistics package. References Methods A multidisciplinary QI project was initiated with input from the ICU, anesthesia and surgical services. A series of PDSA cycles Lane D. Crit Care Med. 2013;41:2015. Starmer AJ. N Engl J Med. 2014;371:1803. S184 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 of IH versus 65.% of OOH discharges. Seventy-eight per cent OOH versus 95% IH patients discharged were reviewed by a doctor within 24 hours. Twenty-nine per cent OOH versus 67% IH patients discharged were reviewed by a consultant within 24 hours. Following discharge a management plan was followed in 94% of IH patients versus 44% OOH, patients had drug charts correctly charted in 100% of IH cases versus 66% OOH and missed/delayed doses were documented in 11.1% of IH cases versus 61% OOH. There was no diff erence between the groups in incidence of clinical deterioration and recognition and follow-up of clinical deterioration. Two patients were readmitted within 48 hours from the OOH group. of IH versus 65.% of OOH discharges. Seventy-eight per cent OOH versus 95% IH patients discharged were reviewed by a doctor within 24 hours. Twenty-nine per cent OOH versus 67% IH patients discharged were reviewed by a consultant within 24 hours. Following discharge a management plan was followed in 94% of IH patients versus 44% OOH, patients had drug charts correctly charted in 100% of IH cases versus 66% OOH and missed/delayed doses were documented in 11.1% of IH cases versus 61% OOH. There was no diff erence between the groups in incidence of clinical deterioration and recognition and follow-up of clinical deterioration. Two patients were readmitted within 48 hours from the OOH group. P525 Adverse events in patients in a Brazilian intensive care unit according to readmission GM Plantier1, CE Bosso1, AC Correa2, BN Azevedo3, JS Alves2, CD Monteiro2, SV Ferreira3, GE Valerio3, JV Moraes3, V Raso3 1Instituto do Coração de Presidente Prudente, Brazil; 2Santa Casa de Presidente Prudente, Brazil; 3Faculdade de Medicina – UNOESTE, Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) Higgins TL, et al. Crit Care Med. 2003;31:45-51. Goldhill DR, et al. Intensive Care Med. 2004;30:1908-13. P525 Adverse events in patients in a Brazilian intensive care unit according to readmission Critical Care 2015, 19(Suppl 1):P525 (doi: 10.1186/cc14605) P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) F Kavanagh, I Browne g National Maternity Hospital, Dublin, Ireland g National Maternity Hospital, Dublin, Ireland y p Critical Care 2015, 19(Suppl 1):P530 (doi: 10.1186/cc14610) y p Critical Care 2015, 19(Suppl 1):P530 (doi: 10.1186/cc14610) Introduction Pregnancy and labour usually progress uneventfully; however, serious complications can occur and develop rapidly, necessitating critical care admission and support. Successive confi dential enquires have highlighted defi ciencies in this area and suboptimal care leading to increased morbidity and mortality [1]. The National Maternity Hospital is the largest maternity hospital in the Republic of Ireland where a total of 8,954 babies were delivered in 2013. It is a stand-alone institution and onsite facilities include a two- bed dedicated anaesthesia lead high-dependency unit. Introduction Hospital costs are a constant concern within health, especially in the ICU. Hospital admissions and average life expectancy have been growing gradually mainly in older and critical patients. This study is aimed to observe the direct costs of patients admitted to an ICU and their relation to the SAPS 3, length of stay in the ICU and fi nal outcome. Methods A retrospective observational study in which the direct costs were studied (materials, medicines, oxygen therapy and hospital fees) for 1,790 ICU patients from November 2013 to November 2014. The readmissions within 48 hours were excluded and also 10% of patients who had the highest and lowest costs. The remaining 1,401 patients were divided by age groups. Methods A retrospective observational study was carried out from January 2011 to January 2014 especially looking at the following parameters: admitting diagnosis, demographics, length of stay and the number of admissions requiring transfer for tertiary-level care. y g g Results Of the patients studied, 54.6% were male. Average age was 57.8 years (18 to 105 years). The biggest ICU average cost was in the group of patients 81 to 90 years old (US$793.00), as well as longest ICU stay (9.25 days), highest SAPS 3 (53.96) and higher ICU and in-hospital mortality (14.29% and 19.25% respectively). This study shows that the direct cost of the ICU stay for older patients was higher than for younger patients. The diff erence was explained by the higher severity measured by SAPS 3 in the older age groups (Figure 1), and the required greater length of stay in the ICU (Figure 2). As might be expected, the mortality in the group of older patients was also signifi cantly higher. References Higgins TL, et al. Crit Care Med. 2003;31:45-51. Goldhill DR, et al. Intensive Care Med. 2004;30:1908-13. S185 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 P528 Reference 1. Special issue: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–2008. The Eighth Report of the Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG. 2011;118 Suppl S1:1-203. 1. Special issue: Saving mothers’ lives: reviewing maternal deaths to make motherhood safer: 2006–2008. The Eighth Report of the Confi dential Enquiries into Maternal Deaths in the United Kingdom. BJOG. 2011;118 Suppl S1:1-203. P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) q g y Results In total, 29,344 deliveries occurred. A total of 376 HDU admissions were recorded in this period, representing 1.28% of all admissions. The average age of patients admitted to the HDU was 34 years. The predominant reasons for admission were hypertensive disease of pregnancy (49.7%), haemorrhage (antepartum/postpartum) (36.4%), sepsis (4.2%) and other reasons (11.1%) including cardiac rhythm disturbances, neurological complications and pre-existing medical disease. In 2013 the average length of stay was 2 days. A total 6.1% of those admitted to HDU required transfer for tertiary-level ICU care in other centres during the study period, this represented 0.07% of all deliveries.i Conclusion This study showed that greater age is associated with higher severity measured by SAPS 3, higher direct costs, and higher mortality both in the ICU and in-hospital environment. Conclusion There is signifi cant demand within our institution for HDU care for our patients, with the number of admissions increasing in 2013. The main admitting diagnoses are hypertensive disease of pregnancy and haemorrhage with an increase in the number of patients being admitted for management of sepsis in 2013. This highlights the increasing awareness, recognition and management of this condition in pregnancy. The increased number of HDU admissions in 2013 could also be explained by the recent introduction of an early warning score for the deteriorating patient in our hospital but this would require further evaluation. The low number of transfers of patients to other tertiary centres underpins the importance of an anaesthesia lead service. Figure 1 (abstract P528). Figure 2 (abstract P528). Figure 1 (abstract P528). Figure 1 (abstract P528). Reference P530 Critical care in a maternity hospital: a retrospective observational study P530 Critical care in a maternity hospital: a retrospective observational study P528 Study of the costs of an ICU according to age groups relating to SAPS 3 gravity, stay and outcomes MB Velasco, MA Leitão, DM Dalcomune, MB Leitão Hospital Meridional S.A., Cariacica, Brazil Critical Care 2015, 19(Suppl 1):P528 (doi: 10.1186/cc14608) P531 Post-traumatic stress symptoms in intensive care staff working in adult and paediatric settings G Colville1, J Hammond1, L Perkins-Porras2 1St George’s Hospital, London, UK; 2St George’s University of London, UK Critical Care 2015, 19(Suppl 1):P531 (doi: 10.1186/cc14611) P534 P534 Mortality in a French military burn centre: a 12-year retrospective study analysing seasonal variations M Boutonnet1, C Dussault2, N Donat1, P Laitselart1, A Cirodde1, J Schaal1, C Hoff mann1, P Jault1, T Leclerc1 1HIA Percy, Clamart, France; 2IRBA, Brétigny sur Orge, France Critical Care 2015, 19(Suppl 1):P534 (doi: 10.1186/cc14614) f Methods After local ethics committee approval, a total of 1,004 patients (2012, n = 492; 2013, n = 454; 2014, n = 58) treated in the ICU in a 2-year period were reviewed. This study included for evaluation 135 patients determined with traumatic or nontraumatic brain damage, with a more than 24-hour stay in the ICU. Reasons for brain damage were determined as brain damage associated with head trauma (Group HT), head trauma accompanying general body trauma (Group HT  + GBT) and spontaneous haemorrhage (Group SH). The type of brain damage was defi ned from the radiological diagnosis (CT and/or MRI) as subarachnoid haemorrhage, intracranial haemorrhage (ICH), subdural haematoma (SDH), epidural haematoma (EDH), skull fracture, brain contusion or a combination of these (COM). Operations were evaluated as performed by the brain surgery department. Introduction Factors associated with mortality in severely burned patients are well known. We aimed to assess the seasonal variations of the mortality of patients admitted to the main French military intensive care burn unit (ICBU) and to determine their relations to seasonal variations of severity or other factors (for example, staffi ng). Methods We performed a retrospective study analysing the medical records of all patients admitted to the ICBU of Percy Military Hospital (France) between January 2000 and December 2011. Statistical analysis was performed with R version 3.1.2. We fi rst conducted univariate analyses with simple logistic regression, then a simple periodic regression for seasonal variations (with and without harmonics for robustness), and fi nally a multivariate logistic regression with periodic terms in order to adjust seasonal variations for known severity factors. Results A total of 1,913 patients were admitted for acute burn injury during the study period, with a male to female ratio of 2.34. Mean total body surface area burned (TBSA) was 23.2% (IQR: 10 to 30) and mean full thickness total body surface area burned (FTBSA) was 13.4% (IQR: 0 to 15). Association with amount of registration and outcome in pediatric severe trauma patients S Ohnishi, N Saito, T Yagi, Y Konda, Y Hara, H Matsumoto Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan Critical Care 2015, 19(Suppl 1):P533 (doi: 10.1186/cc14613) Figure 1 (abstract P531). Main themes of staff descriptions of traumatic events on ICU. Introduction Unexpected trauma is a one of the most major causes of death for children in the world. However, pediatric severe trauma patients are rare and scattered. Although there is a strong association between patient’s volume of trauma center volume and outcomes in adults, such a report is less in children. In this study, we aimed to clarify the relationship of the amount of registration and outcome in pediatric trauma patients. most commonly endorsed: patient death (and particularly untimely deaths on adult units); handling distressed families; and concerns about the quality of care and dealing with staff confl ict (see Figure 1). Conclusion A signifi cant minority of staff reported clinically signifi cant levels of post-traumatic stress related to their work. The facts that post- traumatic stress levels were higher on paediatric units despite their lower rates of mortality and that untimely deaths were frequently mentioned by adult unit staff suggest it may be that untimely deaths are particularly hard to deal with. More research is needed on the prevalence of distress in staff working in these settings. Methods This retrospective study analyzed data of the Japan Trauma Data Bank from 2004 to 2012. We registered pediatric patients aged younger than 12  years with severe multiple or torso trauma (maximum Abbreviated Injury Scale score ≥3 or Injury Severity Score ≥9, and excluded patients with cardiopulmonary arrest on arrival, burn, isolated head or limb injury, lack of data). We divided the facilities into six groups according to every 10 patients registered and compared mortality between each group. Results A total of 1,015 patients from 105 facilities were included in the study. Number of registrations: 0 to 10 patients: 673 patients/68 facilities, 11 to 20: 381/28, 21 to 30: 141/6, 31 to 40: 101/3, 41 to 50: 45/1, 51 to 60: 58/1. Victims of blunt trauma accounted for 98.1%. Median age was 7 (interquartile range: 5 to 10) years, median injury severity score (ISS) was 17 (10 to 26). P533 P533 Association with amount of registration and outcome in pediatric severe trauma patients S Ohnishi, N Saito, T Yagi, Y Konda, Y Hara, H Matsumoto Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan Critical Care 2015, 19(Suppl 1):P533 (doi: 10.1186/cc14613) Distribution and mortality factors of cases with traumatic and nontraumatic brain damage treated in ICU S G k T k N B k G K ö E Ak S K ki Distribution and mortality factors of cases with traumatic and nontraumatic brain damage treated in ICU S Goksu Tomruk, N Bakan, G Karaören, E Akcay, S Keskin Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P532 (doi: 10.1186/cc14612) S Goksu Tomruk, N Bakan, G Karaören, E Akcay, S Keskin Umraniye Research and Training Hospital, Istanbul, Turkey Critical Care 2015, 19(Suppl 1):P532 (doi: 10.1186/cc14612) Introduction Cases of traumatic and nontraumatic brain damage have high rates of morbidity and mortality. In this study of cases being treated in the ICU for a diagnosis of brain damage, it was aimed to evaluate the relationship between mortality and the distribution of reason for and resulting type of brain damage and to determine other factors aff ecting mortality. i Conclusion There was a strong association between amount of registration and outcome. Post-traumatic stress symptoms in intensive care staff working in adult and paediatric settings Figure 2 (abstract P528). Figure 2 (abstract P528). Introduction The objectives of this survey were to establish the prevalence of symptoms of post-traumatic stress in mixed staff groups working in adult and paediatric intensive care settings and to examine the main themes in staff descriptions of the most traumatic event they had experienced at work. p Methods A total of 355 health professionals working on three adult and four paediatric units at two centres were asked to rate their current level of post-traumatic stress symptoms on the Trauma Screening Questionnaire (TSQ).f Results Paediatric/neonatal intensive care staff were more likely to score above the clinical cutoff point for post-traumatic stress symptoms on the TSQ in relation to an incident at work than adult intensive care staff in this sample (PICU n = 33/193 (17%) vs. AICU n = 13/162 (8%), P <0.001). For the 172 staff who provided a description of the most traumatic event they had experienced, the following themes were S186 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P531). Main themes of staff descriptions of traumatic events on ICU. (P <0.01). No statistical diff erence was determined in mortality in terms of age, gender, duration in ICU, surgical treatment or not, or reasons for brain damage (P >0.05). Conclusion There is considerable variation in causes of head injury. From this retrospective study it can be suggested that mortality of neurological/neurosurgical patients, regardless of management method, depends on APACHE II, arrival GCS, number of ventilator-days and type of brain damage. Association with amount of registration and outcome in pediatric severe trauma patients Multivariate analysis adjusted for age, revised trauma score and ISS revealed that the amount of registration (every 10 patients) was independently associated with survival (odds ratio: 1.55, 95% confi dence interval: 1.06 to 2.26, P = 0.023). P534 Simple periodic regression showed a biannual seasonal eff ect on mortality, documented with a 1-year periodic (P <0.01) and a 6-month periodic (P = 0.01) dependency. Multivariate analysis with or without periodic terms identifi ed age, past medical history, TBSA, FTBSA, inhalation, intoxication and admission date as the only factors independently associated with mortality. Conclusion Predictive factors for mortality in our ICBU are in line with the literature. The documented seasonal variations in mortality are fully explained by variations in these severity factors. Complementary analyses are under way to further study a nonlinear age eff ect. Results Baseline liver function was assessed in 1,565 patients. Of those, 522 (33%) exhibited liver dysfunction at baseline. No relationship was found with mortality according to baseline liver dysfunction status at day 28 (HR, 0.847 (95% CI, 0.647 to 1.109); P = 0.2267), day 90 (HR, 0.883 (95% CI, 0.701 to 1.112); P = 0.2892) and day 180 (HR, 0.885 (95% CI, 0.710 to 1.103); P = 0.2761). A total of 403 (26%) patients developed a new liver dysfunction (257/1,043, 25%) or worsened liver dysfunction (146/522, 28%) in the 28-day post-baseline period. The overall median time to develop or to worsen liver dysfunction was 2 (1 to 4) days. Signifi cant relationships between the new onset or the worsening of liver dysfunction post baseline and mortality at day 28 (HR, 1.67 (95% CI, 1.26 to 2.21); P = 0.0004), day 90 (HR, 1.65 (95% CI, 1.30 to 2.09); P <0.0001) and day 180 (HR, 1.57 (95% CI, 1.26 to 1.97); P <0.0001) were found. P534 Inhalation injury was present in 441 (23.1%) cases, intoxication (CO, CN) was present in 88 (4.6%) cases, and associated traumatisms also y g y Results The patients comprised 73.3% males and 26.7% females with a mean age of 40.58 ± 24.14 years (range, 1 to 87 years), mean APACHE II score of 19.07 ± 10.19 (range, 2 to 41), mean GCS of 9.17 ± 4.42 (range, 3 to 15) and 68.1% of whom were discharged and 31.9% were exitus. When the causes of brain damage were evaluated according to the type, the most frequently seen in the HT and HT + GBT groups were COM (37.3%, 42.9%), followed by EDH (13.6%, 28.6%). In the SH group, the most common reason was ICH (43.9%) followed by SDH (24.4%). Directly proportionally, only an increase in APACHE II score increased the mortality risk 1.3-fold (logistic regression analyses, coeffi cient 0.658) but the duration of intubation and ICH ratio was statistically signifi cantly high and GCS was low in the exitus cases, and rates of EDH were determined as high in discharged cases compared with exitus S187 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods All of the patients enrolled in the PROWESS-SHOCK were included. Liver dysfunction at baseline was defi ned by a liver Sequential Organ Function Assessment (SOFA) score >0. The onset of a liver dysfunction post baseline was defi ned as any post-baseline increase of the hepatic SOFA score from 0. The worsening of liver dysfunction post baseline was defi ned as any increase of the hepatic SOFA score from the baseline assessment. The post-baseline period studied extended from study drug infusion to day 28. The main outcome was the mortality at day 180, and the secondary outcomes were the mortality at day 28 and at day 90. Cox proportional hazard models were used to estimate the hazard ratio of death. in 88 (4.6%) cases. The mortality rate was 10.9%. The following factors were associated with mortality in univariate logistic regression: age, body mass index, past medical history, TBSA, FTBSA, intoxication (CO, CN), inhalation injury, fl ame burns, self-infl icted burns (all P <0.0001), sex (P <0.001), and admission date (P <0.01). Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Results One patient was admitted twice during this period. The sample comprised 11 males and 11 females (50%, respectively). Featuring an average age of 52 years, 78% of them were admitted to the ICU because of respiratory failure, 50% were due to diff use alveoli hemorrhage, 36% due to sepsis, 4% hypovolemic shock and fi nally 4% because of tuberculosis. According to the BVAS/WG, 20 patients corresponded to severe disease, one to limited diseases and one to persistent disease. The average ICU length of stay was 20.6 days and as inpatients 43 days. While comparing the SOFA score between alive and deceased patients there was a 0.5-point diff erence (P = 0.077), 63% of the alive patients were diagnosed while they were in the ICU. Plasmapheresis was found to be a protector factor (P <0.05).if Introduction In cancer patients, sepsis is the main cause of admission to the ICU and is associated with elevated mortality rates and healthcare costs. In this population, specifi c factors such as poor functional status and immunosuppression secondary to malignancy and/or antineoplastic treatment contribute to decreased survival. The aim of this study is to identify predictors of mortality in cancer patients admitted to the ICU with septic shock. Methods This is a retrospective study that analyzed predictors of 30- day mortality in 269 patients admitted to the ICU of the Institute of Cancer of State of São Paulo, Brazil, from February 2012 to November 2014. Conclusion The BVAS/WG score was signifi cantly diff erent between alive and deceased patients. Plasmapheresis was found to be a survival predictor. This study has shown that both SOFA and APACHE II scores have no prognostic value in these patients. Results From 1,250 patients admitted to the ICU, 269 patients had the admission diagnosis of septic shock and were analyzed. The mean age was 62 ± 14 years and 152 patients (56.5%) were male. Most patients had solid cancer (93.6%), and 87 patients (34.5%) had gastrointestinal neoplasm. Predictors of 30-day mortality in cancer patients with septic shock E Osawa, C Park, F Bergamin, B Pileggi, J Almeida, R Nakamura, I Duayer, G Queiroz, F Galas, J Ribeiro, I Bispo, J Fukushima, L Hajjar Institute of Cancer of University of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P537 (doi: 10.1186/cc14617) Upon admission, the median SOFA score was 4 (IQR: 2 to 7), median SAPS 3 score was 55 (IQR: 48 to 68) and median lactate level was 1.88 mmol/l (IQR: 1.22 to 3.21). After 48 hours of ICU admission, acute kidney injury (AKI) was diagnosed according to AKIN classifi cation as follows: 202 patients (75.1%) had grade 0, 49 (18.2%) grade 1, seven (2.6%) grade 2 and 11 (4.1%) had grade 3. The 30-day mortality rate was 24.9%. In the univariate analysis, associated variables with 30-day mortality were age, urea and creatinine levels at admission, SOFA score at admission, SAPS 3 score and 48-hour AKI. In multivariate analysis, the predictive factors for 30-day mortality were SOFA score at admission (OR = 1.12; 95% CI: 1.04 to 1.21, P = 0.002) and 48-hour AKI defi ned as AKIN grades 1, 2 and 3 (OR = 2.69; 95% CI: 1.45 to 4.97, P = 0.002). P534 ICU outcomes in patients suff ering granulomatosis with polyangitis C Hernandez-Cardenas1, GL Lugo-Goytia1, MS Sierra Beltran2, G Dominguez Cherit3 1Instituo Nacional de Enfermedades Respiratorias, Mexico DF, Mexico; 2Medica Sur, Mexico DF, Mexico; 3Instituo Nacional de Ciencias Medicas y Nutrición Salvador Zubirán, Mexico DF, Mexico Critical Care 2015, 19(Suppl 1):P535 (doi: 10.1186/cc14615) Introduction This study aims to describe both the clinical course and prognostic factors of patients suff ering granulomatosis with polyangitis (Wegener granulomatosis) (GP) who were admitted to the Salvador Zubirán National Medical Sciences and Nutrition ICU.f Introduction This study aims to describe both the clinical course and prognostic factors of patients suff ering granulomatosis with polyangitis (Wegener granulomatosis) (GP) who were admitted to the Salvador Zubirán National Medical Sciences and Nutrition ICU.f Conclusion The new onset or the worsening of liver dysfunction during the course of septic shock impacts strongly on long-term mortality. Septic patients with liver dysfunction need long-term follow-up. Future research should focus on developing specifi c septic liver therapeutics and new tools for earlier detection of septic liver dysfunction. Reference Methods Twenty-two patients suff ering GP admitted to the ICU, between January 2002 and December 2012, were included. The Acute Physiology and Chronic Health Evaluation (APACHE) II prognostic score scale was used in order to assess the severity of illness on the fi rst ICU day. The Sequential Organ Failure Assessment (SOFA) score was used to measure organ dysfunction, and the Birmingham vasculitis activity score for Wegener granulomatosis (BVAS/WG) was used to assess vasculitis activity. The outcome measurements taken into account were ICU mortality and ICU length of stay. 1. Ranieri et al. N Engl J Med. 2012;366;2055-64. P538 Is admission of hematologic malignancies in the ICU justifi ed? VG Gasparovic, MM Grgic Medic, IG Gornik KBC Zagreb, Croatia Critical Care 2015, 19(Suppl 1):P538 (doi: 10.1186/cc14618) disease remission and 31 (11.2%) had newly diagnosed disease. The ICU mortality rate was 26%, hospital mortality was 35.7% and 6-month mortality was 55.2%. The median number of organ dysfunction was 3 (IQR 2 to 4) and respiratory failure was the leading dysfunction, being present in 209 patients (75.5%). During the ICU stay, 21 patients needed hemodialysis (8%), 69 (25%) needed mechanical ventilation, 162 (58%) used vasoactive agents and 22 (8%) had a decision for limitation of medical treatment. On univariate analysis, risk factors for hospital mortality were acute myeloid leukemia, hospital stay prior to ICU admission >4 days, number of organ dysfunction ≥2, colonization and infection by a multidrug-resistant (MDR) agent, use of mechanical ventilation, use of vasoactive agents and renal replacement therapy. Multivariate analysis revealed that renal replacement therapy (OR  = 6.35 (95% CI: 1.5 to 25.92), P = 0.010), SOFA score (OR = 1.69 (95% CI: 1.38 to 2.06), P <0.001), RDW (OR = 1.27 (95% CI: 1.11 to 1.46), P = 0.001), lactate (OR = 1.04 (95% CI: 1.02 to 1.06), P <0.001), colonization of MDR agent (OR = 10.73 (95% CI: 2.13 to 53.96), P = 0.004) and hospital stay prior to ICU admission >4 days (OR = 4.72 (95% CI: 1.8 to 12.3), P = 0.002) were predictive factors of ICU mortality. P538 Is admission of hematologic malignancies in the ICU justifi ed? VG Gasparovic, MM Grgic Medic, IG Gornik KBC Zagreb, Croatia Critical Care 2015, 19(Suppl 1):P538 (doi: 10.1186/cc14618) Introduction The admission of malignant hematology patients to the ICU is combined with poor prognosis [1]. A young population on one side and serious prognosis on the other are the main characteristics. Do we help? We are analyzing continuously clinical characteristics, treatment, and outcomes of critically ill patients with hematologic malignancies admitted to the medical ICU to identify predictors of adverse outcome [2]. Methods Demographic characteristics, hematologic diagnosis, reasons for ICU admission, transplant status, the presence of neutropenia, and APACHE II and SOFA scores were analyzed. Predictors of ICU mortality were evaluated using univariate analysis. g y Results There was a total of 194 patients (103 male), APACHE II score by admission was 27 ± 8, SOFA 9 ± 3. Acute leukemia (L) in 81 patients (41.8%), chronic L in 19 patients (9.8%), lymphoma in 58 patients (29.9%), and multiple myeloma in 28 patients (14.4%) were the etiology. I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar Institute of Cancer of State of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P539 (doi: 10.1186/cc14619) Introduction In recent decades, therapeutic advances resulted in increased survival of patients with hematologic malignancies. These patients are increasingly admitted to the ICU due to infections, treatment toxicity and decompensation of chronic diseases. The aim of this study is to evaluate ICU, hospital and 6-month mortality in patients with hematological malignancies admitted to the ICU and to identify predictors of ICU mortality. y g Conclusion In our study, ICU and hospital mortality were lower than the pooled mortalities seen in a recent large systematic review [3] – despite our study excluding planned postoperative admissions (who are known to have better outcomes). The 180-day mortality was signifi cantly lower than in a previous study at our own institution [4]. Our study looked at a number of factors that might reasonably be expected to be associated with short-term and long-term outcomes and identifi ed several that were independent predictors of death by 180 days. Methods We performed a retrospective study of 277 consecutive patients with hematological malignancies admitted to the ICU of the Institute of Cancer of State of São Paulo, Brazil, from January 2010 to December 2013. Patient clinical and laboratory characteristics, evaluation of organ dysfunctions and need for hemodialysis, mechanical ventilation and vasoactive agents in the ICU were collected. The primary outcome was ICU mortality. Data were analyzed with univariate and multivariate logistic regression. Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Liver dysfunction is associated with long-term mortality in septic shock N Nesseler1, Y Launey1, C Aninat1, J White2, P Seguin1, Y Mallédant1 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) 1CHU Pontchaillou, Rennes, France; 2Duke University Medical Centre, Durham, NC, USA Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Critical Care 2015, 19(Suppl 1):P536 (doi: 10.1186/cc14616) Introduction Knowledge of the impact of liver dysfunction on mortality during septic shock is limited. However, the liver appears to play a key role during septic illness. To better explore this issue, we investigated the data collected during the Prospective Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis and Septic Shock (PROWESS-SHOCK) trial in which a cohort of 1,697 septic shock patients were constituted [1]. The study aimed to assess the relationship of liver dysfunction at the onset and during the course of septic shock on short-term and long-term mortality. Conclusion In cancer patients with septic shock, SOFA score at admission and acute kidney injury at 48  hours of admission were predictors of 30-day mortality. These fi ndings reinforce the needing of early strategies of diagnosis and therapy in this subset of patients. S188 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Factors associated with short-term and long-term mortality in solid cancer patients admitted to the ICU R Fi h 1 C D i 1 S C i h 2 S Sl 1 L S 1 Factors associated with short-term and long-term mortality in solid cancer patients admitted to the ICU R Fisher1, C Dangoisse1, S Crichton2, S Slanova1, L Starsmore1, T Manickavasagar1, C Whiteley1, M Ostermann1 1Guy’s and St Thomas’ NHS Trust, London, UK; 2King’s College London, UK Critical Care 2015, 19(Suppl 1):P540 (doi: 10.1186/cc14620) Introduction Despite multiple reports demonstrating an improvement in outcomes of critically ill cancer patients admitted to ICUs over the last two decades [1], there is concern that admission policies for patients with cancer are overly restrictive [2]. The purpose of this study was to identify factors associated with mortality in the 180  days following unplanned ICU admission in patients with nonhaematological malignancy. Introduction Despite multiple reports demonstrating an improvement in outcomes of critically ill cancer patients admitted to ICUs over the last two decades [1], there is concern that admission policies for patients with cancer are overly restrictive [2]. The purpose of this study was to identify factors associated with mortality in the 180  days following unplanned ICU admission in patients with nonhaematological malignancy. Conclusion The ICU mortality of critically ill patients with HM is high, particularly in the group on MV. Diff erent factors were independent predictors of mortality, but 46.4% of admitted patients survived because of adequate support possibilities and were transferred back to hematology ward. The ICU admission with organ support is according results important for life saving in this extremely high-risk patient group. g p References Methods We carried out a retrospective analysis of all patients with solid tumours admitted to the Guy’s Critical Care Unit (13-bed level 3 ICU) as an emergency between August 2008 and July 2012. Data were collected regarding patient demographics, type of cancer, reason for referral and organ support required during the ICU stay. References 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 1. Azoulay E, et al. The intensive care support of patients with malignancy: do everything that can be done. Intensive Care Med. 2006;32:3-5. 2. Grgic Medic M, et al. Hematologic malignancies in the medical intensive care unit – outcomes and prognostic factors. Hematology. 2014 [Epub ahead of print]. Results During the 4-year study period there were 356 unplanned admissions of patients with solid cancer (8.3% of all admissions). Three hundred individual patients were admitted and 180-day survival data were available for 293 of these. Mean age at fi rst admission was 65.2  years, 115 (38.3%) were female. The most frequently present malignancies were lung (42.7%), head and neck (17.3%) and renal (6.7%). ICU, hospital and 180-day mortality were 19.1%, 31.0% and 52.2% respectively. Those factors found to be independently associated (in multivariate analysis) with increased risk of 180-day mortality include: metastases (OR = 4.0, 95% CI = 2.1 to 7.6); sepsis on admission (OR = 2.2, 95% CI = 1.2 to 4.1); APACHE II score on admission (OR = 1.06, 95% CI = 1.004 to 1.12); need for inotropes/vasopressors during admission (OR = 2.3, 95% CI = 1.05 to 4.8); and need for renal replacement therapy during admission (OR = 4.65, 1.7 to 12.8). References 1. Mokart D, et al. Intensive Care Med. 2014;40:1570-2. 2. Azoulay E, et al. Ann Intensive Care. 2011;1:5. 3. Puxty K, et al. Intensive Care Med. 2014;40:1409-28. 4. McGrath S, et al. QJM. 2010;103:397-403. P538 Is admission of hematologic malignancies in the ICU justifi ed? VG Gasparovic, MM Grgic Medic, IG Gornik KBC Zagreb, Croatia Critical Care 2015, 19(Suppl 1):P538 (doi: 10.1186/cc14618) Respiratory insuffi ciency, hemodynamic instability, AKI and CNS disturbances were responsible for the admission of 169 patients (87.1%) from the hematology ward to the ICU. In total, 127 patients (59.7%) were mechanically ventilated; 93 required invasive mechanical ventilation (MV). Non-invasive ventilation started in 34 patients and was successful in 14 (6.5%). The ICU mortality rate was 104 patients (53.6%), and the mortality of MV patients was 98 (77.2%). Need for vasopressors at admission, MV, neutropenia, and APACHE II and SOFA scores were identifi ed as independent predictors of fatal outcome. Overall mortality of admitted patients was 53.6% (104 patients), and in ventilated patients was 77.2% (98 patients). Conclusion Patients from our institution have survival rates comparable with data from the literature. Our study suggests that mortality is associated with late ICU admission and colonization of MDR bacteria. P540 P540 Outcomes of patients with hematologic malignancies admitted to the ICU I Duayer, E Osawa, C Park, J Fukushima, J Almeida, R Nakamura, F Galas, L Hajjar Institute of Cancer of State of São Paulo, Brazil Critical Care 2015, 19(Suppl 1):P539 (doi: 10.1186/cc14619) P541 P541 Characteristics and outcomes of lung cancer patients requiring ventilatory support: results from a multinational study AC Toff art1, JF Timsit1, J Salluh2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, B Almeida7, E Azoulay8, M Soares9 1Hopital A. Michalon CHU Grenoble, France; 2Post-graduation Program – Inst. Nacional de Cancer, Rio de Janeiro, Brazil; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton Hospital, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Hopital Saint Louis, Paris, France; 9DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil Critical Care 2015 19(Suppl 1):P541 (doi: 10 1186/cc14621) using Hosmer–Lemeshow goodness of fi t test. Data will be reported as median (range) or proportions (95% confi dence interval). P541 Characteristics and outcomes of lung cancer patients requiring ventilatory support: results from a multinational study AC Toff art1, JF Timsit1, J Salluh2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, B Almeida7, E Azoulay8, M Soares9 1Hopital A. Michalon CHU Grenoble, France; 2Post-graduation Program – Inst. Nacional de Cancer, Rio de Janeiro, Brazil; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton Hospital, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Hopital Saint Louis, Paris, France; 9DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil C iti l C 2015 19(S l 1) P541 (d i 10 1186/ 14621) Results A total of 2,769 patients were included, median age 65 (18 to 100) years and 52% female. Thirty-day mortality was 4.5% and 2.2% were admitted to ICU. Median time to ICU admission was 1 (0 to 70) day. Nursing staff assessed 65% and physicians 26% of admissions. NEWS could be calculated for 85%. Nursing staff had a discriminatory power of 0.865 (0.786 to 0.944) with little variation with experience. Calibration was acceptable, except for the least experienced nurses (<5  years). Physicians had a discriminatory power of 0.789 (0.641 to 0.937), with little variation with experience. Calibration was very good, regardless of experience. NEWS had a discriminatory power of 0.809 (0.727 to 0.891) and poor calibration. There was no signifi cant diff erence in discriminatory power between the three assessments.f Critical Care 2015, 19(Suppl 1):P541 (doi: 10.1186/cc14621) Introduction The aim was to evaluate clinical characteristics and outcomes of patients with lung cancer requiring ventilatory support. References 1. Fuchs L, et al. Intensive Care Med. 2012;38:1654-61. 2. Nielsson MS, et al. Acta Anaesthesiol Scand. 2014;58:19-26. Hospital of South West Jutland, Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P542 (doi: 10.1186/cc14622) Introduction Not all patients in need of critical care arrive in clinical distress and some deteriorate after arrival. Identifying these patients early in their clinical course could potentially improve outcome. The present study was performed with the aim of assessing whether nursing and physician staff were able to identify patients in need of critical care using only clinical judgment and to compare this with the National Early Warning Score (NEWS). P541 Methods Secondary analysis of a prospective multicenter study including patients requiring either invasive (IMV) or non-invasive (NIV) mechanical ventilation for >24 hours admitted to 22 ICUs in six countries from Europe and South America during 2011. We used shared frailty models to identify factors associated with 6-month survival. f y p Conclusion Both nursing staff and physicians were as good as NEWS at identifying patients at increased risk of ICU admission after admission to a medical admission unit. However, both nursing staff and physicians had better calibration (accuracy) than NEWS. P543 Admission to intensive care can be reliably predicted using only clinical judgment Admission to intensive care can be reliably predicted using only clinical judgment M Brabrand Hospital of South West Jutland, Esbjerg, Denmark Critical Care 2015, 19(Suppl 1):P542 (doi: 10.1186/cc14622) M Brabrand Clinical characteristics in surviving and nonsurviving older patients admitted to the ICU Results Out of 449 patients admitted to the ICUs, 239 (small-cell (SCLC) = 34; non-SCLC = 205) required ventilatory support (NIV = 104; IMV = 135). Out of NIV patients, 31 (30%) were subsequently intubated for IMV. Main reasons for ventilatory support were sepsis (n  = 119; among them, 102 patients had pneumonia), airway involvement by tumor (n = 79), ARDS (n = 47) and coma (n = 18). Mean SAPS II score was 54 ± 20 and median SOFA score was 7 (4 to 12) points. Hospital and 6-month mortality rates were 55% and 67%; 94 (39%) patients received treatment limitations in the ICU. Mortality according to ventilatory strategy was 56% for NIV only, 77% for NIV followed by IMV, and 70% for IMV only. In the multilevel model, adjusting for the hospital size, presence of step-down units, type of admission and treatment limitation, performance status (PS) 3 to 4 (HR = 2.25 (95% CI, 1.52 to 3.34)), metastasis (HR = 1.66 (1.18 to 2.33)) and the ventilatory strategy compared with NIV only (HR  = 1.73 (1.02 to 2.92), for NIV followed by IMV; HR = 2.25 (1.51 to 3.35), for IMV only) were associated with increased mortality. Conversely, patients with sepsis had higher survival (HR = 0.67 (0.46 to 0.96)). Introduction In recent years the proportion of older people admitted to the ICU has increased. A variety of clinical and physiological factors are associated with outcome in these patients. The aim of this study is to determine the clinical characteristics associated with survival of ICU mechanically ventilated older patients. Introduction In recent years the proportion of older people admitted to the ICU has increased. A variety of clinical and physiological factors are associated with outcome in these patients. The aim of this study is to determine the clinical characteristics associated with survival of ICU mechanically ventilated older patients. Methods We retrospectively studied 74 patients, aged >65  years, admitted to the ICU who underwent mechanical ventilation. Standard demographic, clinical, and physiologic data were recorded. We examined the signifi cant diff erences in clinical characteristics between survivors and nonsurvivors using the Student t and chi-square tests. Methods We retrospectively studied 74 patients, aged >65  years, admitted to the ICU who underwent mechanical ventilation. Standard demographic, clinical, and physiologic data were recorded. Clinical characteristics in surviving and nonsurviving older patients admitted to the ICU We examined the signifi cant diff erences in clinical characteristics between survivors and nonsurvivors using the Student t and chi-square tests. Results The mean age of patients studied (43 men and 31 women) was 79 ± 6.4 years. The type of admission was surgical 18%, trauma 26% and medical 57%. The ICU mortality of these patients was 57% and it was not associated with gender and cause of admission to ICU. Patients who survived had lower Charlson Comorbidity Index (P <0.05) and shorter duration of mechanical ventilation (P <0.01). The episodes of ventilation-associated pneumonia, sepsis and renal failure were less frequently in survivors (P <0.05). Also, in addition serum iron and cholesterol levels were signifi cantly lower in nonsurvivors (P <0.01). Conclusion The mortality of ICU older patients is high. VAP, sepsis and renal failure are frequent complications in nonsurvivors. Pre-existing comorbidities considerably aff ect mortality. R f g q Results The mean age of patients studied (43 men and 31 women) was 79 ± 6.4 years. The type of admission was surgical 18%, trauma 26% and medical 57%. The ICU mortality of these patients was 57% and it was not associated with gender and cause of admission to ICU. Patients who survived had lower Charlson Comorbidity Index (P <0.05) and shorter duration of mechanical ventilation (P <0.01). The episodes of ventilation-associated pneumonia, sepsis and renal failure were less frequently in survivors (P <0.05). Also, in addition serum iron and cholesterol levels were significantly lower in nonsurvivors (P <0 01) Conclusion In a multinational study, 6-month survival in lung cancer patients requiring ventilatory support is better than perceived a priori. Palliative care should be preferred in patients with poor PS. p p p Acknowledgements Funded by INCA, CNPq and FAPERJ. cholesterol levels were signifi cantly lower in nonsurvivors (P <0.01). Conclusion The mortality of ICU older patients is high. VAP, sepsis and renal failure are frequent complications in nonsurvivors. Pre-existing comorbidities considerably aff ect mortality. References y References References 1. Mokart D, et al. Intensive Care Med. 2014;40:1570-2. 2. Azoulay E, et al. Ann Intensive Care. 2011;1:5. 3. Puxty K, et al. Intensive Care Med. 2014;40:1409-28. 4. McGrath S, et al. QJM. 2010;103:397-403. Results The median age of the population was 57  years and 144 patients (52%) were male. Upon admission, 15 patients (5.4%) had S189 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Frailty predicts increased resource use and postoperative care requirements after revision hip surgery P546 Emergency laparotomy clinical outcome according to patient characteristics, level of postoperative care and time of surgery T Banerjee1, M Templeton2, C Gore2 1St Mary´s Hospital, London, UK; 2Imperial College Healthcare NHS Trust, London, UK Critical Care 2015, 19(Suppl 1):P546 (doi: 10.1186/cc14626) Critical Care 2015, 19(Suppl 1):P546 (doi: 10.1186/cc14626) Introduction Emergency laparotomies have poor outcomes with variable postoperative critical care provision [1-3]. All patients requiring an emergency laparotomy with an estimated risk of death of >10% should go to critical care. Time of surgery should not aff ect standard of care [3,4]. In advance of the National Emergency Laparotomy Audit (NELA) results [2], our objective was to see whether the level of postoperative care and time of surgery aff ect outcome. Results A total of 389 patients with a mean age of 68.7  years were identifi ed. Frail patients were signifi cantly more likely to need vasopressors postoperatively (P  = 0.012). Each increase in frailty score was associated with 0.16 increase in length of stay on the HDU (P  = 0.025). Analysis of patient location at 30  days shows that frail patients stay in hospital longer (P = 0.00). Frail patients also bleed more intraoperatively (P = 0.00 with a coeffi cient value of 239; that is, for every point increase in frailty, average blood loss increases by 239 ml). For each increase by unit of blood transfused, the length of stay increased by 5.3 days (P = 0.000). The use of epidural is not associated with increased need for postoperative vasopressors (P  = 0.598). See Figure 1. g yf Methods Retrospective data were collected across the Imperial NHS trust for all emergency laparotomies over 3 months in 2014: length of stay in days (LOS); mortality; age; ASA; surgery time and postoperative care level, that is ward (L1), high dependency (L2), or ICU (L3). Statistical tests: Mann–Whitney, Pearson correlation (PCC) and multilinear regression analysis. g y Results Seventy-one patients underwent surgery. Overall mortality was 13% and 70% of patients went to a L2/3 bed. More ASA 1/2 patients went to L1 and all ASA 4/5 went to L2/3. Median (IQR) for age was 61 (44 to 67) for L1, 65 (48 to 73) for L2/3 (P = 0.11), LOS was 10 (7 to 16) for L1, 19 (12 to 57) for L2/3 (P = 0.002), and mortality (%) was 0 for L1 and 18 for L2/3. g p References 1. Saunders D, et al. Br J Anaesth. 2012;109:368-75. 1. Saunders D, et al. Br J Anaesth. 2012;109:368-75. 1. Saunders D, et al. Br J Anaesth. 2012;109:368 75. 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. , ; 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. 2. NELA project team. Executive summary, fi rst organisational report of the N ti l E L t A dit L d RC A 2014 2. NELA project team. Executive summary, fi rst organisational report of the National Emergency Laparotomy Audit. London: RCoA; 2014. National Emergency Laparotomy Audit. London: RCoA; 2014. 3. www.ncepod.org.uk/2011poc.htm. 4. www.rcseng.ac.uk/publications/docs/ emergency-surgery-standards-for-unscheduled-care. Methods We prospectively collected data on 339 adult patients admitted to two ICUs in Rwanda between August 2013 and July 2014. We described demographic and presenting characteristics and outcomes. We assessed the discrimination and calibration of the MPMo-III model. Using stepwise selection, we then developed a new logistic model for mortality prediction, the R-MPM. References References 1. Singatullina et al. Abstract. Crit Care. 2014;18 Suppl 1:P49N. 2. Rockwood K, et al. Can Med J. 2005;173:489-95. 1. Singatullina et al. Abstract. Crit Care. 2014;18 Suppl 1:P49N. 2. Rockwood K, et al. Can Med J. 2005;173:489-95. Frailty predicts increased resource use and postoperative care requirements after revision hip surgery For surgery between 08:00 and 17:59, 14 went to L1, 28 to L2/3. Mortality was 5% and LOS 15 (9 to 24). Between 18:00 and 21:59, two went to L1, 10 to L2/3. Mortality was 17% and LOS 22 (8 to 34). Between 22:00 and 07:59, fi ve went to L1, 12 to L2/3. Mortality was 29% and LOS 15 (9 to 36). ASA strongly predicted mortality (P = 0.006, PCC 0.32). There was a negative correlation between postoperative destination and mortality with all deaths happening in those who went to L2/3 (P = 0.038 and PCC –0.25); however, sicker patients may have gone here. There was a strong correlation between mortality and time of surgery, night surgery being a strong predictor of mortality (PCC 0.31, P = 0.008). LOS can be predicted by a combination of ASA, age and care level (P = 0.027); the postoperative care regression coeffi cient was negative (–18.04, SE 10.41) with prolonged LOS in patients admitted to L2/3, which could also be explained by illness severity. g Conclusion Frailty is associated with increased intraoperative resource use and postoperative care requirements independent of choice of anaesthetic technique. This type of surgery should be subject to health economic analysis as demand amongst the frailer surgical population increases. Frailty predicts increased resource use and postoperative care requirements after revision hip surgery A Panickar1, N Singatullina1, J Stubbs1, C Johnson1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Leicester Hospitals, Leicester, UK Critical Care 2015, 19(Suppl 1):P544 (doi: 10.1186/cc14624) A Panickar1, N Singatullina1, J Stubbs1, C Johnson1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Leicester Hospitals, Leicester, UK Critical Care 2015, 19(Suppl 1):P544 (doi: 10.1186/cc14624) y Methods This was a prospective cohort study of all adult patients with a fi rst-time admission to a medical admission unit at a 450-bed regional teaching hospital over a 3-month period in 2010. All subspecialties of internal medicine are present as well as a level 2 ICU. Upon fi rst contact with the patient after arrival, nursing staff and physicians were asked to report their estimation of the probability of ICU admission (0 to 100%). Survival status was extracted from the Danish Civil Registry. All administrative details (including transfers to other hospitals, wards and ICUs) were extracted from the National Patient Registry, both ensuring complete follow-up. The discriminatory power (ability to identify patients at increased risk) was estimated using area under the receiver- operating characteristics curve. Calibration (accuracy) was assessed Introduction There is increasing demand for revision hip surgery in older patients with poor frailty. Our previously submitted work demonstrated that frailty predicts the need for medical review [1]. We reviewed patients for a further 16 months to see whether frailty impacts on care [2]. This is the largest reported study reviewing frailty and the need for organ supports and outcomes in complex orthopaedic surgery. Methods A retrospective note review of all patients from January 2012 to April 2014 undergoing revision hip surgery. Data collected included frailty, comorbidities, operative blood loss, anaesthetic technique and level of organ supports and patient location at 30 days. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 S190 Figure 1 (abstract P544). Frailty versus length of stay. of 8.881. The MPMo-III predicted mortality with area under the curve of 0.720 and Hosmer–Lemeshow chi-square statistic of 16.391, indicating that the predictive risk scores of the MPMo-III were not well calibrated to the Rwandan data. Figure 1 (abstract P544). Frailty versus length of stay. Conclusion The MPMo-III had modest risk prediction capacity in a population of Rwandan ICU patients. The R-MPM is an alternative severity score with fewer and more accessible variables that provides better predictive power. This model needs to be validated in other ICUs. P545 P545 Predicting outcomes in critically ill patients in a resource-poor setting: the Rwanda Mortality Probability Model T Twagirumugabe1, E Riviello2, R Fowler3, V Novack4, A Mueller2, W Kiviri1, V Banner-Goodspeed2, D Talmor2 1University of Rwanda, Kigali, Rwanda; 2Beth Israel Deaconess Medical Center, Boston, MA, USA; 3University of Toronto, ON, Canada; 4Ben-Gurion University of the Negev, Beer Sheva, Israel Critical Care 2015, 19(Suppl 1):P545 (doi: 10.1186/cc14625) Predicting outcomes in critically ill patients in a resource-poor setting: the Rwanda Mortality Probability Model g y y T Twagirumugabe1, E Riviello2, R Fowler3, V Novack4, A Mueller2, W Kiviri1, V Banner-Goodspeed2, D Talmor2 p y y Conclusion Trust mortality is similar to that in the Emergency Laparotomy Network audit [1]. Higher ASA patients are appropriately going to L2/3 care. Baseline health status and time of surgery are the strongest predictors of mortality in emergency laparotomy patients. References Introduction ICU mortality prediction models provide robust tools for research and benchmarking in the developed world, but an ICU mortality prediction model has not been validated in a resource-poor setting. We sought to validate the Mortality Probability Admission Model, version III (MPMo-III) in two public ICUs in Rwanda and to develop a simplifi ed Rwanda Mortality Probability Model (R-MPM) for use in developing countries. P549 P549 Disturbed circadian rhythm in ICU patients as indicated by melatonin levels: a prospective pilot study K Kiss1, I Földesi1, L Kemény1, V Csernus2, Z Molnár1, J Singer3 1University of Szeged, Hungary; 2University of Pécs, Hungary; 3Hungarian Society for Clinical Biostatistics, Hungary Critical Care 2015, 19(Suppl 1):P549 (doi: 10.1186/cc14629) pi y Results Age, platelets, ALT and APACHE II were selected to be included in the new laboratory-based score. The AUC for the score was 0.714, which was higher than each of the individual laboratory parameters. The AUC was increased further to 0.781 by including all 14 variables (age, lactate, FiO2, urea, creatinine, ALT, APACHE II, platelet, bicarbonate, haemoglobin, pH, ionised Ca, carboxyhaemoglobin and albumin), although this improvement was not considered signifi cant as the confi dence intervals of the two scores (4 and 14 variables) overlapped. Conclusion A laboratory-based score was successfully established in ICU patients, revealing an AUC of 0.714 which is comparable with established scores in a similar population. The compilation of the variables to produce a laboratory-based score showed greater prognostic power than individual variables. Model developers require an AUC of >0.7 to be termed useful; however, in order to be used in a clinical setting the AUC must be at least 0.75. Further research including internal and external validation studies must be performed to optimise the model before clinical implementation. Introduction The aim of this prospective pilot study was to test how melatonin levels follow the circadian cycle in ICU patients. There is strong evidence that changes of circadian rhythm are refl ected in melatonin levels with peak levels at dawn and low levels during daytime [1]. The ICU stay is accompanied by disturbed circadian rhythm [2], which could potentially be the result of artifi cial lighting conditions. Methods Melatonin levels were determined in eight medical ICU patients on mechanical ventilation, without brain injury or infection. Arterial blood samples were taken on the day of admission at 18:00 (TE0) and 03:00 in the morning (TM0), then at the same time points 48  hours later (TE1, TM1). Blood samples were centrifuged at 3,000 rpm for 8  minutes, and then serum samples were stored at –80°C. Measurements were performed using a US-CEA908Ge melatonin ELISA kit. For statistical analysis, a binary (yes/no) variable was created from the pairs for each day, assigning ‘Yes’ if the TE values were greater than TM (melatonin peak reversion). Developing a laboratory-based score to predict mortality in patients admitted to the ICU A Iqbal1, I Welters2, R Kolamunnage-Dona3, C Toh3, C Downey2 1Institute of Ageing and Chronic Disease, Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P547 (doi: 10.1186/cc14627) A Iqbal1, I Welters2, R Kolamunnage-Dona3, C Toh3, C Downey2 1Institute of Ageing and Chronic Disease, Liverpool, UK; 2Royal Liverpool University Hospital, Liverpool, UK; 3University of Liverpool, UK Critical Care 2015, 19(Suppl 1):P547 (doi: 10.1186/cc14627) g y p Results Patient median age was 34 (IQR 26 to 49) years; 48.7% were male. Mortality was 50.3%. The variables most predictive of mortality in univariate analyses were: age, sepsis within 24 hours of ICU admission, hypotension or shock at ICU admission, Glasgow Coma Scale score at ICU admission, and heart rate (beats per minute) at ICU admission. Using these fi ve variables, the R-MPM predicted mortality with area under the curve of 0.829 and Hosmer–Lemeshow chi-square statistic Introduction Scoring systems can be used to predict mortality in patients admitted to the ICU. They are produced using variables that are associated with an increased risk of mortality such as patient Introduction Scoring systems can be used to predict mortality in patients admitted to the ICU. They are produced using variables that are associated with an increased risk of mortality such as patient S191 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion The citizens surveyed valued outcomes associated with quality of life and intact neurological function. Mortality and other indices of duration of critical illness were valued less. These preferences need to be confi rmed in larger groups with greater diversity. Future research also needs to further understand these outcome preferences in survivors of critical illness, clinicians, decision-makers, and researchers. Understanding the outcome preferences of pertinent stakeholders and whether these preferences are congruent is imperative to inform the design of future critical care trials. Conclusion The citizens surveyed valued outcomes associated with quality of life and intact neurological function. Mortality and other indices of duration of critical illness were valued less. These preferences need to be confi rmed in larger groups with greater diversity. Future research also needs to further understand these outcome preferences in survivors of critical illness, clinicians, decision-makers, and researchers. Understanding the outcome preferences of pertinent stakeholders and whether these preferences are congruent is imperative to inform the design of future critical care trials. Patient preferences for outcomes in critical care trials (OPTICS): preliminary resultsf p y J Muscedere1, F Lamontagne2, G Boyd1, M Herridge3, S Fleury1, T Sinuff 3 1Queen’s University, Kingston, ON, Canada; 2University of Sherbrooke, QC, Canada; 3University of Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P548 (doi: 10.1186/cc14628) Results Melatonin levels were not normally distributed and were ranging between 9.2 and 23.7  pg/ml at T0 and 11.3 and 17.9  pg/ ml at T1. The median diff erences of the morning and evening serum melatonin levels were: TM0 – TE0 = –2.8 (–3.8 – (–0.2)); TM1 – TE1 = –1.2 (–2.5 – (–0.4)) presented as median (IQR). The proportion of subjects with peak reversals was 92.9% (80.8 to 100.0%). Introduction Although patient-centered outcomes are important to inform therapeutic choices for critically ill patients, patient preferences for outcomes in critical care studies are unknown. It is also unknown whether outcome preferences diff er between researchers, decision- makers and those who have never been critically ill (citizens). The aim of the OPTICS Program is to investigate these preferences. Herein we report the preliminary results for outcomes preferences in citizens. Conclusion Our preliminary data suggest that circadian rhythm disturbances may occur in critically ill patients within 48 hours after admission, and can be detected by inversion of melatonin peaks. Despite the limitations of this study, it may justify the need for larger observational and randomized trials on the eff ect of light on melatonin levels and on outcomes in ICU patients. Methods We recruited and surveyed lay public members without a history of critical illness as to their preferences for outcomes in critical care trials. After an in-person educational session, citizens were asked to rank 11 potential critical care trial outcomes in order of personal preference. Each outcome was also rated for importance on a 7-point Likert scale. Participants were then asked to indicate their agreement with potential tradeoff s between potential outcomes. References 1. Pevet P, et al. J Physiol. 2011;105:170-82. 1. Pevet P, et al. J Physiol. 2011;105:170-82. 2. Castro R, et al. Annual update in Intensive Care and Emergency Medicine. J.-L. Vincent, editor. Berlin: Springer-Verlag; 2011. p. 766-80. 2. Castro R, et al. Annual update in Intensive Care and Emergency Medicine. J.-L. Vincent, editor. Berlin: Springer-Verlag; 2011. p. 766-80. pf p Results The in-person session was attended by 31 citizens whose had a mean age (SD) of 71.6 (5.9) years and 1.5 (1.4) chronic health conditions; 25 (81%) had partially or fully completed post-secondary school education. Of the 11 potential outcomes, the three outcomes ranked of highest importance were: permanent brain dysfunction, quality of life, and requirement for long-term institutional care. The three outcomes ranked of least importance were: duration of hospitalization, death after a prolonged illness, and occurrence of delirium. When rated on a 7-point Likert scale the results were similar. Of the participants, 24/27 (89%) indicated that they would be willing to receive a therapy which was associated with a higher mortality rate but resulted in an improved quality of life in survivors. Conversely, 16/27 (59%) indicated that they would not be willing to receive a therapy which increased the chances of survival but was associated with a reduced quality of life in the survivors. Developing a laboratory-based score to predict mortality in patients admitted to the ICU demographics, physiological measurements and coexisting conditions and can be used to evaluate ICU performance, to stratify patients in clinical trials and to assist in-hospital and healthcare decisions such as resource allocation. The aim of the project was to determine whether a general score derived from routine laboratory parameters could be used to predict mortality rates in patients admitted to the ICU in the UK. Methods P values were calculated using the t test, Mann–Whitney U test and chi-squared test, depending on distribution of data, in order to determine which variables were signifi cantly diff erent in the survivors and nonsurvivors of critical illness. Signifi cant variables were categorised into subgroups according to medically relevant landmarks and univariately analysed by assessing the correlation with mortality. Forward logistic regression models were used to choose the parameters to include in our score. ROC curves illustrated the sensitivity and specifi city of selected variables via their AUC. P549 The proportion of reversals and their 95% confi dence intervals were estimated using a GEE model for repeated binary data, assuming a binomial distribution and log link, and accounting for subject as a repetition factor. All calculations were done in SAS 9.4. P548 Patient preferences for outcomes in critical care trials (OPTICS): preliminary results J Muscedere1, F Lamontagne2, G Boyd1, M Herridge3, S Fleury1, T Sinuff 3 1Queen’s University, Kingston, ON, Canada; 2University of Sherbrooke, QC, Canada; 3University of Toronto, ON, Canada Critical Care 2015, 19(Suppl 1):P548 (doi: 10.1186/cc14628) Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay Both devices can be used to eff ectively monitor and characterize sleep in the ICU environment. g y Methods In this multicenter follow-up study of the Dexamethasone for Cardiac Surgery (DECS) trial, validated self-report questionnaires were sent 1.5 to 4 years after cardiac surgery and ICU treatment to assess symptoms of PTSD and depression, in relation to cumulative life stress (that is, childhood trauma, major stressful life events) and trait anxiety as determinants of psychopathology. Data were available for 1,125 out of 1,244 (90.4%) eligible participants. Mediating and moderating analyses were performed with multivariable linear regression to assess the eff ect of trait anxiety. Subgroup analyses were performed for both sexes. P552 P552 Hospital anxiety and depression after ICU survival: results of a post-ICU aftercare program D Ramnarain, C Slobbe, W Schapendonk, J Van Gorp, I Gnirrip, S Voermans, A Rutten, G Van der Nat, N Van der Lely St.Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P552 (doi: 10.1186/cc14632) Hospital anxiety and depression after ICU survival: results of a post-ICU aftercare program Introduction Although the ICU survival rate has increased in the last decade, the negative eff ects on mental health and related quality of life become more clear. In the literature the prevalence of anxiety and depressive symptoms post ICU ranges from 10 to 43% [1]. Early recognition and treatment of anxiety and depressive symptoms is important because depression caries a risk for suicide, limited quality of life, and delayed return to work. We studied hospital anxiety and depression (HAD) symptoms after ICU discharge. Results Trait anxiety partially mediates the relationship between cumulative life stress and PTSD (β-value reduction from 0.325 to 0.068; P  = 0.000 to P  = 0.003) and fully mediates the association between cumulative life stress and depression (β-value reduction from 0.282 to 0.015; P = 0.000 to P = 0.507). Trait anxiety was not a moderating factor between cumulative life stress and psychopathology. Full mediation of trait anxiety was found in female patients (n = 247), whereas only partial mediation was seen in male patients (n = 878) with regard to PTSD symptoms. As for depression, full mediation was present in both female and male patients. p y p g Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013 for more than 5 days were invited to visit our post-ICU aftercare clinic. Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay L Kok1, M Sep1, D Veldhuijzen2, S Cornelisse1, A Nierich3, J Van der Maaten4, P Rosseel5, J Hofl and6, J Dieleman1, C Vinkers1, L Peelen1, M Joëls1, D Van Dijk1, M Hillegers1 L Kok1, M Sep1, D Veldhuijzen2, S Cornelisse1, A Nierich3, J Van der Maaten4, P Rosseel5, J Hofl and6, J Dieleman1, C Vinkers1, L Peelen1, M Joëls1, D Van Dijk1, M Hillegers1 1University Medical Center Utrecht, the Netherlands; 2Leiden University, the Netherlands; 3Isala Clinics, Zwolle, the Netherlands; 4University Medical Center Groningen, the Netherlands; 5Amphia Hospital, Breda, the Netherlands; 6Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2015, 19(Suppl 1):P550 (doi: 10.1186/cc14630) Introduction ICU survivors are at risk for post-traumatic stress disorder (PTSD) and depression. The development of psychopathology depends partially on stable personality factors such as trait anxiety. Introduction ICU survivors are at risk for post-traumatic stress disorder (PTSD) and depression. The development of psychopathology depends partially on stable personality factors such as trait anxiety. S192 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 distribution was light sleep 62.2% (PAT) and 74.1% (PSG); REM 13.0% (PAT) and 10.7% (PSG); deep sleep 14.5% (PAT) and 7.9% (PSG). Conclusion Both wristwatch-style PAT and miniature PSG devices successfully recorded sleep in ICU patients. Although the simple PAT device overestimated TST, sleep stage times were generally in agreement, especially REM time which correlated strongly. Both devices can be used to eff ectively monitor and characterize sleep in the ICU environment. distribution was light sleep 62.2% (PAT) and 74.1% (PSG); REM 13.0% (PAT) and 10.7% (PSG); deep sleep 14.5% (PAT) and 7.9% (PSG). Among ICU patients a high level of trait anxiety is relatively common and associated with intrusions, a symptom of PTSD. Independently, childhood trauma and stress exposure throughout life have been associated with depression. In cardiac surgery patients admitted to the ICU postoperatively, the eff ect of trait anxiety on the relationship between cumulative life stress and stress-related psychopathology remains unknown. Therefore we aimed to assess the mediating or moderating role of trait anxiety in this at-risk patient population. Conclusion Both wristwatch-style PAT and miniature PSG devices successfully recorded sleep in ICU patients. Although the simple PAT device overestimated TST, sleep stage times were generally in agreement, especially REM time which correlated strongly. Sleep monitoring in ICU patients Sleep monitoring in ICU patients S Namba1, K Hayashi1, T Hirayama1, T Hirayama1, Y Namba1, M Terado1, P Easton2, Y Ujike1 1Okayama University Hospital, Okayama, Japan; 2University of Calgary, AB, Canada Critical Care 2015, 19(Suppl 1):P551 (doi: 10.1186/cc14631) Sleep monitoring in ICU patients S Namba1, K Hayashi1, T Hirayama1, T Hirayama1, Y Namba1, M Terado1, P Easton2, Y Ujike1 1Okayama University Hospital, Okayama, Japan; 2University of Calgary, AB, Canada Critical Care 2015, 19(Suppl 1):P551 (doi: 10.1186/cc14631) Introduction Sleep disruption and deprivation is a continuing problem in the ICU. Strategies to improve sleep are confounded by diffi culties in monitoring and measuring sleep in the ICU; traditional polysomnography cannot be utilized. Practical, non-intrusive diagnostic monitoring of sleep is required. The aims were to test two new ambulatory sleep diagnostic devices to monitor sleep in the ICU, compare sleep data generated by the diff erent devices, and characterize sleep in the ICU. p Methods The devices were: Watch PAT 200 (Itamar), simple wristwatch style, employing peripheral arterial tone and actigraphy to evaluate sleep time and sleep stage by an automatic algorithm (PAT device); and ALICE PDx (Respironics Philips), miniature polysomnographic device utilizing EEG and EMG recordings, requiring post-study sleep technician scoring (PSG device). Nineteen ICU patients provided informed consent (mean age 37 years, two female). Diagnosis of most patients was trauma. Device technical problems terminated one ALICE PDx study and three Watch PAT study; one patient revoked consent. Therefore, 14 patients were recorded successfully in a private room in the ICU, while simultaneously wearing both devices, from 20:00 to 06:00. No patient received sedation. Subjective sleep quality was estimated by the visual analog scale. i References 1. Myhren H, et al. Crit Care. 2010;14:R14. 1. Myhren H, et al. Crit Care. 2010;14:R14. 2. Zimond M, et al. Behav Res Ther. 2003;41:1489-96. 2. Zimond M, et al. Behav Res Ther. 2003;41:1489-96. Trait anxiety mediates stress-related psychopathology after cardiac surgery and ICU stay Six weeks after discharge they received a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale (HADS) questionnaire [2]. Patients were asked to return the questionnaire prior to their visit. All data were analyzed and if the HADS score indicated a clinically signifi cant anxiety or depression, patients were referred to a psychologist for further analyses and treatment. All patient data were analyzed retrospectively. Results Seventy-nine patients, 54 men and 43 women, mean age 57 years. Median APACHE II and IV was 18 and 60 respectively. Median ICU and hospitals days were 9 and 20 respectively. Seventy-six percent were mechanically ventilated with a median of 5 days. Median time after ICU discharge to aftercare visit was 165 days. Patients were divided into three categories: 1, no HAD (45.4%); 2, possible HAD (9.3%); and 3, clinically signifi cant HAD (45.4%). Women compared with men showed signifi cantly more HAD symptoms (26.8% vs. 18.6%, P <0.05). Patients with subarachnoid hemorrhage, neurotrauma and multitrauma patients showed more HAD symptoms. Pain, fatigue, muscle weakness, impairment of daily activity dyspnea, and hoarseness were signifi cantly associated with clinically signifi cant HAD. No association between age and HAD was found. Diagnosis at ICU admission, length of stay, severity of illness, delirium and use of sedatives were not associated with HAD. Conclusion Prevalence of clinically signifi cant post-ICU HAD was 45.4%. Female sex and post-ICU physical complaints – pain, fatigue, muscle weakness, impairment in daily activities, hoarseness and dyspnea  – were signifi cantly associated with HAD. Conclusion In cardiac ICU patients, trait anxiety mediates the infl uence of cumulative life stress on the occurrence of PTSD and depression symptoms. Further prospective research is necessary to establish these factors as reliable measures for the early identifi cation of ICU patients at risk for stress-related psychopathology. Somatic complaints after ICU survival: results of a post-ICU aftercare program p g D Ramnarain, W Schapendonk, I Gnirrip, G Van der Nat, A Rutten, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P554 (doi: 10.1186/cc14634) p g D Ramnarain, W Schapendonk, I Gnirrip, G Van der Nat, A Rutten, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P554 (doi: 10.1186/cc14634) Introduction Critical illness today is well recognized as being associated with new or worsening physical impairment, diminished mental health and cognitive dysfunction. We studied the scope of somatic complaints in ICU survivors 4 to 6 months after ICU treatment. Introduction Critical illness today is well recognized as being associated with new or worsening physical impairment, diminished mental health and cognitive dysfunction. We studied the scope of somatic complaints in ICU survivors 4 to 6 months after ICU treatment. yp yp p Conclusion Irrespective of defi nition using PCL-S or DSM-IV mapping, PTSD was identifi ed in no more than one in 10 survivors of critical illness at either 3 or 12 months post ICU, which is still nearly double the US population past-year PTSD prevalence. In ICU survivors with moderate probability PTSD by PCL-S, the CAPS gold-standard interview is challenging to complete and adds only a small number of diagnoses. However, two in fi ve ICU survivors will develop PTSD subtypes of avoidance or hyperarousal, which both occur twice as frequently as the intrusion subtype. Targeting predominant PTSD subtypes may help optimize treatment strategies for the ICU survivor, such as prolonged exposure and eye movement desensitization and reprocessing for those with the avoidance subtype, and pharmacologic antidepressants targeting the sympathetic nervous system to produce anxiolysis for those with the hyperarousal subtype. Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013, for 5 or more days, were invited to visit our ICU aftercare clinic. Six weeks after ICU discharge a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale questionnaire [1] and Impact of Event Scale Revised questionnaire [2], was sent. Patients were asked to return the questionnaires before visiting our clinic. The main purpose of the post- ICU aftercare was to screen for somatic complaints, mental health and cognitive dysfunction. If necessary, further examination or treatment was advised. All data were retrospectively analyzed. y y Results Ninety-seven patients visited our aftercare program in 2013. P553 Post-traumatic stress disorder after ICU discharge: results of a post-ICU aftercare program D Ramnarain, I Gnirrip, W Schapendonk, A Rutten, G Van der Nat, N Van der Lely St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2015, 19(Suppl 1):P553 (doi: 10.1186/cc14633) Results Both devices calculated total sleep time (TST), but the results were signifi cantly diff erent (P <0.05), with mean TST reported as 443.07 and 270.8 minutes for PAT and PSG devices. VAS correlated tightly with TST calculated by the PSG device (r  = 0.559, P <0.05). Both devices were able to successfully discern diff erent sleep stages, summarized as light sleep, deep sleep, and REM. Measurements of sleep stage were generally in agreement between the two devices; REM sleep time correlated strongly between PAT and PSG devices (P <0.05). Sleep stage Introduction Patients who survive ICU treatment may experience psychological distress for some time after discharge from the ICU. In the literature the reported prevalence of post-traumatic stress disorder (PTSD) ranges from 5 to 64% [1]. We studied PTSD symptoms in relation to ICU factors, demographic data and physical complaints reported by patients 4 to 6 months after ICU discharge. Introduction Patients who survive ICU treatment may experience psychological distress for some time after discharge from the ICU. In the literature the reported prevalence of post-traumatic stress disorder (PTSD) ranges from 5 to 64% [1]. We studied PTSD symptoms in relation to ICU factors, demographic data and physical complaints reported by patients 4 to 6 months after ICU discharge. S193 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Methods Patients who were treated in our ICU from 1 January 2013 until 31 December 2013 for more than 5  days were invited to visit our ICU aftercare clinic. Six weeks after discharge a letter of invitation together with a health-related questionnaire, the Hospital Anxiety and Depression Scale questionnaire and Impact of Event Scale (IES-R) questionnaire, was sent to the patient. Patients were asked to return the questionnaires prior to visiting the aftercare clinic. All data were analyzed and if the IES-R score indicated a possible PTSD, patients were referred to a psychologist for further analyses and treatment. All patient data were analyzed retrospectively. y Referencefi Referencefi 1. Griffi ths J, et al. Intensive Care Med. 2007;33:1506-18. , yp g Results Of the 180 eligible participants at 3  months, PTSD was identifi ed in 10 (6%) using PCL-S scores and 15 (8%) using DSM-IV mapping of the PCL-S. Of the 160 eligible participants at 12 months, PTSD was identifi ed in two (1%) using PCL-S scores and 10 (6%) using DSM-IV mapping of the PCL-S. Of those eligible for CAPS assessments, at 3 months only 13 of 24 (54%) interviews were completed resulting in three extra PTSD diagnoses, and at 12 months only six of 22 (27%) interviews were completed resulting in two extra PTSD diagnoses. At 3 and 12 months, the intrusion subtype was present in 25 (14%) and in 25 (16%), the avoidance subtype was present in 78 (43%) and 60 (38%), and the hyperarousal subtype was present in 82 (46%) and 71 (44%). Conclusion Irrespective of defi nition using PCL-S or DSM-IV mapping, PTSD was identifi ed in no more than one in 10 survivors of critical illness at either 3 or 12 months post ICU, which is still nearly double the US population past-year PTSD prevalence. In ICU survivors with moderate probability PTSD by PCL-S, the CAPS gold-standard interview is challenging to complete and adds only a small number of diagnoses. However, two in fi ve ICU survivors will develop PTSD subtypes of avoidance or hyperarousal, which both occur twice as frequently as the intrusion subtype. Targeting predominant PTSD subtypes may help optimize treatment strategies for the ICU survivor, such as prolonged exposure and eye movement desensitization and reprocessing for those with the avoidance subtype, and pharmacologic antidepressants targeting the sympathetic nervous system to produce anxiolysis for those with the hyperarousal subtype. y Results Of the 180 eligible participants at 3  months, PTSD was identifi ed in 10 (6%) using PCL-S scores and 15 (8%) using DSM-IV mapping of the PCL-S. Of the 160 eligible participants at 12 months, PTSD was identifi ed in two (1%) using PCL-S scores and 10 (6%) using DSM-IV mapping of the PCL-S. Of those eligible for CAPS assessments, at 3 months only 13 of 24 (54%) interviews were completed resulting in three extra PTSD diagnoses, and at 12 months only six of 22 (27%) interviews were completed resulting in two extra PTSD diagnoses. y Referencefi At 3 and 12 months, the intrusion subtype was present in 25 (14%) and in 25 (16%), the avoidance subtype was present in 78 (43%) and 60 (38%), and the hyperarousal subtype was present in 82 (46%) and 71 (44%).i P553 The Pearson chi-squared test was used to compare groups and Cramer’s V analyses was used to examine strength of the association between groups. activity, pain and hoarseness were associated signifi cantly with PTSD and HAD. There was no signifi cant diff erence in somatic complaints between men and women. Conclusion Somatic complaints after ICU discharge are frequently reported in our post-ICU aftercare patients, infl uencing daily performance and quality of life. Patient-centered research and treatment focusing on somatic complaints is of great importance. References 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. 1. Zimond AS, et al. Acta Psychatr Scand. 1983;67:361-70. 2. Creamer M, et al. Behav Res Ther. 2003;41;1489-96. Results Seventy-nine patients, 54 male and 43 women, with mean age 57 years. Median APACHE II and APACHE IV were 18 and 60 respectively. Median ICU days and hospital days were 9 and 20 respectively. Seventy- six percent of patients were mechanically ventilated with a median of 5 days. Median time of ICU discharge to aftercare visit was 165 days. Delirium occurred in 22 (22.7%) patients during ICU treatment. The prevalence of PTSD was 43.3% and was most seen in patients after subarachnoid hemorrhage (SAH) (28.6%). Pain, muscle weakness, fatigue, impairment in daily activity, dyspnea, and hoarseness reported during the ICU aftercare clinic visit were signifi cantly associated with PTSD. There was no signifi cant diff erence in men and women. Sedation, opiates, benzodiazepine, inotropic medication and delirium during ICU treatment were not associated with higher prevalence of PTSD. None of the other demographic data analyzed were signifi cantly associated with PTSD. Post-traumatic stress disorder prevalence and subtypes among survivors of critical illness Post-traumatic stress disorder prevalence and subtypes among survivors of critical illness M Patel1, J Jackson1, A Morandi2, T Girard1, C Hughes1, A Kiehl1, J Thompson1, R Chandrasekhar1, E Ely1, P Pandharipande1 1Vanderbilt University, Nashville, TN, USA; 2Ancelle Hospital, Cremona, Italy Critical Care 2015, 19(Suppl 1):P555 (doi: 10.1186/cc14635) M Patel1, J Jackson1, A Morandi2, T Girard1, C Hughes1, A Kiehl1, J Thompson1, R Chandrasekhar1, E Ely1, P Pandharipande1 1Vanderbilt University, Nashville, TN, USA; 2Ancelle Hospital, Cremona, Italy Critical Care 2015, 19(Suppl 1):P555 (doi: 10.1186/cc14635) Introduction Among North American survivors of critical illness, we aim to describe the prevalence of post-traumatic stress disorder (PTSD), and its subtypes of intrusion, avoidance, and hyperarousal. Methods In this prospective, observational, multicenter cohort study from 2009 to 2010, we screened adults (age ≥18  years) with new- onset respiratory failure, cardiogenic shock, or septic shock, who were admitted to medical and surgical ICUs in four facilities. At 3-month and 12-month follow-ups, high probability of PTSD was defi ned by 17-symptom PTSD Checklist – Event Specifi c Version (PCL-S) score ≥50. Also, PCL-S responses were mapped onto DSM-IV criteria for PTSD. To augment PTSD identifi cation, those with a moderate probability of post-ICU PTSD (PCL-S score ≥35) were further confi rmed with the Clinician Administered PTSD Scale (CAPS) structured interview. Moderate or greater symptoms for each PTSD subtype of intrusion, avoidance, and hyperarousal were categorized. Conclusion Prevalence of PTSD was 43.3% and most seen in patients after SAH, refl ecting the majority of patients treated in our ICU. PTSD was associated signifi cantly with pain, muscle weakness, fatigue, dyspnea, hoarseness and impairment of daily activity after a median 165 days post ICU treatment. Somatic complaints after ICU survival: results of a post-ICU aftercare program Median time after ICU discharge and visit to our after care clinic was 165  days. Twenty-fi ve patients died after ICU discharge. Fifty-four patients were excluded because of various reasons; that is, language barrier, psychiatric illness, mental handicap, hospital admittance elsewhere, great distance. Seventy patients (81.4%) had somatic complaints infl uencing daily performance and quality of life. Fatigue (74.4%), muscle weakness (48.8%), dyspnea (34.9%), impairment of daily activity (81.4%), pain (38.4%) and weight loss (33.3%) were the most frequently reported complaints. Pain was most reported in patients with subarachnoid hemorrhage (27.3%), multitrauma (15.2%) and pneumonia (12.1%). Pain was most localized in the head (15.6%), one or both legs (15.6%), back (10.9%), shoulder (9.3%), hip (9.3%) and thorax (6.3%). Muscle weakness, fatigue, dyspnea, impairment of daily Physiotherapy in the ICU: an evidence-based, expert-driven, practical statement J Sommers1, R Engelbert2, D Dettling1, R Gosselink3, P Spronk4, J Horn 1, F Nollet1, M Van der Schaaf1 1Academical Medical Center, Amsterdam, the Netherlands; 2School of Health, Amsterdam, the Netherlands; 3KU Leuven, Belgium; 4Gelre Hospital, Apeldoorn, the Netherlands Critical Care 2015, 19(Suppl 1):P558 (doi: 10.1186/cc14638) g q y Results A total of 1,743 papers were retrieved, of which 18 studies were eligible for inclusion in the review. Studies had a combined population of 1,970 patients admitted to 38 ICUs from Europe, Asia and North America. Eleven studies were randomized controlled trials (RCTs). Interventions were classifi ed as four groups – music; therapeutic touch; diary and psychotherapeutic interventions. Ten studies found that music interventions were eff ective in the short term; however, follow- up results were limited and some studies were low quality. There was moderate quality evidence from three studies for the eff ectiveness of diary interventions, with medium-term follow-up results. There was mixed-quality evidence for therapeutic touch interventions in the short term from three studies. The two psychotherapeutic interventions studied were of moderate quality, and one showed promising results at 12-month follow-up.fi Introduction Evidence-based, expert-driven, practical statements improve quality and eff ectiveness of the diagnostic and therapeutic process of patient care. Although the eff ectiveness of physiotherapy treatment strategies in ICU patients has been described, statements or guidelines of physiotherapy for ICU patients are not available [1]. Guidelines on safety management and on the diagnostic and therapeutic process may support and guide clinical decision-making leading towards evidence-based tailored care. The aim of this study was to develop an evidence-based statement for the physiotherapy treatment of ICU patients with recommendations for eff ective and safe diagnostic assessment and intervention strategies. Conclusion The evidence for the effi cacy of nonpharmacological interventions to reduce short-term or long-term stress in intensive care patients was of low to moderate quality. Studies included mainly short- term and medium-term follow-up. This highlights the need for larger- scale, better-quality RCTs with longer-term outcome measurement. However, the results indicate that nonpharmacological, including psychological, approaches are likely to be benefi cial for reducing short- term or long-term stress in intensive care patients. Methods For the development of this evidence statement, we used the EBRO method, as recommended by the Dutch Evidence Based Guideline Development Platform [2]. Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 particularly those of working age. More work is required to understand optimal rehabilitation pathways in this patient group. References particularly those of working age. More work is required to understand optimal rehabilitation pathways in this patient group. References effi cacy of such interventions. Previous work has shown that intensive care patients undergo many stressful experiences, which can aff ect their long-term psychological well-being. Studies have demonstrated a high prevalence of depression, anxiety or post-traumatic stress disorder after intensive care admissions. 1. Herridge M, et al. N Engl J Med. 2011;364:1293-304. 2. Corner EJ, et al. Crit Care. 2014;18:R55. Methods A systematic review was carried out according to the Prisma statement. A search was conducted of Medline, Embase and Psychinfo databases. Inclusion criteria included studies of populations of adult patients in mixed or general ICUs. No study designs were excluded, but studies that focused on specifi c disease states were excluded. Included studies were assessed for risk of bias, using a quality checklist. P557 P557 Utilisation of existing community rehabilitation services by critical care survivors CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) Utilisation of existing community rehabilitation services by critical care survivors CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) l Results Three expert-based relevant clinical questions were formulated within the physiotherapy clinical reasoning process and were classifi ed according to the International Classifi cation of Functioning, Disability and Health. In a systematic literature search, 129 studies were identifi ed and assessed for methodological quality and classifi ed according to the level of evidence. The fi nal Evidence Statement consisted of recommendations for physiotherapy in ICU patients including safety criteria, a core set of instruments to assess impairments and activity restrictions and eff ective interventions. CR Soulsby, J McPeake, C Ashcroft, J Kinsella, M Shaw, T Quasim University of Glasgow, UK Critical Care 2015, 19(Suppl 1):P557 (doi: 10.1186/cc14637) Introduction Patients recovering from critical illness suff er many physical and psychological problems during their recovery, including muscle weakness, fatigue, signs and symptoms of PTSD, anxiety and depression [1]. At present, specialist intensive care follow-up and rehabilitation is inconsistent and in many geographical areas is nonexistent. As a result, many survivors of critical illness will require using existing community rehabilitation services [2]. The aim of this present service evaluation was to understand the utilisation of community rehabilitation services by critical care survivors. f Conclusion The Evidence Statement for physiotherapeutic diagnostics and intervention in ICU patients will contribute to the quality of clinical practice by supporting the clinical decision-making process. References 1. Kayambu G, Boots R, Paratz J. Physical therapy for the critically ill in the ICU: a systematic review and meta-analysis. Crit Care Med. 2013;41:1543-54. 2. Burgers JS, van Everdingen JJE. Evidence-based richtlijnontwikkeling in Nederland: the EBRO-platform. Ned Tijdschr Geneeskd. 2004;148:2057-9. Methods A database of acute referrals to community rehabilitation services was retrospectively analysed from 1 May 2014 to 31 October 2014. Age, referring specialty and reason for referral for rehabilitation were documented. This database was cross-checked with the critical care database in Glasgow Royal Infi rmary to identify which individuals had been admitted to critical care during their admission. Physiotherapy in the ICU: an evidence-based, expert-driven, practical statement This method consists of the identifi cation of clinically relevant research questions, followed by a systematic literature search, quality assessment, and summary of the evidence eventually leading to establishing of concept and fi nal recommendations based on feedback from experts. The fi nal recommendations were prepared according to this methodical approach and summarized in fi gures, fl owcharts and appendices. Nonpharmacological interventions to reduce short-term or long-term psychological stress in ICU patients: a systematic review D Wade1, Z Moon2, S Windgassen2, J Weinman2 1University College Hospital, London, UK; 2Kings College London, UK Critical Care 2015, 19(Suppl 1):P556 (doi: 10.1186/cc14636) Introduction A systematic review was performed of studies of nonpharmacological interventions aiming to reduce short-term or long-term stress in intensive care patients, as little is known about the S194 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) i Results A total of 697 hospitals records were analyzed. Patients had on average (SD) 67.8 (13.1) years and the majority of them were males (57%). Our results revealed that 65% of patients in the CICU received phase 4 and 43% of patients in the CICU received phase 5 of the early mobilization protocol. No diff erences in the proportion of patients receiving phase 4 or 5 were found among arrhythmia, coronariopathies and congestive heart failure groups. The only diff erence found was between congestive heart failure group and other cardiovascular pathologies (P  <0.001). The congestive heart failure group was mobilized 5.6 times (95% CI: 2.7 to 11.5) and 3.2 times (95% CI: 1.7 to 5.7) more than the other cardiovascular pathologies group in phase 4 and 5, respectively. Introduction Increasing cost is an important issue in critical care medicine. We tried to analyze in a level 3 care ICU in Kolkata of a tertiary care hospital whether the diff erent payment options (self-paying vs. insurance/corporate paying) do affect the outcome in the critically ill insurance/corporate paying) do aff ect the outcome in the critically ill. Methods Our prospective study included 1,520 patients admitted consecutively to a level 3 care ICU for a period of 20 months. Readmitted patients during the same period were excluded. Payment method was documented for all and divided into two groups as self-paying and insurance/corporate paying. Outcome assessment was done using the APACHE IV model for all cases. Demographic data, number of observed deaths, predicted mortality rate (PMR), standardized mortality ratio (SMR), average length of stay (ALOS), predicted length of stay, and number of discharge against medical advice (DAMA) were documented for each group. Statistical analysis was carried out using unpaired Student t test and P <0.05 was considered signifi cant. Conclusion A considerable proportion of patients was mobilized without any serious complications in the CICU. Our fi ndings suggest that patients diagnosed with arrhythmia, coronariopathies and congestive heart failure can be equally mobilized in an ICU. p gi Results Of 1,520 patients, 995 (65.46%) cases were self-paying while 525 (34.54%) cases were insurance/corporate paying. P559 E l Conclusion In this large retrospective multicenter study, the TISS item associated with the highest risk of death was cardiac arrest and/or countershock. Unexpectedly, the independent eff ect of emergency admission was of comparable magnitude in terms of impact on hospital mortality. Of these, in-ICU cardiac arrest might be amenable to preventive measures and should be studied further. Brazil. The early mobilization protocol consists of fi ve phases: 1  – passive exercises for the unconscious patient; 2  – active exercises associated with respiratory exercises (patient lying on the bed); 3  – phase 2 exercises with the patient sitting on the bed; 4  – phase 2 exercises with the patient sitting on a chair or in a standing position; 5 – phase 4 exercises plus walking. All hospital records from patients, between September 2013 and August 2014, were included in this study. Data extracted from hospital records were: age, gender, diagnosis (arrhythmia, coronariopathies, congestive heart failure and other pathologies), length of stay, number of discharge and number in each phase of the early mobilization protocol. Pearson chi-square test was used to compare the number of mobilizations (phase 4 and 5) per group of diagnoses. Odds ratios were calculated for those comparisons found to be statistically signifi cant (P <0.05). with mortality for pulmonary artery catheter, platelet transfusion and vasoactive drug infusion (one drug) (P >0.05). Conclusion In this large retrospective multicenter study, the TISS item associated with the highest risk of death was cardiac arrest and/or countershock. Unexpectedly, the independent eff ect of emergency admission was of comparable magnitude in terms of impact on hospital mortality. Of these, in-ICU cardiac arrest might be amenable to preventive measures and should be studied further. P561 P560 Need for therapeutic interventions as a predictor of mortality in intensive care Payment options: do they aff ect outcome in the critically ill A Kar, A Datta In the self-paying group, mean age was 59.65 years ± 17.26 SD (median 62), APACHE IV score mean was 62.50 ± 33.61 SD (median 57), average LOS 4.67 days ± 4.29 SD (median 3), PMR was 22.71, 226 observed deaths, 85 cases of DAMA, and SMR was 1.00 (CI  = 0.87 to 1.14). In the insurance/ corporate-paying group, mean age was 61.75 years ± 17.19 SD (median 65), APACHE IV score mean was 58.53 ± 32.94 SD (median 54), average LOS was 5.64 days ± 5.61 SD (median 4), PMR was 21.26, 113 observed deaths, six cases of DAMA, and SMR was 1.01 (CI = 0.83 to 1.21). In the two compared groups, predicted mortality and SMR were not statistically signifi cant (P = 0.2808); however, ALOS in the insurance/ corporate paying group was signifi cantly higher than the self-paying group (P  = 0.0002), mean age of the insurance/corporate paying group was signifi cantly higher than the self-paying group (P = 0.02), and incidence of DAMA is signifi cantly higher in the self-paying group (8.54%) as compared with insurance/corporate paying group (1.14%). Root-cause analysis showed DAMA cases are mostly fi nancial (>95%). Conclusion Statistically signifi cant diff erences in ALOS and DAMA in the two groups are probably due to cost of healthcare not aff ordable to all. Need for therapeutic interventions as a predictor of mortality in intensive care I Ef dij 1 R R j1 S H 2 MB Sk if 1 M R i ik i 3 Need for therapeutic interventions as a predictor of mortality in intensive care I Efendijev1, R Raj1, S Hoppu2, MB Skrifvars1, M Reinikainen3 1HUS, Helsinki, Finland; 2TAYS, Tampere, Finland; 3PKKS, Joensuu, Finland Critical Care 2015, 19(Suppl 1):P560 (doi: 10.1186/cc14640) I Efendijev1, R Raj1, S Hoppu2, MB Skrifvars1, M Reinikainen3 1HUS, Helsinki, Finland; 2TAYS, Tampere, Finland; 3PKKS, Joensuu, Finland Critical Care 2015, 19(Suppl 1):P560 (doi: 10.1186/cc14640) Introduction Various therapeutic interventions needed in critical care may refl ect a high risk of death. We evaluated associations between commonly used interventions and hospital mortality in Finnish ICU patients. Methods We retrieved data on adult patients treated in Finnish ICUs between 2003 and 2013 from the Finnish Intensive Care Consortium database. We used the Therapeutic Intervention Scoring System (TISS-76) for categorizing ICU interventions and the Simplifi ed Acute Physiology Score (SAPS II) for quantifying severity of illness. We excluded readmissions, patients with missing outcome, SAPS II and TISS data. We also excluded very common interventions (arterial line, bolus intravenous medication), very rare ones (prevalence <1%), and interventions only applicable in specifi c populations (intracranial pressure monitoring, intra-aortic balloon assist). We grouped several TISS categories when applicable. We performed a backward stepwise binary logistic regression analysis with TISS items to assess the impact of each intervention on hospital mortality (expressed as odds ratio (OR) with 95% confi dence intervals (CIs)). Age, admission type, and SAPS score (minus age and admission type scores) were adjusted for in the multivariate analysis. P562 P561 Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) Payment options: do they aff ect outcome in the critically ill A Kar, A Datta Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P561 (doi: 10.1186/cc14641) P559 E l Early mobilization according to diagnosis in a Brazilian coronary ICU GS Zavanelli1, SA Padulla1, MR Franco1, RZ Pinto1, LL Faccioli1, DN Barbosa1, DT Neves2, CE Bosso2 1Universidade Estadual Paulista – UNESP, Presidente Prudente, Brazil; 2Instituto do Coração de Presidente Prudente, Brazil Critical Care 2015, 19(Suppl 1):P559 (doi: 10.1186/cc14639) g Results Over this 6-month period 769 patients were referred from their parent specialty for community rehabilitation in North East Glasgow. Thirty-three of the 769 patients (4.3%) referred had a critical care stay during their admission. Of these, eight patients were referred for rehabilitation by orthopaedics, eight by medicine for the older patients, 11 from acute medicine and the remaining six from other specialties. Six of the 769 patients who had a critical care admission were of working age (<1%). Two individuals were admitted to critical care following trauma whilst four had complex social needs prior to their critical care admission. This included an individual with a high body mass index. None of the individuals of working age were referred as a consequence of their critical care stay. Introduction Early mobilization has been advocated to improve muscle function and, consequently, the patient quality of life after discharge. Nevertheless, few studies have explored it in a coronary ICU (CICU). The aims of the present study were to describe the use of an early mobilization protocol in a CICU and to investigate whether diff erent groups of diagnoses respond similarly to this protocol. Introduction Early mobilization has been advocated to improve muscle function and, consequently, the patient quality of life after discharge. Nevertheless, few studies have explored it in a coronary ICU (CICU). The aims of the present study were to describe the use of an early mobilization protocol in a CICU and to investigate whether diff erent groups of diagnoses respond similarly to this protocol. Conclusion This service evaluation demonstrates that very few critical care survivors are referred to community rehabilitation services, Methods This is a retrospective observational study conducted in a medium-sized hospital located in the city of Presidente Prudente, S195 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 with mortality for pulmonary artery catheter, platelet transfusion and vasoactive drug infusion (one drug) (P >0.05). Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P562 (doi: 10.1186/cc14642) Introduction Source of admission to the ICU is of importance. We tried to identify the diff erent sources of ICU admission to a level 3 ICU of a tertiary care hospital in Kolkata and analyze whether the overall patient outcome is aff ected by the diff erent sources of admission. Results We identifi ed 161,134 patients eligible for analysis. The multivariate analysis showed that the highest risk for hospital mortality in all patients was associated with cardiac arrest and/or countershock, OR 2.58 (95% CI = 2.43 to 2.73), SAPS II emergency admission, OR 2.52 (95% CI = 2.32 to 2.74), vasoactive drug infusion (>1 drug), OR 1.66 (95% CI = 1.59 to 1.73) and blood transfusion (a combined TISS item), OR 1.53 (95% CI = 1.44 to 1.63). TISS items associated with the lowest risk of mortality in general population were: active anticoagulation, OR 0.51 (95% CI = 0.49 to 0.53), induced hypothermia, OR 0.68 (95% CI = 0.62 to 0.74) and measurement of cardiac output by any method, OR 0.87 (95% CI = 0.83 to 0.91). All aforementioned associations were statistically signifi cant (P  <0.001). There was no notable association ff Methods Our prospective study included 2,056 patients admitted to a level 3 care ICU over a period of 2 years. Numbers of readmissions were not considered. ICU outcome was analyzed using the APACHE IV model and source of admission to the ICU was documented as either from emergency (ER), from the fl oor or from other hospital. Analysis was carried out between diff erent groups based on admission using unpaired Student t test and P  <0.05 was considered signifi cant. Number of ventilations and the mortality rate in each group were also documented. S196 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Results Of 2,056 admissions, 1,223 cases (59.48%) were from ER, 809 cases (39.35%) were from fl oor and 24 cases (1.16%) were from other hospitals. Association between out-of-hours discharge and mortality in adult patients leaving critical care Association between out-of-hours discharge and mortality in adult patients leaving critical care S Edie, K Burt, J Paddle Royal Cornwall Hospital, Truro, UK Critical Care 2015, 19(Suppl 1):P564 (doi: 10.1186/cc14644) Introduction Out-of-hours (OOH) discharge from critical care is associated with a signifi cantly increased mortality rate in Australasia [1]. In the UK, daytime discharges from critical care are considered a core standard [2]. We sought to assess the impact of OOH discharge from critical care on mortality in a large general ICU, where operational pressures appear to have led to a high rate of OOH discharges. Methods Retrospective data for all patients admitted to our ICU from April 2007 to September 2014 were recorded, using routinely collected data from our databases. Adult patients (>15 years) discharged from their fi rst ICU admission during each hospital stay (episode) were included. Patients that died on the unit and those discharged for palliative care were excluded. Patients transferred to other centres were no longer subject to discharge within our control and were therefore also excluded. Patients discharged directly home from ICU were excluded. We defi ned OOH discharges as those occurring between 22:00 and 06:59, a standard defi nition in UK practice. Mortality status at the time of hospital discharge for each episode was used. We also recorded the readmission rate to ICU. The relative risk (RR) for OOH mortality and readmission was calculated. Statistical signifi cance was accepted at P <0.05. Conclusion The severity of illness index in patients admitted to the ICU from fl oors is signifi cantly higher than emergency admissions. Overall outcome for patients transferred to the ICU from the fl oor is worse based on mortality rate, SMR, and ALOS when compared with the emergency group. Readmission to the ICU: is it a big concern? An analysis A Ahmed, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P563 (doi: 10.1186/cc14643) Readmission to the ICU: is it a big concern? An analysis A Ahmed, A Datta, A Kar Medica Superspecialty Hospital, Kolkata, India Critical Care 2015, 19(Suppl 1):P563 (doi: 10.1186/cc14643) Results Of 4,476 index cases, 714 died on the unit and 80 were discharged for palliative care. A total of 490 patients were excluded for transfer to other centres and discharge directly home. Data were missing for three patients, which left 3,189 records for analysis. In total, 2,711 patients were discharged during daytime hours, of which 145 (5.35%) died. A total of 478 patients were discharged at night, 40 died (8.37%). The RR for OOH mortality was 1.56 (95% CI = 1.12 to 2.19, P = 0.0091). Readmission rate was 5.2% by day, 6.1% at night. The RR for readmission was 1.17 (95% CI = 0.79 to 1.72, P = 0.436). Introduction Readmission to the ICU is an important quality indicator of ICU care. We conducted a prospective study in a level 3 care ICU in Kolkata of a tertiary care hospital to analyze whether there are overall outcome diff erences when comparing the readmission group with the entire group. Conclusion Our data demonstrate an association between critical care discharge time and mortality, to a statistically signifi cant level. Due to the retrospective observational nature of the study, causation cannot be assumed; however, a number of factors may contribute to the increased risk of harm to patients discharged from the ICU at night. Further work will focus on annual OOH mortality trends, thereby gaining an insight into whether bed occupancy demands impact on the necessity for nighttime discharges. Methods Our prospective study included 2,140 patients admitted to a level 3 care ICU over a period of 1 year. The number of readmissions (n = 85) during the same period was also documented. Readmission was defi ned as all patients who were transferred back to the ICU prior to hospital death/discharge during the above period. ICU outcome was calculated using the predictive APACHE IV model. Payment methods were documented as either self-paying or corporate/insurance paying. A comparison analysis between the entire group with the readmission group was done using unpaired Student t test and P  <0.05 was considered statistically signifi cant. Source of ICU admission: does it really matter? A Datta, A Kar, A Ahmed In the ER group, mean APACHE IV was 55.03  ± 31.49 SD (median 50), PMR 16.26, observed deaths 198, ALOS 4.78 days ± 4.83 SD (median 3), SMR 0.995 (CI = 0.86 to 1.14), mean age 60.52 years ± 17.63 SD (median 63), 323 ventilations. In the fl oor group, mean APACHE IV was 65.17 ± 34.40 SD (median 60), PMR 27.03, observed deaths 234, ALOS 5.23 days ± 5.22 SD (median 3), SMR 1.07 (CI = 0.94 to 1.21), mean age 61.38 years ± 15.72 SD (median 64), 302 ventilations. In the other hospital group, mean APACHE IV was 55.29 ± 29.82 SD (median 50), PMR 18.0, observed deaths 2, ALOS 6 days ± 5.85 SD (median 3), SMR 0.46 (CI: 0.23 to 0.88), mean age 56.08 years ± 17.79 SD (median 56.5), six ventilations. During analysis, the other hospital group was omitted because of inadequate sample size. There was statistically signifi cant diff erences in APACHE IV (fl oor >ER, P  <0.0001), PMR (fl oor >ER, P <0.0001), ALOS (fl oor >ER, P = 0.04) noted between the fl oor and ER groups. Number of ventilations (37.33% vs. 26.4%), SMR (1.07 vs. 0.995), and mortality rate (28.92% vs. 16.19%) were also signifi cantly higher for patients admitted from the fl oor. No signifi cant statistical diff erence was observed in age between two groups (P = 0.26). y References 1. Gantner D, et al. Int Care Med. 2014;40:1528-35. 1. Gantner D, et al. Int Care Med. 2014;40:1528-35. 2. Faculty of Intensive Care Medicine and Intensive Care Society. Core standards for intensive care units (2013). http://www.fi cm.ac.uk/standards. 2. Faculty of Intensive Care Medicine and Intensive Care Society. Core standards for intensive care units (2013). http://www.fi cm.ac.uk/standards. y gi Results In the entire group (n = 2,140), mean APACHE IV was 50.34 ± 31.54 SD (median 42), PMR 15.49, observed deaths 327, ALOS 4.05 days ± 4.55 SD (median 3), SMR 0.99 (CI = 0.88 to 1.1), mean age 60.55 years ± 15.68 SD (median 63), 490 ventilations, 72.71% of patients were self-paying while 27.29% of patients were corporate/insurance paying. In the readmission group (n = 85), mean APACHE IV was 77.16 ± 33.72 SD (median 73), PMR 38.89, observed deaths 42, ALOS 5.23 days ± 4.18 SD (median 4), SMR 1.27 (CI = 0.95 to 1.67), mean age 64.79 years ± 14.40 SD (median 67), 43 ventilations, 75.3% of patients were self- paying while 24.7% of patients were corporate/insurance paying. During comparison between the two groups there were statistically signifi cant diff erences, with the readmission group having signifi cantly higher APACHE IV (P <0.0001), PMR (P <0.0001), ALOS (P = 0.002), age (P = 0.005), and SMR (1.27 vs. 0.99) compared with the entire group. Percentage of patients requiring ventilation (50.59% vs. 22.90%) and mortality rate (49.11% vs. 15.28%) were also signifi cantly higher in the readmission group. Readmission was signifi cantly higher in the self- paying group. Root-cause analysis showed most readmissions were due to deteriorating conditions (desaturation, hypotension, sepsis, arrhythmias); however, it was also associated with cases where transfer policy from the ICU was not followed by stakeholders and fi nancial issues were a cause of early transfer. P566 Determination of brain death for adult patients with ECMO I Ceylan, R Iscimen, E Cizmeci, N Kelebek Girgin, F Kahveci Uludag University Faculty of Medicine, Bursa, Turkey Critical Care 2015, 19(Suppl 1):P566 (doi: 10.1186/cc14646) f Results The CFA showed a good fi t indicating factorial validity (CFI: 0.97), reliabilities were from α 0.79 to 0.93 and ICCs were signifi cant (~0.20, P <0.001). HLM revealed that unit-level IL of nurses and residents was positively related to PS (b = 0.34, P <0.001). Being a resident and working in a smaller unit also predicted PS. As expected, unit-level PS was negatively related to individual PFC (b = –0.38, P = 0.025). Further predictors of higher PFC were: being a nurse, having more than 5 years of job experience and higher workload. PS mediated the relationship between unit-level IL and individual PFC (indirect eff ect: –0.13, P <0.001). Additional analyses revealed that attendings’ PFC was negatively related to their perception of residents PS (b = –0.44, P = 0.019). Introduction ECMO support in ARDS is an emerging strategy when conventional treatment modalities fail. ECMO has advantages on oxygenation and circulation but also it has some unfavorable eff ects. The most serious complication is brain death due to cerebrovascular hemorrhage. An apnea test is the most important component in confi rming brain death. For patients supported by ECMO, apnea testing remains challenging. Brain-death diagnosis is often made without an apnea test. p Methods We present two cases who receive V-V ECMO support after progression to ARDS. After initiation of ECMO we used sedation to prevent movement and improve adaptation to mechanical ventilation. Also we used anticoagulation with heparin to prevent thromboembolic events and ECMO circuit occlusion. On daily follow-up we noticed that patients had lost their pupil reactions to light. Their sedation was ceased and a computed brain tomography was performed. Both patients had intracerebral hemorrhage. We decided to determine brain death with apnea tests. We increased ECMO blood fl ow and fi O2 and then decreased sweep gas fl ow and disconnected the patient from mechanical ventilation respectively. In one patient we did not see any spontaneous breathing eff orts after carbon dioxide retention. We concluded that the apnea test was successful and confi rmed brain death. P566 On the other hand, we confi rmed the brain death of the other patient with cerebral angiography due to the occurrence of hypoxia and hypotension during apnea testing. p p Conclusion A sense of PS in an ICU team might reduce futile care by increasing the safety of speaking-up behavior of nurses and residents. PS can be enhanced by attending physicians who practice inclusive leadership behavior to foster autonomy and participation of residents and nurses. End-of-life decisions: how do patients die in the ICU? Introduction One of the main goals of intensive care medicine is to reduce the mortality of critically ill patients. However, it is essential to recognize end-of-life care as an integral component of critical care. Besides survival, the success of intensive care should also include the quality of lives preserved and the quality of dying. The objective of this study was to evaluate the incidence and type of end-of-life decisions (ELD) in critical patients that died in an ICU. y Results We experienced some challenges while determining brain death in patients under ECMO support for ARDS. It is challenging to conduct the apnea testing during ECMO support. Auxiliary tests are required for patients who cannot tolerate the changes needed to conduct the apnea test. With increasing use of ECMO therapies, clinicians may come face to face with more complicated life-ending decisions. p Methods Analysis of all patients included in an ICU running database and who died from 1 November 2013 to 31 October 2014. The following variables were evaluated: age, gender, reason for admission, SAPS II, length of ICU stay and type of ELD. To classify ELD, four concepts were defi ned: ‘Comfort care’, a change from curative therapy to comfort care therapy; ‘Limited therapy’, maintenance of curative therapy but without escalating it (for example, no renal substitution); ‘Decision not to resuscitate’, not to perform advanced life support if cardiac arrest occurs; and ‘Without previous end-of-life decisions’, when there was no prior decision regarding the ELD. Conclusion Current guidelines do not include brain death criteria using supportive therapies such as ECMO and therefore should be updated. References 1. Goswami S, et al. J Cardiothor Vasc Anesth 2013;27:312-4. 2. Jarrah RJ, et al. Pediatr Crit Care Med. 2014;15:38-43. 3. Marasco SF, et al. Heart Lung Circ 2008;17 Suppl:S41–7. 4. Farrah JM, et al. Arch Neurol. 2011;68:1543-9. References 1. Manthous CA, et al. Am J Respir Crit Care Med. 2011;184:17-25. 2. Nembhard IM, et al. J Organ Behav. 2006;27:941-66. P567 Results A total of 507 patients were admitted to the ICU and 132 died (26%). Reasons for admission in those who died were septic shock (47%), post cardiac arrest (13%), cardiogenic shock (8%), and nontraumatic brain bleeding (8%). Fifty-three patients (40%) died after a ‘Comfort care’ decision, 28 patients (21%) after ‘Decision not to resuscitate’ and 14 (11%) after a ‘Limited therapy’ decision. Thirty-seven patients died ‘Without previous end-of-life decisions’. However, specifi cally in this group, when looking for individual records, 32 patients died (86%) in the fi rst 48 hours after the admission and four (11%) had evidence of brain death and were organ donors, which leaves one patient (3%) in whom there was no ELD. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU D Schwarzkopf1, J Felfe2, CS Hartog1, F Bloos1 1Jena University Hospital, Jena, Germany; 2Helmut Schmidt University, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P567 (doi: 10.1186/cc14647) Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) J Hofhuis1, HF Van Stel2, AJ Schrijvers2, JH Rommes1, PE Spronk1 1Gelre Hospitals, Apeldoorn, the Netherlands; 2University Medical Center, Utrecht, the Netherlands Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) , Critical Care 2015, 19(Suppl 1):P565 (doi: 10.1186/cc14645) Introduction Severe critical illness requiring treatment in the ICU may have a serious impact on patients and their families. However, optimal follow-up periods are not defi ned and data on health-related quality of life (HRQOL) before ICU admission as well as those beyond 2-year follow-up are limited. The aim of our study was to assess the impact of ICU stay up to 5 years after ICU discharge. Methods We performed a long-term prospective cohort study in patients admitted >48 hours to a medical–surgical ICU. The Short-Form 36 was used to evaluate HRQOL before admission (by proxy within 48 hours after admission of the patient), at ICU discharge and at 1, 2 and 5 years following ICU discharge (all by patients). Changes in HRQOL were assessed using linear mixed modeling. Conclusion Readmission to the ICU was associated with worse outcome in our study group. Lack of adherence to transfer policy by concerned stakeholders was a concern as well as increasing cost of healthcare. Results We included a total of 749 patients (from 2000 to 2007). At 5  years after ICU discharge, 234 patients could be evaluated. After S197 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 correction for natural decline in HRQOL, the mean scores of four dimensions – physical functioning (P <0.001), physical role (P <0.001), general health (P <0.001) and social functioning (P = 0.003) – were still signifi cantly lower 5  years after ICU discharge compared with their preadmission levels, although eff ect sizes were small (<0.5). Recovery of health-related quality of life in ICU patients: a 5-year prospective cohort study Conclusion Five years after ICU discharge, survivors still perceived a signifi cantly lower HRQOL than their preadmission HRQOL (by proxies), and that of an age-matched general population. Importantly however, after correction for natural decline, the eff ect sizes were small suggesting that patients regain their age-specifi c HRQOL 5 years after their ICU stay. correction for natural decline in HRQOL, the mean scores of four dimensions – physical functioning (P <0.001), physical role (P <0.001), general health (P <0.001) and social functioning (P = 0.003) – were still signifi cantly lower 5  years after ICU discharge compared with their preadmission levels, although eff ect sizes were small (<0.5). report less perceived futile care (PFC). We also expected that attending physicians’ inclusive leadership (IL), which invites nurses’ and residents’ participation [2], would decrease PFC and that PS mediates this relationship. Methods The hypotheses were tested in a cross-sectional, multicenter paper-and-pencil survey addressing medical staff on participating ICUs. A total of 22 ICUs and four intermediate care units were included in the sample and 73 attendings, 147 residents and 659 nurses participated in the study (52% participation). Psychometric properties were tested by confi rmatory factor analysis (CFA), Cronbach’s α and intraclass correlations (ICC). A series of hierarchical linear models (HLM) were conducted to test the study hypotheses separately among nurses/residents and attendings. IL and PS were entered as unit-level predictors (mean values per unit). Covariates were demographics, working hours per week, workload and unit size (number of staff ). Mediation eff ects were tested.i f Conclusion Five years after ICU discharge, survivors still perceived a signifi cantly lower HRQOL than their preadmission HRQOL (by proxies), and that of an age-matched general population. Importantly however, after correction for natural decline, the eff ect sizes were small suggesting that patients regain their age-specifi c HRQOL 5 years after their ICU stay. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU D Schwarzkopf1, J Felfe2, CS Hartog1, F Bloos1 1Jena University Hospital, Jena, Germany; 2Helmut Schmidt University, Hamburg, Germany Critical Care 2015, 19(Suppl 1):P567 (doi: 10.1186/cc14647) Introduction Psychological safety (PS), for example safety of speaking up, fosters team learning and prevents treatment errors on the ICU [1]. Since speaking up might also prevent excessive and inappropriate (futile) care for patients, we hypothesized that teams with higher PS Conclusion In this study, ‘Comfort care’ was the main ELD, which is in line with the concept that ELD are essential to ensure that care provided is S198 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 examine the association of palliative care screening criteria with adverse patient outcomes. consistent with quality of life and death. The apparent large proportion of patients ‘Without previous end-of-life decisions’ was due to patients who died in the fi rst 48 hours after ICU admission corresponding to conditions refractory to treatment. Additionally, this study also draws our attention to better plan ICU admissions and hospital outreach in order to reduce early ICU mortality. consistent with quality of life and death. The apparent large proportion of patients ‘Without previous end-of-life decisions’ was due to patients who died in the fi rst 48 hours after ICU admission corresponding to conditions refractory to treatment. Additionally, this study also draws our attention to better plan ICU admissions and hospital outreach in order to reduce early ICU mortality. Methods We performed an observational unicentric study on a 12-bed, medical emergency department intensive care unit (EDICU). A three- item palliative care screen was developed from consensus reports. A senior critical care physician screened patients upon admission using these questions during a 10-week period. The questions were: does this patient suff er from a life-limiting disease (end-stage lung, liver, heart or kidney disease, severe neurological disability, extreme frailty, locally advanced or metastatic cancer, advanced-stage AIDS). If the answer to the fi rst question is yes, we proceed to the next one: do you believe this patient will survive to hospital discharge? Answers to those questions were recorded, SAPS III was calculated and all patients were followed until death, discharge or transfer to another center. Diff erences in mortality and SAPS III score between groups were examined using a Student’s t test. Proportions were compared using chi-square test. References Table 1 (abstract P569). Respondents advocating withdrawal for the patient Withdrawal self (%) Withdrawal family (%) Day 3 71 67 Day 7 83 76 Day 28 96 88 Day 42 98 97 Table 1 (abstract P569). Respondents advocating withdrawal for the patient 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. 1. Nelson JE, et al. Crit Care Med. 2013;41:2318-27. 2. Weissman DE, et al. J Palliat Med. 2011;14:17-23. Parents’ return to the hospital after the death of their children: importance of palliative care after death p p G Halal, PL Lago, J Piva, M Halal p g Critical Care 2015, 19(Suppl 1):P572 (doi: 10.1186/cc14652 Introduction To analyze the perception of parents regarding their return to the hospital where their children died to participate in a conversation with doctors and to analyze the feelings of parents about their participation in a study evaluating the care provided in the moments leading up to the death of children. p Withdrawal self (%) Withdrawal family (%) Day 3 16 10 Day 7 23 15 Day 14 25 19 Day 42 42 29 Methods A descriptive exploratory qualitative study. The study sites were the pediatric ICUs of the Hospital São Lucas and Hospital de Clinicas de Porto Alegre. Fifteen parents of children who died in the PICUs studied participated in the study. Data collection occurred in 2010 and was conducted through semistructured interviews. Data were analyzed using thematic content analysis. The research was approved by the research ethics committees of both hospitals. Conclusion Of the ICU physicians who would withdraw care for their patient, the majority would also want the same for themselves. The disparity between decision to continue to treat the patients versus treating self or family increased with increasing length of stay. Reference Results The ability to return to the hospital and talk to medical assistants was considered by parents as a positive and enlightening opportunity. Parents who participated in the study understood this moment as an opportunity to be heard and demonstrated the intention to contribute with their experiences in order to improve care in the hospitals studied. Conclusion We conclude that there is a need to implement measures to provide palliative care to parents after the death of their children. It is necessary to consider the possibility of providing families with follow-up meetings with the multidisciplinary team after the death of children. 1. Korones DN. What would you do if it were your kid? N Engl J Med. 2013;369:1291-3. Making it safe to speak up about futile care: a multiperspective survey on leadership, psychological safety and perceived futile care in the ICU P <0.05 was considered statistically signifi cant. P569 Do intensivists prognosticate patients diff erently from themselves or their loved ones? 1. Piva J, Lago P, Othero J, Garcia PC, Fiori R, Fiori H, et al. Evaluating end of life practices in ten Brazilian paediatric and adult intensive care units. J Med Ethics. 2010;36:344-8. Do intensivists prognosticate patients diff erently from themselves or their loved ones? S Gupta, C Green, R Tiruvoipati, J Botha S Gupta, C Green, R Tiruvoipati, J Botha Peninsula Health, Frankston, Australia Critical Care 2015, 19(Suppl 1):P569 (doi: 10.1186/cc14649) Introduction There is a paucity of data about whether our treatment philosophy is diff erent for our patients as compared with what we would have wanted for ourselves, or while acting as surrogate decision- makers for our loved ones. y gi Results During the period, 191 patients were admitted to the EDICU, from which 151 had complete data and follow-up. A total of 63 patients (41.7%) suff ered from a life-limiting disease and were evaluated as having a high probability of death in 1 year. This group was further divided between 35 patients who in the moment of initial screening were expected to die in this hospital admission and 28 patients who were believed to survive to discharge. Comparison between these two groups showed patients believed to die at this hospital admission had higher SAPS III scores (66.9 vs. 59, P = 0.010) and hospital mortality (48.6% vs. 10.7%, P = 0.001). Methods An anonymous survey was sent to all the members of Australia and New Zealand Intensive Care Society and the College of Intensive Care Medicine (CICM). The fi rst section comprised a hypothetical case scenario spanning over 6 weeks of ICU stay for a patient. At four diff erent stages of the ICU stay, responders were requested to answer multiple- choice questions regarding the philosophy of treatment, based on their perceived prognosis of the patient at that particular time. The following two sections contained the same set of questions with the hypothetical scenario of responders acting as surrogate decision-makers for the patient and that of responders being patients themselves, in the same situation. The responses were compared amongst three sections at each stage using the chi-square test. Conclusion A high percentage of patients admitted to our EDICU have life-limiting disease and might benefi t from palliative care. These patients can be identifi ed using simple screening questions at admission and positive answers to those questions can be associated with worse outcomes. Results A total of 115 responses were received from the fellows of CICM. The results are presented in Tables 1 and 2. P573 How readable are our Patient Information Sheets? L Strachan, M Booth Glasgow Royal Infi rmary, Glasgow, UK Critical Care 2015, 19(Suppl 1):P573 (doi: 10.1186/cc14653) Results Sixty-eight knowledge to care gaps were proposed, rated and revised by the committee over three rounds of review, resulting in 13 priorities for improvement. Then, 1,103 providers (62% response rate) representing nurses, respiratory therapists, allied health professionals and physicians evaluated the priorities and rated nine as necessary. In multivariable logistic regression analyses, provider (profession, experience and teaching status of ICU) and knowledge to care gap characteristics (strength of supporting evidence, potential to benefi t the patient, potential to improve patient/family experience, and potential to decrease costs) were associated with priorities rated as necessary. After disseminating the results to all network members, 627 responded (35% response rate) and indicated that the priorities were reasonable choices for quality improvement initiatives (87%), that they were highly supportive of working on initiatives targeting the priorities (61%) and would be willing to act as local champions for the initiatives (n = 92 individuals). Introduction We often need to obtain consent for clinical studies in the ICU. Participant Information Sheets (PIS) can be diffi cult to understand. A recent French publication [1] supports our hypothesis that PIS have poor readability scores. y Methods Protocols submitted for ethics approval between 2008 and 2009 were obtained with permission from the Scotland A Ethics Research Committee. Ethical approval was not required for this observational study. All header, footers, diagrams and tables were removed. Readability scoring was performed using the Flesch Reading Ease and Flesch–Kincaid (FK) grades. Statistical analysis using Excel and MiniTab was then performed. The readability of these documents was compared with everyday documents – newspaper articles, politicians’ speeches [2] and standard contract agreements. p g Results A total of 104 protocols containing 209 PIS were reviewed. Of these, 99 (47%) were written for patients, 56 (27%) for GPs, 26 (12%) for relatives, 17 (8%) for carers, fi ve (2%) for legal representatives and six (3%) were summary sheets only. Sixty-seven (64%) of these protocols were submitted by academic institutions (for example, university or health boards) and 37 (36%) by pharmaceutical companies. Results are expressed as the median and 25th and 75th percentiles. P573 The word count and number of pages were higher for those PIS submitted by pharmaceutical companies compared with academic institutions: 1,561 (471; 5,167) versus 1,177 (626.5; 1,559.8) with P <0.05 and 4 (2; 10) versus 3 (2; 4) with P <0.05 respectively. The Flesch Reading Ease (63 (56; 69) vs. 60 (52.6; 65.4)) and FK grades (3 (5.4; 7.2) vs. 6.8 (6; 7.6)) were similar for both groups. Further subanalysis demonstrated that PIS designed for GPs had a lower word count, lower Flesch and higher FK grade compared with those for patients – the diff erence in Flesch and FK grade were compared using a Mann–Whitney test and were statistically signifi cant. Conclusion Our research approach engaged a diverse group of stakeholders to identify nine priorities for improving the quality and value of care provided to critically ill patients. This methodology can be used to engage stakeholders and identify priorities for quality improvement in other healthcare systems and domains. Additional work is required to reconcile provider/decision-maker and patient/ family priorities. References 1. Piva J, Lago P, Othero J, Garcia PC, Fiori R, Fiori H, et al. Evaluating end of life practices in ten Brazilian paediatric and adult intensive care units. J Med Ethics. 2010;36:344-8. Introduction A high percentage of patients admitted to ICUs fulfi ll one or more criteria for palliative care. There are currently few comprehensive studies in critical care settings that have set out to 2. Lago PM, Nilson C, Pedro Piva J, Vieira AC, Halal MG, de Carvalho Abib GM, et al. Nurses’ participation in the end-of-life process in two paediatric intensive 2. Lago PM, Nilson C, Pedro Piva J, Vieira AC, Halal MG, de Carvalho Abib GM, et al. Nurses’ participation in the end-of-life process in two paediatric intensive S199 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 care units in Brazil. Int J Palliat Nurs. 2011;17:264, 267-70. 3. Abib El Halal GM, Piva JP, Lago PM, El Halal MG, Cabral FC, Nilson C, et al. Parents’ perspectives on the deaths of their children in two Brazilian paediatric intensive care units. Int J Palliat Nurs. 2013;19:495-502. Methods Using a modifi ed RAND/UCLA Appropriateness Methodology, a committee of 38 providers and decision-makers representing a population-based clinical network of adult (n = 14) and pediatric (n = 2) medical–surgical ICUs in Alberta, Canada (population 4 million) serially proposed, rated and revised potential knowledge to care gaps as priorities for improvement. The priorities developed by the committee were sent to the network’s 1,790 frontline providers to rate their importance. The fi nal list of priorities that were rated as important was disseminated to all network members for feedback. p H Stelfox1, E McKenzie1, S Bagshaw2, M Gill1, P Oxland1, D Boulton1, D Oswell1, M Potestio1, D Niven1 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P575 (doi: 10.1186/cc14655) y gi Conclusion The FK grade is equivalent to US school grade level. The US government advises all policies produced should have a FK grade of <9. Our study suggests that protocols submitted to the ethics committee are easy to read, comparing favourably with broadsheet journalism and standard contract, for example loan contract. However, the average reading age in the UK is 9 years [3], suggesting participants may struggle with the information provided. References Introduction With increasing emphasis on patient and family-centred care, it follows that patients and their family members should be included when priorities for improving care are established. We therefore used a novel methodology that employs former patients and family members as researchers to describe the experiences of critically ill patients and their families with ICUs and to identify opportunities for improvement. journalism and standard contract, for example loan contract. However, the average reading age in the UK is 9 years [3], suggesting participants may struggle with the information provided. References p Methods Using the patient engagement framework developed by Marlett and Emes, we engaged four former patients and family members trained in qualitative research methods to conduct and analyse semistructured focus groups and interviews with adult patients who had recovered from critical illness and family members of both surviving and deceased patients. Participants were recruited from 13 ICUs in Alberta, Canada. Focus groups and interviews were recorded, transcribed and analysed using phenomenological reduction. Data collection continued until thematic saturation was reached. 1. Menoni V. The readability of information and consent forms in cli research in France. PLos One. 2010;5:e10576. Menoni V. The readability of information and consent forms in clinic research in France. PLos One. 2010;5:e10576. 2. http://www.britishpoliticalspeech.org/speech-archive.l 3. Gillies K. Patient information leafl ets for UK randomised controls. Trial. 2014;15:62. Using patient researchers to understand patient and family experiences in ICUs p H Stelfox1, E McKenzie1, S Bagshaw2, M Gill1, P Oxland1, D Boulton1, D Oswell1, M Potestio1, D Niven1 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P575 (doi: 10.1186/cc14655) Survey of visiting hours in critical care units in English trauma centres One hundred per cent were able have questions answered satisfactorily. Linked to the FS-ICU, we have seen marked improvements in decision-making and satisfaction. Methods A telephone survey on visiting times was conducted in 53 adult critical care units in trauma centres in England. A list of trauma centres was obtained from the NHS England website. All critical care units (other than obstetric high dependency units and coronary care units, unless part of a cardiothoracic critical care unit) within each hospital were surveyed. Each respondent was asked about the visiting hours, whether children were allowed to visit and how many visitors to a bed space. Conclusion We have shown progressive improvement over 3  years across all domains. Marked improvement in information provision and decision-making support from 53% to 96% over 3  years since introducing the FWR correlates with the improved overall satisfaction (Figure 1). Interestingly FWR is more helpful than relatives anticipated. The FWR was very well received and our results suggest an unrecognised need is being met. Because this was a pragmatic study, we feel this is a true representation of family satisfaction. It is encouraging that communication, information and decision-making support continue to improve. They have become embedded in the fabric of our critical care practice and lead to marked improvement in satisfaction for families. Reference Results Fifty-three units with between four and 75 beds and covering the whole of England were surveyed: there was a 100% response rate. Visiting hours varied between hospitals and between units within the same hospital. Nine units (17%) had open visiting hours, although most gave advice on times to avoid such as nursing handover. The majority of units (44.83%) operated restricted visiting with a median (range) of 6 (2 to 9) hours. All units allowed a maximum of two visitors to the bedspace. Children were allowed in nine units without restriction, the remaining units advised that it may not be appropriate for children to visit and it was at the discretion of the parents and medical staff . 1. Wall et al. Refi nement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit Care Med. 2007;35:271-9. f Conclusion The majority of adult critical care units in England, including our own, have restricted visiting policies. Reference 1. Berti D, et al. Intensive Care Med. 2007;33:1060-5. 1. Berti D, et al. Intensive Care Med. 2007;33:1060-5. Survey of visiting hours in critical care units in English trauma centres Visiting policies are a source of debate amongst staff in intensive care with concerns about open visiting including increased workload and interruptions to normal routine [1]. This is consistent with the views of staff at our own unit who, in appreciative enquiry, have expressed mixed opinions about extending visiting times. Extending visiting times is only part of a wider project to improve the way relatives experience intensive care whilst ensuring both medical and nursing staff feel supported, creating an environment for optimal communication. P574 Stakeholder engagement to identify priorities for improving the quality and value of care provided to critically ill patients H Stelfox1, D Niven1, S Bagshaw2, E McKenzie1, M Potestio1, F Clement1, D Zygun2 1University of Calgary, AB, Canada; 2University of Alberta, Edmonton, AB, Canada Critical Care 2015, 19(Suppl 1):P574 (doi: 10.1186/cc14654) Results Thirty-two participants including patients (n = 11) and family members (n = 21) participated in fi ve focus groups (n = 23 participants) and eight interviews (n  = 9 participants). Participants articulated themes refl ecting important components of care organised across three phases of the ICU experience; admission to ICU, daily care in ICU and after ICU discharge. Admission to ICU comprised three themes: patient and family transition into ICU, patient and family disorientation upon admission to ICU and preferred staff actions to help patients/ family adapt to the ICU. The daily care phase of ICU consisted of fi ve themes: honouring patient’s voices, needing to know, making decisions, culture in ICU and medical care. The experience after ICU discharge comprised two themes: transition from ICU to a hospital ward and long-term eff ects of critical illness. Participants identifi ed fi ve Critical Care 2015, 19(Suppl 1):P574 (doi: 10.1186/cc14654) Introduction Healthcare systems do not make optimal use of evidence, which results in suboptimal patient care. Large amounts of scientifi c evidence are generated but not implemented into patient care (knowledge to care gap). We sought to identify and prioritize knowledge to care gaps in critical care medicine as opportunities to improve quality and value in care. S200 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Figure 1 (abstract P577). Trends in family satisfaction. priorities for improvement: provide families with a guide/navigator; educate providers about the fragility of family trust; improve provider communication skills; inform patients about the long-term eff ects of critical illness; and develop strategies to facilitate continuity of care between providers. p Conclusion Patients and family members are an untapped resource and engaging them as researchers is a viable strategy to identify opportunities for quality improvement that are patient and family centred. Survey of visiting hours in critical care units in English trauma centres Figure 1 (abstract P577). Trends in family satisfaction. E Taylor, N Bunker E Taylor, N Bunker The Royal London Hospital, London, UK round (FWR), to enhance and standardise communication and improve satisfaction. Following introduction of the FWR we have audited family satisfaction using the validated FS-ICU questionnaire [1]. round (FWR), to enhance and standardise communication and improve satisfaction. Following introduction of the FWR we have audited family satisfaction using the validated FS-ICU questionnaire [1]. Methods This was a prospective study of relatives’ satisfaction for patients completing their critical care episode. The questionnaire was completed anonymously and data collected. This was a pragmatic study, no changes were made to communication strategies. Results There is a high degree of satisfaction across all domains of the FS-ICU including treatment of family and provision of information (Figure 1). One hundred per cent found FWR to be helpful, only 55% had anticipated this. Fifteen per cent changed their perception of critical care. It enabled 15% to raise new concerns. One hundred per cent were able have questions answered satisfactorily. Linked to the FS-ICU, we have seen marked improvements in decision-making and satisfaction. Introduction The purpose of this study was to assess the visiting restrictions placed on families visiting adult patients on critical care units within trauma hospitals in England. Whilst it is well recognised that high-quality care for patients is of paramount importance, we should also be aware that supporting patients’ families off ers long- term benefi ts for patient, family and hospital. In our own unit we are reviewing whether we could adopt a more fl exible attitude to visiting times and assessing how to provide a more welcoming environment to relatives. To inform our own review and in order to develop a best practise approach, we surveyed all of the major trauma centres in England. Methods This was a prospective study of relatives’ satisfaction for patients completing their critical care episode. The questionnaire was completed anonymously and data collected. This was a pragmatic study, no changes were made to communication strategies. Results There is a high degree of satisfaction across all domains of the FS-ICU including treatment of family and provision of information (Figure 1). One hundred per cent found FWR to be helpful, only 55% had anticipated this. Fifteen per cent changed their perception of critical care. It enabled 15% to raise new concerns. 1. Berti D, et al. Intensive Care Med. 2007;33:1060-5. P578 Bereavement care in UK ICUs: a national survey M Berry1, E Brink2, V Metaxa2 1Imperial Healthcare Trust, London, UK; 2King’s College Hospital, London, UK Critical Care 2015, 19(Suppl 1):P578 (doi: 10.1186/cc14658) Introduction For the families of critically ill patients, the death of a loved one in the ICU is often an unexpected and traumatic event, characterised by diffi cult decisions regarding withholding or withdrawing life-sustaining therapy. Increasingly the importance of bereavement care (BC) in the ICU is being acknowledged, although reports continue to highlight the inadequacies around end-of life care in the critical care environment. In 1998, the Intensive Care Society (ICS) published guidelines mapping out BC in the ICU [1]. We aimed to compare BC in ICUs across England against the recommendations set out by the ICS. P577 Evolving outcomes in the ICU: family ward rounds improve satisfaction year on year R Handslip, A Molokhia University Hospital Lewisham, London, UK Critical Care 2015, 19(Suppl 1):P577 (doi: 10.1186/cc14657) y Methods All adult ICUs in England were contacted over a 2-week period, using a standardised questionnaire based on the nine domains identifi ed by the ICS. All answers were collected anonymously using SurveyMonkey®. An 80% compliance rate was deemed acceptable. Results From the 148 ICUs identifi ed, 113 answered the questionnaire (76%). Forty-three per cent of the responders had access to training in BC and in communication skills, and 54% had a named member of staff responsible for training, writing, auditing and developing the y Methods All adult ICUs in England were contacted over a 2-week period, using a standardised questionnaire based on the nine domains identifi ed by the ICS. All answers were collected anonymously using SurveyMonkey®. An 80% compliance rate was deemed acceptable.i R Handslip, A Molokhia Introduction Patient satisfaction is a crucial part of clinical care and there is now increasing recognition of the importance of family involvement and satisfaction in the provision of care for the critically ill. Since 2012 our unit has introduced a consultant-led family ward Results From the 148 ICUs identifi ed, 113 answered the questionnaire (76%). Forty-three per cent of the responders had access to training in BC and in communication skills, and 54% had a named member of staff responsible for training, writing, auditing and developing the S201 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Critical Care 2015, Volume 19 Suppl 1 http://ccforum.com/supplements/19/S1 Conclusion This is the fi rst national audit of BC in the ICU since the initial ICS guideline publication. Even though most ICUs provided relatives with information around the time of death, training, auditing and adequate facilities do not meet the recommended standards. The lack of adherence is defi nitely multifactorial and requires further research. In the meantime, vigorous implementation of these guidelines is warranted in order to ensure optimal care for the bereaved families. Reference BC policy. When asked about the presence of a written BC policy only 45% responded positively, and even less (19%) had provisions for audit and development of the service. . Intensive Care Society (1998). http://www.ics.ac.uk/ics-homepage/ guidelines-and-standards/. P577 Information to staff about cultural and religious rites around the time of death, and to relatives on what to do after a death was available in 81% and 96% respectively. The general practitioner was informed of the deaths taking place in the ICU in 77% of the cases. In more than 70% of the participating ICUs, eff orts were made to ensure privacy of the grieving relatives and to have dedicated follow-up facilities for the bereaved. Even though staff support programmes were recognised as paramount, only 54% of the ICUs had formal ones set up.
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Fibrin clot properties independently predict adverse clinical outcome following acute coronary syndrome: a PLATO substudy
European heart journal
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Aims Acute coronary syndrome • Fibrin clot • Lysis time • Biomarker * Corresponding author. Tel: þ44 114 226 9133, Fax: þ44 114 226 6159, Email: r.f.storey@sheffield.ac.uk V C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Cardiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. European Heart Journal (2018) 39, 1078–1085 doi:10.1093/eurheartj/ehy013 European Heart Journal (2018) 39, 1078–1085 doi:10.1093/eurheartj/ehy013 CLINICAL RESEARCH European Heart Journal (2018) 39, 1078–1085 doi:10.1093/eurheartj/ehy013 Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 Wael Sumaya1, Lars Wallentin2,3, Stefan K. James2,3, Agneta Siegbahn3,4, Katja Gabrysch3, Maria Bertilsson3, Anders Himmelmann5, Ramzi A. Ajjan6, and Robert F. Storey1* Wael Sumaya1, Lars Wallentin2,3, Stefan K. James2,3, Agneta Siegbahn3,4, Katja Gabrysch3, Maria Bertilsson3, Anders Himmelmann5, Ramzi A. Ajjan6, and Robert F. Storey1* 1Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK; 2Department of Medical Sciences, Cardiology, Uppsala University, Uppsala, Sweden; 3Uppsala Clinical Research Center, Uppsala University, Dag Hammarskjo¨lds va¨g 38, SE-752 37 Uppsala, Sweden; 4Department of Medical Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden; 5AstraZeneca Research and Development, Gothenburg, Sweden; and 6Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK Received 6 September 2017; revised 27 October 2017; editorial decision 9 January 2018; accepted 10 January 2018; online publish-ahead-of-print 29 January 2018 See page 1086 for the editorial comment on this article (doi: 10.1093/eurheartj/ehy118) Aims To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syn- drome (ACS). ................................................................................................................................................................................................... Methods and results Plasma samples were collected at hospital discharge from 4354 ACS patients randomized to clopidogrel or ticagre- lor in the PLATelet inhibition and patient Outcomes (PLATO) trial. A validated turbidimetric assay was employed to study plasma clot lysis time and maximum turbidity (a measure of clot density). One-year rates of cardiovascular (CV) death, spontaneous myocardial infarction (MI) and PLATO-defined major bleeding events were assessed after sample collection. Hazard ratios (HRs) were estimated using Cox proportional hazards models. After adjusting for CV risk factors, each 50% increase in lysis time was associated with CV death/spontaneous MI [HR 1.17, 95% confi- dence interval (CI) 1.05–1.31; P < 0.01] and CV death alone (HR 1.36, 95% CI 1.17–1.59; P < 0.001). Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death (HR 1.24, 95% CI 1.03–1.50; P = 0.024). After adjustment for other prognostic biomarkers (leukocyte count, high-sensitivity C-reactive protein, high-sensitivity troponin T, cystatin C, N-terminal pro B-type natriuretic peptide, and growth differentiation factor- 15), the association with CV death remained significant for lysis time (HR 1.2, 95% CI 1.01–1.42; P = 0.042) but not for maximum turbidity. These associations were consistent regardless of randomized antiplatelet treatment (all interaction P > 0.05). Neither lysis time nor maximum turbidity was associated with major bleeding events. ................................................................................................................................................................................................... Conclusion Fibrin clots that are resistant to lysis independently predict adverse outcome in ACS patients. Novel therapies tar- geting fibrin clot properties might be a new avenue for improving prognosis in patients with ACS. Wael Sumaya1, Lars Wallentin2,3, Stefan K. James2,3, Agneta Siegbahn3,4, Katja Gabrysch3, Maria Bertilsson3, Anders Himmelmann5, Ramzi A. Ajjan6, and Robert F. Storey1* Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death (HR 1.24, 95% CI 1.03–1.50; P = 0.024). After adjustment for other prognostic biomarkers (leukocyte count, high-sensitivity C-reactive protein, high-sensitivity troponin T, cystatin C, N-terminal pro B-type natriuretic peptide, and growth differentiation factor- 15), the association with CV death remained significant for lysis time (HR 1.2, 95% CI 1.01–1.42; P = 0.042) but not for maximum turbidity. These associations were consistent regardless of randomized antiplatelet treatment (all interaction P > 0.05). Neither lysis time nor maximum turbidity was associated with major bleeding events. ................................................................................................................................................................................................... Conclusion Fibrin clots that are resistant to lysis independently predict adverse outcome in ACS patients. Novel therapies tar- geting fibrin clot properties might be a new avenue for improving prognosis in patients with ACS.                                                                                                                                                                                                                     Keywords Acute coronary syndrome • Fibrin clot • Lysis time • Biomarker To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syn- drome (ACS). To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syn- Wael Sumaya1, Lars Wallentin2,3, Stefan K. James2,3, Agneta Siegbahn3,4, Katja Gabrysch3, Maria Bertilsson3, Anders Himmelmann5, Ramzi A. Ajjan6, and Robert F. Storey1*                                                                                                                                                                                                                     Keywords Acute coronary syndrome • Fibrin clot • Lysis time • Biomarker To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syn- drome (ACS). To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syn- Aims To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syn- drome (ACS). ................................................................................................................................................................................................... Methods and results Plasma samples were collected at hospital discharge from 4354 ACS patients randomized to clopidogrel or ticagre- lor in the PLATelet inhibition and patient Outcomes (PLATO) trial. A validated turbidimetric assay was employed to study plasma clot lysis time and maximum turbidity (a measure of clot density). One-year rates of cardiovascular (CV) death, spontaneous myocardial infarction (MI) and PLATO-defined major bleeding events were assessed after sample collection. Hazard ratios (HRs) were estimated using Cox proportional hazards models. After adjusting for CV risk factors, each 50% increase in lysis time was associated with CV death/spontaneous MI [HR 1.17, 95% confi- dence interval (CI) 1.05–1.31; P < 0.01] and CV death alone (HR 1.36, 95% CI 1.17–1.59; P < 0.001). Acute coronary syndrome • Fibrin clot • Lysis time • Biomarker Statistical methods Biomarker levels were natural log-transformed before analysis. Baseline patient characteristics, medical history, and biomarkers were compared across quartile groups of each of the fibrin variables. Continuous data are presented as medians and interquartile ranges and compared using Kruskal–Wallis tests. Categorical data are presented as numbers and per- centages and compared using v2 tests. The primary outcome of interest was the composite of CV death and spontaneous myocardial infarction (MI). Secondary outcomes were CV death alone, spontaneous MI alone, stroke, all-cause mortality, definite or probable stent thrombosis accord- ing to Academic Research Consortium criteria, PLATO-defined major bleeding, and PLATO-defined bleeding unrelated to coronary artery bypass graft surgery (CABG) (see Supplementary material online for bleeding definitions). Kaplan–Meier curves were derived to compare event rates across the four quartile groups of each of the fibrin clot varia- bles. Cox-proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Hazard ratios are expressed per 50% increase in fibrin variable level when assessed as con- tinuous variables or compared to the lowest quartile of the fibrin variable when assessed as categorical variables. Two models were used for adjust- ment. Model 1 included randomized treatment, age, gender, body mass index, smoking history, hypertension, dyslipidaemia, DM, CKD, ST- elevation ACS and previous MI, congestive heart failure (CHF), revascu- larisation, ischaemic stroke, or PAD.25–27 Model 2 included all variables in Model 1 (excluding CKD) and the following inflammatory and prognostic biomarkers: CRP, white blood cell count, cystatin C, NT-proBNP, tropo- nin T, and GDF-15. The assumptions of proportional hazards were assessed visually by calculating Schoenfeld residuals. To assess the prog- nostic value of fibrin clot properties, Harrell’s C-index was estimated and compared to a clinical predictive model (Model 1) without the addition of fibrin clot variables using likelihood ratio tests. The efficacy and safety of ticagrelor compared with clopidogrel according to fibrin clot proper- ties was assessed using a Cox proportional hazards model that included randomized treatment, continuous fibrin variable level using restricted cubic splines, and randomized treatment by fibrin variable interaction. The effect of fibrin clot properties on clinical outcome in relation to pre- sentation was also assessed using a Cox proportional hazards model that included presentation, continuous fibrin variable level using restricted cubic splines, and presentation-by-fibrin-variable interaction. P-values <0.05 from two-tailed tests were considered statistically significant. Statistical methods Due to the exploratory nature of this study, P-values were not adjusted for The majority of previous studies in this area used a cross-sectional retrospective design and large-scale longitudinal studies are lacking. We, therefore, aimed to study fibrin clot properties in plasma sam- ples collected from ACS patients at hospital discharge and explore the relationship between those characteristics and subsequent clini- cal outcomes. Introduction low-dose anticoagulant therapy (rivaroxaban), have all resulted in improved outcomes but increased the risk of major bleeding events.1–3 Adverse events, including cardiovascular (CV) death, remain com- mon following acute coronary syndrome (ACS). Intensive antithrom- botic therapies, including potent P2Y12 inhibitors and the addition of There is marked overlap between risk factors for ischaemic and bleeding events.4,5 Consequently, tailoring therapy to achieve the ed from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 rsity of Leeds Library user ly 2018 1079 Adverse fibrin clot and clinical outcome in ACS ‘sweet spot’ of mitigating ischaemia whilst maintaining effective hae- mostasis is an ongoing challenge and readily available biomarkers to aid the decision process are lacking. Spontaneous major bleeding events are associated with a similar prognosis to ischaemic events6 and, in patients undergoing percutaneous coronary intervention (PCI), major bleeding events independently predict major CV events.7 activator 83ng/mL, calcium chloride 7.5 mM, and thrombin 0.03U/mL; final concentrations). After shaking for 2 s, plates were read at 340 nm every 12s at 37C until lysis in all samples was achieved. Quality control samples were included in all plates. This method has previously been vali- dated.12,22–24 Studied variables included lysis time (time taken for turbid- ity to drop by 50% from maximum as a measure of lysis potential) and maximum turbidity (turbidity refers to the scattering of light as a measure of fibrin clot density). The co-efficient of variation was 8.3% for lysis time and 3.8% for maximum turbidity. All analyses were performed blinded to clinical outcome and clinical characteristics. Following ACS, treatment includes aspirin and a P2Y12 inhibitor.8,9 Although contemporary therapy effectively targets platelets,10,11 around 20% of patients suffer recurrent events within 12months.5 This dual antiplatelet treatment strategy largely spares the protein arm of coagulation, which leads to fibrin formation. Other biochemical analyses Plasma samples obtained at randomization were used to determine other biomarker levels, as previously reported.21,25–27 Briefly, N-terminal pro B-type natriuretic peptide (NT-proBNP), high-sensitivity troponin T, cys- tatin C, C-reactive protein (CRP), and growth differentiation factor-15 (GDF-15) were measured using sandwich immunoassays. Differential blood count was determined on EDTA-anticoagulated blood samples at randomization. Patients with thrombotic conditions demonstrate unfavourable fibrin clot structure.12–14 High-risk conditions, such as diabetes melli- tus (DM), chronic kidney disease (CKD), and peripheral artery dis- ease (PAD), have all shown associations with compact fibrin clots and resistance to fibrinolysis.15–17 Factors contributing to this pheno- type can include levels of clotting and lysis proteins, disease-specific post-translational changes to fibrinogen, and genetic determi- nants.18,19 Assessment of fibrin clot offers a functional evaluation of the impact of all these factors on the protein arm of coagulation. Study population and patient samples Study population and patient samples y p p p p The PLATelet inhibition and patient Outcomes (PLATO) trial was an international multi-centre, double-blind, randomized controlled trial of ticagrelor compared with clopidogrel in 18 624 moderate- to high-risk ACS patients. The study design and results have previously been pub- lished.1,20 Baseline patient characteristics, including medical and medica- tion history, were recorded at baseline. Study visits were performed at 1, 3, 6, 9, and 12months. In the PLATO biomarker sub-study,21 a subset of 4354 patients provided blood at hospital discharge, which was used for this analysis in order to avoid the effects of anticoagulant therapy used as part of the initial ACS management. Plasma was obtained from citrate- anticoagulated venous blood samples and stored initially at –20C prior to transfer to Uppsala Clinical Research Centre for storage at –80C. All study patients provided written informed consent according to a proto- col approved by local research ethics committees at participating centres. For our analyses, frozen plasma samples were transferred to the University of Sheffield and stored at –80C until analysis. .................................. Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 biomarkers A total of 4354 patients were included in this study. Table 1 and Supplementary material online, Table S1 summarise the clinical char- acteristics and biomarkers across the quartile groups of both lysis time and maximum turbidity. Neither lysis time nor maximum turbidity appeared to be affected by haemoglobin or haematocrit. Maximum turbidity and lysis time had a modest correlation (Spearman correlation co-efficient 0.375, P < 0.001). The prevalence of DM, hypertension, CKD, and female sex signifi- cantly increased with increasing lysis time quartile group. Although the differences were not significant, the highest quartile group of lysis time appeared to have the highest prevalence of PAD and CHF. Fibrin clot assessment Human thrombin was obtained from Merck Biosciences, recombinant tis- sue plasminogen activator from Technoclone, calcium chloride dehydrate and Tris from Fisher Scientific, and sodium chloride from Sigma Aldrich. High-throughput turbidimetric analysis was performed in flat-bottomed, polystyrene 96-well plates (Greiner) using a dedicated Multiskan FC (Thermo Scientific) plate reader. Permeation buffer solution (100mM sodium chloride, 50mM Tris, and pH 7.4) was used for dilution. Twenty five lL aliquots of plasma (in duplicates) were mixed with 75lL lysis mix and clots were formed by adding 50lL activation mix (tissue plasminogen 1080 W. Sumaya et al. ................................................................ increasing quartile group, the levels of troponin T, NT-proBNP, CRP, and white blood cell count increased. Growth differentiation factor- 15 and cystatin-C levels were also highest in the highest quartile group. multiple testing. All statistical analyses were performed at Uppsala Clinical Research Centre using R statistics software (Version 3.3.2; R Foundation for Statistical Computing, Vienna, Austria). Similar to lysis time, the prevalence of DM was highest in the high- est quartile group of maximum turbidity. There was a higher percent- age of female patients in the lowest maximum turbidity quartile group. The relationship between maximum turbidity and the other biomarkers was also pronounced (see Supplementary material online, Table S1). Fibrin clot properties and clinical outcome During follow-up, 125 (2.9%) patients had CV death, 183 (4.2%) patients had spontaneous MI, and 41 (0.94%) patients had stroke. There were 145 all-cause deaths (3.3%). Amongst those treated with PCI (4335), 38 (0.88%) had definite or probable stent thrombosis. Patients in the shortest lysis time quartile group tended to be older but the absolute difference in mean age was small. Strong associations were also observed between lysis time and other biomarkers: with ....................................................................................................................................... .................................................................................................................................................................................................................... variables). CHF, congestive heart failure; CKD, chronic kidney disease; CRP, C-reactive protein; GDF, growth differentiation factor; MI, myocardial infarction; NT-proBNP, N-terminal pro b-type natriuretic peptide; PAD, peripheral artery disease; STE-ACS, ST-elevation acute coronary syndrome. Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 Values are medians (IQRs) for continuous data and n (%) for categorical data. P-values calculated using v2 test (categorical variables) or Kruskal–Wallis test (continuous variables). CHF, congestive heart failure; CKD, chronic kidney disease; CRP, C-reactive protein; GDF, growth differentiation factor; MI, myocardial infarction; NT-proBNP, N-terminal pro Values are medians (IQRs) for continuous data and n (%) for categorical data. P-values calculated using v2 test (categorical variables) or Kruskal–Wallis test (continuous variables). Values are medians (IQRs) for continuous data and n (%) for categorical data. P-values calculated using v2 test (categorical variables) variables). or continuous data and n (%) for categorical data. P-values calculated using v2 test (categorical variables) or Kruskal–Wallis test (continu Fibrin clot properties and clinical outcome Table 1 Baseline clinical characteristics and biomarkers across lysis time quartile groups Variables Lysis time (s) quartile group P-value Q1 (<564; n 5 1098) Q2 (564–696; n 5 1108) Q3 (696–888; n 5 1066) Q4 (>888; n 5 1082) Demographics and medical history Age (years) 63 (55–72) 61 (54–70) 61 (53–70) 61 (53–70) <0.001 Female 242 (22.0) 318 (28.7) 271 (25.4) 442 (40.9) <0.001 Body mass index (kg/m2) 27.0 (24.5–29.7) 27.3 (24.9–30.1) 27.8 (25.3–30.8) 28.6 (25.6–31.8) <0.001 Current smoker 392 (35.7) 450 (40.6) 404 (37.9) 349 (32.3) <0.001 Hypertension 673 (61.3) 717 (64.7) 711 (66.7) 764 (70.6) <0.001 Hyperlipidaemia 453 (41.3) 462 (41.7) 456 (42.8) 469 (43.3) 0.744 Diabetes mellitus 206 (18.8) 221 (19.9) 242 (22.7) 305 (28.2) <0.001 Previous MI 216 (19.7) 215 (19.4) 214 (20.1) 201 (18.6) 0.843 Previous CHF 53 (4.8) 59 (5.3) 58 (5.4) 79 (7.3) 0.068 Previous stroke 34 (3.1) 42 (3.8) 35 (3.3) 40 (3.7) 0.783 PAD 62 (5.6) 68 (6.1) 63 (5.9) 80 (7.4) 0.345 CKD 29 (2.6) 42 (3.8) 27 (2.5) 49 (4.5) 0.028 Type of ACS STE-ACS 504 (45.9) 522 (47.1) 510 (47.8) 486 (44.9) 0.536 Biomarkers Troponin T (ng/L) 129 (35–453) 151 (37–511) 177 (46–582) 210 (47–755) <0.001 NT-proBNP (pmol/L) 387 (119–992) 386 (129–1088) 389 (131–1033) 469 (139–1433) 0.009 Cystatin C (mg/L) 0.80 (0.65–0.97) 0.80 (0.66–0.96) 0.81 (0.66–0.97) 0.86 (0.69–1.05) <0.001 GDF-15 (ng/L) 1503 (1095–2058) 1446 (1108–2092) 1509 (1182–2064) 1584 (1150–2254) 0.003 CRP (mg/L) 2.4 (1.1–5.8) 3.1 (1.4–7.6) 3.9 (1.8–9.8) 5.2 (2.2–13.5) <0.001 White cell count (109/L) 8.7 (6.9–10.9) 9.1 (7.3–11.4) 9.7 (7.8–11.9) 9.9 (7.8–12.5) <0.001 Haemoglobin (g/L) 141 (131–151) 142 (132–151) 142 (132–152) 141 (130–151) 0.144 Haematocrit (L/L) 0.41 (0.39–0.44) 0.42 (0.39–0.44) 0.42 (0.39–0.45) 0.42 (0.38–0.44) 0.265 V l di (IQR ) f ti d t d (%) f t i l d t P l l l t d i 2 t t ( t i l i bl ) K k l W lli t t ( ti Table 1 Baseline clinical characteristics and biomarkers across lysis time quartile groups Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 1081 Adverse fibrin clot and clinical outcome in ACS ......................... PLATO-defined major bleeding occurred in 256 (5.9%) patients with 96 (2.2%) patients having non-CABG-related major bleeding. increased risk of CV death/spontaneous MI (HR 1.48, 95% CI 1.06– 2.06; P = 0.027) and CV death alone (HR 1.92, 95% CI 1.19–3.1; P < 0.001). Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 Fibrin clot properties and clinical outcome As a continuous variable, each 50% increase in lysis time was associated with increased risk of CV death/spontaneous MI (HR 1.17, 95% CI 1.05–1.31; P = 0.006) and CV death alone (HR 1.36, 95% CI 1.17–1.59; P < 0.001). This association remained significant for lysis time after adjustment for inflammatory and prognostic biomarkers (see Supplementary material online, Table S2). Similarly, each 50% Rates of the composite outcome of CV death and spontaneous MI were higher in the highest quartile groups of both lysis time and maxi- mum turbidity compared with rates in the lowest quartile groups (Figure 1). This was primarily driven by higher rates of CV death in the highest quartile groups (Figure 2). After adjustment for CV risk factors (Model 1), the highest quartile group of lysis time was associated with Figure 1 Relationship between fibrin clot parameters and 1-year cumulative event rate of cardiovascular death or spontaneous myocardial infarc- tion. The Kaplan–Meier curves for cumulative event rate of the combined outcome of cardiovascular death or spontaneous myocardial infarction per quartile group of lysis time (A) and maximum turbidity (B). Figure 1 Relationship between fibrin clot parameters and 1-year cumulative event rate of cardiovascular death or spontaneous myocardial infarc- tion. The Kaplan–Meier curves for cumulative event rate of the combined outcome of cardiovascular death or spontaneous myocardial infarction per quartile group of lysis time (A) and maximum turbidity (B). Figure 1 Relationship between fibrin clot parameters and 1-year cumulative event rate of cardiovascular death or spontaneous myocardial infarc- tion. The Kaplan–Meier curves for cumulative event rate of the combined outcome of cardiovascular death or spontaneous myocardial infarction per quartile group of lysis time (A) and maximum turbidity (B). Figure 2 Relationship between fibrin clot parameters and 1-year cumulative event rate of cardiovascular death. The Kaplan–Meier curves for 1-year cumulative event rate of cardiovascular death per quartile group of lysis time (A) and maximum turbidity (B). Figure 2 Relationship between fibrin clot parameters and 1-year cumulative event rate of cardiovascular death. The Kaplan–Meier curves for 1-year cumulative event rate of cardiovascular death per quartile group of lysis time (A) and maximum turbidity (B). Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 1082 W. Sumaya et al. Fibrin clot properties and clinical outcome increase in maximum turbidity was associated with CV death alone (HR 1.24, 95% CI 1.03–1.50; P = 0.024) but this association was no longer significant after adjustment for inflammatory and prognostic biomarkers (see Supplementary material online, Table S3). Findings for all-cause mortality were similar to those for CV death (see Supplementary material online, Tables S2 and S3). clot properties on the association between randomized treatment and major bleeding (Figure 3). Similarly, the association between fibrin clot properties and CV death was present irrespective of subtype of ACS presentation (all interaction P > 0.05). A subset of patients received low-molecular weight heparin on either the day of sampling or the day before (see Supplementary material online, Tables S4 and S5). This treatment did not affect the prognostic value of fibrin clot parameters (all interaction P > 0.25). There was no clear association with rates of stent thrombosis and stroke but event rates were low (data not shown). Neither lysis time nor maximum turbidity was able to predict major bleeding events (see Supplementary material online, Tables S2 and S3). Further char- acterisation of bleeding events is provided in the Supplementary mate- rial online, Tables S6 to S8. Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 Incremental prognostic value of lysis time Model performance to predict the composite outcome of CV death/ sMI significantly improved when lysis time was added to a clinical pre- dictive model: C-index 0.67 (0.637–0.703) for Model 1 þ lysis time vs. 0.665 (0.631–0.698) for Model 1 only, P = 0.007. Prediction of CV There was no significant impact of fibrin clot properties on the CV mortality reduction with ticagrelor compared with clopidogrel (inter- action P > 0.7) (Figure 3). There was also no significant impact of fibrin re 3 Relationship between fibrin clot parameters and 1-year rates of cardiovascular death or major bleeding according to randomized treat- group. One-year rates of cardiovascular death (A and B) or major bleeding (C and D) in relation to maximum turbidity (A and C) or lysis time D), transformed using restricted cubic splines, according to randomized treatment with clopidogrel (C, pink lines) or ticagrelor (T, blue lines). d areas represent 95% confidence intervals. Vertical lines indicate quartiles. Figure 3 Relationship between fibrin clot parameters and 1-year rates of cardiovascular death or major bleeding according to randomized treat- ment group. One-year rates of cardiovascular death (A and B) or major bleeding (C and D) in relation to maximum turbidity (A and C) or lysis time (B and D), transformed using restricted cubic splines, according to randomized treatment with clopidogrel (C, pink lines) or ticagrelor (T, blue lines). Shaded areas represent 95% confidence intervals. Vertical lines indicate quartiles. 1083 Adverse fibrin clot and clinical outcome in ACS death also significantly improved: C-index 0.7 (0.649–0.75) for Model 1 þ lysis time vs. 0.69 (0.642–0.741) for Model 1 only, P < 0.001. death also significantly improved: C-index 0.7 (0.649–0.75) for Model 1 þ lysis time vs. 0.69 (0.642–0.741) for Model 1 only, P < 0.001. approach that might have less impact on haemostasis, particularly if the aim was to normalise lysis potential.18 Studying fibrin clot properties is attractive for many reasons. First, fibrinogen conversion and cross-linking of fibrin fibres to form a sta- ble network is a key step in the formation of an obstructive vascular thrombus. Second, thrombotic occlusion of coronary arteries could represent a failure of the protective endogenous thrombolytic mech- anisms to lyse clots before they become occlusive. Incremental prognostic value of lysis time Third, this assay provides a functional and simple assessment of the complex interac- tions between different clotting/lysis factors and other plasma pro- teins and takes into account both quantitative and qualitative changes in coagulation factors that may affect fibrinolytic efficiency.18,28 Fourth, this is a relatively cheap, easy and reproducible test to per- form and, importantly, fibrin clots that resist lysis might give us mech- anistic insights into recurrent events. Discussion We have shown, in a large population of ACS patients treated with contemporary therapies and followed up for up to 1 year, that fibrin clot properties independently predict the risk of spontaneous MI and CV death following initial in-hospital management. Importantly, lysis time predicted worse outcome after adjusting for other established or new prognostic biomarkers, thus indicating the potential for a fur- ther biomarker that provides insight into prognosis following ACS. The association between lysis time and the levels of the established biomarkers also raises the possibility that variability in fibrin clot properties might contribute to the association of these other bio- markers with thrombotic events (Take home figure). Given that antith- rombotic therapy is mainly centred around antiplatelet agents following the acute phase, our data support the hypothesis that at least a subgroup of patients might benefit from additional therapy that aims at improving lysis potential. For example, further work could explore whether anticoagulant therapy, in combination with a platelet P2Y12 receptor antagonist, offers an advantage over dual anti- platelet therapy in those with adverse fibrin clot properties. Other novel therapies that specifically target proteins implicated in impaired lysis, such as complement C3 or plasmin inhibitor, is another Previous studies, using different assays in whole blood, have shown a positive association between prolonged lysis and CV death.29,30 Another study using thromboelastography in plasma showed similar results.31 However, these were small studies with limited event rates and assessment of clotting and lysis in whole blood cannot reliably dif- ferentiate between the cellular and protein components of thrombus formation. The relationships between adverse fibrin clot dynamics and some clinical characteristics demonstrated in our work are consistent with evidence obtained from smaller observational, cross-sectional Take home Figure High-risk conditions are associated with increased thrombotic potential through a variety of mechanisms Dual antiplatele Take home Figure High-risk conditions are associated with increased thrombotic potential through a variety of mechanisms. Dual antiplatelet therapy successfully targets platelet reactivity. However, hypofibrinolysis remains unaffected and constitutes a potential therapeutic target. LDL-C, low-density lipoprotein cholesterol; PAI-1, plasminogen activation inhibitor-1; TAFI, thrombin activatable fibrinolysis inhibitor. Take home Figure High-risk conditions are associated with increased thrombotic potential through a variety of mechanisms. Dual antiplatelet therapy successfully targets platelet reactivity. However, hypofibrinolysis remains unaffected and constitutes a potential therapeutic target. LDL-C, low-density lipoprotein cholesterol; PAI-1, plasminogen activation inhibitor-1; TAFI, thrombin activatable fibrinolysis inhibitor. Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 Funding This analysis has been supported by the British Heart Foundation (FS/15/ 82/31824 clinical research training fellowship for W.S.). The PLATO trial, including plasma sample storage, was funded and supported by AstraZeneca. The relationship between maximum turbidity and worse out- comes lost significance after adjusting for other biomarkers. Fibrinogen levels have a greater effect on maximum turbidity com- pared with lysis time24,37 (see also Supplementary material online, Figure S5) and fibrinogen levels go hand-in-hand with inflammatory markers, particularly CRP.38 Conflict of interest: L.W.: Institutional Research Grants; AstraZeneca, Bristol-Myers Squibb/Pfizer, GlaxoSmithKline, Roche Diagnostics, Boehringer Ingelheim, Merck & Co, Roche Diagnostics. Consultant: AstraZeneca, Abbott, Bristol-Myers Squibb/Pfizer, GlaxoSmithKline, Boehringer Ingelheim. Travel support: AstraZeneca, Bristol-Myers Squibb/Pfizer, GlaxoSmithKline, Boehringer Ingelheim. S.K.J.: Institutional Research Grants; AstraZeneca, Medtronic, The Medicines Company. Consultant; AstraZeneca, Medtronic, Janssen, Bayer. Honoraria; AstraZeneca, The Medicines Company. A.S.: Institutional Research Grants; AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb/Pfizer, GlaxoSmithKline, Roche Diagnostics. K.G. and M.B.: Institutional Research Grant; AstraZeneca. A.H.: Employment; AstraZeneca. R.A.A.: Institutional Research Grants; Abbott, Bayer, Eli Lilly, NovoNordisk, Roche, Takeda. Honoraria/education support/Consultant; Abbott, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, GlaxoSmithKline, Merck Sharp & Dohme, NovoNordisk, Takeda. R.F.S.: Research Grants; AstraZeneca, PlaqueTec. Honoraria; AstraZeneca. Consultant/Advisory Board; AstraZeneca, Actelion, Avacta, Bayer, Bristol Myers Squibb/Pfizer, Idorsia, Novartis, The Medicines Company. All other authors nothing to disclose. Interestingly, fibrin clot density and resistance to lysis increased with increasing levels of NT-proBNP and troponin T. The exact molecular mechanism for this association is difficult to ascertain. However, higher troponin T and NT-proBNP reflect larger infarcts and these are associated with a greater inflammatory response, which might account for some of the prothrombotic changes. NT-proBNP has been shown to add prognostic value regardless of the degree of necrosis after ACS.39 Our findings, therefore, point to an additional mechanism, beyond the increased risks of death from heart failure and arrhythmia associated with left ventricular systolic dysfunction, whereby NT-proBNP is associated with worse outcome, as a conse- quence of more dense fibrin clots that resist lysis leading to increased risk of atherothrombosis. Turbidimetric analysis of fibrin clots requires trained laboratory per- sonnel, and therefore, is not suitable as a bedside test. Similar to other clotting assays, results might be influenced by high-level anticoagulant therapy and significant liver conditions, which make results difficult to interpret in those scenarios. A limitation to this study is that it only pro- vides a ‘snapshot’ assessment of fibrin clot characteristics at hospital discharge (median 6days). Discussion Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 1084 W. Sumaya et al. ......................................................................................... ........................................................................ studies. For example, DM, CKD, and PAD, all high-risk conditions for cardiac ischaemia, have shown associations with adverse fibrin clot characteristics.15–17 properties might be a new avenue for improving clinical outcomes in patients with ACS. properties might be a new avenue for improving clinical outcomes in patients with ACS. Available biomarkers have added little incremental prognostic value beyond a clinical predictive model and therefore their clinical use has been limited in secondary prevention of CV disease.32 Although the added prognostic value of lysis time is also modest, identifying patients with adverse fibrin clot properties might help direct therapy beyond the current approach of antiplatelet therapy in these patients. References 1. Wallentin L, Becker RC, Budaj A, Cannon CP, Emanuelsson H, Held C, Horrow J, Husted S, James S, Katus H, Mahaffey KW, Scirica BM, Skene A, Steg PG, Storey RF, Harrington RA; PLATO Investigators, Freij A, Thorsen M. Ticagrelor versus clopidogrel patients with acute coronary syndromes. N Engl J Med 2009; 361:1045–1057. 2. Wiviott SD, Braunwald E, McCabe CH, Montalescot G, Ruzyllo W, Gottlieb S, Neumann FJ, Ardissino D, De Servi S, Murphy SA, Riesmeyer J, Weerakkody G, Gibson CM, Antman EM; TRITON-TIMI 38 Investigators. 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Funding It is established that internal fibrinolytic activity has a circadian rhythm largely driven by variations in plasmino- gen activator inhibitor—1 activity.40 Unfortunately sampling times are not available in our database but samples were collected during office working hours. The clear relationship with DM and biomarker levels, which were all measured at baseline, reassure us that the influence of circadian variation is likely to be marginal. Future analyses will seek to assess the stability of this phenotype over time and how the relation- ship with clinical outcome could change in a stable patient cohort. Acknowledgements We are grateful to Ebba Bergman, PhD, Uppsala Clinical Research Centre, for publication management support and to Birgitta Ho¨gberg, Uppsala Clinical Research Centre, for support with provision of the plasma samples. 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Leander K, Blomback M, Wallen H, He S. Impaired fibrinolytic capacity and increased fibrin formation associate with myocardial infarction. Thromb Haemost 2012;107:1092–1099. 14. Undas A, Zalewski J, Krochin M, Siudak Z, Sadowski M, Pregowski J, Dudek D, Janion M, Witkowski A, Zmudka K. Altered plasma fibrin clot properties are associated with in-stent thrombosis. Arterioscler Thromb Vasc Biol 2010;30: 276–282. 33. Thomas MR, Outteridge SN, Ajjan RA, Phoenix F, Sangha GK, Faulkner RE, Ecob R, Judge HM, Khan H, West LE, Dockrell DH, Sabroe I, Storey RF. Platelet P2Y12 inhibitors reduce systemic inflammation and its prothrombotic effects in an experimental human model. Arterioscler Thromb Vasc Biol 2015;35:2562–2570. 15. Scott DJ, Prasad P, Philippou H, Rashid ST, Sohrabi S, Whalley D, Kordowicz A, Tang Q, West RM, Johnson A, Woods J, Ajjan RA, Ariens RA. Clot architecture is altered in abdominal aortic aneurysms and correlates with aneurysm size. Arterioscler Thromb Vasc Biol 2011;31:3004–3010. 34. Undas A, Plicner D, Ste˛pien E, Drwiła R, Sadowski J. Altered fibrin clot structure in patients with advanced coronary artery disease: a role of C-reactive protein, lipoprotein(a) and homocysteine. Downloaded from https://academic.oup.com/eurheartj/article-abstract/39/13/1078/4829678 by University of Leeds Library user on 10 July 2018 Conclusions Despite strong relationships with clinical risk factors, particularly DM, and inflammatory and other prognostic biomarkers, the resistance of fibrin clots to lysis independently predicts CV death following ACS. These findings suggest that novel therapies targeting fibrin clot 5. Jernberg T, Hasvold P, Henriksson M, Hjelm H, Thuresson M, Janzon M. Cardiovascular risk in post-myocardial infarction patients: nationwide real world data demonstrate the importance of a long-term perspective. Eur Heart J 2015; 36:1163–1170. 1085 Adverse fibrin clot and clinical outcome in ACS 40. 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Recovering complete plant root system architectures from soil via X-ray μ-Computed Tomography
Plant methods
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* Correspondence: tony.pridmore@nottingham.ac.uk 1Centre for Plant Integrative Biology, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK 2School of Computer Science, University of Nottingham, Jubilee Campus, Nottingham NG8 1BB, UK Full list of author information is available at the end of the article : Root systems architecture, 3D, X-ray Computed Tomography, Image analysis, Root phenotyping Keywords: Root systems architecture, 3D, X-ray Computed Tomography, Image analysis, Root p et. al [3]) and aeroponic (Zobel et. al [4]) techniques, and often rely on artificial growth media such as semitranspar- ent nutrient agar [5,6], gellan gum [7] or transparent soil (Downie et. al [8]). Though image analysis is made more tractable, questions are raised regarding the effect these environments might have on root development. Recovering complete plant root system architectures from soil via X-ray μ-Computed Tomography Stefan Mairhofer1,2, Susan Zappala1,3, Saoirse Tracy1,3, Craig Sturrock1,3, Malcolm John Bennett1,3, Sacha Jon Mooney1,3 and Tony Paul Pridmore1,2* © 2013 Mairhofer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 PLANT METHODS Open Access © 2013 Mairhofer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creativ Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: X-ray micro-Computed Tomography (μCT) offers the ability to visualise the three-dimensional structure of plant roots growing in their natural environment – soil. Recovery of root architecture descriptions from X-ray CT data is, however, challenging. The X-ray attenuation values of roots and soil overlap, and the attenuation values of root material vary. Any successful root identification method must both explicitly target root material and be able to adapt to local changes in root properties. RooTrak meets these requirements by combining the level set method with a visual tracking framework and has been shown to be capable of segmenting a variety of plant roots from soil in X-ray μCT images. The approach provides high quality root descriptions, but tracks root systems top to bottom and so omits upward-growing (plagiotropic) branches. Results: We present an extension to RooTrak which allows it to extract plagiotropic roots. An additional backward- looking step revisits the previous image, marking possible upward-growing roots. These are then tracked, leading to efficient and more complete recovery of the root system. Results show clear improvement in root extraction, without which key architectural traits would be underestimated. Conclusions: The visual tracking framework adopted in RooTrak provides the focus and flexibility needed to separate roots from soil in X-ray CT imagery and can be extended to detect plagiotropic roots. The extended software tool produces more complete descriptions of plant root structure and supports more accurate computation of architectural traits. Background In addition, observations are limited to the boundary surface of the rhizotron, and so reveal only a small fraction of the root architecture. X-ray micro-Computed Tomography (μCT) provides an attractive alternative. X-ray μCT is a non-destructive imaging technique that can visualize the internal struc- ture of opaque objects. μCT scanners acquire a series of projections from different angles, measuring the attenu- ation of ionizing radiation passing through the target object. These projections are combined to reconstruct a three-dimensional data set. Data values recorded at each voxel reflect the density of the imaged material and are usually mapped to greyscale intensity values for visualization purposes [14]. μCT is not subject to the constraints facing light-based imaging techniques and enables non-invasive, non-destructive imaging of roots growing in soil. RooTrak has been shown to be capable of separating root systems from their soil environment in μCT images, and recovering root system architecture traits. The tracking technology upon which RooTrak is based [22] allows the target object to split, allowing the root to branch as it “moves” down the soil column. RooTrak, however, considers image slices in fixed top to bottom order, making upward-growing laterals problematic. Though many researchers have shown μCT to be an efficient tool with which to visualize root systems [15], the automatic extraction of quantitative descriptions of root architecture from the resulting data sets presents challenges. First, the X-ray attenuation values of plant roots and the organic matter present in soil overlap. This makes it impossible to specify a root/soil classifica- tion criterion based on attenuation alone; some add- itional information is needed. Heeraman et al. [16] provide that information during a training stage. Here, selected voxels are manually assigned to different classes representing the components contained in the scanned specimen, effectively building a model of the X-ray attenuation data expected from each material. This model is then used to interpret the remainder of the data, separating roots from soil. The approach is, how- ever, sensitive to noise and heavily reliant on the user’s contribution. Downward-growing laterals cause the tracked root ob- ject to split, so that the initial single root becomes many, each following its own, visible, path through the remain- der of the image sequence (Figure 1a). Upward growing (plagiotropic) roots, however, appear before, and not after, their connection to the primary root. Background The way roots develop in soil can have a critical effect on plant growth and impacts crop yield, which is vital to ef- forts to ensure food security [1,2]. This has prompted the development of a variety of methods for characterising root systems. Many of these require plants to be grown in artificial environments designed to increase the visibility of, and so ease the process of imaging, their roots. Con- trolled environment methods include hydroponic (Price The most common method used to study the root sys- tems of plants grown in their natural soil environment is root washing [9,10]. This, however, often leads to the underestimation of fine roots through breakage during the washing process. The three-dimensional, spatial dis- tribution of the root system is also lost, limiting the architectural traits that can be recovered. Rhizotrons and minirhizotrons [11–13] allow roots to be imaged * Correspondence: tony.pridmore@nottingham.ac.uk 1Centre for Plant Integrative Biology, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK 2School of Computer Science, University of Nottingham, Jubilee Campus, Nottingham NG8 1BB, UK Full list of author information is available at the end of the article Page 2 of 7 Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 volume as a stack of cross-sectional images and follow- ing and extracting root objects as they appear to move through them. Visual tracking algorithms build and maintain models of the likely motion and appearance of the target object, using the motion model to predict where the object of interest will appear in the next image and the appearance model to locate it. By adopting a simple motion model [22] capturing the knowledge that roots are connected, RooTrak’s tracking framework focuses analysis on the root. The only user input required is a single mouse click indicating the root in the first image. Updating the appearance model during tracking allows RooTrak to adapt to local changes in root greyscale while distinguishing root from non-root materials with similar intensities. from within the soil, and have been used extensively, but artificially restrict the direction of root growth to two di- mensions. Introduction of the artificial boundary may even affect local soil properties, making conditions near the rhizotron different to those elsewhere in the field, which in turn might impact on root growth. Background Unless they are long enough to appear at the top of the image stack and are marked by the user, RooTrak will simply be un- aware of their existence (Figure 1b). The problem is not restricted to root branches, but arises whenever roots grow upwards. Many root systems contain some roots that grow plagiotropically as they explore the soil for water and nutrients (Nakamoto, [23]. In what follows we describe an extended RooTrak which captures plagiotropic roots, producing more complete root system descriptions and so improved measurement of root system architecture traits. A further problem is that the X-ray attenuation values of root material vary, as a result of differential water re- tention and changes in the density of root material with age. A model of root attenuation built from measure- ments at the top of the data volume will not be effective at the bottom, and vice versa. This has a profound effect on the performance of threshold-based methods, which have been widely used but require error-correcting post- processing [17–21]. Implementation RooTrak relies upon the level set method [24]. The user marks a single point at the top of the root system in the first image, and a novel variant [22] of the level set ap- proach is applied. The effect is to perform a local seg- mentation of the image, marking pixels around the start point with sufficiently similar grey values. A connected component algorithm is applied to group adjacent marked pixels together, and the distribution of grey levels within the connected component is recorded. This distribution constitutes a model of the X-ray attenuation of nearby root material. Any successful root identification method must both explicitly target root material and be able to adapt to local changes in root properties. Recently, RooTrak [22] adopted a tracking-based strategy, viewing the data Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 Page 3 of 7 Mairhofer et al. Plant Methods 2013, 9:8 a b Figure 1 a) When tracking roots from top to bottom of the image sequence RooTrak’s tracking mechanism allows targets to split, successfully recovering branched architectures. b) Plagiotropic roots, however, are overlooked. They only appear in the image sequence before they join the primary. a b b Figure 1 a) When tracking roots from top to bottom of the image sequence RooTrak’s tracking mechanism allows targets to split, successfully recovering branched architectures. b) Plagiotropic roots, however, are overlooked. They only appear in the image sequence before they join the primary. This first root object is then used to initialise analysis of the next slice (image). It is assumed that the root will appear at a similar position, and with similar size, shape and attenuation properties in the following image. The level set method is initialised with the previous object’s size, shape and position information, and deforms its ini- tial segment description to identify a new region with a similar attenuation distribution. The connected compo- nent algorithm is applied again, a new attenuation model computed, and the process continues through the image stack. Care must, however, be taken to ensure that the attenuation model is only updated using reliable root ob- jects. For further details see Mairhofer et al. [22]. objects are identified, i.e. more connected components are reported when approaching an image from below than were seen from above, we consider the new objects to be potential upward growing roots, and mark them as such (Figure 1b). Implementation Processing then continues downwards (i.e. with image n + 1) until the entire stack has been tra- versed. The result at this point, following a single com- pleted traversal of the image stack, is as produced by the original RooTrak, but with markers indicating possible backward growing roots. To complete the root system description, RooTrak then tracks upwards from each marker. Markers are ex- amined in fixed order, from the lowest in the stack to the highest. These tracking operations may generate fur- ther markers, indicating downward growing roots that are connected to the primary root not directly, but via an upward growing root segment. When all upward growing markers have been processed, RootTrak again moves down the stack, tracking from newly reported downward markers. This process is repeated, alternating directions, until all targets are lost and no markers re- main (Figure 2). Note that only the first pass must examine the entire image stack. Subsequent processing focuses on detected markers and each pass only con- siders images in which a previously undetected plagio- tropic root is expected to be visible. When the root branches, the set of pixels identified by the level set becomes separated and the connected com- ponent algorithm will identify more than one compo- nent. This is a key feature of the level set approach, and one which allows different attenuation models to be associated with, and used to extract, different root branches. RooTrak therefore adapts its attenuation model to suit both different root branches, and to reflect changes in attenuation along each branch. Output is a voxel-based representation of the root, from which a variety of traits are recovered. The tracker, however, only includes root material that is directly connected to a known root object and visible as it proceeds down the stack. Results and discussion To address this, an additional step has been intro- duced to RooTrak, allowing it to ‘look back’ for plagio- tropic roots. After all root objects have been identified in image n, and before advancing to image n + 1, RooTrak revisits image n-1. This second examination of image n-1 is initialized with the root objects extracted from image n. If the root objects detected when looking back at image n-1 were all found on the forward pass, no plagiotropic roots are present. If, however, additional μCT data was acquired at The University of Nottingham using a Phoenix Nanotom X-ray CT scanner. Scanning resolution was 23.91 μm, X-ray voltage 110 kV and current 180 μA. 1200 projections were used in each case. Figure 3 shows results obtained from tomato plants (Solanum lycopersicum L) grown in clay loam (Figure 3a,b) and loamy sand (Figure 3c,d) for 10 days. A Newport series Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 Mairhofer et al. Plant Methods 2013, 9:8 Page 4 of 7 a b c d e f Figure 2 Extraction of a simple, artificially generated, plagiotropic root by RooTrak. a. The primary root is extracted and one upward growing section marked on the first pass through the stack. Subsequent processing focusses on the marked branch, extracting a complete description following five further tracking stages (b-f). b d Figure 2 Extraction of a simple, artificially generated, plagiotropic root by RooTrak. a. The primary root is extracted and one upward growing section marked on the first pass through the stack. Subsequent processing focusses on the marked branch, extracting a complete description following five further tracking stages (b-f). loamy sand (brown soil) and a Worcester series clay loam soil (argillic pelosol) from the University of Nottingham farm at Bunny, Nottinghamshire, UK (52.52°N, 1.07°W) were air-dried and sieved to <2 mm. These soil textures are commonly found in UK fields. Stacks of 1,388 × 1,404 × 1,313 and 1,356 × 1,352 × 1,712 pixel images were analysed to generate Figures 3a,b and c,d respectively. Figure 3a,c show root architectures recovered before, and Figure 3b,d after the extensions to RooTrak described here. Arrows in- dicate previously omitted root material recovered by the ex- tended version. RooTrak requires the user to set two parameters (see [22] for details). Results and discussion Values of α = 0.606 and β = 0.246 were used to recover Figure 3a,b while α = 0.608 and β = 0.368 during generation of Figure 3c,d. and c. 9.25% for Tomato 2 respectively, compared to the original version of RooTrak. It is worth noting that these plants were examined at a very early growth stage so one would expect higher detection values in a more mature plant. Besides the geometrical properties (volume and surface area) of the root system, we also wanted to assess struc- tural differences in the descriptions produced the two dif- ferent versions of RooTrak. To express and quantify the difference, we measured the maximum width as well as the volume of the convex hull enclosing the root system. The results are shown in Table 1. The maximum width was obtained by projecting all voxels to a single x-y plane and then calculating the minimum enclosing circle using Welzl’s algorithm [25]. For Tomato 1 (Figure 3a,b), the maximum width remained the same, since the additional root segments were mostly located near or around the pri- mary root. For Tomato 2 (Figure 3c,d), on the other hand, there was a slight increase in the maximum width. A big- ger difference between the two versions can be seen in the volume enclosed by the convex hull. The convex hull was computed using the QuickHull algorithm [26] in which volume is estimated using Monte Carlo Integration [27]. Table 1 compares root volume and surface area esti- mates computed from the data shown in Figure 1. Volume was calculated by counting the number of voxels and multiplying by voxel size cubed. Surface area is obtained by extracting the iso-surface as a mesh of triangles and summing the areas of all triangles in the mesh. There is a clear increase in identified root material. The extended RooTrak recorded an increase of c. 16.62% in root volume and c. 6.20% in surface area for Tomato 1 and c. 3.89% Page 5 of 7 Page 5 of 7 Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 Mairhofer et al. Plant Methods 2013, 9:8 to be tracked, RooTrak is not required to go through the entire image stack again in its search for opposite di- rected roots. Conclusions Though the proportion of plagiotropic branches varies widely, most root systems are likely to contain some pla- giotropic roots (Nakamoto, [23]). Understanding of the factors affecting angle of growth is incomplete, but there is evidence that both internal (hormonal) and external conditions (pH, temperature, oxygen and nutrient concen- tration) have a role to play [28]. Additional plagiotropic growth may result from disease, in particular the hairy root disease caused by Agrobacterium rhizogenes [29]. Figure 3 3D visualization using volume ray-casting of data extracted by RooTrak before (a, c) and after (b, d) the extensions described here. Arrows mark the additional roots detected. The reason for observing a bigger difference is that the maximum width is a one-dimensional measurement, while the convex hull, in contrast, is a function of all three di- mensions. For the samples, Tomato 1 (Figure 3a,b) and Tomato 2 (Figure 3c,d) there was an increase of 48.41% and 10.08% respectively in volume. y g g The original RooTrak [22] allows 3D descriptions of gravitropic roots growing in soil to be recovered from X-ray CT data. RooTrak adopts a visual tracking frame- work that is less sensitive to the natural ambiguity of X-ray attenuation data than previous approaches, and so allows a more flexible and adaptive search for roots. While previous threshold-based techniques are more rigid, and hence may not be robust in highly heterogeneous soil environments, they are usually easier to apply in higher dimensions, and capable of dealing with plagiotropism. In contrast, the greater adaptability of RooTrak’s tracking approach comes at the cost of a fixed search direction and so requires an explicit mechanism for the extraction of plagiotropic roots. The extension of RooTrak to deal with The time needed by the original version of RooTrak to process a CT stack depends on image size, number of images, and amount of root material (see [22] for more details). Through the additional “backward-looking” step introduced here, the time required to process an image stack is doubled at best. This is because every image has to be visited at least twice; during the normal forward traversal and while looking backward (the additional step). The effort of looking for markers, however, has its advantages. Results and discussion a b c d Figure 3 3D visualization using volume ray-casting of data extracted by RooTrak before (a, c) and after (b, d) the extensions described here. Arrows mark the additional roots detected. b a Note also that the extended RooTrak’s two stage (mark, then track) structure allows it to report the pro- portion of the root system which grows upward. It can also identify points at which direction of growth changes. This may be of value in itself, allowing new traits such as average length of upward/downward grow- ing sections, angles between them etc. to be recovered. Alternatively, these changes in direction might indicate significant changes in soil properties, to which the root is responding. X-ray CT provides simultaneous imaging of both root and soil: detected changes in root direction could be used to target analysis of related soil features. Though changes in growth direction could be identified following extraction of a full geometric description of the root system architecture from RooTrak’s segmenta- tion, the ability to recover them directly during segmen- tation avoids significant amounts of processing. References 1. de Dorlodot S, Forster B, Pagès L, Price A, Tuberosa R, Draye X: Root system architecture: opportunities and constraints for genetic improvement of crops. Trends Plant Sci 2007, 12:474–481. 1. de Dorlodot S, Forster B, Pagès L, Price A, Tuberosa R, Draye X: Root system architecture: opportunities and constraints for genetic improvement of crops. Trends Plant Sci 2007, 12:474–481. 2. Postma JA, Lynch JP: Root cortical aerenchyma enhances the growth of maize on soils with suboptimal availability of nitrogen, phosphorus, and potassium. 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Plant Soil 1999, 217:183–193. 13. Vamerali T, Ganis A, Bona S, Mosca G: An approach to minirhizotron root image analysis. Plant Soil 1999, 217:183–193. RooTrak is open source and available from SourceForge via www.rootrak.net. The tool is written in C++ and in- cludes Visual Studio 2008 project files to compile for Windows. The Qt 4.8 framework is required for the compilation of the source code. RooTrak incorporates a volume rendering tool which displays the root system using GLSL, and so requires a GLSL compliant graphics card. 14. Mooney SJ: Three-dimensional visualization and quantification of soil macroporosity and water flow patterns using Computed Tomography. Soil Use Manage 2002, 18:142–151. 14. Mooney SJ: Three-dimensional visualization and quantification of soil macroporosity and water flow patterns using Computed Tomography. Soil Use Manage 2002, 18:142–151. 15. Mooney SJ, Pridmore TP, Helliwell J, Bennett MJ: Developing X-ray Computed Tomography to non-invasively image 3-D root systems architecture in soil. Plant and Soil 2012, 352:1–22. 15. Mooney SJ, Pridmore TP, Helliwell J, Bennett MJ: Developing X-ray Computed Tomography to non-invasively image 3-D root systems architecture in soil. Plant and Soil 2012, 352:1–22. 16. Heeraman D, Hopmans J, Clausnitzer V: Three dimensional imaging of plant roots in situ with x-ray Computed Tomography. Plant Soil 1997, 189:167–179. 16. Heeraman D, Hopmans J, Clausnitzer V: Three dimensional imaging of plant roots in situ with x-ray Computed Tomography. Plant Soil 1997, 189:167–179. 17. Kaestner A, Schneebeli M, Graf F: Visualizing three-dimensional root networks using Computed Tomography. Geoderma 2006, 136:459–469. Authors’ contributions SM d i d d i l SM designed and implemented the extended RooTrak software. SZ and ST provided advice on plant preparation and growth and CS captured the image data. SJM, MJB and TPP devised the research, provided analysis and ib d h i l All h d d d h fi l SM designed and implemented the extended RooTrak software. SZ and ST provided advice on plant preparation and growth and CS captured the d d d d h h d d l d 19. Lontoc-Roy M, Dutilleul P, Prasher SO, Liwen H, Smith DL: Computed Tomography scanning for three-dimensional imaging and complexity analysis of developing root systems. Can J Botany 2005, 83:1434–1442. provided advice on plant preparation and growth and CS captured the image data. SJM, MJB and TPP devised the research, provided analysis and contributed to the article. All authors read and approved the final manuscript. 20. Perret J, Al-Belushi M, Deadman M: Non-destructive visualization and quantification of roots using Computed Tomography. Soil Biol Biochem 2007, 39:391–399. Competing interests Th h d l h p g The authors declare that they have no competing interests. 18. Lontoc-Roy M, Dutilleul P, Prasher SO, Liwen H, Brouillet T, Smith DL: Advances in the acquisition and analysis of ct scan data to isolate a crop root system from the soil medium and quantify root system complexity in 3-d space. Geoderma 2006, 137:231–241. Acknowledgements 21. Pierret A, Capowiez Y, Moran CJ, Kretzschmar A: X-ray Computed Tomography to quantify tree rooting spatial distributions. Geoderma 1999, 90:307–326. This work was supported by the University of Nottingham Inter-disciplinary Doctoral Training Centre in Integrative Biology (PhD studentship to S.M.) and Biotechnology and Biological Science Research Council and Engineering and Physical Sciences Research Council Centre for Integrative Systems Biology program funding to the Centre for Plant Integrative Biology. 22. Mairhofer S, Zappala S, Tracy SR, Sturrock C, Bennett M, Mooney SJ, Pridmore T: RooTrak: automated recovery of three-dimensional plant root architecture in soil from X-ray Micro-Computed Tomography Images using visual tracking. Plant Physiol 2012, 158:561–569. 23. Nakamoto T, Oyanagi A: The direction of growth of seminal roots of Triticum aestivum L. and experimental modification thereof. Ann Bot 1994, 73:363–367. Conclusions Once located the extraction can be contin- ued from each marker and stop when no objects are left Table 1 Measured root volume and surface area using the original RooTrak and its extension RooTrak original version RooTrak extended version Tomato 1 (Figure 1a) Tomato 2 (Figure 1c) Tomato 1 (Figure 1b) Tomato 2 (Figure 1d) Volume (mm3) 21.17 34.17 24.69 35.50 Surface area (mm2) 260.76 407.18 276.94 444.85 Max. width (mm) 50.63 54.63 50.63 55.58 Convex hull (mm3) 1623.90 3057.78 2410.18 3366.24 Table 1 Measured root volume and surface area using the original RooTrak and its extension olume and surface area using the original RooTrak and its extension Page 6 of 7 Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 plagiotropic roots allows the tracking methodology to be applied to the full range of root architectures and will, we believe, allow higher quality root descriptions to be obtained than was previously possible. RooTrak’s tracking framework has been extended to allow both gravitropic and plagiotropic branches to be segmented and described, allowing RooTrak to produce more complete root descriptions, and so more accurate whole root system traits. Plagiotropic branches are distin- guished from downward growing, gravitropic roots dur- ing the segmentation process, providing opportunities to compute new comparative (gravitropic vs plagiotro- pic) measures without potentially expensive, higher- level recognition of plagiotropic growth. 25. Welzl E: In Smallest enclosing disks (balls and ellipsoids). Edited by Maurer H. Berlin: New Results and New Trends in Computer Science, Lecture Notes in Computer Science, Springer-Verlag; 1991:359–370. 555. Author details 1 1Centre for Plant Integrative Biology, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK. 2School of Computer Science, University of Nottingham, Jubilee Campus, Nottingham NG8 1BB, UK. 3School of Biosciences, University of Nottingham, Sutton Bonington Campus, Nottingham LE12 5RD, UK. 24. Sethian JA: In Level Set Methods and Fast Marching Methods: Evolving Interfaces in Computational Geometry, Fluid Mechanics, Computer Vision, and Materials Science (Cambridge . . . on Applied and Computational Mathematics), Ed 2. New York, NY, USA: Cambridge University Press; 1999. y 25. Welzl E: In Smallest enclosing disks (balls and ellipsoids). Edited by Maurer H. Berlin: New Results and New Trends in Computer Science, Lecture Notes in Computer Science, Springer-Verlag; 1991:359–370. 555. 25. Welzl E: In Smallest enclosing disks (balls and ellipsoids). Edited by Maurer H. Berlin: New Results and New Trends in Computer Science, Lecture Notes in Computer Science, Springer-Verlag; 1991:359–370. 555. Received: 21 January 2013 Accepted: 12 March 2013 Published: 20 March 2013 Received: 21 January 2013 Accepted: 12 March 2013 Published: 20 March 2013 Page 7 of 7 Page 7 of 7 Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 Mairhofer et al. Plant Methods 2013, 9:8 http://www.plantmethods.com/content/9/1/8 26. Barber CB, Dobkin DP, Huhdanpaa H: The quickhull algorithm for convex hulls. ACM T Math Software 1996, 22:469–483. 27. Rubinstein RY: Simulation and the Monte Carlo Methods. San Francisco: John Wiley and Sons, Inc.; 1981. 28. Rufelt H: The course of the geotropic reaction of wheat roots. Physiologica Plantarum 1957, 10:231–247. 29. Christey MC, Sinclair BK, Braun RH, Wyke L: Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation. Plant Cell Rep 1997, 16:587–593. doi:10.1186/1746-4811-9-8 Cite this article as: Mairhofer et al.: Recovering complete plant root system architectures from soil via X-ray μ-Computed Tomography. Plant Methods 2013 9:8. doi:10.1186/1746-4811-9-8 Cite this article as: Mairhofer et al.: Recovering complete plant root system architectures from soil via X-ray μ-Computed Tomography. Plant Methods 2013 9:8. 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Published on: Nov 14, 2021 License: Creative Commons Attribution 4.0 International License (CC-BY 4.0) Hagia Sophia San Antonio Review • Photo by Raimond Klavins on Unsplash Photo by Raimond Klavins on Unsplash Byzantium is but the shadow of a whisper that holds your hollow ghost like the dead hold  their bones your eyes are dry, for life has escaped through cracks that were supposed Hagia Sophia San Antonio Review • to show pilgrims parts of a heaven that hoped to be yours   But suns set and moons rose, so many of them that when people stopped counting they thought it was an eternity   but you knew better, the crescent holds no numbers, no notes, no chimes   Yet, it peeled your name off of the surface of time  – letter by letter   I saw them drip  one by one    like grains of sand through the tight throat of an hourglass and stood wondering, if you’ll recognize it when I sigh it to you   Instead, you shrink inside halos to show pilgrims parts of a heaven that hoped to be yours But suns set and moons rose, so many of them that when people stopped counting they thought it was an eternity I saw them drip like grains of sand through the tight throat of an hourglass and stood wondering, if you’ll recognize it when I sigh it to you Instead, you shrink inside halos 3 Hagia Sophia San Antonio Review • Christ is without a cross Christ is without a cross Who will be saved now? Who will be saved now? I dreamt my great grandfather  in front of your gates - weeping his tears wet my eyes his hands held my heart in his throat and I heard the soil sob with souls that wander through six hundred years  of unsang troparions   then, I thought of the time when God so loved the world that he gave  his only son   Saint John couldn’t imagine what God would do if he had to give  his only daughter   Aida Bode is a Pushcart-nominated Albanian p published online and in print. Visit her websit Aida Bode is a Pushcart-nominated Albanian poet and writer, whose works have been published online and in print. Visit her website for her extensive publications. 4
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ERROR: type should be string, got "https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The global impact of the transport sectors on aerosol and climate\nunder the Shared Socioeconomic Pathways (SSPs)\nMattia Righi, Johannes Hendricks, and Sabine Brinkop\nDeutsches Zentrum für Luft- und Raumfahrt (DLR), Institut für Physik der Atmosphäre, Oberpfaffenhofen, Germany\nCorrespondence: Mattia Righi (mattia.righi@dlr.de) The global impact of the transport sectors on aeroso\nunder the Shared Socioeconomic Pathways (SSPs) The transport-induced impacts on aerosol mass and number concentrations result in a present-day radiative forc-\ning of −164, −145 and −64 mW m−2, for land transport, shipping and aviation, respectively, with a dominating contribution\nby aerosol-cloud interactions. These forcings represent a marked offset to the CO2 warming from the transport sectors and are therefore very relevant for climate policy. The projections under the SSPs show that the impact of the transport sectors on\n15\naerosol and climate are generally consistent with the narratives underlying these scenarios: the lowest impacts of transport on\nboth aerosol and climate are simulated under SSP1, especially for the land transport sector, while SSP3 is generally character-\nized by the largest effects. Notable exceptions to this picture, however, exist, as the emissions of other anthropogenic sectors\nalso contribute to the overall aerosol concentrations and thus modulate the relevance of the transport sectors in the different\nscenarios, not always consistently with their underlying storyline. On a qualitative level, the results for present-day mostly\n20\nconfirm the findings of our previous assessment for the year 2000, which used a predecessor version of the same model and are therefore very relevant for climate policy. The projections under the SSPs show that the impact of the transport sectors on\n15\naerosol and climate are generally consistent with the narratives underlying these scenarios: the lowest impacts of transport on\nboth aerosol and climate are simulated under SSP1, especially for the land transport sector, while SSP3 is generally character-\nized by the largest effects. Notable exceptions to this picture, however, exist, as the emissions of other anthropogenic sectors\nalso contribute to the overall aerosol concentrations and thus modulate the relevance of the transport sectors in the different are therefore very relevant for climate policy. The projections under the SSPs show that the impact of the transport sectors on\n15\naerosol and climate are generally consistent with the narratives underlying these scenarios: the lowest impacts of transport on\nboth aerosol and climate are simulated under SSP1, especially for the land transport sector, while SSP3 is generally character-\nized by the largest effects. Notable exceptions to this picture, however, exist, as the emissions of other anthropogenic sectors\nalso contribute to the overall aerosol concentrations and thus modulate the relevance of the transport sectors in the different scenarios, not always consistently with their underlying storyline. The global impact of the transport sectors on aeroso\nunder the Shared Socioeconomic Pathways (SSPs) Abstract. A global aerosol-climate model is applied to quantify the impact of the transport sectors (land transport, shipping\nand aviation) on aerosol and climate. Global simulations are performed for present-day (2015), based on the emission inventory\nof the Climate Model Intercomparison Project Phase 6 (CMIP6), and for near-term (2030) and mid-term (2050) future projec-\ntions, under the Shared Socioeconomic Pathways (SSPs). The results for present-day show that land transport emissions have a large impact on near-surface concentrations of black carbon and aerosol nitrate over the most populated areas of the globe,\n5\nbut with contrasting patterns in terms of relative contributions between developed and developing countries. In spite of the\nrecently introduced regulations to limit the fuel sulphur content in the shipping sector, shipping emissions are still responsible\nfor a considerable impact on aerosol sulfate near-surface concentrations, about 0.5 to 1 µg m−3 in the most travelled regions,\nwith significant effects also on continental air pollution and in the northern polar regions. Aviation impacts on aerosol mass a large impact on near-surface concentrations of black carbon and aerosol nitrate over the most populated areas of the globe,\n5\nbut with contrasting patterns in terms of relative contributions between developed and developing countries. In spite of the\nrecently introduced regulations to limit the fuel sulphur content in the shipping sector, shipping emissions are still responsible\nfor a considerable impact on aerosol sulfate near-surface concentrations, about 0.5 to 1 µg m−3 in the most travelled regions,\nwith significant effects also on continental air pollution and in the northern polar regions. Aviation impacts on aerosol mass are found to be quite small, of the order of a few nanograms per cubic meter, while this sector considerably affects particle\n10\nnumber concentrations, contributing up to 20–30% of the upper tropospheric particle number concentration at the northern\nmid-latitudes. The transport-induced impacts on aerosol mass and number concentrations result in a present-day radiative forc-\ning of −164, −145 and −64 mW m−2, for land transport, shipping and aviation, respectively, with a dominating contribution\nby aerosol-cloud interactions. These forcings represent a marked offset to the CO2 warming from the transport sectors and are found to be quite small, of the order of a few nanograms per cubic meter, while this sector considerably affects particle\n10\nnumber concentrations, contributing up to 20–30% of the upper tropospheric particle number concentration at the northern\nmid-latitudes. The global impact of the transport sectors on aeroso\nunder the Shared Socioeconomic Pathways (SSPs) On a qualitative level, the results for present-day mostly\n20\nconfirm the findings of our previous assessment for the year 2000, which used a predecessor version of the same model and\nthe CMIP5 emissions data. Some important quantitative differences are found, which can mostly be ascribed to the improved\nrepresentation of aerosol background concentrations in the present study. scenarios, not always consistently with their underlying storyline. On a qualitative level, the results for present-day mostly\n20\nconfirm the findings of our previous assessment for the year 2000, which used a predecessor version of the same model and\nthe CMIP5 emissions data. Some important quantitative differences are found, which can mostly be ascribed to the improved\nrepresentation of aerosol background concentrations in the present study. 1 1 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 1\nIntroduction These compounds have\na detrimental effect on the air quality, while some of them can also impact climate (Fiore et al., 2012; von Schneidemesser Although the climate policies mostly target the emissions of CO2 and other greenhouse gases, the combustion processes\n35\npowering vehicles, ships and aircraft are also responsible for the emissions of several types of short-lived pollutants, such as\nnitrogen oxides (NOx=NO+NO2), carbon monoxide (CO), non-methane hydrocarbons (NMHC), sulphur dioxide (SO2) and\naerosol particles in form of black carbon (BC), organic carbon (OC) and primary aerosol sulfate (SO4). These compounds have\na detrimental effect on the air quality, while some of them can also impact climate (Fiore et al., 2012; von Schneidemesser et al., 2015). For example, NOx and NMHC are precursors of ozone, which is a well-known greenhouse gas (e.g. Stevenson\n40\net al., 2013; Mertens et al., 2018), while aerosol and their precursors can alter the planetary radiation balance via their direct\ninteraction with solar radiation (absorption and scattering, leading to warming and cooling, respectively; Boucher et al., 2013). Perhaps most importantly, aerosol can further impact the climate by interacting with clouds and modifying their radiative prop-\nerties, resulting in an overall cooling effect (Bellouin et al., 2020). Air pollution reduction measures targeting these compounds may therefore affect both air quality and climate, with beneficial effects for both in some cases (e.g. when targeting BC, which\n45\nhas a warming effect on climate and a detrimental impact on air quality; Bond et al., 2013), and trade-offs in other cases, like\nsulphur reduction policies, which improve air quality but reduce the cooling impact due the aerosol indirect effect on clouds\n(Fuglestvedt et al., 2009). Furthermore, these policies are implemented at different scales, with land transport being mostly\ntargeted at the national or regional (e.g. European) level, while shipping and aviation are regulated at the international level, may therefore affect both air quality and climate, with beneficial effects for both in some cases (e.g. when targeting BC, which\n45\nhas a warming effect on climate and a detrimental impact on air quality; Bond et al., 2013), and trade-offs in other cases, like\nsulphur reduction policies, which improve air quality but reduce the cooling impact due the aerosol indirect effect on clouds\n(Fuglestvedt et al., 2009). Furthermore, these policies are implemented at different scales, with land transport being mostly\ntargeted at the national or regional (e.g. 1\nIntroduction With a share of 14% (8.5 Pg) of the total CO2-equivalent emissions in 2018, the transport sectors are major contributors to\n25\nclimate change (Lamb et al., 2021). This share is dominated by land transport (including road and rail transport, and inland\nnavigation), which is responsible for 80.4% of it, while international shipping and aviation (international and domestic) account\nfor the remaining 8.4% and 11.2%, respectively. Moreover, the transport sectors are characterized by an approximately steady\ngrowth in emissions of greenhouse gases, at a rate of about 2% per year in the last three decades, mostly driven by developed countries (North America and Europe) and, since the beginning of the century, by the fast growing economies of East Asia\n30\n(Lamb et al., 2021). Traffic volumes are projected to grow further in the coming decades (Girod et al., 2013; Sims et al., 2014),\nmodulated by the current efforts to decarbonise this sector in order to achieve the goals of the Paris Agreement (Pietzcker et al.,\n2014; Creutzig et al., 2015; Esmeijer et al., 2020) and by the uncertain recovery path from the COVID-19 pandemic, which\nsignificantly affected the recent global emissions (Liu et al., 2020; Le Quéré et al., 2020). countries (North America and Europe) and, since the beginning of the century, by the fast growing economies of East Asia\n30\n(Lamb et al., 2021). Traffic volumes are projected to grow further in the coming decades (Girod et al., 2013; Sims et al., 2014),\nmodulated by the current efforts to decarbonise this sector in order to achieve the goals of the Paris Agreement (Pietzcker et al.,\n2014; Creutzig et al., 2015; Esmeijer et al., 2020) and by the uncertain recovery path from the COVID-19 pandemic, which\nsignificantly affected the recent global emissions (Liu et al., 2020; Le Quéré et al., 2020). Although the climate policies mostly target the emissions of CO2 and other greenhouse gases, the combustion processes\n35\npowering vehicles, ships and aircraft are also responsible for the emissions of several types of short-lived pollutants, such as\nnitrogen oxides (NOx=NO+NO2), carbon monoxide (CO), non-methane hydrocarbons (NMHC), sulphur dioxide (SO2) and\naerosol particles in form of black carbon (BC), organic carbon (OC) and primary aerosol sulfate (SO4). 1\nIntroduction European) level, while shipping and aviation are regulated at the international level, via the International Maritime Organization (IMO) and the International Civil Aviation Organization (ICAO), respectively, al-\n50\nthough regional policies are also applied in shipping, for instance the implementation of the Sulphur Emissions Control Areas\n(SECAs; Buhaug et al., 2009; Smith et al., 2014). To fully characterize the impact of short-lived pollutants from the transport\nsectors it is therefore necessary to consistently account for changes in the atmospheric composition and climate impact and, at\nthe same time, to address regional dependencies. In this study, we focus on the impact of transport-induced aerosol and aerosol precursor species. We update the assessments\n55\nof Righi et al. (2013, 2015, 2016) and improve them on three major aspects: In this study, we focus on the impact of transport-induced aerosol and aerosol precursor species. We update the assessments\n55\nof Righi et al. (2013, 2015, 2016) and improve them on three major aspects: In this study, we focus on the impact of transport-induced aerosol and aerosol precursor species. We update the assessments\n55\nof Righi et al. (2013, 2015, 2016) and improve them on three major aspects: 2 2 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 1. We use the recent CMIP6 emission inventory (Hoesly et al., 2018; van Marle et al., 2017), thus replacing the CMIP5\ninventory (Lamarque et al., 2010) applied in our previous studies and now consider the year 2015 instead of 2000 as\na reference for present-day conditions. 1. We use the recent CMIP6 emission inventory (Hoesly et al., 2018; van Marle et al., 2017), thus replacing the CMIP5\ninventory (Lamarque et al., 2010) applied in our previous studies and now consider the year 2015 instead of 2000 as\na reference for present-day conditions. 1. We use the recent CMIP6 emission inventory (Hoesly et al., 2018; van Marle et al., 2017), thus replacing the CMIP5\ninventory (Lamarque et al., 2010) applied in our previous studies and now consider the year 2015 instead of 2000 as\na reference for present-day conditions. 2. We implement the Shared Socioeconomic Pathways (SSPs; O’Neill et al., 2017) for the future projections, which are\na new generation of scenarios developed in support of the IPCC Sixth Assessment Report (Gidden et al., 2019). They\nare socio-economic scenarios that include assumptions on several indicators (e.g. 1\nIntroduction population, wealth, urbanisation, etc.)\nto describe the ability of the society to cope with climate change and are further combined with the climate pathways\nbased on the narratives of Representative Concentration Pathways (RCPs; van Vuuren et al., 2011) for climate policies,\nresulting in a matrix of possible futures. 60 65 3. We apply a newer version of the global chemistry-climate model EMAC (ECHAM-MESSy Atmospheric Chemistry;\nJöckel et al., 2010), featuring an updated aerosol scheme (MADE3, i.e. 3rd version of the Modal Aerosol Dynamics for\nEurope adapted for global applications; Kaiser et al., 2014) and a state-of-the-art two-moment cloud scheme (Kuebbeler\net al., 2014) accounting for aerosol-cloud interactions for all cloud phases (liquid, mixed-phase and ice). This model\nconfiguration has been thoroughly tuned and evaluated by Kaiser et al. (2019) and Righi et al. (2020), demonstrating its\nability to represent key aspects of aerosol, cloud and radiation processes. 70 Being key targets of decarbonisation and air pollution control policies, the transport sectors are subject of active research. Lund et al. (2014a) applied a global chemistry-transport model to quantify the effect of short-lived pollutants on climate when\nreplacing conventional diesel fuels with biofuels in the EU road transport sector and found a 15% and 80% reduction of Being key targets of decarbonisation and air pollution control policies, the transport sectors are subject of active research. Lund et al. (2014a) applied a global chemistry-transport model to quantify the effect of short-lived pollutants on climate when\nreplacing conventional diesel fuels with biofuels in the EU road transport sector and found a 15% and 80% reduction of radiative forcing (RF) when replacing 20% and 100% of diesel with biofuels, respectively. Lund et al. (2014b) considered\n75\nthe climate impact of black carbon from road diesel engines in 2010 and 2050 following a current-legislation scenario and\nfound that Europe gives the largest contribution in 2010, while South and East Asia dominate in 2050, as a result of different\nimplementation of reduction measures. The potential global relevance of local policies on land-based transport was highlighted\nby Hendricks et al. (2018), who estimated the climate impact of German land-based transport using a combination of modelling techniques and found that this is dominated by CO2, while the effect of short-lived pollutants is negligible. 1\nIntroduction This paper is organised as follows: Section 2 briefly describes the EMAC model with the aerosol submodel MADE3 and\nthe method used to estimate the impact of the transport sectors. A detailed analysis of the emission inventories for present-day climate is projected to remain relevant throughout the century, with strong regional variations. Hence, re-assessing the impact\n115\nof transport on aerosol concentrations, also with a focus on regional effects, is essential to understand the scope of the air\npollution measures covered by the SSPs narratives and the resulting climate impacts. This paper is organised as follows: Section 2 briefly describes the EMAC model with the aerosol submodel MADE3 and\nthe method used to estimate the impact of the transport sectors. A detailed analysis of the emission inventories for present-day climate is projected to remain relevant throughout the century, with strong regional variations. Hence, re-assessing the impact\n115\nof transport on aerosol concentrations, also with a focus on regional effects, is essential to understand the scope of the air\npollution measures covered by the SSPs narratives and the resulting climate impacts. This paper is organised as follows: Section 2 briefly describes the EMAC model with the aerosol submodel MADE3 and\nthe method used to estimate the impact of the transport sectors. A detailed analysis of the emission inventories for present-day This paper is organised as follows: Section 2 briefly describes the EMAC model with the aerosol submodel MADE3 and\nthe method used to estimate the impact of the transport sectors. A detailed analysis of the emission inventories for present-day\nand the future projections is provided in Sect. 3, focusing on the share of the three transport sectors on the total emissions of\n120\naerosol and precursors. The impact of transport on the aerosol concentration is discussed in Sect. 4, while Section 5 deals with\nthe climate impacts. A summary of the conclusions of this study is provided in Sect. 6. the method used to estimate the impact of the transport sectors. A detailed analysis of the emission inventories for present-day\nand the future projections is provided in Sect. 3, focusing on the share of the three transport sectors on the total emissions of\n120\naerosol and precursors. The impact of transport on the aerosol concentration is discussed in Sect. 4, while Section 5 deals with\nthe climate impacts. A summary of the conclusions of this study is provided in Sect. 6. 1\nIntroduction This effect was also simulated by Gettelman and Chen (2013), who however considered generally\n100\nsmaller particle sizes for aerosol sulfate in their assumptions, thus finding a larger RF in the range −164 to −23 mW m−2. A later study by Kapadia et al. (2016) supported these estimates and again found a reduction of the indirect effect under\na scenario with low aviation FSC, while Matthes et al. (2021) showed that the impact of aviation aerosol on low clouds may\nbe dependent on the cruise altitude of the global fleet. Aviation emissions of soot are also potentially impacting ice clouds (Hendricks et al., 2005, 2011; Penner et al., 2018), but this effect is very uncertain and strongly dependent on the assumed\n105\nice nucleating abilities of soot. In the model configuration adopted in the present study, we conservatively assume a very low\nice nucleating efficiency for aviation soot, so that its impact on climate via the interaction with natural cirrus clouds may be\nconsidered negligible (Righi et al., 2021). Despite active research on the impact of the transport sectors on atmosphere and climate, the literature on the impact of (Hendricks et al., 2005, 2011; Penner et al., 2018), but this effect is very uncertain and strongly dependent on the assumed\n105\nice nucleating abilities of soot. In the model configuration adopted in the present study, we conservatively assume a very low\nice nucleating efficiency for aviation soot, so that its impact on climate via the interaction with natural cirrus clouds may be\nconsidered negligible (Righi et al., 2021). Despite active research on the impact of the transport sectors on atmosphere and climate the literature on the impact of (Hendricks et al., 2005, 2011; Penner et al., 2018), but this effect is very uncertain and strongly dependent on the assumed\n105\nice nucleating abilities of soot. In the model configuration adopted in the present study, we conservatively assume a very low\nice nucleating efficiency for aviation soot, so that its impact on climate via the interaction with natural cirrus clouds may be\nconsidered negligible (Righi et al., 2021). 1\nIntroduction 95\nAs shown in Righi et al. (2013, hereafter R13), FSC is also critical for the aviation sector, as sulfate particles produced in\nthe aircraft exhaust plume could be transported downwards and potentially impact low level clouds, resulting in a significant\n(albeit quite uncertain) aerosol indirect effect. R13 estimated this effect to be between −70 and −15 mW m−2, depending on\nthe assumptions on the size of the emitted sulphate particles, but found no statistically significant effect when a low FSC was 2018) and its impacts on the regional and global climate. 95\nAs shown in Righi et al. (2013, hereafter R13), FSC is also critical for the aviation sector, as sulfate particles produced in\nthe aircraft exhaust plume could be transported downwards and potentially impact low level clouds, resulting in a significant\n(albeit quite uncertain) aerosol indirect effect. R13 estimated this effect to be between −70 and −15 mW m−2, depending on\nthe assumptions on the size of the emitted sulphate particles, but found no statistically significant effect when a low FSC was assumed for the aircraft fleet. This effect was also simulated by Gettelman and Chen (2013), who however considered generally\n100\nsmaller particle sizes for aerosol sulfate in their assumptions, thus finding a larger RF in the range −164 to −23 mW m−2. A later study by Kapadia et al. (2016) supported these estimates and again found a reduction of the indirect effect under\na scenario with low aviation FSC, while Matthes et al. (2021) showed that the impact of aviation aerosol on low clouds may\nbe dependent on the cruise altitude of the global fleet. Aviation emissions of soot are also potentially impacting ice clouds assumed for the aircraft fleet. This effect was also simulated by Gettelman and Chen (2013), who however considered generally\n100\nsmaller particle sizes for aerosol sulfate in their assumptions, thus finding a larger RF in the range −164 to −23 mW m−2. A later study by Kapadia et al. (2016) supported these estimates and again found a reduction of the indirect effect under\na scenario with low aviation FSC, while Matthes et al. (2021) showed that the impact of aviation aerosol on low clouds may\nbe dependent on the cruise altitude of the global fleet. Aviation emissions of soot are also potentially impacting ice clouds assumed for the aircraft fleet. 1\nIntroduction They also note,\n80\nhowever, that this might be different in other regions or countries, depending on the specific composition of the fleet and on\nits age, and also on the photochemical activity which varies considerably around the globe. The climate impact of ozone from\nglobal land-transport and shipping was quantified by Mertens et al. (2018), who applied an innovative tagging method to track\nthe emissions from specific sectors in a global model simulation and thus isolate the climate impact with better accuracy than in standard perturbation approaches. This resulted in higher land-transport- and shipping-induced concentrations of surface-level\n85\nozone and in a RF of 92 and 62 mW m−2, for the two sectors respectively. The shipping sector is particularly interesting due to\nthe new regulations introduced by MARPOL 73/78 (the International Convention for the Prevention of Pollution from Ships) in\nits Annex VI to globally limit fuel sulphur content (FSC) in shipping (Buhaug et al., 2009) and which took full effect in 2020. Sofiev et al. (2018), Kontovas (2020) and Bilsback et al. (2020) showed how reducing FSC leads to reduced SO2 emissions and lower shipping-induced aerosol concentrations: while this is beneficial for air quality and reduces premature mortality, it may\n90 3 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. also remove the cooling from the aerosol effects, hence accelerating global warming. Measurements of ship engine exhaust,\nhowever, show that particle number emission factors in low-sulphur fuels are not significantly decreased compared to standard\nheavy fuel oil (Kuittinen et al., 2021), implying that shipping would remain an important source of particle number even after\nthe MARPOL regulations. Another policy-relevant issue is the opening of Arctic routes in the near future (Stephenson et al.,\n2018) and its impacts on the regional and global climate. 95 also remove the cooling from the aerosol effects, hence accelerating global warming. Measurements of ship engine exhaust,\nhowever, show that particle number emission factors in low-sulphur fuels are not significantly decreased compared to standard\nheavy fuel oil (Kuittinen et al., 2021), implying that shipping would remain an important source of particle number even after\nthe MARPOL regulations. Another policy-relevant issue is the opening of Arctic routes in the near future (Stephenson et al.,\n2018) and its impacts on the regional and global climate. 95 2018) and its impacts on the regional and global climate. 1\nIntroduction Despite active research on the impact of the transport sectors on atmosphere and climate, the literature on the impact of Despite active research on the impact of the transport sectors on atmosphere and climate, the literature on the impact of\nspecific sectors in the context of the SSPs is still limited and a detailed analysis on the role of transport-induced aerosol is still\n110\nlacking. Lund et al. (2019) assessed the anthropogenic aerosol forcing in a subset of the SSP scenarios until the end of the\ncentury, concluding that the air pollution control measures assumed in these scenarios span a much broader range of emissions\nthan in the previous RCPs. Lund et al. (2020) used a climate response emulator to calculate the temperature response of various\nshort-lived compounds under the SSPs and compared it to the one of CO2, showing that the role of short-lived compounds on specific sectors in the context of the SSPs is still limited and a detailed analysis on the role of transport-induced aerosol is still\n110\nlacking. Lund et al. (2019) assessed the anthropogenic aerosol forcing in a subset of the SSP scenarios until the end of the\ncentury, concluding that the air pollution control measures assumed in these scenarios span a much broader range of emissions\nthan in the previous RCPs. Lund et al. (2020) used a climate response emulator to calculate the temperature response of various\nshort-lived compounds under the SSPs and compared it to the one of CO2, showing that the role of short-lived compounds on climate is projected to remain relevant throughout the century, with strong regional variations. Hence, re-assessing the impact\n115\nof transport on aerosol concentrations, also with a focus on regional effects, is essential to understand the scope of the air\npollution measures covered by the SSPs narratives and the resulting climate impacts. Thi\ni\ni\nd\nf ll\nS\nti\n2 b i fl\nd\nib\nth EMAC\nd l\nith th\nl\nb\nd l MADE3\nd climate is projected to remain relevant throughout the century, with strong regional variations. Hence, re-assessing the impact\n115\nof transport on aerosol concentrations, also with a focus on regional effects, is essential to understand the scope of the air\npollution measures covered by the SSPs narratives and the resulting climate impacts. 1\nIntroduction and the future projections is provided in Sect. 3, focusing on the share of the three transport sectors on the total emissions of\n120\naerosol and precursors. The impact of transport on the aerosol concentration is discussed in Sect. 4, while Section 5 deals with\nthe climate impacts. A summary of the conclusions of this study is provided in Sect. 6. 4 4 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 2\nModel and model simulations In this assessment we apply the global model EMAC with the aerosol submodel MADE3. EMAC is a chemistry and cli-\nmate model featuring several submodels to simulate tropospheric and middle atmospheric processes and their interaction with\n125\noceans, land, and human influences (Jöckel et al., 2010). EMAC is based on the second version of the Modular Earth Submodel\nSystem (MESSy) to link multi-institutional computer codes. The core atmospheric model is the ECHAM5 (fifth generation Eu-\nropean Centre Hamburg) general circulation model (Roeckner et al., 2006). Aerosols microphysics is represented by the aerosol\nsubmodel MADE3 (Kaiser et al., 2014), which considers aerosol sulfate, ammonium, nitrate, sea salt, particulate organic mat- ter, black carbon, mineral dust, and aerosol water. These species are distributed in nine aerosol modes, resulting from the\n130\ncombination of three size classes (Aitken, accumulation, and coarse) with three particle mixing states (soluble, insoluble and\nmixed particles). The representation of aerosol mass and number concentrations in MADE3 has been evaluated in detail in\nKaiser et al. (2019), using observational data from various station networks around the globe, from aircraft campaigns, and\nfrom satellite observations. ter, black carbon, mineral dust, and aerosol water. These species are distributed in nine aerosol modes, resulting from the\n130\ncombination of three size classes (Aitken, accumulation, and coarse) with three particle mixing states (soluble, insoluble and\nmixed particles). The representation of aerosol mass and number concentrations in MADE3 has been evaluated in detail in\nKaiser et al. (2019), using observational data from various station networks around the globe, from aircraft campaigns, and\nfrom satellite observations. For the present study, we apply the model configuration developed by Righi et al. (2020, 2021), which further includes an\n135\nexplicit representation of aerosol-radiation and aerosol-cloud interactions. Cloud microphysics and precipitation are dealt with\nby the CLOUD submodel, which in this configuration considers aerosol effects on warm, mixed-phase and cirrus clouds based\non the two-moment cloud scheme by Kuebbeler et al. (2014). Cloud-radiation and aerosol-radiation interactions are simulated\nby the submodels CLOUDOPT and RAD (Dietmüller et al., 2016), respectively. As the focus of this study lies on the radiative For the present study, we apply the model configuration developed by Righi et al. (2020, 2021), which further includes an\n135\nexplicit representation of aerosol-radiation and aerosol-cloud interactions. 2\nModel and model simulations Cloud microphysics and precipitation are dealt with\nby the CLOUD submodel, which in this configuration considers aerosol effects on warm, mixed-phase and cirrus clouds based\non the two-moment cloud scheme by Kuebbeler et al. (2014). Cloud-radiation and aerosol-radiation interactions are simulated\nby the submodels CLOUDOPT and RAD (Dietmüller et al., 2016), respectively. As the focus of this study lies on the radiative effects of aerosol and clouds, the concentrations of the radiatively active gases CO2, CH4, N2O, O3 and chlorofluorocarbons\n140\nare prescribed using globally averaged values for the year 2015. The studies by Kaiser et al. (2019) and Righi et al. (2020)\ndemonstrated the model’s ability to reproduce the relevant observed aerosol properties both close to the surface and in the\nmiddle and upper troposphere, as well as the main cloud and radiative properties. This configuration of EMAC is therefore\nsuited to the scope of the present study. effects of aerosol and clouds, the concentrations of the radiatively active gases CO2, CH4, N2O, O3 and chlorofluorocarbons\n140\nare prescribed using globally averaged values for the year 2015. The studies by Kaiser et al. (2019) and Righi et al. (2020)\ndemonstrated the model’s ability to reproduce the relevant observed aerosol properties both close to the surface and in the\nmiddle and upper troposphere, as well as the main cloud and radiative properties. This configuration of EMAC is therefore\nsuited to the scope of the present study. The chosen model resolution is T42L41, corresponding to a horizontal resolution of about 2.8\n◦× 2.8\n◦in latitude and longi-\n145\ntude (roughly 300 km at the Equator), with 41 non-equidistant vertical levels from the ground to about 5 hPa, mostly centered\nwithin the troposphere. All simulations cover a period of 11 years, between 2005 and 2015, with the first year used as spin-up\nand hence not included in the analysis. The model meteorology (temperature, winds and logarithm of the surface pressure) is\nnudged using the ERA-Interim reanalysis data for the same time period (Dee et al., 2011). The chosen model resolution is T42L41, corresponding to a horizontal resolution of about 2.8\n◦× 2.8\n◦in latitude and longi-\n145\ntude (roughly 300 km at the Equator), with 41 non-equidistant vertical levels from the ground to about 5 hPa, mostly centered\nwithin the troposphere. 2\nModel and model simulations We consider the results to be statistically significant when their As shown by R13, the perturbation method outlined above could lead to some inaccuracies in the estimated effects, due\nto the non-linearities in the involved processes, which affect in particular the secondary aerosol species. Alternative methods,\nsuch as for example tagging (Grewe, 2013; Rieger et al., 2018; Mertens et al., 2018), are hard to apply to the aerosol phase,\nespecially for secondary species, due to the difficulties in tracking the chemical processes taking place both in the gas phase and in the liquid phase (e.g., aerosol sulfate formation). Furthermore, the bulk of the climate effects estimated here are expected\n165\nto result from aerosol interactions with clouds, which cannot be isolated with the tagging approach, due to the prominent role\nof feedbacks. and in the liquid phase (e.g., aerosol sulfate formation). Furthermore, the bulk of the climate effects estimated here are expected\n165\nto result from aerosol interactions with clouds, which cannot be isolated with the tagging approach, due to the prominent role\nof feedbacks. 2\nModel and model simulations All simulations cover a period of 11 years, between 2005 and 2015, with the first year used as spin-up\nand hence not included in the analysis. The model meteorology (temperature, winds and logarithm of the surface pressure) is\nnudged using the ERA-Interim reanalysis data for the same time period (Dee et al., 2011). 150 As in R13, the effects of the transport sectors are quantified using the perturbation approach, in which a reference simulation\n150\nis compared to a sensitivity one where the emissions of a given sector are completely neglected. In the following sections, we\nshow both the absolute change (A) due to the emissions from the given sector and their relative contribution (R) to a specific\nquantity (e.g., burden or concentration). Considering, for instance, the land transport sector, these are defined as: As in R13, the effects of the transport sectors are quantified using the perturbation approach, in which a reference simulation\n150\nis compared to a sensitivity one where the emissions of a given sector are completely neglected. In the following sections, we\nshow both the absolute change (A) due to the emissions from the given sector and their relative contribution (R) to a specific\nquantity (e.g., burden or concentration). Considering, for instance, the land transport sector, these are defined as: A = REF −NOLAND\n(1) A = REF −NOLAND\n(1) (1) A = REF −NOLAND 5 5 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 155 (2) R = (REF −NOLAND)/REF where REF is the reference simulation and NOLAND the sensitivity simulation with land transport emissions switched off. The\nsame applies to the other two sectors, i.e. shipping and aviation. The statistical significance of A and R is verified by means\nof a two-tailed t-test with respect to the interannual variability. We consider the results to be statistically significant when their\nconfidence level is above 95%. 160 where REF is the reference simulation and NOLAND the sensitivity simulation with land transport emissions switched off. The\nsame applies to the other two sectors, i.e. shipping and aviation. The statistical significance of A and R is verified by means\nof a two-tailed t-test with respect to the interannual variability. 3\nEmissions of aerosol and precursors from the transport sectors The model simulations performed in this study are driven by the anthropogenic and open burni The model simulations performed in this study are driven by the anthropogenic and open burning emission datasets developed\nin support of the CMIP6 Project (Hoesly et al., 2018; van Marle et al., 2017). The emission data cover the historical period from\n170\nthe pre-industrial time (1750) to present-day (2014), and future projections until the year 2100. Here, we consider a present\nday case (2015) and two projections, for a near- (2030) and mid-term (2050) future, both under three SSPs: SSP1 (van Vuuren\net al., 2017), SSP2 (Fricko et al., 2017), and SSP3 (Fujimori et al., 2017). Although more SSPs are available in this inventory,\nthe three SSPs analysed here are fairly representative of possible futures, including: a sustainability scenario (SSP1) with in support of the CMIP6 Project (Hoesly et al., 2018; van Marle et al., 2017). The emission data cover the historical period from\n170\nthe pre-industrial time (1750) to present-day (2014), and future projections until the year 2100. Here, we consider a present\nday case (2015) and two projections, for a near- (2030) and mid-term (2050) future, both under three SSPs: SSP1 (van Vuuren\net al., 2017), SSP2 (Fricko et al., 2017), and SSP3 (Fujimori et al., 2017). Although more SSPs are available in this inventory,\nthe three SSPs analysed here are fairly representative of possible futures, including: a sustainability scenario (SSP1) with strong technology development and environmental concerns; a middle-of-the-road scenario (SSP2) reflecting a continuation\n175\nof historical trends in many aspects; and a regionally heterogeneous scenario (SSP3) characterized by slow economic growth,\nweak technology development and, most importantly for the present assessment, by a high level of air pollutant emissions,\nincluding aerosol. We do not consider the long-term future after 2050, as the projections beyond this time horizon are affected\nby large uncertainties in their driving forces (such as demographic changes, economic growth and technological development; see Bauer et al., 2017). The reason for choosing 2015 (from the SSP2 scenario) as the reference for the present day, instead of\n180\nthe last year of the historical CMIP6 time-series (2014), is to have consistent emissions from the same database, i.e. the SSPs. Note, however, that the small differences between 2014 and 2015 are negligible for the scope of the present assessment. 3\nEmissions of aerosol and precursors from the transport sectors A series of specific assumptions corresponds to each of the SSP narratives, concerning, for instance, population and eco-\nnomic growth, urbanisation level, policy implementation, technological advancements, availability of resources, human and societal developments, etc. These assumptions are then elaborated by integrated assessment models to produce emission pro-\n185\njections for greenhouse gases, short-lived gases and aerosol particles. Note that in their reference version, the SSPs storylines societal developments, etc. These assumptions are then elaborated by integrated assessment models to produce emission pro-\n185\njections for greenhouse gases, short-lived gases and aerosol particles. Note that in their reference version, the SSPs storylines 6 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. do not assume any implementation of climate policies. In the matrix framework of the SSPs (van Vuuren et al., 2014), climate\npolicies are included at a later stage, when the reference scenarios are combined with mitigation scenarios using the same\napproach as in the RCPs (Moss et al., 2010; van Vuuren et al., 2011), i.e., targeting a given anthropogenic RF in the year 2100. do not assume any implementation of climate policies. In the matrix framework of the SSPs (van Vuuren et al., 2014), climate\npolicies are included at a later stage, when the reference scenarios are combined with mitigation scenarios using the same\napproach as in the RCPs (Moss et al., 2010; van Vuuren et al., 2011), i.e., targeting a given anthropogenic RF in the year 2100. Among the various combinations of the SSPs with forcing pathways (Gidden et al., 2019), we choose here SSP1-1.9 (targeting\n190\nan anthropogenic RF of 1.9 W m−2 in 2100 and matching the 1.5◦C goal of the Paris Agreement), SSP2-4.5 (RF of 4.5 W m−2\nin 2100, close to the previous RCP4.5 scenario), and SSP3-7.0 (RF of 7.0 W m−2 in 2100). For simplicity, in the following we\nwill refer to the SSPs just by their number and skip the RF indication, as there is no ambiguity. do not assume any implementation of climate policies. In the matrix framework of the SSPs (van Vuuren et al., 2014), climate\npolicies are included at a later stage, when the reference scenarios are combined with mitigation scenarios using the same\napproach as in the RCPs (Moss et al., 2010; van Vuuren et al., 2011), i.e., targeting a given anthropogenic RF in the year 2100. 3\nEmissions of aerosol and precursors from the transport sectors Shipping and aviation contributions are generally small, but one should note that ships and\n200\naircraft mostly travel in regions which are otherwise hardly affected by anthropogenic emissions, hence their local contribution\nto atmospheric aerosol concentrations can be relevant even though the emissions are low. Future emissions in 2030 and 2050 strongly decrease with respect to present day in SSP1, consistently with the narrative\nunderlying this scenario. According to van Vuuren et al. (2017), SSP1 is characterized by a rapid decline in costs for clean especially for land transport). Shipping and aviation contributions are generally small, but one should note that ships and\n200\naircraft mostly travel in regions which are otherwise hardly affected by anthropogenic emissions, hence their local contribution\nto atmospheric aerosol concentrations can be relevant even though the emissions are low. Future emissions in 2030 and 2050 strongly decrease with respect to present day in SSP1, consistently with the narrative\nunderlying this scenario. According to van Vuuren et al. (2017), SSP1 is characterized by a rapid decline in costs for clean technologies, favoring a rapid transition to electric vehicles, and by an increase in public transport and car sharing. It further\n205\nassumes the worldwide implementation of strict air-quality policies, currently only applied in OECD countries. The high share\nof electric vehicles (up to 75%) leads to a strong decrease in NOx emissions in SSP1, whose contribution to the total is reduced\nfrom 26% in 2015 to 16% in 2030 and further down to 6.7% in 2050, while the contribution of aviation remains constant around\n2% and the shipping one is only reduced in 2050, due to the known challenges in electrifying these sectors. A similar trend can technologies, favoring a rapid transition to electric vehicles, and by an increase in public transport and car sharing. It further\n205\nassumes the worldwide implementation of strict air-quality policies, currently only applied in OECD countries. The high share\nof electric vehicles (up to 75%) leads to a strong decrease in NOx emissions in SSP1, whose contribution to the total is reduced\nfrom 26% in 2015 to 16% in 2030 and further down to 6.7% in 2050, while the contribution of aviation remains constant around\n2% and the shipping one is only reduced in 2050, due to the known challenges in electrifying these sectors. A similar trend can be seen for the other species. 3\nEmissions of aerosol and precursors from the transport sectors The reduction in SO2 emissions is stronger than for other species in SSP1, mostly driven by the\n210\ndecrease in the non-traffic anthropogenic emissions, which, however, maintain a roughly constant share around 85–90% of the\ntotal. The shipping sector contribution to SO2 remains relatively high until 2030, decreasing only slightly from 9% in 2015 to\n7% in 2030. This could be due to the phase-out of traditional (low-sulphur) biofuels in 2030 in this scenario and to the lack of\nalternative technologies before 2050, when the share is strongly reduced to 1.7%. be seen for the other species. The reduction in SO2 emissions is stronger than for other species in SSP1, mostly driven by the\n210\ndecrease in the non-traffic anthropogenic emissions, which, however, maintain a roughly constant share around 85–90% of the\ntotal. The shipping sector contribution to SO2 remains relatively high until 2030, decreasing only slightly from 9% in 2015 to\n7% in 2030. This could be due to the phase-out of traditional (low-sulphur) biofuels in 2030 in this scenario and to the lack of\nalternative technologies before 2050, when the share is strongly reduced to 1.7%. be seen for the other species. The reduction in SO2 emissions is stronger than for other species in SSP1, mostly driven by the\n210\ndecrease in the non-traffic anthropogenic emissions, which, however, maintain a roughly constant share around 85–90% of the\ntotal. The shipping sector contribution to SO2 remains relatively high until 2030, decreasing only slightly from 9% in 2015 to\n7% in 2030. This could be due to the phase-out of traditional (low-sulphur) biofuels in 2030 in this scenario and to the lack of\nalternative technologies before 2050, when the share is strongly reduced to 1.7%. In contrast to the optimistic SSP1 scenario, the SSP2 narrative (Fricko et al., 2017) assumes a significant use of fossil\n215\nfuel in the future, combined with less stringent air pollution reduction measures. This leads to an only moderate reduction in\nthe emissions of short-lived compounds in the future. The share of NOx emissions by land-transport and shipping remains\napproximately constant until 2050 (around 25% and 14%, respectively), while for aviation it almost doubles in 2030 (from\n1.9% to 3.4%) and triples in 2050 (5.3%). 3\nEmissions of aerosol and precursors from the transport sectors Among the various combinations of the SSPs with forcing pathways (Gidden et al., 2019), we choose here SSP1-1.9 (targeting\n190\nan anthropogenic RF of 1.9 W m−2 in 2100 and matching the 1.5◦C goal of the Paris Agreement), SSP2-4.5 (RF of 4.5 W m−2 Among the various combinations of the SSPs with forcing pathways (Gidden et al., 2019), we choose here SSP1-1.9 (targeting\n190\nan anthropogenic RF of 1.9 W m−2 in 2100 and matching the 1.5◦C goal of the Paris Agreement), SSP2-4.5 (RF of 4.5 W m−2\nin 2100, close to the previous RCP4.5 scenario), and SSP3-7.0 (RF of 7.0 W m−2 in 2100). For simplicity, in the following we\nwill refer to the SSPs just by their number and skip the RF indication, as there is no ambiguity. The total emissions of aerosol and precursor gases from all sectors in the CMIP6 inventory are summarized in Fig 1 for Among the various combinations of the SSPs with forcing pathways (Gidden et al., 2019), we choose here SSP1-1.9 (targeting\n190\nan anthropogenic RF of 1.9 W m−2 in 2100 and matching the 1.5◦C goal of the Paris Agreement), SSP2-4.5 (RF of 4.5 W m−2\nin 2100, close to the previous RCP4.5 scenario), and SSP3-7.0 (RF of 7.0 W m−2 in 2100). For simplicity, in the following we\nwill refer to the SSPs just by their number and skip the RF indication, as there is no ambiguity. The total emissions of aerosol and precursor gases from all sectors in the CMIP6 inventory are summarized in Fig. 1, for The total emissions of aerosol and precursor gases from all sectors in the CMIP6 inventory are summarized in Fig. 1, for\nthe present-day (2015) and the three SSPs for the years 2030 and 2050. The total emissions of the anthropogenic non-transport\n195\nsectors (comprising energy, industry, residential and commercial, agriculture, solvents production and application, and waste)\ndominate the emission budget of all species in all scenarios, with relative contributions over 90% to NH3 emissions (Fig. 1c)\nmostly due to the agriculture sector. The transport sectors are particularly relevant for NOx emissions (Fig. 1a), with large\nrelative contributions from land transport and shipping (mainly driven by emissions from diesel engines) and BC (Fig. 1e, especially for land transport). 3\nEmissions of aerosol and precursors from the transport sectors This is related to the increase in air traffic volumes combined with the lack of In contrast to the optimistic SSP1 scenario, the SSP2 narrative (Fricko et al., 2017) assumes a significant use of fossil\n215\nfuel in the future, combined with less stringent air pollution reduction measures. This leads to an only moderate reduction in\nthe emissions of short-lived compounds in the future. The share of NOx emissions by land-transport and shipping remains\napproximately constant until 2050 (around 25% and 14%, respectively), while for aviation it almost doubles in 2030 (from\n1.9% to 3.4%) and triples in 2050 (5.3%). This is related to the increase in air traffic volumes combined with the lack of In contrast to the optimistic SSP1 scenario, the SSP2 narrative (Fricko et al., 2017) assumes a significant use of fossil\n215\nfuel in the future, combined with less stringent air pollution reduction measures. This leads to an only moderate reduction in\nthe emissions of short-lived compounds in the future. The share of NOx emissions by land-transport and shipping remains\napproximately constant until 2050 (around 25% and 14%, respectively), while for aviation it almost doubles in 2030 (from\n1.9% to 3.4%) and triples in 2050 (5.3%). This is related to the increase in air traffic volumes combined with the lack of efficient alternative technologies for emissions reduction in this sector, which only in SSP1 is counteracted by a reduction in\n220\nair traffic. Similar trends can be seen for the other species in Fig. 1, with the exception of SO2 for shipping. In this case, a very efficient alternative technologies for emissions reduction in this sector, which only in SSP1 is counteracted by a reduction in\n220\nair traffic. Similar trends can be seen for the other species in Fig. 1, with the exception of SO2 for shipping. In this case, a very 7 7 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 3\nEmissions of aerosol and precursors from the transport sectors 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n25\n50\n75\n100\n125\n150\n175\n[Tg(NO2) yr\n1]\n29\n14\n2.2\n40\n15\n26\n14\n1.9\n44\n13\n16\n15\n2.1\n47\n20\n23\n14\n3.4\n46\n13\n27\n11\n2.6\n48\n11\n6.7\n2.1\n2.1\n57\n33\n26\n14\n5.3\n40\n15\n29\n7.4\n3.3\n49\n11\n(a) - NOx emissions\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n200\n400\n600\n800\n1000\n[Tg(CO) yr\n1]\n21\n0.1\n<0.1\n34\n45\n14\n<0.1\n<0.1\n45\n41\n12\n0.1\n0.1\n36\n52\n15\n<0.1\n0.1\n48\n36\n15\n<0.1\n<0.1\n52\n33\n5.0\n<0.1\n0.1\n37\n58\n18\n<0.1\n0.1\n43\n39\n15\n<0.1\n0.1\n53\n32\n(b) - CO emissions\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n20\n40\n60\n80\n[Tg(NH3) yr\n1]\n1.0\n<0.1\n<0.1\n75\n24\n0.6\n<0.1\n<0.1\n92\n7.4\n0.6\n<0.1\n<0.1\n94\n4.9\n0.5\n<0.1\n<0.1\n94\n5.8\n0.7\n<0.1\n<0.1\n93\n6.0\n0.6\n<0.1\n<0.1\n95\n4.6\n0.4\n<0.1\n<0.1\n94\n6.0\n0.7\n<0.1\n<0.1\n93\n6.5\n(c) - NH3 emissions\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n20\n40\n60\n80\n100\n[Tg(SO2) yr\n1]\n4.0\n10\n0.2\n82\n3.8\n3.2\n9.1\n0.3\n85\n2.4\n1.6\n7.0\n0.8\n86\n5.1\n2.9\n3.1\n0.5\n91\n2.8\n4.4\n6.6\n0.4\n86\n2.2\n0.6\n1.7\n1.3\n88\n8.1\n3.7\n0.4\n0.9\n91\n4.1\n5.0\n3.4\n0.5\n89\n2.3\n(d) - SO2 emissions\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n2\n4\n6\n8\n10\n[Tg(BC) yr\n1]\n17\n1.7\n<0.1\n45\n35\n14\n1.7\n0.1\n65\n19\n12\n2.6\n0.2\n53\n33\n15\n1.9\n0.2\n62\n21\n14\n1.3\n0.1\n68\n16\n5.1\n0.9\n0.1\n44\n50\n19\n0.3\n0.3\n56\n24\n16\n0.9\n0.2\n67\n15\n(e) - BC emissions\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n10\n20\n30\n40\n[Tg(OC) yr\n1]\n4.0\n0.4\n<0.1\n29\n67\n1.6\n0.3\n<0.1\n49\n49\n0.6\n0.6\n<0.1\n40\n59\n1.2\n0.4\n<0.1\n53\n46\n1.7\n0.3\n<0.1\n58\n40\n0.2\n0.5\n<0.1\n34\n65\n1.3\n<0.1\n<0.1\n49\n50\n1.8\n0.3\n<0.1\n60\n38\n(f) - OC emissions\nLand transport\nShipping\nAviation\nOther anthrop. 3\nEmissions of aerosol and precursors from the transport sectors Open burning\nFigure 1. Total emissions of each species from the different sectors in the CMIP6/SSP inventory, compared with the year 2000 emissions\nfrom the CMIP5 inventory (Lamarque et al., 2010) used in Righi et al. (2013). The numbers on the left of each bar indicate the relative\ncontribution (in percent) of each sector to the total. OC emissions are multiplied by a factor 1.4 in the model to obtain particulate organic\nmatter (POM) emissions, as required by the MADE3 aerosol submodel. Furthermore, a small fraction (∼2%, depending on the sector) of the\nSO2 emitted mass is emitted as primary aerosol sulfate in the model (see Kaiser et al., 2019, for more details). Note that the vertical scales\ndiffer among the panels. 3\nEmissions of aerosol and precursors from the transport sectors 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n200\n400\n600\n800\n1000\n[Tg(CO) yr\n1]\n21\n0.1\n<0.1\n34\n45\n14\n<0.1\n<0.1\n45\n41\n12\n0.1\n0.1\n36\n52\n15\n<0.1\n0.1\n48\n36\n15\n<0.1\n<0.1\n52\n33\n5.0\n<0.1\n0.1\n37\n58\n18\n<0.1\n0.1\n43\n39\n15\n<0.1\n0.1\n53\n32\n(b) - CO emissions\n(d)\nSO\nemissions 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n25\n50\n75\n100\n125\n150\n175\n[Tg(NO2) yr\n1]\n29\n14\n2.2\n40\n15\n26\n14\n1.9\n44\n13\n16\n15\n2.1\n47\n20\n23\n14\n3.4\n46\n13\n27\n11\n2.6\n48\n11\n6.7\n2.1\n2.1\n57\n33\n26\n14\n5.3\n40\n15\n29\n7.4\n3.3\n49\n11\n(a) - NOx emissions\n(c)\nNH\nemissions 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n20\n40\n60\n80\n100\n[Tg(SO2) yr\n1]\n4.0\n10\n0.2\n82\n3.8\n3.2\n9.1\n0.3\n85\n2.4\n1.6\n7.0\n0.8\n86\n5.1\n2.9\n3.1\n0.5\n91\n2.8\n4.4\n6.6\n0.4\n86\n2.2\n0.6\n1.7\n1.3\n88\n8.1\n3.7\n0.4\n0.9\n91\n4.1\n5.0\n3.4\n0.5\n89\n2.3\n(d) - SO2 emissions 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n20\n40\n60\n80\n[Tg(NH3) yr\n1]\n1.0\n<0.1\n<0.1\n75\n24\n0.6\n<0.1\n<0.1\n92\n7.4\n0.6\n<0.1\n<0.1\n94\n4.9\n0.5\n<0.1\n<0.1\n94\n5.8\n0.7\n<0.1\n<0.1\n93\n6.0\n0.6\n<0.1\n<0.1\n95\n4.6\n0.4\n<0.1\n<0.1\n94\n6.0\n0.7\n<0.1\n<0.1\n93\n6.5\n(c) - NH3 emissions\n( )\nC\ni\ni 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n2\n4\n6\n8\n10\n[Tg(BC) yr\n1]\n17\n1.7\n<0.1\n45\n35\n14\n1.7\n0.1\n65\n19\n12\n2.6\n0.2\n53\n33\n15\n1.9\n0.2\n62\n21\n14\n1.3\n0.1\n68\n16\n5.1\n0.9\n0.1\n44\n50\n19\n0.3\n0.3\n56\n24\n16\n0.9\n0.2\n67\n15\n(e) - BC emissions\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n0\n10\n20\n30\n40\n[Tg(OC) yr\n1]\n4.0\n0.4\n<0.1\n29\n67\n1.6\n0.3\n<0.1\n49\n49\n0.6\n0.6\n<0.1\n40\n59\n1.2\n0.4\n<0.1\n53\n46\n1.7\n0.3\n<0.1\n58\n40\n0.2\n0.5\n<0.1\n34\n65\n1.3\n<0.1\n<0.1\n49\n50\n1.8\n0.3\n<0.1\n60\n38\n(f) - OC emissions\nLand transport\nShipping\nAviation\nOther anthrop. 3\nEmissions of aerosol and precursors from the transport sectors Open burning Figure 1. Total emissions of each species from the different sectors in the CMIP6/SSP inventory, compared with the year 2000 emissions\nfrom the CMIP5 inventory (Lamarque et al., 2010) used in Righi et al. (2013). The numbers on the left of each bar indicate the relative\ncontribution (in percent) of each sector to the total. OC emissions are multiplied by a factor 1.4 in the model to obtain particulate organic\nmatter (POM) emissions, as required by the MADE3 aerosol submodel. Furthermore, a small fraction (∼2%, depending on the sector) of the\nSO2 emitted mass is emitted as primary aerosol sulfate in the model (see Kaiser et al., 2019, for more details). Note that the vertical scales\ndiffer among the panels. 8 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. marked decrease in the share of this sector is evident in SSP2, decreasing from 9.1% in 2015 to 3.1% in 2030 and 0.4% in\n2050, representing the lowest share of this sector among the three investigated scenarios. SSP3 represents a more pessimistic scenario, implementing weak air pollution reduction measures, combined with continued\ndependency on fossil fuels and slow implementation of new technologies (Fujimori et al., 2017). This scenario further considers\n225\na late saturation in transport demand, combined with a low electrification level (up to 10%). This leads to an increase in the\nland-transport share of NOx which, together with the increase in the other non-traffic anthropogenic sources, drives the overall\nincrease of NOx emissions in SSP3 in 2030. The total NOx emissions remain then approximately constant until 2050, thanks\nto the reduced share of the shipping sector and the shift to alternative fuels. Due to the rapid shift to high speed transport modes\nassumed in SSP3 the share of aviation increases as well from 1 9% in 2015 to 2 6% in 2030 and 3 3% in 2050 although this\n230 SSP3 represents a more pessimistic scenario, implementing weak air pollution reduction measures, combined with continued\ndependency on fossil fuels and slow implementation of new technologies (Fujimori et al., 2017). This scenario further considers\n225\na late saturation in transport demand, combined with a low electrification level (up to 10%). This leads to an increase in the\nland-transport share of NOx which, together with the increase in the other non-traffic anthropogenic sources, drives the overall\nincrease of NOx emissions in SSP3 in 2030. 3\nEmissions of aerosol and precursors from the transport sectors The total NOx emissions remain then approximately constant until 2050, thanks\nto the reduced share of the shipping sector and the shift to alternative fuels. Due to the rapid shift to high speed transport modes assumed in SSP3, the share of aviation increases as well, from 1.9% in 2015 to 2.6% in 2030 and 3.3% in 2050, although this\n230\nincrease is less marked than in SSP2. An analogous trend is also evident for SO2, BC and OC emissions, although the latter is\npartly compensated by a reduction of open burning emissions in 2030 and 2050. Since in the present work we use an updated inventory (CMIP6/SSP) with respect to our previous assessment (CMIP5/RCP;\nR13), in Fig. 1 we also compare the total emissions between these two inventories. Note that not only the inventory was ),\ng\np\ny\ny\nupdated, but we also considered a different baseline year for present-day conditions (2015 instead of 2000). A general increase\n235\nin the total emissions can be seen, mostly driven by the anthropogenic non-transport sectors, while only the emissions of\nCO (Fig. 1b) and SO2 (Fig. 1d) slightly decrease. The transport sectors mostly show a decrease in emissions between 2000\nand 2015, both in absolute and relative terms, with the notable exception of NOx (Fig. 1a) and BC (Fig. 1e), whose share,\nhowever, decreased from 29% to 26% and from 17% to 14%, respectively. This indicates that, although the overall growth updated, but we also considered a different baseline year for present-day conditions (2015 instead of 2000). A general increase\n235\nin the total emissions can be seen, mostly driven by the anthropogenic non-transport sectors, while only the emissions of\nCO (Fig. 1b) and SO2 (Fig. 1d) slightly decrease. The transport sectors mostly show a decrease in emissions between 2000\nand 2015, both in absolute and relative terms, with the notable exception of NOx (Fig. 1a) and BC (Fig. 1e), whose share,\nhowever, decreased from 29% to 26% and from 17% to 14%, respectively. This indicates that, although the overall growth updated, but we also considered a different baseline year for present-day conditions (2015 instead of 2000). A general increase\n235\nin the total emissions can be seen, mostly driven by the anthropogenic non-transport sectors, while only the emissions of\nCO (Fig. 1b) and SO2 (Fig. 1d) slightly decrease. 3\nEmissions of aerosol and precursors from the transport sectors The transport sectors mostly show a decrease in emissions between 2000\nand 2015, both in absolute and relative terms, with the notable exception of NOx (Fig. 1a) and BC (Fig. 1e), whose share,\nhowever, decreased from 29% to 26% and from 17% to 14%, respectively. This indicates that, although the overall growth of land transport volumes leads of course to an increase in emissions of these two highly relevant species for this sector, the\n240\nintroduction of air pollution control measures (e.g., on diesel engines) in the developed countries helped to reduce the relative\nimpact of the sector on the global emission budget. Another interesting case is SO2 (Fig. 1d) from shipping, which shows\na decrease in both absolute and relative terms between 2000 and 2015. This is a consequence of the limits on shipping FSC\nintroduced starting from 2010 in the MARPOL Annex VI (Buhaug et al., 2009). These measures also affected shipping NOx of land transport volumes leads of course to an increase in emissions of these two highly relevant species for this sector, the\n240\nintroduction of air pollution control measures (e.g., on diesel engines) in the developed countries helped to reduce the relative\nimpact of the sector on the global emission budget. Another interesting case is SO2 (Fig. 1d) from shipping, which shows\na decrease in both absolute and relative terms between 2000 and 2015. This is a consequence of the limits on shipping FSC\nintroduced starting from 2010 in the MARPOL Annex VI (Buhaug et al., 2009). These measures also affected shipping NOx of land transport volumes leads of course to an increase in emissions of these two highly relevant species for this sector, the\n240\nintroduction of air pollution control measures (e.g., on diesel engines) in the developed countries helped to reduce the relative\nimpact of the sector on the global emission budget. Another interesting case is SO2 (Fig. 1d) from shipping, which shows\na decrease in both absolute and relative terms between 2000 and 2015. This is a consequence of the limits on shipping FSC\nintroduced starting from 2010 in the MARPOL Annex VI (Buhaug et al., 2009). These measures also affected shipping NOx emissions, which however still grew between 2000 and 2015 (Fig. 1a), but keeping a stable share of 14% of the total. 3\nEmissions of aerosol and precursors from the transport sectors The\n245\nemissions from the aviation sector increase for all species between 2000 and 2015, which is expected since emission reduction\nmeasures in this sector are over-compensated by the high growth rates in traffic volumes during this period. An important\nimprovement in the CMIP6 inventory is the consideration of the emissions from more species in the aviation sector, while in\nCMIP5 only NOx and BC were available. In R13 we nevertheless included SO2 emissions from aviation, deriving them from\nBC emissions by scaling with the respective emission factors of the two species\n250 emissions, which however still grew between 2000 and 2015 (Fig. 1a), but keeping a stable share of 14% of the total. The\n245\nemissions from the aviation sector increase for all species between 2000 and 2015, which is expected since emission reduction\nmeasures in this sector are over-compensated by the high growth rates in traffic volumes during this period. An important\nimprovement in the CMIP6 inventory is the consideration of the emissions from more species in the aviation sector, while in\nCMIP5 only NOx and BC were available. In R13 we nevertheless included SO2 emissions from aviation, deriving them from emissions, which however still grew between 2000 and 2015 (Fig. 1a), but keeping a stable share of 14% of the total. The\n245\nemissions from the aviation sector increase for all species between 2000 and 2015, which is expected since emission reduction\nmeasures in this sector are over-compensated by the high growth rates in traffic volumes during this period. An important\nimprovement in the CMIP6 inventory is the consideration of the emissions from more species in the aviation sector, while in\nCMIP5 only NOx and BC were available. In R13 we nevertheless included SO2 emissions from aviation, deriving them from BC emissions by scaling with the respective emission factors of the two species. 250\nThe emission inventories applied here only provide data for the emitted mass of each compounds, whereas the MADE3\naerosol submodel requires emission fluxes for both mass and number. The latter is derived by assuming typical log-normal size\ndistributions for the emitted particles in each sector, based on measurements when available. Further assumptions are made to\nassign the emitted mass to the various modes of MADE3. This follows the same approach of Kaiser et al. (2019), summarized BC emissions by scaling with the respective emission factors of the two species. 3\nEmissions of aerosol and precursors from the transport sectors 250\nThe emission inventories applied here only provide data for the emitted mass of each compounds, whereas the MADE3\naerosol submodel requires emission fluxes for both mass and number. The latter is derived by assuming typical log-normal size\ndistributions for the emitted particles in each sector, based on measurements when available. Further assumptions are made to\nassign the emitted mass to the various modes of MADE3. This follows the same approach of Kaiser et al. (2019), summarized BC emissions by scaling with the respective emission factors of the two species. 250\nThe emission inventories applied here only provide data for the emitted mass of each compounds, whereas the MADE3\naerosol submodel requires emission fluxes for both mass and number. The latter is derived by assuming typical log-normal size\ndistributions for the emitted particles in each sector, based on measurements when available. Further assumptions are made to\nassign the emitted mass to the various modes of MADE3. This follows the same approach of Kaiser et al. (2019), summarized in Table 1. For the open burning sector, we follow the AeroCom recommendations (Dentener et al., 2006) and assign the aerosol\n255\nemissions of primary sulfate (SO4), BC and OC to the accumulation mode. For the anthropogenic non-traffic sector, we use the in Table 1. For the open burning sector, we follow the AeroCom recommendations (Dentener et al., 2006) and assign the aerosol\n255\nemissions of primary sulfate (SO4), BC and OC to the accumulation mode. For the anthropogenic non-traffic sector, we use the in Table 1. For the open burning sector, we follow the AeroCom recommendations (Dentener et al., 2006) and assign the aerosol\n255\nemissions of primary sulfate (SO4), BC and OC to the accumulation mode. For the anthropogenic non-traffic sector, we use the 9 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 1. Summary of the emission setup used for the model simulations in this study, together with the log-normal size distribution param-\neters applied to derive number emission fluxes from the mass. MF is the mass fraction in the Aitken (akn) and accumulation (acc) mode. D (geometric mean diameter) and σ (standard deviation) are the parameters of the log-normal distribution. See Kaiser et al. (2019) and\nreferences therein for a detailed discussions about these parameters. 3\nEmissions of aerosol and precursors from the transport sectors Sector\nSpecie\nMFakn [%]\nDakn [nm]\nσakn\nMFacc [%]\nDacc [nm]\nσacc\nOpen burning\nSO4\n–\n–\n–\n100\n80\n1.8\nBC/POM\n–\n–\n–\n100\n80\n1.8\nAntrop. non-traffic\nSO4\n–\n–\n–\n–\n–\n–\nBC/POM\n–\n–\n–\n100\n138\n1.59\nLand transport\nSO4\n100\n30\n1.80\n–\n–\n–\nBC/POM\n10\n58\n1.58\n90\n138\n1.59\nShipping\nSO4\n10\n70\n1.45\n90\n260\n1.25\nBC/POM\n10\n70\n1.45\n90\n260\n1.25\nAviation\nSO4\n91\n25\n1.55\n9\n150\n1.65\nBC/POM\n91\n25\n1.55\n9\n150\n1.65 parameters measured by Birmili et al. (2009) in the urban background of Berlin, hence assuming an aged aerosol population. This assigns the emissions to the accumulation mode only. Note that no primary SO4 emissions are considered for this sector. We use the same parameters also for land transport, but in this case we assign 10% of the emitted mass to the Aitken mode, representative of a fresh aerosol population in the urban areas close to the emission sources. Primary SO4 emissions from land\n260\ntransport follow again the AeroCom recommendations, assigning the emitted mass to Aitken mode only (note, however, that\nthe land transport emissions of SO4 are relatively low due to the low-sulphur content of gasoline and diesel fuels used for land\ntransport vehicles in most regions of the world). For shipping, we used the parameters measured by Petzold et al. (2008) in ship\nplumes. As for the land transport sector, this assumes a 10/90% split of emitted mass between Aitken and accumulation mode, respectively, but with much larger particles in the accumulation mode (260 nm vs. 138 nm). As noted by Petzold et al. (2008)\n265\nand R13, due to a more efficient mixing, the aging process is particularly effective in the marine boundary layer, leading to\na relatively fast growth of particles in ship plumes. In-situ measurements in actual plumes are also used to derive the parameters\nfor the aviation emissions (Petzold et al., 1999). Due to the high burning efficiency of aircraft turbines, the emitted BC and\nOC particles are small, with an Aitken mode at 25 nm median diameter comprising about 90% of the emitted mass. Here we respectively, but with much larger particles in the accumulation mode (260 nm vs. 138 nm). As noted by Petzold et al. 3\nEmissions of aerosol and precursors from the transport sectors (2008)\n265\nand R13, due to a more efficient mixing, the aging process is particularly effective in the marine boundary layer, leading to\na relatively fast growth of particles in ship plumes. In-situ measurements in actual plumes are also used to derive the parameters\nfor the aviation emissions (Petzold et al., 1999). Due to the high burning efficiency of aircraft turbines, the emitted BC and\nOC particles are small, with an Aitken mode at 25 nm median diameter comprising about 90% of the emitted mass. Here we conservatively assume the same size distribution also for primary SO4, although their actual size is much more uncertain and\n270\nsome studies (e.g. Kärcher et al., 2007) show that small, nucleation-size sulfate particles of the order of a few nanometers are\npresent in aircraft plumes during the dispersion phase. conservatively assume the same size distribution also for primary SO4, although their actual size is much more uncertain and\n270\nsome studies (e.g. Kärcher et al., 2007) show that small, nucleation-size sulfate particles of the order of a few nanometers are\npresent in aircraft plumes during the dispersion phase. 10 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4\nTransport impact on aerosol concentrations Fig. 2 shows the impact of land transport, shipping and aviation on the burdens of various aerosol species and of aerosol\nparticle number. To obtain more representative values, the burdens are calculated integrating the aerosol concentrations over\n275\nthe domains relevant to the respective sector. For land transport and shipping, the two lowermost model layers (from the surface\nto an altitude of about 250 m) are considered, over the continents and over the oceans, respectively. Aviation concentrations are\nintegrated globally at the typical cruise altitudes between about 8 and 12 km. This approach is consistent with the one adopted\nin R13, thus allowing for a direct comparison of the results. In the following, we analyse the impact of the emissions from the three sectors on the burdens and concentrations of relevant aerosol species, for the present-day (2015) and the three SSP\n280\nscenarios in 2030 and 2050. We also compare with the results for the year 2000 obtained with the CMIP5 inventory assessed\nby R13. For a correct interpretation of the concentration values discussed in the following, it should be noted that these are\nlarge-scale mean concentrations, which are smaller than the peak concentrations occurring close to the emission sources (e.g.,\nat kerbside). Such peak values are highly relevant for assessing air pollution effects, but cannot be resolved by large-scale\nclimate models which rely on mean concentrations driving large-scale climate effects. 285 the three sectors on the burdens and concentrations of relevant aerosol species, for the present-day (2015) and the three SSP\n280\nscenarios in 2030 and 2050. We also compare with the results for the year 2000 obtained with the CMIP5 inventory assessed\nby R13. For a correct interpretation of the concentration values discussed in the following, it should be noted that these are\nlarge-scale mean concentrations, which are smaller than the peak concentrations occurring close to the emission sources (e.g.,\nat kerbside). Such peak values are highly relevant for assessing air pollution effects, but cannot be resolved by large-scale\nclimate models which rely on mean concentrations driving large-scale climate effects. 4\nTransport impact on aerosol concentrations 285 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n1.8\n2.9\n2.7\n2.5\n1.3\n0.2\n2.1\n1.6\n2.1\n6.3\n2.2\n0.02\n0.8\n0.3\n0.5\n3.3\n1.1\n0.005\n1.8\n1.0\n0.9\n6.1\n1.9\n0.01\n2.4\n1.7\n2.2\n8.0\n2.8\n0.02\n0.2\n0.09\n0.2\n1.1\n0.3\n0.001\n1.7\n1.0\n0.8\n7.8\n2.4\n0.01\n2.9\n2.0\n2.1\n9.0\n3.1\n0.03\n(a) - Land transport (absolute impact)\n-2\n0\n2\n4\n6\n8\n10\n12\nmass [Gg] / number [1026 particles]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n0.2\n0.3\n9.4\n1.1\n1.0\n0.01\n0.3\n0.3\n6.3\n6.9\n1.6\n0.003\n0.2\n0.2\n2.4\n4.9\n0.6\n0.001\n0.2\n0.3\n2.9\n6.5\n0.7\n0.001\n0.2\n0.3\n4.8\n6.0\n1.2\n0.002\n0.04\n0.2\n0.4\n0.5\n0.08\n0.001\n0.03\n0.03\n1.5\n5.9\n0.4\n-0.001\n0.1\n0.2\n2.9\n4.1\n0.7\n0.001\n(b) - Shipping (absolute impact)\n-2\n0\n2\n4\n6\n8\n10\n12\nmass [Gg] / number [1026 particles]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n0.1\n0.000\n3.2\n-1.1\n-0.2\n12.3\n0.3\n1.0\n7.1\n0.1\n1.7\n1.1\n0.1\n0.4\n5.2\n0.7\n1.7\n1.3\n0.3\n1.2\n8.9\n1.7\n2.8\n1.5\n0.4\n1.4\n10.0\n0.1\n2.5\n1.5\n0.09\n0.6\n5.8\n2.1\n2.3\n1.4\n0.3\n1.2\n10.4\n4.5\n4.1\n1.9\n0.5\n1.6\n11.9\n0.8\n3.3\n1.7\n(c) - Aviation (absolute impact)\n-2\n0\n2\n4\n6\n8\n10\n12\nmass [Gg] / number [1026 particles]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n19.5\n3.5\n3.5\n28.6\n4.8\n12.8\n18.5\n2.2\n3.0\n24.6\n8.1\n9.1\n16.3\n0.6\n1.6\n17.0\n6.9\n3.4\n19.4\n1.5\n1.4\n22.9\n7.0\n7.4\n19.3\n2.2\n3.2\n25.6\n9.2\n10.7\n7.6\n0.2\n0.7\n7.7\n3.3\n1.0\n23.8\n1.6\n1.6\n29.0\n11.0\n7.9\n21.9\n2.5\n3.0\n26.2\n9.4\n11.9\n(d) - Land transport (relative contribution)\n-5\n0\n5\n10\n15\n20\n25\n30\n[%]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n6.0\n1.4\n13.6\n24.0\n6.9\n3.3\n4.8\n1.0\n9.0\n24.5\n8.3\n1.5\n6.8\n1.1\n5.8\n23.5\n5.9\n0.8\n5.0\n1.0\n4.5\n24.0\n4.2\n0.8\n3.5\n0.8\n6.8\n20.1\n6.2\n1.1\n2.7\n1.1\n1.2\n3.7\n1.1\n0.9\n0.9\n0.1\n2.9\n23.0\n2.9\n-0.4\n2.5\n0.7\n4.0\n13.9\n3.6\n0.8\n(e) - Shipping (relative contribution)\n-5\n0\n5\n10\n15\n20\n25\n30\n[%]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n1.8\n0.000\n2.9\n-4.8\n-0.6\n11.5\n4.6\n2.1\n4.3\n0.1\n2.6\n6.2\n4.1\n1.0\n4.7\n0.6\n2.7\n8.9\n5.8\n2.5\n5.7\n2.0\n4.0\n8.7\n5.8\n2.7\n5.9\n0.2\n3.5\n8.4\n4.1\n1.8\n5.5\n1.6\n3.5\n9.2\n6.8\n2.7\n7.8\n3.8\n5.7\n12.0\n6.6\n3.1\n6.8\n0.8\n4.3\n9.7\n(f) - Aviation (relative contribution)\n-5\n0\n5\n10\n15\n20\n25\n30\n[%]\nFigure 2. 4\nTransport impact on aerosol concentrations Absolute impact (a-c) and relative contribution (d-f) of the three transport sectors to the burdens of various aerosol species and o\nparticle number. Changes which are not statistically significant at a 95% confidence level are shown in gray. Note the different units for mass\n(Gg) and number burdens (1026 particles) in panels (a-c). 4\nTransport impact on aerosol concentrations 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n1.8\n2.9\n2.7\n2.5\n1.3\n0.2\n2.1\n1.6\n2.1\n6.3\n2.2\n0.02\n0.8\n0.3\n0.5\n3.3\n1.1\n0.005\n1.8\n1.0\n0.9\n6.1\n1.9\n0.01\n2.4\n1.7\n2.2\n8.0\n2.8\n0.02\n0.2\n0.09\n0.2\n1.1\n0.3\n0.001\n1.7\n1.0\n0.8\n7.8\n2.4\n0.01\n2.9\n2.0\n2.1\n9.0\n3.1\n0.03\n(a) - Land transport (absolute impact)\n-2\n0\n2\n4\n6\n8\n10\n12\nmass [Gg] / number [1026 particles]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n0.2\n0.3\n9.4\n1.1\n1.0\n0.01\n0.3\n0.3\n6.3\n6.9\n1.6\n0.003\n0.2\n0.2\n2.4\n4.9\n0.6\n0.001\n0.2\n0.3\n2.9\n6.5\n0.7\n0.001\n0.2\n0.3\n4.8\n6.0\n1.2\n0.002\n0.04\n0.2\n0.4\n0.5\n0.08\n0.001\n0.03\n0.03\n1.5\n5.9\n0.4\n-0.001\n0.1\n0.2\n2.9\n4.1\n0.7\n0.001\n(b) - Shipping (absolute impact)\n-2\n0\n2\n4\n6\n8\n10\n12\nmass [Gg] / number [1026 particles]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n0.1\n0.000\n3.2\n-1.1\n-0.2\n12.3\n0.3\n1.0\n7.1\n0.1\n1.7\n1.1\n0.1\n0.4\n5.2\n0.7\n1.7\n1.3\n0.3\n1.2\n8.9\n1.7\n2.8\n1.5\n0.4\n1.4\n10.0\n0.1\n2.5\n1.5\n0.09\n0.6\n5.8\n2.1\n2.3\n1.4\n0.3\n1.2\n10.4\n4.5\n4.1\n1.9\n0.5\n1.6\n11.9\n0.8\n3.3\n1.7\n(c) - Aviation (absolute impact)\n-2\n0\n2\n4\n6\n8\n10\n12\nmass [Gg] / number [1026 particles] 2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n19.5\n3.5\n3.5\n28.6\n4.8\n12.8\n18.5\n2.2\n3.0\n24.6\n8.1\n9.1\n16.3\n0.6\n1.6\n17.0\n6.9\n3.4\n19.4\n1.5\n1.4\n22.9\n7.0\n7.4\n19.3\n2.2\n3.2\n25.6\n9.2\n10.7\n7.6\n0.2\n0.7\n7.7\n3.3\n1.0\n23.8\n1.6\n1.6\n29.0\n11.0\n7.9\n21.9\n2.5\n3.0\n26.2\n9.4\n11.9\n(d) - Land transport (relative contribution)\n-5\n0\n5\n10\n15\n20\n25\n30\n[%]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n6.0\n1.4\n13.6\n24.0\n6.9\n3.3\n4.8\n1.0\n9.0\n24.5\n8.3\n1.5\n6.8\n1.1\n5.8\n23.5\n5.9\n0.8\n5.0\n1.0\n4.5\n24.0\n4.2\n0.8\n3.5\n0.8\n6.8\n20.1\n6.2\n1.1\n2.7\n1.1\n1.2\n3.7\n1.1\n0.9\n0.9\n0.1\n2.9\n23.0\n2.9\n-0.4\n2.5\n0.7\n4.0\n13.9\n3.6\n0.8\n(e) - Shipping (relative contribution)\n-5\n0\n5\n10\n15\n20\n25\n30\n[%]\n2000\nCMIP5\n2015\nCMIP6\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\nBC\nPOM\nSO4\nNO3\nNH4\nParticle\nnumber\n1.8\n0.000\n2.9\n-4.8\n-0.6\n11.5\n4.6\n2.1\n4.3\n0.1\n2.6\n6.2\n4.1\n1.0\n4.7\n0.6\n2.7\n8.9\n5.8\n2.5\n5.7\n2.0\n4.0\n8.7\n5.8\n2.7\n5.9\n0.2\n3.5\n8.4\n4.1\n1.8\n5.5\n1.6\n3.5\n9.2\n6.8\n2.7\n7.8\n3.8\n5.7\n12.0\n6.6\n3.1\n6.8\n0.8\n4.3\n9.7\n(f) - Aviation (relative contribution)\n-5\n0\n5\n10\n15\n20\n25\n30\n[%] Figure 2. 4\nTransport impact on aerosol concentrations Absolute impact (a-c) and relative contribution (d-f) of the three transport sectors to the burdens of various aerosol species and of\nparticle number. Changes which are not statistically significant at a 95% confidence level are shown in gray. Note the different units for mass\n(Gg) and number burdens (1026 particles) in panels (a-c). Figure 2. Absolute impact (a-c) and relative contribution (d-f) of the three transport sectors to the burdens of various aerosol species and of\nparticle number. Changes which are not statistically significant at a 95% confidence level are shown in gray. Note the different units for mass\n(Gg) and number burdens (1026 particles) in panels (a-c). 11 11 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.1\nLand transport The contribution of land transport to present-day aerosol burdens is particularly large for BC and aerosol nitrate (NO3), see\nFig. 2a. The continental near-surface burden of BC is 2.1 Gg in 2015, contributing 19% of the total, similar to the value for\nthe year 2000. The nitrate burden from land transport, on the other hand, is larger in 2015 than in R13 (6.3 vs. 2.5 Gg), but\nits relative contribution is slightly lower (25% vs. 29%). This is consistent with the difference in NOx (a nitrate precursor)\n90\nemissions between CMIP5 and CMIP6 (Fig. 1a). The contribution of land transport to present-day aerosol burdens is particularly large for BC and aerosol nitrate (NO3), see\nFig. 2a. The continental near-surface burden of BC is 2.1 Gg in 2015, contributing 19% of the total, similar to the value for\nthe year 2000. The nitrate burden from land transport, on the other hand, is larger in 2015 than in R13 (6.3 vs. 2.5 Gg), but\nits relative contribution is slightly lower (25% vs. 29%). This is consistent with the difference in NOx (a nitrate precursor)\n290\nemissions between CMIP5 and CMIP6 (Fig. 1a). In the future scenarios, the absolute impact of land transport on BC is strongly reduced in SSP1 and SSP2 in 2030 and\nfurther in 2050, but only in SSP1 this corresponds to a reduction in the relative contributions. On the one hand, this shows\nthat the optimistic SSP1 scenario implements stringent air quality control policies that can effectively reduce land-transport- 290 In the future scenarios, the absolute impact of land transport on BC is strongly reduced in SSP1 and SSP2 in 2030 and\nfurther in 2050, but only in SSP1 this corresponds to a reduction in the relative contributions. On the one hand, this shows\nthat the optimistic SSP1 scenario implements stringent air quality control policies that can effectively reduce land-transport- induced near-surface concentrations of BC. On the other hand, the higher degree of electrification of this sector in SSP1 (van\n295\nVuuren et al., 2017, up to 75%) compared to SSP2 (Fricko et al., 2017, up to 50%) implies strong reductions of BC from land\ntransport, but not from other sectors, hence decreasing the relative contribution of land transport itself. Similar conclusions\nhold for nitrate, where again only SSP1 is successful in decreasing the land-transport-induced burden both in absolute and\nrelative term, with a very large decrease in 2050. 4.1\nLand transport This is reduced in SSP1 and SSP2 in 2030 and 2050, while it keeps increasing Transport-induced changes in particle number burden are relevant as well, not only for air quality, but also for climate,\n300\nsince aerosol particles can act as cloud condensation nuclei in warm clouds if they grow to a sufficient size. An increase in their\nconcentration can therefore lead to a change in the microphysical and radiative properties of these clouds (Boucher et al., 2013). Land transport provides a significant contribution to particle number, with 9% of near-surface particle number burden over the\ncontinents attributable to this sector in 2015. This is reduced in SSP1 and SSP2 in 2030 and 2050, while it keeps increasing in SSP3. Note that the impacts on land-transport-induced particle number concentrations is strongly related to the assumptions\n305\non the size distribution of emitted particles. As shown in R13, assuming size distribution parameters representative of either\nyoung or aged particle populations upon emissions can change the resulting impacts on number concentration by about one\norder of magnitude. As discussed in Sect. 1 and Table 1, here we assume an aged distribution for particles emitted by land\ntransport, representative of air masses on large spatial scales and therefore more consistent with the representation in the coarse in SSP3. Note that the impacts on land-transport-induced particle number concentrations is strongly related to the assumptions\n305\non the size distribution of emitted particles. As shown in R13, assuming size distribution parameters representative of either\nyoung or aged particle populations upon emissions can change the resulting impacts on number concentration by about one\norder of magnitude. As discussed in Sect. 1 and Table 1, here we assume an aged distribution for particles emitted by land\ntransport, representative of air masses on large spatial scales and therefore more consistent with the representation in the coarse resolution of a global model. 310\nThe geographical distribution of land-transport-induced surface-level BC concentration is shown in Fig. 3. In 2015 (Fig. 3b),\nthe largest absolute impacts are found over China, India and the Arabian Peninsula, with concentrations changes of about\n1 µg m−3. Large values are also simulated over Eastern Europe and in some spots in South Africa and South America, in cor-\nrespondence of the largest urban areas. Western Europe and Eastern U.S. are characterized by generally lower land-transport- resolution of a global model. 4.1\nLand transport induced near-surface concentrations of BC. On the other hand, the higher degree of electrification of this sector in SSP1 (van\n295\nVuuren et al., 2017, up to 75%) compared to SSP2 (Fricko et al., 2017, up to 50%) implies strong reductions of BC from land\ntransport, but not from other sectors, hence decreasing the relative contribution of land transport itself. Similar conclusions\nhold for nitrate, where again only SSP1 is successful in decreasing the land-transport-induced burden both in absolute and\nrelative term, with a very large decrease in 2050. induced near-surface concentrations of BC. On the other hand, the higher degree of electrification of this sector in SSP1 (van\n295\nVuuren et al., 2017, up to 75%) compared to SSP2 (Fricko et al., 2017, up to 50%) implies strong reductions of BC from land\ntransport, but not from other sectors, hence decreasing the relative contribution of land transport itself. Similar conclusions\nhold for nitrate, where again only SSP1 is successful in decreasing the land-transport-induced burden both in absolute and\nrelative term, with a very large decrease in 2050. Transport-induced changes in particle number burden are relevant as well, not only for air quality, but also for climate,\n300\nsince aerosol particles can act as cloud condensation nuclei in warm clouds if they grow to a sufficient size. An increase in their\nconcentration can therefore lead to a change in the microphysical and radiative properties of these clouds (Boucher et al., 2013). Land transport provides a significant contribution to particle number, with 9% of near-surface particle number burden over the\ncontinents attributable to this sector in 2015. This is reduced in SSP1 and SSP2 in 2030 and 2050, while it keeps increasing Transport-induced changes in particle number burden are relevant as well, not only for air quality, but also for climate,\n300\nsince aerosol particles can act as cloud condensation nuclei in warm clouds if they grow to a sufficient size. An increase in their\nconcentration can therefore lead to a change in the microphysical and radiative properties of these clouds (Boucher et al., 2013). Land transport provides a significant contribution to particle number, with 9% of near-surface particle number burden over the\ncontinents attributable to this sector in 2015. 4.1\nLand transport 310\nThe geographical distribution of land-transport-induced surface-level BC concentration is shown in Fig. 3. In 2015 (Fig. 3b),\nthe largest absolute impacts are found over China, India and the Arabian Peninsula, with concentrations changes of about\n1 µg m−3. Large values are also simulated over Eastern Europe and in some spots in South Africa and South America, in cor-\nrespondence of the largest urban areas. Western Europe and Eastern U.S. are characterized by generally lower land-transport- induced BC concentrations. This pattern roughly matches the results for 2000 with the CMIP5 inventory (R13), although with\n315\nlarger values, especially in the Middle East and in Eastern and Southern Asia. In relative terms (Fig. 3c), however, the contri-\nbution of land transport is confirmed to be relatively small in Eastern and Southern Asia, amounting to only 10 to 20% of the\nsurface-level BC concentration in these countries, due to the prevalence of emissions from other sectors, like energy production\nor household. A somewhat opposite effect is the large relative contribution over the Arabian Peninsula, with 60–70% of BC 12 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.1\nLand transport 2015\na\nBackground concentration\nb\nAbsolute impact\nc\nRelative contribution\n0.005\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on BC\n2030 SSP1\nd\nBackground concentration\ne\nAbsolute impact\nf\nRelative contribution\n0.005\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on BC\n2030 SSP2\ng\nBackground concentration\nh\nAbsolute impact\ni\nRelative contribution\n0.005\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on BC\n2030 SSP3\nj\nBackground concentration\nk\nAbsolute impact\nl\nRelative contribution\n0.005\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on BC\n2050 SSP1\nm\nBackground concentration\nn\nAbsolute impact\no\nRelative contribution\n0.005\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on BC\n2050 SSP2\np\nBackground concentration\nq\nAbsolute impact\nr\nRelative contribution\n0.005\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on BC\n2050 SSP3\ns\nBackground concentration\nt\nAbsolute impact\nu\nRelative contribution\n0.005\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on BC\nLand transport impact on surface-level BC concentration. 4.1\nLand transport The left panel show the background concentration for r\nd right panels show the land-transport-induced absolute impacts (Eq. 1) and the relative contributions (Eq. 2), resp\nre the impacts are not significant to a 95% confidence level are masked out in gray. Absolute impact\nLand transport impact on BC [%] Figure 3. Land transport impact on surface-level BC concentration. The left panel show the background concentration for reference. The\nmiddle and right panels show the land-transport-induced absolute impacts (Eq. 1) and the relative contributions (Eq. 2), respectively. Grid\npoints where the impacts are not significant to a 95% confidence level are masked out in gray. 13 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.1\nLand transport 2015\na\nBackground concentration\nb\nAbsolute impact\nc\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-2000\n-1000\n-500\n-200\n-100\n -50\n -20\n 0\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on particle number\n2030 SSP1\nd\nBackground concentration\ne\nAbsolute impact\nf\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-2000\n-1000\n-500\n-200\n-100\n -50\n -20\n 0\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on particle number\n2030 SSP2\ng\nBackground concentration\nh\nAbsolute impact\ni\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-2000\n-1000\n-500\n-200\n-100\n -50\n -20\n 0\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on particle number\n2030 SSP3\nj\nBackground concentration\nk\nAbsolute impact\nl\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-2000\n-1000\n-500\n-200\n-100\n -50\n -20\n 0\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on particle number\n2050 SSP1\nm\nBackground concentration\nn\nAbsolute impact\no\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-2000\n-1000\n-500\n-200\n-100\n -50\n -20\n 0\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on particle number\n2050 SSP2\np\nBackground concentration\nq\nAbsolute impact\nr\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-2000\n-1000\n-500\n-200\n-100\n -50\n -20\n 0\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on particle number\n2050 SSP3\ns\nBackground concentration\nt\nAbsolute impact\nu\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-2000\n-1000\n-500\n-200\n-100\n -50\n -20\n 0\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nLand transport impact on particle number\n4. 4.1\nLand transport S3), which could be related to an enhanced aerosol removal via wet deposition. This effect, however, is consistent with the storyline of this scenario, assuming large differences between developed and developing countries. 335\nSimilar conclusions can be drawn for the other species (Figs. S1–S6), although in relative terms only nitrate (Fig. S1) and\nparticle number concentrations (Fig. 4) show substantial impacts from land transport in 2015 and in the future projections. A remarkable feature is the land-transport-induced decrease in surface-level aerosol sulfate concentration over some parts of\nIndia and China (Fig. S3), which could be related to an enhanced aerosol removal via wet deposition. This effect, however, is small in relative terms (< 10%). 340\nWith respect to the RCPs scenarios, the projections for the land-transport sectors in the SSPs appear to be more coherent\nwith their narratives in terms of short-lived pollutants, while this was not always the case in the RCPs. Righi et al. (2015),\nfor instance, found that the RCP scenarios with the most stringent climate policies were often characterized by larger impacts\non aerosol concentrations, hence showing an anti-correlation between their ranking and the resulting impact on short-lived species. This is not the case in the SSP scenarios: SSP1 is by far the greenest scenario in terms of air pollution impact, while\n345\nSSP2 and SSP3 show only limited changes in 2030 and 2050 with respect to the present-day situation, consistently with their\nunderlying storylines and long-term climate projection. species. This is not the case in the SSP scenarios: SSP1 is by far the greenest scenario in terms of air pollution impact, while\n345\nSSP2 and SSP3 show only limited changes in 2030 and 2050 with respect to the present-day situation, consistently with their\nunderlying storylines and long-term climate projection. 4.1\nLand transport As in Fig. 3, but for the land transport impact on surface-level particle number concentration. Absolute impact\nLand transport impact on particle number Figure 4. As in Fig. 3, but for the land transport impact on surface-level particle number concentration. concentration due to land-transport in these regions, following the comparatively low BC emissions from other sectors in this\n320\nregion. This sector remains quite relevant for air pollution also in the developed countries, with contributions of 30 to 50% in\nEurope and in the U.S. These numbers are lower than in 2000, also in terms of absolute land-transport-induced concentration 14 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. changes, revealing the effectiveness of the air pollution control measures and the technical improvements in this sector over\nthe 15 years. Fig. 3c also reveals the importance of the long-range transport of polluted air masses from the continents to the\noceans, especially in the subtropics and southern mid-latitudes. 325 changes, revealing the effectiveness of the air pollution control measures and the technical improvements in this sector over\nthe 15 years. Fig. 3c also reveals the importance of the long-range transport of polluted air masses from the continents to the oceans, especially in the subtropics and southern mid-latitudes. 325\nAs expected, the land-transport-induced BC concentrations are strongly reduced in SSP1 in 2030 (Fig. 3e) and become\nalmost negligible in 2050 (Fig. 3n), with only India and the Arabian Peninsula showing concentration changes larger than\n0.05 µg m−3, although the relative contributions in the Arabian Peninsula still remain high (Fig. 3f,o). This is consistent with\nthe narrative of this scenario, considering a worldwide transition to a more sustainable future with reduced inequality across oceans, especially in the subtropics and southern mid-latitudes. 325\nAs expected, the land-transport-induced BC concentrations are strongly reduced in SSP1 in 2030 (Fig. 3e) and become\nalmost negligible in 2050 (Fig. 3n), with only India and the Arabian Peninsula showing concentration changes larger than\n0.05 µg m−3, although the relative contributions in the Arabian Peninsula still remain high (Fig. 3f,o). This is consistent with\nthe narrative of this scenario, considering a worldwide transition to a more sustainable future with reduced inequality across the narrative of this scenario, considering a worldwide transition to a more sustainable future with reduced inequality across\ncountries. 4.1\nLand transport In SSP2, on the contrary, only the developed countries show a noticeable decrease in BC concentration in 2030\n330\nand 2050, both in absolute and relative terms, while developing countries remain on a similar level as in 2015 (Fig. 3h,i). In\n2050, land transport is still the major source of BC pollution in many regions of the world, including the Arabian Peninsula,\nNorth Africa, and Central and South America (Fig. 3k,l). SSP3 shows no remarkable differences with respect to SSP2 in 2030\n(Fig. 3q,r), whereas it is characterized by an increased land-transport-induced concentration in 2050 (Fig. 3t,u), which is again\ni\ni h h\nli\nf hi\ni\ni\nl\ndiff\nb\nd\nl\nd\nd d\nl\ni\ni\n335 countries. In SSP2, on the contrary, only the developed countries show a noticeable decrease in BC concentration in 2030\n330\nand 2050, both in absolute and relative terms, while developing countries remain on a similar level as in 2015 (Fig. 3h,i). In\n2050, land transport is still the major source of BC pollution in many regions of the world, including the Arabian Peninsula,\nNorth Africa, and Central and South America (Fig. 3k,l). SSP3 shows no remarkable differences with respect to SSP2 in 2030\n(Fig. 3q,r), whereas it is characterized by an increased land-transport-induced concentration in 2050 (Fig. 3t,u), which is again countries. In SSP2, on the contrary, only the developed countries show a noticeable decrease in BC concentration in 2030\n330\nand 2050, both in absolute and relative terms, while developing countries remain on a similar level as in 2015 (Fig. 3h,i). In\n2050, land transport is still the major source of BC pollution in many regions of the world, including the Arabian Peninsula,\nNorth Africa, and Central and South America (Fig. 3k,l). SSP3 shows no remarkable differences with respect to SSP2 in 2030\n(Fig. 3q,r), whereas it is characterized by an increased land-transport-induced concentration in 2050 (Fig. 3t,u), which is again consistent with the storyline of this scenario, assuming large differences between developed and developing countries. 335\nSimilar conclusions can be drawn for the other species (Figs. S1–S6), although in relative terms only nitrate (Fig. S1) and\nparticle number concentrations (Fig. 4) show substantial impacts from land transport in 2015 and in the future projections. A remarkable feature is the land-transport-induced decrease in surface-level aerosol sulfate concentration over some parts of\nIndia and China (Fig. 4.2\nShipping efficient take up of ultra-fine particles by larger aerosol in ship plumes, which occurs within a time scale of the order of only\n365\n1 hour (Petzold et al., 2008; Righi et al., 2011). This process is implicitly considered in our assumptions on the size distribution\nof ship-emitted particles (see Sect. 1 and Table 1), with 90% of shipping emissions released in the accumulation mode, and\nleads to the small impacts on particle burden. These are further reduced in the future scenarios, in line with the reductions in\nthe mass burden of the most relevant species from this sector. Fig. 5 depicts the geographical distributions of shipping-induced near-surface sulfate concentrations. In 2015 (Fig. 5b), the\n370\nlargest ship-induced concentrations are simulated over the Northern Hemisphere, especially in the Eastern Atlantic Ocean, in\nthe Northern Indian Ocean and in the Mediterranean, with values around 1 µg m−3. Significant changes of 0.05–0.1 µg m−3\nare also simulated over land in many coastal regions of the Northern Hemisphere, confirming the relevance of this sector for\ncontinental air pollution (Eyring et al., 2010). This pattern closely matches the distribution simulated in R13 for the year 2000. Fig. 5 depicts the geographical distributions of shipping-induced near-surface sulfate concentrations. In 2015 (Fig. 5b), the\n370\nlargest ship-induced concentrations are simulated over the Northern Hemisphere, especially in the Eastern Atlantic Ocean, in\nthe Northern Indian Ocean and in the Mediterranean, with values around 1 µg m−3. Significant changes of 0.05–0.1 µg m−3\nare also simulated over land in many coastal regions of the Northern Hemisphere, confirming the relevance of this sector for\ncontinental air pollution (Eyring et al., 2010). This pattern closely matches the distribution simulated in R13 for the year 2000. Fig. 5 depicts the geographical distributions of shipping-induced near-surface sulfate concentrations. In 2015 (Fig. 5b), the\n370\nlargest ship-induced concentrations are simulated over the Northern Hemisphere, especially in the Eastern Atlantic Ocean, in\nthe Northern Indian Ocean and in the Mediterranean, with values around 1 µg m−3. Significant changes of 0.05–0.1 µg m−3\nare also simulated over land in many coastal regions of the Northern Hemisphere, confirming the relevance of this sector for\ncontinental air pollution (Eyring et al., 2010). This pattern closely matches the distribution simulated in R13 for the year 2000. The relative contribution of shipping to surface-level sulfate concentration (Fig. 5c), however, is generally lower than in R13. 4.2\nShipping Such a low impact is a consequence of the very\nefficient take up of ultra-fine particles by larger aerosol in ship plumes, which occurs within a time scale of the order of only\n365\n1 hour (Petzold et al., 2008; Righi et al., 2011). This process is implicitly considered in our assumptions on the size distribution\nof ship-emitted particles (see Sect. 1 and Table 1), with 90% of shipping emissions released in the accumulation mode, and\nleads to the small impacts on particle burden. These are further reduced in the future scenarios, in line with the reductions in\nthe mass burden of the most relevant species from this sector. which are all characterized by a reduced impact of sulfate burden. In 2030, the relative contribution of shipping to near-surface\noceanic sulfate burden is reduced to 6–7% in all scenarios, and further to 1–4% in 2050, with the largest (smallest) decrease\nin SSP1 (SSP3), hence consistent with the scenario narratives. MARPOL also introduced emission limits on NOx for all new\nship engines built after year 2000 and above a certain power output. These regulations, however, seem to have a limited impact\n360\non the resulting nitrate burdens, which are only slightly reduced in 2030 for all scenarios. Stronger reductions in near-surface\nnitrate burden become effective in 2050, especially in SSP1 (4%) and SSP3 (14%), while they remain high (23%) in SSP2,\nconsistent with the high share of shipping NOx emissions in this scenario (Fig. 1). The shipping impact on particle number\nburden is quite small, about 1.5% in 2015, in line with the results of R13. Such a low impact is a consequence of the very\nefficient take up of ultra-fine particles by larger aerosol in ship plumes which occurs within a time scale of the order of only\n365 360 efficient take up of ultra-fine particles by larger aerosol in ship plumes, which occurs within a time scale of the order of only\n365\n1 hour (Petzold et al., 2008; Righi et al., 2011). This process is implicitly considered in our assumptions on the size distribution\nof ship-emitted particles (see Sect. 1 and Table 1), with 90% of shipping emissions released in the accumulation mode, and\nleads to the small impacts on particle burden. These are further reduced in the future scenarios, in line with the reductions in\nthe mass burden of the most relevant species from this sector. 4.2\nShipping As in the year 2000, the shipping sector is characterized by its large contributions to the burdens of nitrate and sulfate (SO4, see As in the year 2000, the shipping sector is characterized by its large contributions to the burdens As in the year 2000, the shipping sector is characterized by its large contributions to the burdens of nitrate and sulfate (SO4, see Fig. 2b) over the oceans. The impact of shipping on near-surface nitrate burden over the oceans in 2015 (6.9 Gg) is significantly\n350\nlarger than in R13 for the year 2000 (1.1 Gg), but it remains stable in relative terms at about 25%. Sulfate pollution, on the other\nhand, is smaller than in 2000 (6.3 vs. 9.4 Gg) and its relative contribution is reduced to 9% compared to the 14% value reported\nin R13. As mentioned above, this can be related to the introduction of the MARPOL Annex VI regulations on the maximum\nFSC in shipping. Although fully implemented in 2020, a first step towards the reduction was taken in 2012, reducing the maximum allowed FSC by mass from 4.5% to 3.5% globally (Buhaug et al., 2009). Stricter limits were applied in the SECAs,\n355\nreducing the cap from 1.5% to 1% in 2010 and to 0.1% in 2015. This reduction trend appears to continue in the future scenarios, 15 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. which are all characterized by a reduced impact of sulfate burden. In 2030, the relative contribution of shipping to near-surface\noceanic sulfate burden is reduced to 6–7% in all scenarios, and further to 1–4% in 2050, with the largest (smallest) decrease\nin SSP1 (SSP3), hence consistent with the scenario narratives. MARPOL also introduced emission limits on NOx for all new\nship engines built after year 2000 and above a certain power output. These regulations, however, seem to have a limited impact\n360\non the resulting nitrate burdens, which are only slightly reduced in 2030 for all scenarios. Stronger reductions in near-surface\nnitrate burden become effective in 2050, especially in SSP1 (4%) and SSP3 (14%), while they remain high (23%) in SSP2,\nconsistent with the high share of shipping NOx emissions in this scenario (Fig. 1). The shipping impact on particle number\nburden is quite small, about 1.5% in 2015, in line with the results of R13. 4.2\nShipping 375\nIn particular, lower contributions are found over the Northern Pacific Ocean (20–40%, due to the lower absolute impact of\nshipping in this region than in R13) and over the Southern Atlantic (10–30%, due to the higher background concentration of\nsulfate in this region than in R13). An interesting feature is the large contribution of shipping to the sulfate concentration over\nthe Northern Atlantic Ocean, between the coasts of Greenland and Scandinavia: this is due to the increased emissions in the polar regions in the CMIP6 dataset for 2015 compared to the CMIP5 for 2000, in particular along the Northern Sea Route,\n380\nwhich has become more travelled in recent years following the depletion of the Arctic ice. The shipping impacts are reduced in 2030 in all scenarios, although the geographical distribution remains essentially the\nsame. This is because the SSPs do not consider any significant change in the route displacement, but just apply a scaling of\nthe traffic activity across the existing routes according to a global spatial matrix (Feng et al., 2020). SSP1 and SSP2 (Fig. 5e,h) project similar ship-induced concentrations, with the highest values reduced to 0.2–0.5 µg m−3. In relative terms, however,\n385\nthis translates in a higher impact in SSP1, due to the cleaner background following the reductions in the emissions from other\nanthropogenic sectors. In SSP3 (Fig. 5k), the concentration changes in 2030 are still relatively high compared to 2015, although\nwith a smaller relative contribution (Fig. 5l). The shipping sector has only a negligible impact on sulfate pollution in 2050 in\nSSP1 and SSP2 (Fig. 5n,o,q,r), while it remains relevant in SSP3, with large induced concentrations, around 0.2–0.5 µg m−3 in the Northern Indian Ocean and around the Mediterranean, but with a relative contribution reduced to a maximum of 20%\n390\nover the Northern Atlantic and Northern Pacific Ocean (Fig. 5t,u). 16 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.2\nShipping 2015\na\nBackground concentration\nb\nAbsolute impact\nc\nRelative contribution\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n 10\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n-0.01\n 0\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on SO4\n2030 SSP1\nd\nBackground concentration\ne\nAbsolute impact\nf\nRelative contribution\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n 10\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n-0.01\n 0\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on SO4\n2030 SSP2\ng\nBackground concentration\nh\nAbsolute impact\ni\nRelative contribution\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n 10\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n-0.01\n 0\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on SO4\n2030 SSP3\nj\nBackground concentration\nk\nAbsolute impact\nl\nRelative contribution\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n 10\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n-0.01\n 0\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on SO4\n2050 SSP1\nm\nBackground concentration\nn\nAbsolute impact\no\nRelative contribution\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n 10\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n-0.01\n 0\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on SO4\n2050 SSP2\np\nBackground concentration\nq\nAbsolute impact\nr\nRelative contribution\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n 10\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n-0.01\n 0\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on SO4\n2050 SSP3\ns\nBackground concentration\nt\nAbsolute impact\nu\nRelative contribution\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n 5\n 10\n[ g m\n3]\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n-0.01\n 0\n0.01\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n[ g m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on SO4\nFigure 5. 4.2\nShipping As in Fig. 3, but for the shipping impact on surface-level SO4 concentration. Absolute impact\nShipping impact on SO4 Figure 5. As in Fig. 3, but for the shipping impact on surface-level SO4 concentration. The impact on nitrate (Fig. S7) shows a similar geographic distribution, but a much different future evolution. The shipping-\ninduced nitrate concentrations remain considerably high through 2030 and 2050, with very large relative contributions around\nand above 70% over most of the northern hemispheric oceans and along the major southern hemispheric routes. This reveals the 17 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.2\nShipping Absolute impact\nShipping impact on particle number 2015\na\nBackground concentration\nb\nAbsolute impact\nc\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-100\n -50\n -20\n -10\n -5\n -2\n 0\n 2\n 5\n 10\n 20\n 50\n 100\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on particle number\n2030 SSP1\nd\nBackground concentration\ne\nAbsolute impact\nf\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-100\n -50\n -20\n -10\n -5\n -2\n 0\n 2\n 5\n 10\n 20\n 50\n 100\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on particle number\n2030 SSP2\ng\nBackground concentration\nh\nAbsolute impact\ni\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-100\n -50\n -20\n -10\n -5\n -2\n 0\n 2\n 5\n 10\n 20\n 50\n 100\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on particle number\n2030 SSP3\nj\nBackground concentration\nk\nAbsolute impact\nl\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-100\n -50\n -20\n -10\n -5\n -2\n 0\n 2\n 5\n 10\n 20\n 50\n 100\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on particle number\n2050 SSP1\nm\nBackground concentration\nn\nAbsolute impact\no\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-100\n -50\n -20\n -10\n -5\n -2\n 0\n 2\n 5\n 10\n 20\n 50\n 100\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on particle number\n2050 SSP2\np\nBackground concentration\nq\nAbsolute impact\nr\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-100\n -50\n -20\n -10\n -5\n -2\n 0\n 2\n 5\n 10\n 20\n 50\n 100\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on particle number\n2050 SSP3\ns\nBackground concentration\nt\nAbsolute impact\nu\nRelative contribution\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n1000\n2000\n[cm\n3]\n-100\n -50\n -20\n -10\n -5\n -2\n 0\n 2\n 5\n 10\n 20\n 50\n 100\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nShipping impact on particle number\n6. 4.2\nShipping As in Fig. 3, but for the shipping impact on surface-level particle number concentration. -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nr\n \n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nu\nRelative contribution\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%] [cm\n3] Figure 6. As in Fig. 3, but for the shipping impact on surface-level particle number concentration. challenges in reducing the emissions from NOx in the shipping sectors, contrary to the sulfate which can be more effectively\n395\ncontrolled by reducing the shipping FSC. Nitrate impacts are effectively reduced only in the green SSP1 scenario in 2050. Another aspect to be taken into account is the competition between the formation pathways of ammonium sulfate and ammo- challenges in reducing the emissions from NOx in the shipping sectors, contrary to the sulfate which can be more effectively\n395\ncontrolled by reducing the shipping FSC. Nitrate impacts are effectively reduced only in the green SSP1 scenario in 2050. Another aspect to be taken into account is the competition between the formation pathways of ammonium sulfate and ammo- 18 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. nium nitrate for available ammonia in the lower troposphere, with ammonium sulfate formation being favoured. A reduction\nof sulfate concentration makes more ammonia available for the formation of ammonium nitrate, leading to enhanced nitrate\nformation, hence partly compensating the reduction in sulfate. Being the largest contributors to shipping pollution, sulfate and\n400\nnitrate also influence the impact of this sector on particle number concentrations (Fig. 6). Here again the largest impacts are\nfound over the northern hemispheric oceans and in the southern high latitude regions, progressively reducing in 2030 and 2050. Over the southern extra-tropics shipping induces a reduction of the particle number in most scenarios: this could be due to the\nrelatively large size of ship-induced particles inhibiting the nucleation of new particles typical of the pristine environments of\nthese regions and thus limiting or removing a key source of particle number. 405 4.3\nAviation Compared to the other two sectors, the impact of aviation on mass burdens is small (Fig. 2c), even though this is calculated\nin the altitude range of 8 to 12 km where most of the aviation emissions are released. Aviation contributes 0.3 Gg to the\nupper-tropospheric BC burden in 2015, almost three times more than in R13. The contribution is larger also in relative terms (5% versus 2%). This could be due to an increase in BC emissions between 2000 and 2015, but also to the improved model\n410\nvertical resolution adopted in the present study (41 levels) with respect to R13 (19 levels). The L41 resolution has been\nspecifically designed to resolve the upper troposphere and the related process in higher detail and has been shown to be\nsuitable in several modelling studies targeting this region (Dietmüller et al., 2014; Bock and Burkhardt, 2016; Righi et al.,\n2020). The coarse vertical resolution used in R13 may therefore have resulted in an underestimated impact of aviation-induced BC in the upper troposphere. This appears to be the case also for sulfate, which shows an aviation-induced upper-tropospheric\n415\nburden of 7.1 Gg, a factor of more than two larger than in R13. This is also larger in relative terms (4% vs. 3%). Despite the\nincreased aviation impact on the mass burden, the impact on number is lower by about one order of magnitude than in R13\n(1.1 × 1026 vs. 1.2 × 1027 particles) and about the half in relative terms (6% vs. 12%), although identical size distributions for\nthe emitted particles have been assumed in the two studies. Besides the aforementioned improvement in the vertical resolution, BC in the upper troposphere. This appears to be the case also for sulfate, which shows an aviation-induced upper-tropospheric\n415\nburden of 7.1 Gg, a factor of more than two larger than in R13. This is also larger in relative terms (4% vs. 3%). Despite the\nincreased aviation impact on the mass burden, the impact on number is lower by about one order of magnitude than in R13\n(1.1 × 1026 vs. 1.2 × 1027 particles) and about the half in relative terms (6% vs. 12%), although identical size distributions for\nthe emitted particles have been assumed in the two studies. Besides the aforementioned improvement in the vertical resolution, BC in the upper troposphere. 4.3\nAviation Aviation impact on zonally averaged BC concentration. The left panel show the background concentration for reference\nmiddle and right panels show the land-transport-induced absolute impacts and the relative contributions, respectively. Grid points whe\ndifferences are not significant at a 95% confidence level are masked out in gray Note that in contrast to the other figures the units\nhttps://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.3\nAviation -80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2015\na\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nb\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nc\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP1\nd\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ne\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nf\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP2\ng\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nh\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ni\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP3\nj\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nk\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nl\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP1\nm\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nn\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\no\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP2\np\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nq\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nr\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP3\ns\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nt\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nu\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\nAviation impact on zonally averaged BC concentration. 4.3\nAviation This appears to be the case also for sulfate, which shows an aviation-induced upper-tropospheric\n415\nburden of 7.1 Gg, a factor of more than two larger than in R13. This is also larger in relative terms (4% vs. 3%). Despite the\nincreased aviation impact on the mass burden, the impact on number is lower by about one order of magnitude than in R13\n(1.1 × 1026 vs. 1.2 × 1027 particles) and about the half in relative terms (6% vs. 12%), although identical size distributions for\nthe emitted particles have been assumed in the two studies. Besides the aforementioned improvement in the vertical resolution, the improved representation of the aerosol dynamics in the new version of the MADE aerosol submodel adopted here (MADE3;\n420\nKaiser et al., 2019) could explain these differences, as revealed, for instance, by the much lower background concentration of\naerosol particles (Fig. 8a) with respect to the previous assessment. The EMAC model system has also been updated with respect\nto the previous assessment and now uses the second version of the MESSy interface, comprising numerous update in several\nkey submodels, such as for example the scavenging submodel SCAV (Tost et al., 2006). Aviation-induced upper-tropospheric BC burden keeps growing until 2030 in SSP2 and SSP3, and further in 2050 for SSP3. Aviation-induced BC burden decreases\n425\nonly in the SSP1 scenarios, with a factor of more than two reduction in 2030 and further down in 2050. In relative terms\nthese changes are less marked, due to the corresponding changes in background BC concentrations. The sulfate burden follows\na similar path, with a weak decrease in SSP1 and a steady increase in SSP2 and SSP3. This shows that the a desulphurisation\nof the aviation sector is not considered as an emission reduction measure in these scenarios, although it is relatively easy to implement, as it does not require technical modifications nor does it need to cope with the long lifetime of the commercial fleet\n430\n(e.g. Unger, 2011; Moore et al., 2017). 4.3\nAviation 19 -80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2015\na\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nb\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nc\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP1\nd\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ne\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nf\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP2\ng\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nh\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ni\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP3\nj\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nk\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nl\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP1\nm\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nn\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\no\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP2\np\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nq\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nr\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP3\ns\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nt\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nu\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\nFigure 7. 4.3\nAviation Aviation impact on zonally averaged BC concentration. The left panel show the background concentration for referen\nmiddle and right panels show the land-transport-induced absolute impacts and the relative contributions, respectively. Grid points w\ndiff\nt i\nifi\nt t\n95%\nfid\nl\nl\nk d\nt i\nN t th t i\nt\nt t th\nth\nfi\nth\ni\nhttps://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.3\nAviation -80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2015\na\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nb\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nc\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP1\nd\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ne\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nf\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP2\ng\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nh\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ni\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP3\nj\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nk\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nl\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP1\nm\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nn\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\no\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP2\np\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nq\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nr\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP3\ns\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nt\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nu\nRelative contribution\n0.0001\n0.0002\n0.0005\n0.001\n0.002\n0.005\n0.01\n0.02\n0.05\n 0.1\n[ g m\n3]\n -2\n -1\n-0.5\n-0.2\n-0.1\n-0.05\n-0.02\n 0\n0.02\n0.05\n 0.1\n 0.2\n 0.5\n 1\n 2\n[ng m\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on BC\nFigure 7. 4.3\nAviation -80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2015\na\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nb\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nc\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP1\nd\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ne\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nf\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP2\ng\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nh\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ni\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP3\nj\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nk\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nl\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP1\nm\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nn\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\no\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP2\np\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nq\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nr\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP3\ns\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nt\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nu\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number -80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2015\na\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nb\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nc\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP1\nd\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ne\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nf\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP2\ng\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nh\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\ni\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2030 SSP3\nj\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nk\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nl\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP1\nm\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nn\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\no\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP2\np\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nq\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nr\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nPressure [hPa]\n2050 SSP3\ns\nBackground concentration\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nt\nAbsolute impact\n-80\n-60\n-40\n-20\n0\n20\n40\n60\n80\nLatitude [degrees]\n1000\n900\n800\n700\n600\n500\n400\n300\n200\n100\nu\nRelative contribution\n 100\n 200\n 300\n 400\n 500\n 600\n 700\n 800\n 900\n1000\n[cm\n3]\n-500\n-200\n-100\n -50\n -20\n -10\n -5\n 0\n 5\n 10\n 20\n 50\n 100\n 200\n 500\n[cm\n3]\n -70\n -60\n -50\n -40\n -30\n -20\n -10\n 0\n 10\n 20\n 30\n 40\n 50\n 60\n 70\n[%]\nAviation impact on particle number\nre 8. 4.3\nAviation The left panel show the background concentration for ref\nd right panels show the land-transport-induced absolute impacts and the relative contributions, respectively. Grid poin\ns are not significant at a 95% confidence level are masked out in gray. Note that, in contrast to the other figures, the\nhanges are nanogram per cubic meter. Figure 7. Aviation impact on zonally averaged BC concentration. The left panel show the background concentration for reference. The\nmiddle and right panels show the land-transport-induced absolute impacts and the relative contributions, respectively. Grid points where the\ndifferences are not significant at a 95% confidence level are masked out in gray. Note that, in contrast to the other figures, the units of the\nabsolute changes are nanogram per cubic meter. 20 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.3\nAviation 7h,q), while they increase in relative terms (Fig. 7i,l), up to\n50–60% in 2050. This is the scenario with the highest relative contribution in the future: even though SSP3 shows much higher\n3 overall pattern is still comparable. The future aviation impacts decrease only in the SSP1 scenario, while maintaining a similar\n440\nzonal distribution. Aviation-induced BC concentrations are reduced to 0.2–0.5 ng m−3 in 2030 and further down to 0.1 ng m−3\nover most of the northern latitudes in this scenario (Fig. 7e,n), with minor changes in relative terms (Fig. 7f,o). In SSP2, the\nabsolute impact remains approximately constant until 2050 (Fig. 7h,q), while they increase in relative terms (Fig. 7i,l), up to\n50–60% in 2050. This is the scenario with the highest relative contribution in the future: even though SSP3 shows much higher absolute impacts (around 1–2 ng m−3, Fig. 7k,t), the cleanest background emissions in SSP2 make the aviation sector more\n445\nimpacting in this scenario. Similar considerations can be made for the aviation-induced impacts on sulfate (Fig. S15), while the\nnitrate impacts (Fig. S13) show an interesting dual structure, with concentration increases between 5 and 20 ng m−3 throughout\nthe whole troposphere in northern mid-latitudes and significant increases in the upper troposphere at latitudes above 40◦N. As\nmentioned in Sect. 4.2, this is due to the competition between ammonium sulfate and ammonium nitrate for available ammonia, absolute impacts (around 1–2 ng m−3, Fig. 7k,t), the cleanest background emissions in SSP2 make the aviation sector more\n445\nimpacting in this scenario. Similar considerations can be made for the aviation-induced impacts on sulfate (Fig. S15), while the\nnitrate impacts (Fig. S13) show an interesting dual structure, with concentration increases between 5 and 20 ng m−3 throughout\nthe whole troposphere in northern mid-latitudes and significant increases in the upper troposphere at latitudes above 40◦N. As\nmentioned in Sect. 4.2, this is due to the competition between ammonium sulfate and ammonium nitrate for available ammonia, which is particularly distinct in the upper troposphere due to the low availability of ammonia (e.g. Unger et al., 2013). 450\nDespite the relatively small impact on mass concentrations, of the order of a few nanograms per cubic meter, the aviation\nsector significantly increase the particle number concentration in the upper troposphere. 4.3\nAviation As in Fig. 7, but for the aviation impact on zonally averaged particle number concentration. Figure 8. As in Fig. 7, but for the aviation impact on zonally averaged particle number concentration. The spatial distributions of aviation-induced BC concentrations are depicted in Fig. 7 as zonal mean maps. In 2015 (Fig. 7b),\nthe largest changes (0.5–1 ng m−3) are simulated for the Northern Hemisphere at latitudes > 40◦N at the typical cruise levels\nof 200–350 hPa. Similarly high concentrations are simulated at lower levels, possibly driven by short- and medium-range 21 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. flight over the continents, and close to the surface at mid-latitudes, related to landing and take-off and airport activities. The\n435\nSouthern Hemisphere concentrations are only weakly affected by aviation, with induced changes in BC concentrations below\n0.05 ng m−3. As inferred from the analysis of the burdens, these values are significantly higher as those reported in R13. This\nis also the case for the relative contribution of aviation to BC concentration (Fig. 7c), peaking at 30–40% at high latitude in the\nNorthern Hemisphere at 200–300 hPa. This is higher than the ∼5% value reported in R13 and shifted northward, although the flight over the continents, and close to the surface at mid-latitudes, related to landing and take-off and airport activities. The\n435\nSouthern Hemisphere concentrations are only weakly affected by aviation, with induced changes in BC concentrations below\n0.05 ng m−3. As inferred from the analysis of the burdens, these values are significantly higher as those reported in R13. This\nis also the case for the relative contribution of aviation to BC concentration (Fig. 7c), peaking at 30–40% at high latitude in the\nNorthern Hemisphere at 200–300 hPa. This is higher than the ∼5% value reported in R13 and shifted northward, although the Northern Hemisphere at 200 300 hPa. This is higher than the\n5% value reported in R13 and shifted northward, although the\noverall pattern is still comparable. The future aviation impacts decrease only in the SSP1 scenario, while maintaining a similar\n440\nzonal distribution. Aviation-induced BC concentrations are reduced to 0.2–0.5 ng m−3 in 2030 and further down to 0.1 ng m−3\nover most of the northern latitudes in this scenario (Fig. 7e,n), with minor changes in relative terms (Fig. 7f,o). In SSP2, the\nabsolute impact remains approximately constant until 2050 (Fig. 4.3\nAviation This is due to the high efficiency of\nthe combustion processes in aircraft engines, which lead to the formation of smaller BC particles compared to other sectors\n(Petzold et al., 1999). Furthermore, sulphur dioxide in aircraft plumes can drive efficient new particle formation in the upper which is particularly distinct in the upper troposphere due to the low availability of ammonia (e.g. Unger et al., 2013). 450\nDespite the relatively small impact on mass concentrations, of the order of a few nanograms per cubic meter, the aviation\nsector significantly increase the particle number concentration in the upper troposphere. This is due to the high efficiency of\nthe combustion processes in aircraft engines, which lead to the formation of smaller BC particles compared to other sectors\n(Petzold et al., 1999). Furthermore, sulphur dioxide in aircraft plumes can drive efficient new particle formation in the upper troposphere, contributing to particle number concentrations. Aviation-induced particle number concentrations as high as 200–\n455\n500 cm−3 are found in the upper-tropospheric northern mid-latitudes in 2015 (Fig. 8b). This is about one order of magnitude\nlower than in R13. The aviation impact also propagates to lower levels, with induced changes of a few tens of particles per\ncubic centimeter, with potential impacts on low level clouds. The strongest relative contribution of aviation to particle number\nconcentration, however, remains confined in the upper troposphere at mid-latitudes, with values of 20–30%, in line with R13. troposphere, contributing to particle number concentrations. Aviation-induced particle number concentrations as high as 200–\n455\n500 cm−3 are found in the upper-tropospheric northern mid-latitudes in 2015 (Fig. 8b). This is about one order of magnitude\nlower than in R13. The aviation impact also propagates to lower levels, with induced changes of a few tens of particles per\ncubic centimeter, with potential impacts on low level clouds. The strongest relative contribution of aviation to particle number\nconcentration, however, remains confined in the upper troposphere at mid-latitudes, with values of 20–30%, in line with R13. This means that both the aviation impact and the background are lower in this study than in R13. This could be due to the\n460\nimproved representation of the nucleation process in MADE3, which assumes a larger size of 10 nm for newly formed particles\nwith respect to its predecessor version, in order to better reproduce the observations in the upper troposphere (Kaiser et al.,\n2019). 4.3\nAviation As a consequence, the nucleation process leads to the formation of a lower number of particles (see again Fig. 8a). The aviation impact on particle number concentrations remain large in all future scenarios. The impacts in the lower tropo- The aviation impact on particle number concentrations remain large in all future scenarios. The impacts in the lower tropo\nsphere, in particular, increase to values larger than 10 cm−3 in all SSPs, with potential consequences for the aerosol interactions\n465\nwith low clouds. Interestingly, the relative contribution show a counter-intuitive development, with the highest values in SSP1\nand SSP2 (> 60%) and smaller in SSP3. This can be explained with the to relatively long lifetime of the fleet in the aviation\nsector, leading to a slower implementation of technical and policy measures in this sector, which therefore reacts less promptly\nthan the other sectors in determining the background concentrations. In SSP1, for example, background concentrations are very sphere, in particular, increase to values larger than 10 cm−3 in all SSPs, with potential consequences for the aerosol interactions\n465\nwith low clouds. Interestingly, the relative contribution show a counter-intuitive development, with the highest values in SSP1\nand SSP2 (> 60%) and smaller in SSP3. This can be explained with the to relatively long lifetime of the fleet in the aviation\nsector, leading to a slower implementation of technical and policy measures in this sector, which therefore reacts less promptly\nthan the other sectors in determining the background concentrations. In SSP1, for example, background concentrations are very 22 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. efficiently reduced in 2030 and 2050 (Fig. 8d,m), while aviation follows a less efficient reduction path (Fig. 8e,n), resulting\n470\nin a higher relative contribution to overall pollution in the future than in SSP3 (Fig. 8f,o). Note that, according to Feng et al. (2020), the geographical distribution of aviation emissions in the CMIP6 inventory remains the same as in 2015 in all SSPs\nand is simply scaled to match the global emissions. 5\nClimate impacts The climate impact of the transport sectors is depicted in terms of all-sky and clear-sky aerosol RF in Fig. 9. The first considers\n475\nthe changes in radiative fluxes on both cloudy and cloud-free model grid-boxes in each model time-step, whereas the latter is\na diagnostic quantity calculated ignoring the effect of clouds on the radiative fluxes. The comparison of these two quantities\nhelps understanding the role of aerosol-cloud interactions in aerosol-induced radiative forcing. The climate impact of the transport sectors is depicted in terms of all-sky and clear-sky aerosol RF in Fig. 9. The first considers\n475\nthe changes in radiative fluxes on both cloudy and cloud-free model grid-boxes in each model time-step, whereas the latter is\na diagnostic quantity calculated ignoring the effect of clouds on the radiative fluxes. The comparison of these two quantities\nhelps understanding the role of aerosol-cloud interactions in aerosol-induced radiative forcing. Aerosol from the land transport sector accounts for a RF of −164 mW m−2 in 2015 (Fig. 9a), representing the largest\nimpact among the three sectors. This estimate is considerably larger than the value of −12 mW m−2 reported in R13. Although\n480\nboth studies assume the same size distribution for the particles emitted by land transport (based on Birmili et al., 2009), here\nwe further assume that primary sulfate aerosol particle from land transport are emitted in the Aitken mode with a geometric\nmean diameter of 30 nm (see Table 1). This results in a larger land-transport-induced impact on particle number concentration\n(Fig. 4b,c) than in R13, especially in relative terms, with land-transport contributions to particle number well above 20–30% impact among the three sectors. This estimate is considerably larger than the value of −12 mW m−2 reported in R13. Although\n480\nboth studies assume the same size distribution for the particles emitted by land transport (based on Birmili et al., 2009), here\nwe further assume that primary sulfate aerosol particle from land transport are emitted in the Aitken mode with a geometric\nmean diameter of 30 nm (see Table 1). This results in a larger land-transport-induced impact on particle number concentration\n(Fig. 4b,c) than in R13, especially in relative terms, with land-transport contributions to particle number well above 20–30% in several regions of the world. 5\nClimate impacts Aerosol from shipping exerts a RF of −145 mW m−2 in the year 2015 (Fig. 9b), which is slightly smaller than the RF\n495\nof −181 mW m−2 found in R13. This is a consequence of the assumption on the size distribution of ship-emitted particles\nin this study: although we use the same size distribution parameters by Petzold et al. (2008), we assigned only 10% of the\nemitted mass to the Aitken mode rather than 20% as in R13, hence assuming a more aged population. This reduces the\nnumber of emitted particles and in turn the impact of these particles on clouds and the RF. Another reason, as discussed in Aerosol from shipping exerts a RF of −145 mW m−2 in the year 2015 (Fig. 9b), which is slightly smaller than the RF\n495\nof −181 mW m−2 found in R13. This is a consequence of the assumption on the size distribution of ship-emitted particles\nin this study: although we use the same size distribution parameters by Petzold et al. (2008), we assigned only 10% of the\nemitted mass to the Aitken mode rather than 20% as in R13, hence assuming a more aged population. This reduces the\nnumber of emitted particles and in turn the impact of these particles on clouds and the RF. Another reason, as discussed in Aerosol from shipping exerts a RF of −145 mW m−2 in the year 2015 (Fig. 9b), which is slightly smaller than the RF\n495\nof −181 mW m−2 found in R13. This is a consequence of the assumption on the size distribution of ship-emitted particles\nin this study: although we use the same size distribution parameters by Petzold et al. (2008), we assigned only 10% of the\nemitted mass to the Aitken mode rather than 20% as in R13, hence assuming a more aged population. This reduces the\nnumber of emitted particles and in turn the impact of these particles on clouds and the RF. Another reason, as discussed in Sect. 4.2, is the lower shipping-induced aerosol sulfate in this study, following the introduction of the MARPOL regulations\n500\nof FSC starting from 2010. Since sulfate has a remarkable contribution to cloud condensation nuclei, its reduction affects the\nRF impact via the aerosol indirect effect. As for land-transport, the shipping RF is significant at the northern mid-latitudes Sect. 5\nClimate impacts This is also due to a different representation of the aerosol particle background in the present\n485\nstudy, which is generally lower than in R13 (Fig. 4a) and in better agreement with observations (Kaiser et al., 2019). The zonal\nprofiles show that the effect is particularly strong in the northern mid-latitudes (Fig. S19a), where most emissions are released. The RFs simulated in the future scenarios clearly match the narratives underlying the SSPs. In SSP1, the land-transport RF is\nstrongly reduced (in absolute terms) to −28 mW m−2 in 2030 and becomes statistically non-significant in 2050. A less marked in several regions of the world. This is also due to a different representation of the aerosol particle background in the present\n485\nstudy, which is generally lower than in R13 (Fig. 4a) and in better agreement with observations (Kaiser et al., 2019). The zonal\nprofiles show that the effect is particularly strong in the northern mid-latitudes (Fig. S19a), where most emissions are released. The RFs simulated in the future scenarios clearly match the narratives underlying the SSPs. In SSP1, the land-transport RF is\nstrongly reduced (in absolute terms) to −28 mW m−2 in 2030 and becomes statistically non-significant in 2050. A less marked reduction in the climate impact of land transport characterizes SSP2, with a RF of −122 mW m−2 in 2030 and −106 mW m−2\n490\nin 2050. Consistently with the narrative of the SSP3 scenario, an increase in RF is simulated here, with a stronger land-\ntransport-induced cooling effect of −194 mW m−2 in 2030 and −205 mW m−2 in 2050. In all cases, the all-sky RF dominates\nover the clear-sky component, revealing that the land-transport-induced climate impact mostly results from the interactions\nbetween aerosol and clouds, thus confirming the results of R13 and Righi et al. (2015). reduction in the climate impact of land transport characterizes SSP2, with a RF of −122 mW m−2 in 2030 and −106 mW m−2\n490\nin 2050. Consistently with the narrative of the SSP3 scenario, an increase in RF is simulated here, with a stronger land-\ntransport-induced cooling effect of −194 mW m−2 in 2030 and −205 mW m−2 in 2050. In all cases, the all-sky RF dominates\nover the clear-sky component, revealing that the land-transport-induced climate impact mostly results from the interactions\nbetween aerosol and clouds, thus confirming the results of R13 and Righi et al. (2015). 5\nClimate impacts 4.2, is the lower shipping-induced aerosol sulfate in this study, following the introduction of the MARPOL regulations\n500\nof FSC starting from 2010. Since sulfate has a remarkable contribution to cloud condensation nuclei, its reduction affects the\nRF impact via the aerosol indirect effect. As for land-transport, the shipping RF is significant at the northern mid-latitudes 23 2015\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n200\n150\n100\n50\n0\nRF [mW m\n2]\n(a) - Land transport RF\nall-sky\nclear-sky\n2015\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n200\n150\n100\n50\n0\nRF [mW m\n2]\n(b) - Shipping RF\nall-sky\nclear-sky\n2015\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n200\n150\n100\n50\n0\nRF [mW m\n2]\n(c) - Aviation RF\nall-sky\nclear-sky\nFigure 9. All-sky (solid) and clear-sky (hatched) RF of land transport, shipping and aviation in the year 2015 and for the three SSP scenarios\nin 2030 and 2050. The error bars represent the 95% confidence interval and refer to the all-sky RF. The crosses indicate non-significant\nresults. https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 2015\n2030\nSSP1\n2030\nSSP2\n2030\nSSP3\n2050\nSSP1\n2050\nSSP2\n2050\nSSP3\n200\n150\n100\n50\n0\n(c) - Aviation RF\nall-sky\nclear-sky Figure 9. All-sky (solid) and clear-sky (hatched) RF of land transport, shipping and aviation in the year 2015 and for the three SSP scenarios\nin 2030 and 2050. The error bars represent the 95% confidence interval and refer to the all-sky RF. The crosses indicate non-significant\nresults. (Fig. S19b), but has an additional very strong component in the southern hemisphere, where ship traffic is still relevant along\na few routes and, most importantly, aerosol and precursors are released in a pristine background (Fig. 6a), where the cloud a few routes and, most importantly, aerosol and precursors are released in a pristine background (Fig. 6a), where the cloud\nsusceptibility can be high. The shipping-induced negative RF is reduced in all scenarios in 2030, mostly for SSP1 and SSP2\n505\n(from −145 mW m−2 to −99 and −95 mW m−2, respectively), while in SSP3 the reduction is marginal (to −131 mW m−2). 5\nClimate impacts As discussed above, this is due to the larger relative impacts of aviation on particle number concen-\ntration in the current study, following an improved representation of background particles, in particular the ones generated by contrary, projects a non-significant aerosol climate effect from shipping in this year, consistent with the large reductions in\n510\nshipping SO2 emissions in this scenario (see Sect. 3 and Fig. 1d) and still relevant background sulfate pollution (Fig. 5p). The impact of aviation-induced aerosol in 2015 is quantified as −64 mW m−2, a factor of about four larger than the estimate\nin R13 (−15 mW m−2). As discussed above, this is due to the larger relative impacts of aviation on particle number concen-\ntration in the current study, following an improved representation of background particles, in particular the ones generated by the new particle formation process (Kaiser et al., 2019). Although other modelling groups have confirmed these large aviation\n515\nRF (Gettelman and Chen, 2013; Kapadia et al., 2016), the effect of aviation-induced aerosol on liquid clouds is still considered\nquite speculative (Lee et al., 2021), as large uncertainties exist in the size distribution of emitted particles and in the effective-\nness of the downward transport of aviation-induced particles from typical flight altitudes (8–12 km) to the lower atmospheric\nlayers, where they could potentially impact liquid phase clouds by acting as condensation nuclei. All SSPs project a steadily increasing impact of the aviation RF, with the strongest growth in SSP2, reaching a RF of −126 mW m−2 in 2050. As argued\n520\nin Righi et al. (2016), the long lifetime of the aviation fleet and the tight constraints on safety and costs make the introduction\nof mitigation measures in aviation more challenging than in other sectors. In all scenarios the mitigation measures do not lead\nto a reduction of the overall aviation emissions, which are instead only modulated by different growth rates in overall traffic\nvolumes. increasing impact of the aviation RF, with the strongest growth in SSP2, reaching a RF of −126 mW m−2 in 2050. As argued\n520\nin Righi et al. (2016), the long lifetime of the aviation fleet and the tight constraints on safety and costs make the introduction\nof mitigation measures in aviation more challenging than in other sectors. 5\nClimate impacts In\n2050, the greenest SSP1 scenario has the largest climate impact (−120 mW m−2), comparable to SSP3 (−110 mW m−2). This\nmight appear counterintuitive, but can be explained with the much lower particle number background in this clean scenario\n(Fig. 6d), such that even a relatively small amount of shipping-induced particles leads to a large RF effect. SSP2, on the\ncontrary, projects a non-significant aerosol climate effect from shipping in this year, consistent with the large reductions in\n510 susceptibility can be high. The shipping-induced negative RF is reduced in all scenarios in 2030, mostly for SSP1 and SSP2\n505\n(from −145 mW m−2 to −99 and −95 mW m−2, respectively), while in SSP3 the reduction is marginal (to −131 mW m−2). In\n2050, the greenest SSP1 scenario has the largest climate impact (−120 mW m−2), comparable to SSP3 (−110 mW m−2). This\nmight appear counterintuitive, but can be explained with the much lower particle number background in this clean scenario\n(Fig. 6d), such that even a relatively small amount of shipping-induced particles leads to a large RF effect. SSP2, on the contrary, projects a non-significant aerosol climate effect from shipping in this year, consistent with the large reductions in\n510\nshipping SO2 emissions in this scenario (see Sect. 3 and Fig. 1d) and still relevant background sulfate pollution (Fig. 5p). The impact of aviation-induced aerosol in 2015 is quantified as −64 mW m−2, a factor of about four larger than the estimate\nin R13 (−15 mW m−2). As discussed above, this is due to the larger relative impacts of aviation on particle number concen-\ntration in the current study, following an improved representation of background particles, in particular the ones generated by contrary, projects a non-significant aerosol climate effect from shipping in this year, consistent with the large reductions in\n510\nshipping SO2 emissions in this scenario (see Sect. 3 and Fig. 1d) and still relevant background sulfate pollution (Fig. 5p). The impact of aviation-induced aerosol in 2015 is quantified as −64 mW m−2, a factor of about four larger than the estimate\nin R13 (−15 mW m−2). 5\nClimate impacts In all scenarios the mitigation measures do not lead\nto a reduction of the overall aviation emissions, which are instead only modulated by different growth rates in overall traffic\nvolumes. 24 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 6\nConclusions and outlook\n525 The results for the transport-induced aerosol mass concentrations generally confirm the findings of R13, although\nsome major differences exist, especially in the geographical distribution of the transport impacts. These are essentially due to the updated emission inventory used as an input to the model simulations and the different year under investigation (2015 vs. 540\n2000), but also to the improvement of the model. Substantial differences, on the other hand, are found in terms of simulated\ntransport-induced impacts on aerosol number concentration. This is because the new MADE3 model version adopted here is\ncharacterized by a generally lower background concentration of particles, as a result of different assumptions on the particles\nsize distribution upon emissions by anthropogenic sources (including transport) and an improved representation of the aging of the updated emission inventory used as an input to the model simulations and the different year under investigation (2015 vs. 540\n2000), but also to the improvement of the model. Substantial differences, on the other hand, are found in terms of simulated\ntransport-induced impacts on aerosol number concentration. This is because the new MADE3 model version adopted here is\ncharacterized by a generally lower background concentration of particles, as a result of different assumptions on the particles\nsize distribution upon emissions by anthropogenic sources (including transport) and an improved representation of the aging of the updated emission inventory used as an input to the model simulations and the different year under investigation (2015 vs. 540\n2000), but also to the improvement of the model. Substantial differences, on the other hand, are found in terms of simulated\ntransport-induced impacts on aerosol number concentration. This is because the new MADE3 model version adopted here is\ncharacterized by a generally lower background concentration of particles, as a result of different assumptions on the particles\nsize distribution upon emissions by anthropogenic sources (including transport) and an improved representation of the aging of newly-formed particles via the nucleation process. The extensive evaluation performed by Kaiser et al. (2019) and Righi et al. 545\n(2020) shows that the new model version provides a better representation of the aerosol background and the climate mean-state\nof the key cloud and radiation variables compared with observations. 6\nConclusions and outlook\n525 In this study, the global impact of the transport sectors (land transport, shipping and aviation) on aerosol and the resulting\nclimate effects for present-day (2015) and three future projections for 2030 and 2050 have been assessed using the most recent\nCMIP6 emission inventory and the SSPs scenarios as an input to a state-of-the-art global aerosol-climate model, including\ndetailed aerosol microphysics and an explicit representation of aerosol-radiation and aerosol-cloud interactions. The results have been compared with the findings of our previous assessments (Righi et al., 2013, 2015, 2016), which used a predecessor\n530\nversion of the same model and the earlier CMIP5 emissions data for the year 2000 combined with the RCP projections for\n2030. Black carbon and nitrate are found to be highly relevant pollutants from land transport, which contributes 15–20% to the near-\nsurface concentrations of these species. The shipping sector is also an important source of nitrate pollution, responsible for more have been compared with the findings of our previous assessments (Righi et al., 2013, 2015, 2016), which used a predecessor\n530\nversion of the same model and the earlier CMIP5 emissions data for the year 2000 combined with the RCP projections for\n2030. Black carbon and nitrate are found to be highly relevant pollutants from land transport, which contributes 15–20% to the near-\nsurface concentrations of these species. The shipping sector is also an important source of nitrate pollution, responsible for more than 20% of its near-surface concentration over the oceans, and still contributes significantly to aerosol sulfate pollution, despite\n535\nthe introduction of control measures for fuel sulphur content in shipping by MARPOL since 2010. The aviation contribution to\nthe aerosol mass concentrations in the upper-troposphere is generally small (below 5%), while it is larger for particle number\n(5–10%). The results for the transport-induced aerosol mass concentrations generally confirm the findings of R13, although\nsome major differences exist, especially in the geographical distribution of the transport impacts. These are essentially due to than 20% of its near-surface concentration over the oceans, and still contributes significantly to aerosol sulfate pollution, despite\n535\nthe introduction of control measures for fuel sulphur content in shipping by MARPOL since 2010. The aviation contribution to\nthe aerosol mass concentrations in the upper-troposphere is generally small (below 5%), while it is larger for particle number\n(5–10%). 6\nConclusions and outlook\n525 This results in larger RF effects for land transport and\naviation with respect to the previous assessment, while a smaller RF is simulated for shipping, thanks also to the reduced sulfate\nimpacts following the MARPOL regulations on fuel sulphur content. newly-formed particles via the nucleation process. The extensive evaluation performed by Kaiser et al. (2019) and Righi et al. 545\n(2020) shows that the new model version provides a better representation of the aerosol background and the climate mean-state\nof the key cloud and radiation variables compared with observations. This results in larger RF effects for land transport and\naviation with respect to the previous assessment, while a smaller RF is simulated for shipping, thanks also to the reduced sulfate\nimpacts following the MARPOL regulations on fuel sulphur content. The mid- and long-term projections of the transport effects under the SSPs are generally consistent with the narratives\n550\nunderlying the scenarios. SSP1 is characterized by the lowest impacts, with a strong decrease of the land transport effects,\na slight decrease for shipping and a lower increase than the other scenarios for aviation. The climate impact of shipping and\naviation in this scenario are still relatively high despite a strong reduction in their emissions, which can be ascribed to the very\nlow background concentrations, which is mostly controlled by the emissions of the other anthropogenic sectors. The results for The mid- and long-term projections of the transport effects under the SSPs are generally consistent with the narratives\n550\nunderlying the scenarios. SSP1 is characterized by the lowest impacts, with a strong decrease of the land transport effects,\na slight decrease for shipping and a lower increase than the other scenarios for aviation. The climate impact of shipping and\naviation in this scenario are still relatively high despite a strong reduction in their emissions, which can be ascribed to the very\nlow background concentrations, which is mostly controlled by the emissions of the other anthropogenic sectors. The results for the SSP2 scenario confirm its middle-of-the-road nature, which is evident especially in the impacts of the land transport sector. 555\nA peculiarity of this scenario it the low climate impact of shipping, due to low emissions from this sector combined with still\nelevated aerosols levels induced by other sectors, and the relatively large impact of aviation, whose emissions in this scenario\nare still high compared to other sectors. 6\nConclusions and outlook\n525 their consequence on the atmospheric composition, for example hydrogen (Popa et al., 2015), and new particle types resulting\n580\nfrom non-combustion processes (Grigoratos and Martini, 2014; Timmers and Achten, 2016), whose impact is projected to\nbecome increasingly important with the progressive electrification of the land transport sector. their consequence on the atmospheric composition, for example hydrogen (Popa et al., 2015), and new particle types resulting\n580\nfrom non-combustion processes (Grigoratos and Martini, 2014; Timmers and Achten, 2016), whose impact is projected to\nbecome increasingly important with the progressive electrification of the land transport sector. Code and data availability. MESSy is continuously developed and applied by a consortium of institutions. The usage of MESSy and access\nto the source code is licensed to all affiliates of institutions which are members of the MESSy Consortium. Institutions can become members Code and data availability. MESSy is continuously developed and applied by a consortium of institutions. The usage of MESSy and access\nto the source code is licensed to all affiliates of institutions which are members of the MESSy Consortium. Institutions can become members Code and data availability. MESSy is continuously developed and applied by a consortium of institutions. The usage of MESSy and access\nto the source code is licensed to all affiliates of institutions which are members of the MESSy Consortium. Institutions can become members\nof the MESSy Consortium by signing the MESSy Memorandum of Understanding. More information can be found on the MESSy Consor-\n585\ntium Website (http://www.messy-interface.org, last access: 22 November 2022). The model configuration discussed in this paper is based on\nEMAC version 2.55. The exact setup used to produce the result of this paper is archived at the German Climate Computing Center (DKRZ)\nand can be made available to members of the MESSy community upon request. The output of the model simulations discussed in this work\nwill be made available via DOI in the final version of this paper. of the MESSy Consortium by signing the MESSy Memorandum of Understanding. More information can be found on the MESSy Consor-\n585\ntium Website (http://www.messy-interface.org, last access: 22 November 2022). The model configuration discussed in this paper is based on\nEMAC version 2.55. The exact setup used to produce the result of this paper is archived at the German Climate Computing Center (DKRZ)\nand can be made available to members of the MESSy community upon request. 6\nConclusions and outlook\n525 The prominent role of the background has further consequences for\nthe development and application of climate response models (e.g., Grewe and Stenke, 2008; Wild et al., 2012; Dahlmann et al.,\n2016; Rieger and Grewe, 2022) to assess the transportation impacts of aerosol, implying that the global model simulations for training these models shall be performed of varying background conditions on the region of interest. 575\nOn the policy side, a further challenge is posed by the trade-offs connected with the implementation of specific measures. One\nprominent example is the reduction in fuel sulfur content in shipping to improve air quality in the coastlines, that at the same\ntime leads to a reduction of the ship-induced cooling via the aerosol indirect-effect, in turn reducing the offset of greenhouse\nwarning (Quaas et al., 2022). Future modelling studies will also need to deal with the growing impacts of alternative fuels and training these models shall be performed of varying background conditions on the region of interest. 575\nOn the policy side, a further challenge is posed by the trade-offs connected with the implementation of specific measures. One\nprominent example is the reduction in fuel sulfur content in shipping to improve air quality in the coastlines, that at the same\ntime leads to a reduction of the ship-induced cooling via the aerosol indirect-effect, in turn reducing the offset of greenhouse\nwarning (Quaas et al., 2022). Future modelling studies will also need to deal with the growing impacts of alternative fuels and training these models shall be performed of varying background conditions on the region of interest. 575\nOn the policy side, a further challenge is posed by the trade-offs connected with the implementation of specific measures. One\nprominent example is the reduction in fuel sulfur content in shipping to improve air quality in the coastlines, that at the same\ntime leads to a reduction of the ship-induced cooling via the aerosol indirect-effect, in turn reducing the offset of greenhouse\nwarning (Quaas et al., 2022). Future modelling studies will also need to deal with the growing impacts of alternative fuels and\ntheir consequence on the atmospheric composition, for example hydrogen (Popa et al., 2015), and new particle types resulting\n580\nfrom non-combustion processes (Grigoratos and Martini, 2014; Timmers and Achten, 2016), whose impact is projected to\nbecome increasingly important with the progressive electrification of the land transport sector. 6\nConclusions and outlook\n525 The representation of the background is also a modelling challenge and our results the other hand, the representation of the aerosol concentration background in the model can play a significant role in charac-\n565\nterizing the impact of the perturbation introduced by the emissions of the specific sectors. This is particularly the case when\nanalysing the scenario projections, as the potential reductions in the emissions for a given sector needs to be set in the context of\nthe emission changes in other sectors. Policy measures cannot therefore be evaluated only focusing on a single sector, but have\nto be considered as part of a larger picture. The representation of the background is also a modelling challenge and our results suggest that an accurate representation of the aerosol background concentrations is important and can significantly affect the\n570\nestimated impacts, as it is clear when comparing the outcome of the present study with the result of the previous assessment\nobtained with a predecessor version of the same model. The prominent role of the background has further consequences for\nthe development and application of climate response models (e.g., Grewe and Stenke, 2008; Wild et al., 2012; Dahlmann et al.,\n2016; Rieger and Grewe, 2022) to assess the transportation impacts of aerosol, implying that the global model simulations for suggest that an accurate representation of the aerosol background concentrations is important and can significantly affect the\n570\nestimated impacts, as it is clear when comparing the outcome of the present study with the result of the previous assessment\nobtained with a predecessor version of the same model. The prominent role of the background has further consequences for\nthe development and application of climate response models (e.g., Grewe and Stenke, 2008; Wild et al., 2012; Dahlmann et al.,\n2016; Rieger and Grewe, 2022) to assess the transportation impacts of aerosol, implying that the global model simulations for suggest that an accurate representation of the aerosol background concentrations is important and can significantly affect the\n570\nestimated impacts, as it is clear when comparing the outcome of the present study with the result of the previous assessment\nobtained with a predecessor version of the same model. 6\nConclusions and outlook\n525 The strong growth of pollution characterizing SSP3 is remarkable, showing steadily the SSP2 scenario confirm its middle-of-the-road nature, which is evident especially in the impacts of the land transport sector. 555\nA peculiarity of this scenario it the low climate impact of shipping, due to low emissions from this sector combined with still\nelevated aerosols levels induced by other sectors, and the relatively large impact of aviation, whose emissions in this scenario\nare still high compared to other sectors. The strong growth of pollution characterizing SSP3 is remarkable, showing steadily the SSP2 scenario confirm its middle-of-the-road nature, which is evident especially in the impacts of the land transport sector. 555\nA peculiarity of this scenario it the low climate impact of shipping, due to low emissions from this sector combined with still\nelevated aerosols levels induced by other sectors, and the relatively large impact of aviation, whose emissions in this scenario\nare still high compared to other sectors. The strong growth of pollution characterizing SSP3 is remarkable, showing steadily 25 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. growing impacts in 2030 and 2050 with respect to 2015, especially in the land transport sector. These results show that the\nimpact of the transport sectors under the SSPs are more consistent with the respective storylines than in the predecessor RCP\n560\nscenarios used in (Righi et al., 2015, 2016). From the analysis of the simulations in this study we can conclude that the challenges in quantifying the impact of the trans-\nportation sectors on aerosol and climate are twofold. On the one hand, reliable emission inventories are essential for a correct\nquantification of the resulting impacts: this concerns not only the total emitted mass, but also its geographical distribution. On the other hand, the representation of the aerosol concentration background in the model can play a significant role in charac-\n565\nterizing the impact of the perturbation introduced by the emissions of the specific sectors. This is particularly the case when\nanalysing the scenario projections, as the potential reductions in the emissions for a given sector needs to be set in the context of\nthe emission changes in other sectors. Policy measures cannot therefore be evaluated only focusing on a single sector, but have\nto be considered as part of a larger picture. Financial support. This study was supported by the DLR transport research program (TraK and DATAMOST projects).\n600 6\nConclusions and outlook\n525 The output of the model simulations discussed in this work\nwill be made available via DOI in the final version of this paper. 26 https://doi.org/10.5194/esd-2022-52\nPreprint. Discussion started: 4 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Author contributions. MR conceived the study, designed and performed the simulations, analysed the data and wrote the paper. JH conceived\n590\nthe study, contributed to the simulation design and to the text. SB prepared the CMIP6/SSP emission data used to drive the model simulations\nand contributed to the text. Competing interests. The authors declare no competing interests. Competing interests. The authors declare no competing interests. Competing interests. The authors declare no competing interests. Acknowledgements. We are grateful to Axel Lauer (DLR) for his valuable comments on an earlier version of this manuscript, and to Christof\nBeer, Patrick Jöckel and Mariano Mertens (DLR) for helpful suggestions. The processing of the CMIP6/SSP emissions for usage in EMAC\n595\nhas greatly benefited from the efforts by Markus Kunze (Freie Universität Berlin) and Phoebe Graf (DLR). MR would also like to thank\nManuel Schlund (DLR), for his competent help with the matplotlib Python library. The model simulations and data analysis for this work\nused the resources of the Deutsches Klimarechenzentrum (DKRZ) granted by its Scientific Steering Committee (WLA) under project ID\nbd0080. Beer, Patrick Jöckel and Mariano Mertens (DLR) for helpful suggestions. The processing of the CMIP6/SSP emissions for usage in EMAC\n595\nhas greatly benefited from the efforts by Markus Kunze (Freie Universität Berlin) and Phoebe Graf (DLR). MR would also like to thank\nManuel Schlund (DLR), for his competent help with the matplotlib Python library. The model simulations and data analysis for this work\nused the resources of the Deutsches Klimarechenzentrum (DKRZ) granted by its Scientific Steering Committee (WLA) under project ID\nbd0080. Beer, Patrick Jöckel and Mariano Mertens (DLR) for helpful suggestions. The processing of the CMIP6/SSP emissions for usage in EMAC\n595\nhas greatly benefited from the efforts by Markus Kunze (Freie Universität Berlin) and Phoebe Graf (DLR). MR would also like to thank\nManuel Schlund (DLR), for his competent help with the matplotlib Python library. 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Lett., 38, n/a–n/a,\n835\nhttps://doi.org/10.1029/2011gl049289, https://doi.org/10.1029%2F2011gl049289, 2011. Unger, N.: Global climate impact of civil aviation for standard and desulfurized jet fuel, Geophys. Res. Lett., 38, n/a–n/a,\n835\nhttps://doi.org/10.1029/2011gl049289, https://doi.org/10.1029%2F2011gl049289, 2011. Unger, N., Zhao, Y., and Dang, H.: Mid-21st century chemical forcing of climate by the civil aviation sector, Geophys. Res. Lett., 40, Unger, N.: Global climate impact of civil aviation for standard and desulfurized jet fuel, Geophys. Res. Lett., 38, n/a–n/a,\n835\nhttps://doi.org/10.1029/2011gl049289, https://doi.org/10.1029%2F2011gl049289, 2011. Unger, N., Zhao, Y., and Dang, H.: Mid-21st century chemical forcing of climate by the civil aviation sector, Geophys. Res. Lett., 40,\n641–645, https://doi.org/10.1002/grl.50161, https://doi.org/10.1002%2Fgrl.50161, 2013. Unger, N., Zhao, Y., and Dang, H.: Mid-21st century chemical forcing of climate by the civil aviation sector, Geophys. Res. Lett., 40,\n641–645, https://doi.org/10.1002/grl.50161, https://doi.org/10.1002%2Fgrl.50161, 2013. van Marle, M. J. E., Kloster, S., Magi, B. I., Marlon, J. R., Daniau, A.-L., Field, R. D., Arneth, A., Forrest, M. Knorr, W., Lasslop, G., Li, F., Mangeon, S., Yue, C., Kaiser, J. W., and van der Werf, G. R.: Historic global biomass burning emissions\n840\nfor CMIP6 (BB4CMIP) based on merging satellite observations with proxies and fire models (1750–2015), Geosci. Model Dev., 10,\n3329–3357, https://doi.org/10.5194/gmd-10-3329-2017, https://doi.org/10.5194%2Fgmd-10-3329-2017, 2017. References A., Zeng,\nG., Hess, P., Duncan, B. N., Bergmann, D. J., Szopa, S., Jonson, J. E., Keating, T. J., and Zuber, A.: Modelling future changes in surface\nozone: a parameterized approach, Atmos. Chem. Phys., 12, 2037–2054, https://doi.org/10.5194/acp-12-2037-2012, 2012. 34"
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https://www.ocl-journal.org/articles/ocl/pdf/2016/01/ocl150045-s.pdf
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Understanding the effects of docosahexaenoic acid (DHA) supplementation during pregnancy on multiple outcomes from the DOMInO trial
Oilseeds and fats, crops and lipids/OCL. Oilseeds & fats crops and lipids
2,015
cc-by
3,114
OCL 2016, 23(1) D105 c M. Makrides, Published by EDP Sciences 2015  DOI: 10.1051/ocl/2015045 OCL Oilseeds & fats Crops and Lipids Available online at: www.ocl-journal.org Research Article – Dossier Open Access Understanding the effects of docosahexaenoic acid (DHA) supplementation during pregnancy on multiple outcomes from the DOMInO trial Maria Makrides1,2,3, 1 2 3 Child Nutrition Research Centre, Women’s and Children’s Hospital, Flinders Medical Centre and Women’s and Children’s Health Research Institute, 72 King William Road, North Adelaide SA 5006, Adelaide, Australia Discipline of Pediatrics, University of Adelaide, Australia South Australian Health and Medical Research Institute, Adelaide, Australia Received 17 June 2015 – Accepted 29 July 2015 Abstract – Docosahexaenoic acid (DHA) has been postulated to extend the period of gestation, increase birth weight, enhance neurodevelopment and reduce the risk of allergic disease. Because of its large sample size and relatively broad inclusion criteria, the DOMInO (DHA to Optimise Mother Infant Outcome) trial offers the opportunity to explore the effect of prenatal DHA supplementation of multiple outcomes. Overall, the DOMInO trial showed that prenatal DHA supplementation increases the length of gestation, reduces the risk of early preterm and low birth weight, has little or no effect on maternal postpartum depression and early childhood neurodevelopment but reduces the risk of atopic eczema and sensitisation in the first year of life. The clinical utility of prenatal DHA supplementation of reducing early birth is important and requires further investigation. Keywords: DHA / pregnancy / child development / child allergies Résumé – Comprendre les effets de la supplémentation en acide docosahexaénoïque (DHA) pendant la grossesse sur de multiples paramètres à partir de l’étude DOMIn0. Il a été envisagé que l’acide docosahexaénoïque (DHA) puisse prolonger la période de gestation, augmenter le poids de naissance, renforcer le développement neurologique et réduire le risque de maladie allergique. En raison de la grande taille de son échantillon et de critères d’inclusion relativement larges, l’étude DOMInO (DHA to Optimise Mother Infant Outcome) offre l’occasion d’explorer l’effet d’une supplémentation prénatale en DHA sur de multiples paramètres. Dans l’ensemble, l’étude DOMInO a montré que la supplémentation prénatale en DHA augmente la durée de la gestation, réduit le risque de prématurité précoce et de faible poids de naissance, a peu ou pas d’effet sur la dépression maternelle post-partum et sur le développement neurologique durant la petite enfance, mais réduit le risque d’eczéma atopique et de sensibilisation dans la première année de la vie. L’utilité clinique d’une supplémentation prénatale en DHA afin de réduire les naissances avant terme est importante et nécessite une enquête plus approfondie. Mots clés : DHA / grossesse / développement de l’enfant / allergies de l’enfant 1 Introduction The n-3 long chain polyunsaturated fatty acid (LCPUFA), docosahexaenoic acid (DHA), has multiple biochemical and physiological roles in many organ systems. This is perhaps not surprising as DHA is an integral part of all cell membranes. It also naturally follows that DHA has a number of postulated clinical effects, especially in the perinatal period which is a period characterized by anabolic change and organ growth. Some of the postulated effects of DHA in the perinatal period are to extend gestational length, enhance early childhood neuro development and to ameliorate or prevent the development of allergic disease. While various clinical studies have evaluated the effect of DHA supplementation during pregnancy, it has been difficult to elucidate the extent of benefit for general populations as different studies have been specifically designed to assess particular outcomes. For example, many studies designed to assess the effect of DHA supplementation during pregnancy on childhood neurodevelopmental outcomes have excluded children born prematurely and do not contain data relating to the duration of pregnancy. Because of its large sample size and relatively broad inclusion criteria, the DOMInO (DHA to Optimise Mother Infant Outcome) trial offers the Correspondence: maria.makrides@sa.gov.au This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dossier LIPIDS AND BRAIN Lipides et cerveau M. Makrides: OCL 2016, 23(1) D105 opportunity to explore the effect of prenatal DHA supplementation of multiple outcomes. The purpose of this paper is to bring together the core outcomes of the DOMInO trial reported to date and consider the current evidence as well as the research gaps for further investigation. 2 The DOMInO trial The DOMInO trial is a multi-center, double-blind randomized controlled trial that was originally designed to test the effect of DHA supplementation during the last half of pregnancy on reducing the symptoms of post-partum depression and enhancing early childhood development (DOMInO Trial: ACTRN12605000569606). The DOMInO trial is the largest DHA supplementation study in pregnancy reported to date, and the methods have been previously published (Makrides et al., 2010). To summarize, women at 5 study sites around Australia, with singleton pregnancies less than 21 weeks gestation were invited to participate (between October 2005– January 2008). Women were excluded if they were already taking a supplement containing DHA, their fetus had a known major abnormality, they had a bleeding disorder in which fish oil was contraindicated, were taking anticoagulant therapy, had a documented history of drug or alcohol abuse, were participating in another fatty acid trial, were unable to give written or informed consent or if English was not the main language spoken at home. Enrolled women were assigned a unique study number corresponding to allocation of a treatment pack through a computer driven telephone randomization service according to an independently generated randomization schedule, stratified by center and parity. Women allocated to the DHA group were asked to consume three 500 mg capsules per day of DHA-rich fish oil providing ∼800 mg/d DHA and 100 mg/d of eicosapentaenoic acid (EPA) (Incromega 500 TG, Croda Chemicals, East Yorkshire, England) and women in the control group were asked to consume three 500 mg vegetable oil (rapeseed, sunflower and palm oil) capsules (without DHA) per day, from study entry until birth. All capsules were identical in size, shape and color and donated by Efamol, Surrey, England. A total of 2399 women were randomized into the DOMInO trial. Of these, 1197 were randomly assigned to treatment with DHA and 1202 to control treatment. Outcome measures for the DOMInO trial included major pregnancy and birth outcomes, maternal symptoms of post-partum depression, as well as neurodevelopment and allergic disease in early childhood for specific subgroups of children. head circumference was defined as below the 90th percentile for birth weight, length, and head circumference for the corresponding gestational age and sex. Perinatal death was defined as stillbirth or death within the first 28 days of life. Postpartum Depression was assessed using the selfreported Edinburgh Postnatal Depression Scale (EPDS) questionnaire in English at six weeks and six months Postpartum (Cox et al., 1987). Validation studies indicate high sensitivity (68–95%) and high specificity (78–96%) of the EPDS against a clinical psychiatric diagnosis of PPD (Cox et al., 1987; Boyce et al., 1993; Murray and Carothers, 1990). A score of more than 12 on the EPDS is widely used to indicate a probable depressive disorder (Hiscock and Wake, 2001; Murray, 1992). Neurodevelopment at 18 months and 4 years: a subset of infants, consisting of 630 randomly selected term infants and 96 preterm infants, from two centres in Adelaide were selected for neurodevelopmental assessments at 18 months and 4 years of age. Assessments were performed by study psychologists who were blinded to treatment group allocation and previous results. Infants were assessed with the Bayley Scales of Infant and Toddler Development, Third Edition (BayleyIII) at 18 months (Bayley, 2006). Child neurodevelopment at 4 years was assessed using the General Conceptual Ability (GCA) score of the Differential Ability Scales Second Edition (DAS II) (Elliot, 2007). The Bayley-III and DAS II have age-standardized scores with a mean of 100 and SD of 15 (range 50–150). Standardized scores more than one SD below the mean (< 85) were considered delayed. For children born preterm, their corrected age was used to standardize test scores. Allergy assessments at 1 and 3 years of age: infants whose mothers were enrolled at either of two centres in Adelaide and who had a first-degree relative with medically diagnosed allergic disease (asthma, allergic rhinitis, eczema) were eligible for the allergy follow-up. Medically diagnosed atopic (or IgE associated) disease at 1 and 3 years, defined as a positive skin prick test together with clinical symptoms of allergy (eczema, asthma, allergic rhinitis or food allergy) was assessed by blinded study doctors (Palmer et al., 2013). A total of n = 706 women enrolled, with n = 368 women in the intervention group and n = 338 women in the control group. At 1 year of age, the food allergens tested were whole hens’ egg, cows’ milk, wheat, tuna and peanut, and the aeroallergens tested were ryegrass pollen, olive tree pollen, Alternaria tenuis, cat hair and house dust mite (Dermatophagoides pteronyssinus). At 3 years of age, the same allergens were tested with the addition of two foods (cashew nut and sesame seed) and one aeroallergen (house dust mite, Dermatophagoides farinae). 3 Outcome assessments Pregnancy outcome data were collected via a blinded review of women’s and infant’s medical records including duration of gestation, the frequency of preterm birth < 37 weeks’ gestation and early preterm birth < 34 weeks gestation, gestational diabetes mellitus (GDM), preeclampsia and pregnancy induced hypertension (Makrides et al., 2010; Zhou et al., 2012). Other outcomes included small for gestational age for weight, length, and head circumference was defined as below the 10th percentile for birth weight, length, and head circumference, and large for gestational age for weight, length, and 4 Outcome data from the DOMInO trial Pregnancy outcomes: there were fewer very preterm births (< 34 weeks’ gestation) in the DHA group compared with the control group (1.09% vs. 2.25%; adjusted RR, 0.49; 95% CI, 0.25–0.94; P = 0.03) and this was associated with fewer infants requiring admission to neonatal intensive care in the DHA group (1.75% vs. 3.08%, RR 0.57; 95% CI 0.34–0.97; P = 0.04) (Makrides et al., 2010). However, DHA supplementation resulted in more postterm births requiring obstetric intervention (inductions or cesarean deliveries) compared D105, page 2 of 4 with the control group (17.59% vs. 13.72%; adjusted RR, 1.28; 95% CI, 1.06–1.54; P = 0.01) (Makrides et al., 2010). This overall shift in the length of gestation resulted in a higher mean birth weight (68 g, 95% CI, 23–114 g; P = 0.003) and fewer infants of low birth weight (3.41% vs. 5.27%; adjusted RR, 0.65; 95% CI, 0.44–0.96; P = 0.03) in the DHA group compared with the control group. Mean birth weight z scores (corrected for gestational age and sex) did not differ between groups, strongly indicating that group differences in birth size were largely a function of gestational age at birth (Makrides et al., 2010). No statistically significant differences in the RR of small for gestational age or large for gestational age for weight, length, or head circumference were found between the groups. Five infants had brain injury at birth and 5 had neonatal convulsion in the control group compared with none in the DHA rich fish-oil group (P = 0.03). There were 12 perinatal deaths among infants of control mothers and 3 among infants of the fish-oil – supplemented mothers (P = 0.03). No differences were observed between the groups in other pregnancy or perinatal complications, including the incidence of GDM, PE, pregnancy induced hypertension, and neonatal hypoglycaemia, resuscitation at birth or other neonatal complications (Zhou et al., 2012). The tantalising data suggesting that supplementation with 800 mg of DHA per day can reduce the risk of early preterm birth by about 50% is consistent with the Cochrane systematic review that was completed before the inclusion of the DOMInO trial and included trials that largely supplemented pregnant women with marine oil dominant in EPA rather than DHA. While n-3 LCPUFA supplementation during pregnancy appears to increase the length of gestation, the clinical utility of our findings and their incorporation into clinical practice guidelines requires further work. Postpartum Depression: the percentage of women with depressive symptoms (EPDS score > 12) during the first 6 months postpartum did not differ between the DHA and control groups (9.67% vs. 11.19%; adjusted RR, 0.85; 95% CI, 0.70–1.02; P = 0.09) (Makrides et al., 2010). Depressive symptoms were more common among women with a previous or current diagnosis of depression at trial entry, but did not differ between groups. The percentage of women with a new medical diagnosis for depression during the trial or a diagnosis requiring treatment also did not differ between groups (Makrides et al., 2010). Overall, our data do not support DHA supplementation during pregnancy as a strategy to prevent post-partum depression. Neurodevelopment of children at 18 months and 4 years: at 18 months of age, mean cognitive composite scores (adjusted mean difference, 0.01; 95% CI, −1.36 to 1.37; P = 0.99) and mean language composite scores (adjusted mean difference, −1.42; 95% CI, −3.07 to 0.22; P = 0.09) of children in the DHA group did not differ from children in the control group, although fewer children from the DHA group had cognitive scores indicating delayed cognitive development compared with controls (Makrides et al., 2010). At 4 years of age, 703 were eligible for follow-up and 646 (91.9%; n = 313 in DHA group and n = 333 in control group) were included in the analysis. Mean GCA scores of children born to women in the DHA group did not differ from children from women in the control group (adjusted mean difference 0.29, 95% CI –1.35 to 1.93, P = 0.73) and there was no difference in the proportion of children with impaired (GCA < 85) or advanced (GCA > 115) performance between the groups (Makrides et al., 2014). These data support the most recent systematic review assessing the effect of DHA interventions during pregnancy on the neurodevelopmental outcomes of children (Gould et al., 2013) and indicates that in largely well-nourished populations DHA supplementation has negligible benefit on longer term developmental outcomes. This is perhaps not surprising as the vast majority of children would have experienced the full benefit of in utero DHA transfer until term birth. Allergy: our data showed that there was no significant difference in the overall percentage of children with IgE mediated allergic disease in the first 3 years of life between the DHA and control groups (64/368 (17.3%) vs. 76/338 (22.6%); adjusted relative risk 0.78; 95% CI 0.58–1.06; P = 0.11) (Palmer et al., 2013). At 1 year of age, the percentage of infants diagnosed as having atopic eczema was lower in the DHA group (26/368 (7%) vs. 39/338 (12%); unadjusted relative risk 0.61, 0.38 to 0.98, P = 0.04; adjusted relative risk 0.64, 0.40 to 1.02, P = 0.06) and fewer infants were sensitised to egg in the DHA group (34/368 (9%) vs. 52/338 (15%); unadjusted relative risk 0.61, 0.40 to 0.91, P = 0.02; adjusted relative risk 0.62, 0.41 to 0.93, P = 0.02) (Palmer et al., 2012). However, these findings did not persist at 3 years of age. It may be that the effects were diluted as the children grow by other environmental factors or that any effect of n-3 LCPUFA, like DHA, in the prenatal period are smaller than originally anticipated. The non-significant risk reductions of up to 22% may still be of public health significance however, as the burden and cost of allergic disease on affected families are high, and fish oil intervention is safe and relatively cheap. In conclusion, our DOMInO study with a relatively typical and large sample of pregnant women has demonstrated that DHA supplementation during the last half of pregnancy significantly increases the duration of gestation and that this results in a reduction in the number infants who are born very preterm and low birth weight. The importance of this finding is of potential global importance because of the high morbidity associated with early preterm birth. It is interesting to note that the direct longer term effects of prenatal supplementation were either not evident (such as the reduction in postpartum depression or enhanced neurodevelopment in the children) or relatively short term (such as the reduction in atopic eczema at 1 but not 3 years of age). It may be that the effect on these longer term outcomes is small and therefore not easily detectable and not clinically significant or that longer periods of supplementation may be needed. References Bayley N. 2006. Bayley Scales of infant and toddler development, 3rd ed. TX: Pearson Education, Inc. Boyce P, Stubbs J, Todd A. 1993. The Edinburgh Postnatal Depression Scale: validation for an Australian sample. Aust. N.Z. J. Psychiatry 27: 472–476. Cox JL, Holden JM, Sagovsky R. 1987. Detection of postnatal depression. Development of the 10-item Edinburgh Postnatal Depression Scale Br. J. Psychiatry 150: 782–786. D105, page 3 of 4 Dossier M. Makrides: OCL 2016, 23(1) D105 M. Makrides: OCL 2016, 23(1) D105 Elliott CD. 2007. Differential ability scales second edition. PsychCorp, Pearson. Gould JF, Smithers LG, Makrides M. 2013. The effect of maternal omega-3 LCPUFA supplemention during pregnancy on early childhood cognitive and visual development: a systematic review and meta-analysis of randoomized controlled trials. Am. J. Clin. Nutr. 97: 531–544. Hiscock H, Wake M. 2001. Infant sleep problems and postnatal depression: A community-based study. Pediatrics 107: 1317–1322. Makrides M, Gibson RA, McPhee AJ, Yelland L, Quinlivan J, Ryan P. 2010. Effect of DHA supplementation during pregnancy on maternal depression and neurodevelopment of young children: a randomized controlled trial. J. Am. Med. Assoc. 304: 1675–1683. Makrides M, Gould JF, Gawlik NR, et al. 2014. Four-year follow-up of children born to women in a randomized trial of prenatal DHA supplementation. J. Am. Med. Assoc. 311: 1802–1804. Murray L. 1992. The impact of postnatal depression on infant development. J. Child. Psychol. Psyc. 33: 543–561. Murray L, Carothers AD. 1990. The validation of the Edinburgh Postnatal Depression Scale on a community sample. Br. J. Psychiatry 157: 288–290. Palmer DJ, Sullivan T, Gold MS, et al. 2012. Effect of n-3 long chain polyunsaturated fatty acid supplementation in pregnancy on infants’ allergies in first year of life: randomised controlled trial. B.M.J. 344: e184. Palmer DJ, Sullivan T, Gold MS, et al. 2013. Randomized controlled trial of fish oil supplementation in pregnancy on childhood allergies. Allergy 68: 1370–1376. Zhou SJ, Yelland L, McPhee AJ, Quinlivan J, Gibson RA, Makrides M. 2012. Fish-oil supplementation in pregnancy does not reduce the risk of gestational diabetes or preeclampsia. Am. J. Clin. Nutr. 95: 1378–1384. Cite this article as: Maria Makrides. Understanding the effects of docosahexaenoic acid (DHA) supplementation during pregnancy on multiple outcomes from the DOMInO trial. OCL 2016, 23(1) D105. D105, page 4 of 4
https://openalex.org/W4389976406
https://www.ijfmr.com/papers/2023/6/6646.pdf
English
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An Investigation into the Reasons of Conflicts Between Learners and Teachers: A Case of Three Selected Secondary Schools in Zambezi District
International Journal For Multidisciplinary Research
2,023
cc-by-sa
3,660
Mungulo Kuwema1, Malvern Kanyati2 Rockview University, Lusaka, Zambia Abstract The purpose of the study was to investigate the reasons of conflicts between learners and teachers. The objectives of the study were to identify the teachers’ weakness in coming into conflicts with learners, to establish learners’ weakness in coming into conflicts with teachers and to find out views from the community about the reasons of conflicts between learners and teachers. Data was collected from forty respondents from three selected secondary schools in Zambezi district. The study used descriptive survey design and data was collected using interviews, questionnaires and focus groups guides. Random sampling was used to determine the participant. The findings revealed that there are some conflicts between learners and teachers emanating from over socialization. The study therefore, recommends upholding of professionalism among teachers and well discipled learners for quality education delivery. Keywords: Reasons, Conflicts, Learner, Teachers International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Volume 5, Issue 6, November-December 2023 Research Objectives 1. To identify the teachers’ weakness in coming into conflicts with learners, 1. To identify the teachers’ weakness in coming into conflicts with learners, To establish learners’ weakness in coming into conflicts with teachers and, 2. To establish learners’ weakness in coming into conflicts with teachers and, 3. To find out views from the community about the common reasons of conflicts between learners and teachers. 3. To find out views from the community about the common reasons of conflicts between learners and teachers. Purpose of the Study The purpose of the study was to investigate the causes of conflicts between learners and teachers in selected secondary schools in Zambezi district. Significance of the Study These findings of the study is useful or helpful to Principals, Inspectors, Head teachers, Lectures and teachers in understanding the conflicts between learners and teachers and then, appreciate the role of staff and students in its promotion. Statement of the Problem Conflicts will continuously occur since they are essential to human nature and are obligatory for moral and emotional development and found in schools. Since conflict arises recurrently in schools, it follows that educators need to acclimatize at handling it positively. It’s critical to deal with conflict head-on and find solutions that are both integrative and constructive while building cooperative connections. Thus, the study sought to investigate the causes of conflicts between learners and teachers. International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com valued, and individual learners and teachers are heard and included in the conversation—all so that educational activity can proceed. valued, and individual learners and teachers are heard and included in the conversation—all so that educational activity can proceed. Theoretical Framework Conflict theory states that tensions and conflicts arise when resources, status, and power are unevenly distributed between groups in society and that these conflicts become the engine for social change. In this context, Marx’s conflict approach was developed in 19th century by Karl Marx (1818-83). Marx argued wealth and power were unequally distributed in society and sought to explain how one minority group in society maintained its dominance over the majority (working –class). INTRODUTION School conflicts is an occurrence that has been witnessed in most secondary schools. School conflicts disrupt school programs and lowers performance of the school. School conflicts occurs in different forms such as strikes, riots, unrests among others. School conflict is defined as the disagreement between individuals or groups regarding ideas, interests, principles, and values within the school community, perceiving the parties their interests as excluded, although they may not be (Perez-Serrano and Perez-de-Guzman, 2011). Conflict is an essential and unavoidable human phenomenon because where there is human interaction, there is a likelihood of personal likes and dislikes. These agreements and disagreements among individuals and groups in the school sometimes lead to conflict. Conflicts are neither constructive non-disruptive but the ways these are handled in the school make them positive or negative (Isabu, 2017). Conflict is inevitable. It results from the interplay of differences between people. Since people differ in many ways, in their cultural backgrounds and assumptions, their personal styles, their worldviews and perspectives, and their hopes and aspirations, there will frequently be places where they rub against each other. This is as true in school communities as in other places in the social world. Schools do not need to aim for an environment in which conflict is never manifest, so much as an environment in which conflict is handled and managed effectively, so that differences are respected, competing cultural perspectives are Volume 5, Issue 6, November-December 2023 IJFMR23066646 IJFMR23066646 1 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com valued, and individual learners and teachers are heard and included in the conversation—all so that educational activity can proceed. rnational Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com When people share common environments, the development of conflicts is inevitable, and it could be argued that this is perfectly normal. In fact, it is not possible to imagine a conflict-free organization, since every organization is made up of people with different ideas, thoughts, and emotions. Therefore, it is normal for rivalry, dispute, and conflicting needs and goals to emerge, when people are required to work and coexist with other stakeholders of an organization. Conflicts are inherent in the life of individuals, teams, and organizations, and this should be expected, because conflicts are associated with interaction. The concept of conflict has existed since the dawn of humanity. The issue of conflicts has dominated organizational life since the 1970s, especially in the United States, followed by its spread all over the world (Doğan, 2016). According to Tjosvold (2017), conflicts play a key part in organizational thinking, supervision, performance, and management. In this context, it has been acknowledged that conflicts are part of everyday organizational life Various definitions of the concept of conflict are provided in literature. According to these definitions, conflict can be described as a dispute that emerges on either a personal or team level, and affects negatively the fulfilment of the social, psychological, and physical needs of individuals, as it is argued by Eren and Peker & Aytürk (as it cited in Yaraş & Gündüzalp, 2021). Although there is no single definition that is commonly accepted by researchers, the concept of interdependence among individuals and/or groups of individuals should be taken into consideration, since it is essential for conflict to occur (Deutsch, 2013; Manesis, Vlachou, & Mitropoulou, 2019; Rahim, 2001; Robbins, 2014). Actually, conflict is a difference in goals among individuals, groups of individuals, and organizations (Hocker & Wilnat, 2015). It is an interactive situation that is based on differences among individuals and groups of individuals (Shahin, 2019). Therefore, conflict is a social phenomenon that is experienced as a result of dispute that arises in the context of interpersonal relationships (Rahim, 2013). Research Design The study implored a descriptive design that adopted a qualitative method. This allowed for more flexible strategies of data collection in order to answer the research questions (Musonda, 2019). Research Sites The study was strictly carried out in Zambezi district of North Western Province. The population target was all the secondary schools in Zambezi. rnational Journal for Multidisciplinary Research (IJFMR) At organizational level (workplace environment), conflicts are attributed to differences or disagreements that refer to attitudes and opinions and views, as well as needs and values, roles and tactics, conflicting interests in a project/task, and also disputes that are caused by rivalry, errors/mistakes or ignorance, and negative behaviours and reactions of organizational stakeholders, as well as absenteeism, excessive complaining, etc. (Saiti, 2015). In more detail, in schools, negative situations that are due to the qualitative and quantitative characteristics of families, students, support staff, principals, and teachers, and which emerge during the fulfilment of pre-set goals, are the basis of organizational conflicts, according to İnandı, Tunç, and Gündüz (as it is cited in Yaraş & Gündüzalp, 2021). LITERATURE REVIEW Schools are no longer for children; half the learners now in secondary schools are sexually mature. However, although sexual development is an important part of adolescence, it is often not seen as an integral part of schooling (Lindsay Geoff, 2013). Thompson (2015) observes that learners view schools as operating on assumption of distrust. The teachers have no faith in learners and they have no faith in teachers. The consequence of this distrust is a self-fulfilling prophecy in learners. Kings (2014) made another important contribution to distrust viewed by learners between them and authority. The result of this study showed that poor communication between staff and learners, failure to provide learners with opportunities to exercise responsibility, failure to interpret the reasons and purpose of the school rules by the authority and failure to be trusted as human beings who are sensible, trigger reasons of conflicts between learners and teachers. Volume 5, Issue 6, November-December 2023 IJFMR23066646 2 2 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Data Analysis y The research data was analyzed qualitatively as semi structured interviews and questionnaires were used as data collection instruments. Thematic approach was used, where data analysis started with the categorization of themes from the semi structured interviews and questionnaire (Smith, 2013). Population, Sample and Sampling Procedure The study was carried out on 40 participants. These included 22 learners, 6 teachers, 6 community members and 6 school managers from Blue, Orange and White selected secondary schools in Zambezi. Volume 5, Issue 6, November-December 2023 IJFMR23066646 3 Ethical Issues The researcher got permission from the community members, the District Education Board Secretary and school authorities. The respondents were assured of the confidentiality of the data collected and pseudo names were used in the study. FINDINGS AND DISCUSSIONS Male learners having relationships with female teachers In agreement with the saying which gives: Love is blind; Love conquer is all, some young female teachers straight from college may surprisingly fall in love with some older boys in grade 12, especially if the boy is from a well to do family. When this comes to the knowledge of many learners, they turn their teachers into objects and that if they can manage to compete with teachers for a girl-friend, what more if they were in employment? Male teachers will definitely react very sharply to this. They will, rust out at both the female teachers and boys. The boys, helped by their girls, would treat the reaction as an act of open jealous and conflicts arise. Teacher teaching his/her age mates Unlike in the past when most teachers would be older than almost all the learners, nowadays, teachers, especially straight from college may be young. Naturally, antagonism between the older learners and the young teachers arise. Older learners would argue: what can these young men or ladies tell/teach us? On the other hand, young teachers would argue: Are we responsible for your late completing of school? These results into a spiral of conflicts Male teachers having relationships with female learners Most female learners in secondary school have reached adolescence stage. During this stage, they would want to experience love affairs with people older than them because they find the boys with whom they are classmates, childish and in experienced. To this, some girls may try to entail their male teachers who in turn, will get trapped. When this gets to the knowledge of the boys, they lose respect towards their teachers and conflicts arise. Learners having too much knowledge of teachers’ private life If learners know too much about their teachers’ private life, especially the negative things about their teachers, conflicts arise. For instance, a teacher known for drinking too much or having a lot of relationships with girls would in no way be listened to whenever he talks against issues of sex and beer to the learners. Drinking from the same places with learners Whenever a teacher steps into a drinking place where learners are also present, learners would run away. Alternatively, if they have got money, learners would buy beer for their teachers. If this habit continues, learners lose respect for their teachers by denouncing them at school to their fellow learners saying: we buy beer for him/them; he/they is/are cash-strapped. In cases where the teacher does not know the learner and the learners are aware that the teacher does not know them, they don’t run away but rather continue to drink. The look at what type of beer to that being drunk by their teachers, learners may look down upon him, denouncing to their fellow learners at school. Some teachers may be hated by learners for being tough on the learners found in drinking places. Later conflicts start. FINDINGS AND DISCUSSIONS FINDINGS AND DISCUSSIONS Table 1: Records compiled from the answers in the questionnaires and interviews. SUSPECTED REASON OF CONLICT NUMBER FOR NUMBER AGAINST PERCEN TAGE FOR (%) PERCEN TAGE AGAINST (%) Teachers having love affairs with female learners 30 08 85 10 Learners having love with female teachers. 05 35 05 90 Teaching his/her age mates 35 10 75 25 Drinking from the same places with learners 25 15 63 38 Having too much knowledge of teachers’ private life 28 12 60 30 Teachers having showdowns with learners 20 20 50 50 Source: Field data Records compiled from the answers in the questionnaires and interviews. Source: Field data The study reviewed that, there were more people about (85%) who agreed or rotted for the idea that teachers have love affairs with female pupils while only few (10%) were against. The reason as to whether pupils had love with female teachers was rotted against by most interviewed people (90%). Only a hand full rotted for. The reason as to whether pupils had love with female teachers was rotted against by most interviewed people (90%). Only a hand full rotted for. There are more teachers teaching their age mates (75%) and (25%) rotted against (i.e. old teachers) There are a good number of teachers drinking together from the same places with pupils (63%) and only a few were against. There are more teachers teaching their age mates (75%) and (25%) rotted against (i.e. old teachers) There are a good number of teachers drinking together from the same places with pupils (63%) and only a few were against. Pupils have too much knowledge of their teachers’ private affairs (60%) than those who rotted against. On teachers having showdowns with pupils, the scores were (50%): (50%) who rotted for and/or against. The research which sought to find out the reasons of conflicts between learners and teachers was carried out at Blue, Orange and White schools in Zambezi district and below are some of the points of conflicts the researcher got from the interviews and questionnaires. Volume 5, Issue 6, November-December 2023 IJFMR23066646 4 CONCLUSION Indiscipline among learners in secondary schools is influenced by poor parenting of the learner or individual stress. According to Gangelos (2017) said that these factors causing learners to become irresponsible to their behaviours does leads to disrespectful behaviours and this calls for teachers to stop them from those behaviours. Conflict is a common problem among the lifestyle of people and therefore it is part and parcel of life. According to Platinum (2014) school managers should find a way of solving a conflict because it has an impact according to how it is approached and the resolutions. Conflicts arise because of the following issues; needs not met, poor communication, lack of compromise, prejudice, intolerance, ignorance, lack of empathy, lack of resources, beliefs and power struggles. Conflict occurs in every school. If handled effectively, conflict can create a good learning experience. If handled ineffectively, conflict can quickly escalate to physical and emotional violence. RECOMMENDATIONS 1. The ministry of education should provide induction workshops for newly appointed Deputy Head teachers to equip them with managerial skills to enable them handle conflict among learners and teachers. 1. The ministry of education should provide induction workshops for newly appointed Deputy Head teachers to equip them with managerial skills to enable them handle conflict among learners and teachers. 2. School strategic plans policies on discipline management should be enhanced with the help of the Guidance and Counseling department for behavior modification. 2. School strategic plans policies on discipline management should be enhanced with the help of the Guidance and Counseling department for behavior modification. 3. Code of conduct procedures and professionalism guidelines and documents should be availed to teachers. Also, school regulation policies should be made known to every learner to have a disciplined environment. Poor communication network Poor communication network is highly rated as one of the main causes of conflict in schools (Iwuagwu, 2011). School principals who introduce innovation without first educating the teachers and students of the nature and foreseen benefits of the innovation in the school cannot expect genuine cooperation and participation from them. And this can lead to conflict. Also, when decisions are not disseminated effectively, sources of conflict can this be created for or by teachers and students. According to Awuor (2008) fixed rules, lacks of dialogues and authoritative administration increases unrest cases of the students thus this leads to school conflict which affects performance whether individually as a learner and the school. According to Johdi and Apitree (2012) the provision of resources in schools they are not equitably shared among learners and teachers thus leading to student and teachers fight for those scarce resources leading to school conflict. According to Mubika and Thodiana (2010) school consists of different staff members who have different beliefs values, age, cultural background, goals and attitudes so different personal conflicts leads to school conflict because every individual fight to be recognized as a winner at different levels of their understanding. Teachers having showdowns with their learners in class Some teachers may try to embarrass their learners in the presence of their girl friend or boyfriends. This, to some learners would take lightly. They may retort and conflicts arise. Volume 5, Issue 6, November-December 2023 Volume 5, Issue 6, November-December 2023 IJFMR23066646 5 REFERENCES 1. Argyropoulou, E. (2018). Conflict management at school unit. Investigation of principals’ and educators’ perceptions of primary education at third district of Athens (Post Graduate Thesis). Patras, Greece: Hellenic Open University (in Greek). 2. Awan, A. G., & Anjum, K. (2015). Cost of high employees turnover rate in oil industry of Pakistan. Information and Knowledge Management. 2. Awan, A. G., & Anjum, K. (2015). Cost of high employees turnover rate in oil industry of Pakistan. Information and Knowledge Management. Volume 5, Issue 6, November-December 2023 IJFMR23066646 6 E-ISSN: 2582-2160 ● Website: www.ijfmr.com 3. Cain, G., & du Plessis, A. (2013). Teachers' perceptions of the consequences of interpersonal conflict: a case study in three primary schools. Problems of Management in the 21st Century. 4. Catana, L. (2015). Conflicts between teachers: Causes and effects (conference paper). In Proceedings of Central and Eastern European Conference "New Approaches in Social and Humanistic Sciences" at Chișinău, Republic of Moldo. 4. Catana, L. (2015). Conflicts between teachers: Causes and effects (conference paper). In Proceedings of Central and Eastern European Conference "New Approaches in Social and Humanistic Sciences" at Chișinău, Republic of Moldo. 5. Choi, Y. J., & Lee, K. H. (2014). The Influence of Teachers' Development and Organizational Conflicts on School Organizational Efficiency. Journal of Fisheries and Marine Sciences Education. 6. De Dreu, C. K. (2018). The virtue and vice of workplace conflict: Food for (pessimistic) thought. Journal of Organizational Behavior: The International Journal of Industrial, Occupational and Organizational Psychology and Behavior. 6. De Dreu, C. K. (2018). The virtue and vice of workplace conflict: Food for (pessimistic) thought. Journal of Organizational Behavior: The International Journal of Industrial, Occupational and Organizational Psychology and Behavior. 7. De Dreu, C. K., & Van de Vliert, E. (Eds.). (2017). Using conflict in organizations. Sage. 7. De Dreu, C. K., & Van de Vliert, E. (Eds.). (2017). Using conflict in organizations 8. Dogan, S. (2016). Conflicts management model in school: A mixed design study. Journal of Education and Learning. 8. Dogan, S. (2016). Conflicts management model in school: A mixed design study. Journal of Education and Learning. 9. Deutsch, M. (2013). The resolution of conflict: Constructive and destructive processes. Boston, MS, USA: Yale University Press. 9. Deutsch, M. (2013). The resolution of conflict: Constructive and destructive processes. Boston, MS, USA: Yale University Press. 10. Everard, K. B., & Morris, G. (2019). Effective Educational Management. Patras, Greece: Hellenic Open University (in Greek). 10. Everard, K. B., & Morris, G. (2019). Effective Educational Management. Patras, Greece: Hellenic Open University (in Greek). 11. Fassoulis, K. (2016, May). The creative communication as a tool of conflict management in a school environment. In Proceedings of the 3rd Greek Conference of the EMIEKEK on ‘‘Critical and creative thought in education: Theory and Practice’’ (p.p.14-16). Athens, Greece (in Greek) 11. Fassoulis, K. (2016, May). The creative communication as a tool of conflict management in a school environment. International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ E-ISSN: 2582-2160 ● Website: www.ijfmr.com In Proceedings of the 3rd Greek Conference of the EMIEKEK on ‘‘Critical and creative thought in education: Theory and Practice’’ (p.p.14-16). Athens, Greece (in Greek) 12. Filippou, D. (2016). Conflicts among educators and principals of primary education and the role of principal in conflicts management: Investigation of conflicts during the secret ballot of educators’ boards for the election of school units’ principals (Post Graduate Thesis). Patras, Greece: Hellenic Open University (in Greek). 12. Filippou, D. (2016). Conflicts among educators and principals of primary education and the role of principal in conflicts management: Investigation of conflicts during the secret ballot of educators’ boards for the election of school units’ principals (Post Graduate Thesis). Patras, Greece: Hellenic Open University (in Greek). Volume 5, Issue 6, November-December 2023 IJFMR23066646 Volume 5, Issue 6, November-December 2023 7
https://openalex.org/W2170433184
https://kau.diva-portal.org/smash/get/diva2:639744/FULLTEXT01
English
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Quality of Life with Flotation Therapy for a Person Diagnosed with Attention Deficit Disorder, Atypical Autism, PTSD, Anxiety and Depression
Open journal of medical psychology
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ABSTRACT The aim of this single-subject study was to report experiences from one and a half years of regular floating as described by a person with neuropsychiatric and mental health disorders. Floating, or Flotation Restricted Environmental Stimula- tion Technique, involves relaxation and sensory deprivation by means of resting in a tank with highly salted and body-tempered water. The subject, a 24-year-old woman diagnosed with attention deficit hyperactivity disorder, atypi- cal autism, post-traumatic stress disorder, anxiety and depression floated regularly for one and a half years. Interviews regarding her experiences were analyzed and the main findings involved a subjective sense of improved quality of life, wellbeing and healthy behavior. There were no negative effects from treatment. Results suggest that floating may have beneficial therapeutic effects on mental health. Further studies that evaluate the efficacy and possible effects of floating with regard to mental health are needed. Keywords: Flotation; Relaxation; Mental Health; Neuropsychiatric Disorders Keywords: Flotation; Relaxation; Mental Health; Neuropsychiatric Disorders Received April 4, 2013; revised May 11, 2013; accepted May 21, 2013 Received April 4, 2013; revised May 11, 2013; accepted May 21, 2013 Copyright © 2013 Anette Kjellgren et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. *Corresponding author. Open Journal of Medical Psychology, 2013, 2, 134-138 doi:10.4236/ojmp.2013.23020 Published Online July 2013 (http://www.scirp.org/journal/ojmp) Open Journal of Medical Psychology, 2013, 2, 134-138 doi:10.4236/ojmp.2013.23020 Published Online July 2013 (http://w Copyright © 2013 SciRes. 1. Introduction substance use disorders, depression, anxiety [2-4], stress, overweight, sleep disturbances and poor physical health in general [5-7]. Mental illness and impairment are often treated with single-target therapy aiming to alleviate iso- lated dysfunctions and complaints. ADHD first-line treat- ment involves pharmacological central stimulant therapy with documented effects on core symptoms but also negative effects, e.g. headache, nausea and insomnia [8]. Treatment for autistic dysfunctions typically includes behavioral therapy as well as medication for anxious tendencies and careful regulation of environmental de- mands [9]. A common finding is that single core symp- toms may be effectively treated with medications [10] but secondary symptoms of both ADHD, autistic and comorbid disorders also require a more general ap- proach including sometimes non-traditional therapies for symptom-relief, healthy life-changes, quality of life and everyday functioning [8,9]. Alternative treatments are the subject of a growing number of studies. Iron and magne- sium supplementation [11], EEG feedback [12] massage, The rapid increment of mental disorders among younger ages calls for new therapeutic approaches to mental health. Current methods of treatment are not always use- ful, and may inflict unpleasant side effects and be inef- fective from a long-time perspective. Conditions of neu- ropsychiatric and severe mood disorders with additional psychiatric comorbidity are especially challenging to treat with conventional methods and the illness is even likely to escalate from ineffective treatment. For espe- cially children and young adults with neuropsychiatric disorders, non-effective treatments are strong predictors of additional health and mental problems and cumulated, these disorders may become even more difficult to treat effectively [1]. For example, simultaneous and unsuc- cessfully treated autism and Attention Deficit Hyperac- tivity Disorder (ADHD) constitute a risk-factor for de- velopment of Post-Traumatic Stress Disorder (PTSD), OJMP Copyright © 2013 SciRes. A. KJELLGREN ET AL. 135 tion, starting at 18 mg for five weeks, 36 mg for two weeks and 54 mg for eight weeks. Floating was self-ini- tiated at the end of the 54 mg period as an attempt to ease the headache she felt was a side effect from the medicine. After a few flotation sessions, a self-initiated decrease of stimulants took place through consultation with her phy- sician, starting with 36 mg for the first three days, 18 mg for three more days and then stimulants was completely put out. She terminated all prescribed drugs at close to one month after starting with flotation. Copyright © 2013 SciRes. 2.2. The Interviews The aim of this qualitative single-subject study was to report the experiences from long-term regular flotation tank therapy made by a 24-year-old female subject with psychiatric and neuropsychiatric disorders. The first interview was conducted at the respondents flotation centre, it prolonged for 74 minutes and was re- corded on a mini-disc. The interview was semi-structured with questions like: how come you started floating, how do you experience floating, has your life somehow been affected by floating, has your experience of floating changed over time? A one-year follow-up was performed to understand more about the experiences from long- lasting flotation and handwritten notes were taken. The respondent was informed that all of the material would be treated confidentially, that some of the quotations would be included in the presentation of the study later on, and that she had the right to terminate the interview and participating in the study at any time. The study fol- lowed the ethical standards for research involving Hu- man subjects according to the Helsinki declaration [20]. Also, a short follow-up interview was performed again after two additional years. 2.1. Case Presentation The respondent was a 24-year-old woman from Sweden. Her background involved severe social and academic problems that had started and persisted throughout ele- mentary school. In high school, she was recommended for the individual program, i.e., special education meth- odology, due to bad grades. After two years, she dropped out of high school in order to live with a man in another town. He repeatedly abused her and used violence against her. She managed to leave him and to return to her hometown. She was depressed and anxious, and suf- fered from social phobia and easily became anxious in social situations. She had a strong fear of talking and was often afraid of saying something wrong and of being clumsy. After two years of sick-leave she was thoroughly assessed and diagnosed with Attention Deficit Hyperac- tivity Disorder predominantly inattentive subtype (DSM- IV; 314.00) and with atypical autism (DSM-IV; 299:80) by a neuropsychiatric specialist-team according to the criterions of the DSM-IV [19]. When she initiated float- ing, she suffered from PTSD (due to the earlier episode of assault), high stress load, fatigue, social phobia, anxi- ety, recurring episodes of depression, muscle tension pain and general stiffness. 1. Introduction relaxation and EMG biofeedback, meditation and vesti- bular stimulation yield promising pilot study data, essen- tial fatty acids have not yet demonstrated satisfying re- sults in clinical controlled trials but interesting case-con- trol data [13]. In this qualitative single-subject study we investigate experiences from a therapy known as Flotation-Re- stricted Environmental Stimulation Technique, i.e., Flo- tation-REST, or floating, which provides sensory isola- tion and deep relaxation by means of laying in a tank with highly salted and body-tempered water. Floating has shown promising results for e.g. fibromyalgia, muscle tension pain, whiplash associated disorders as well as on mental disorders and complaints like stress, burn-out depression, depression, anxiety and ADHD [14-17]. For example, a female subject with neuropsychiatric disor- ders and burn-out depression experienced symptom- relief, increased well-being, focus, energy, calm and op- timism after a series of flotation sessions [18]. At week one, flotation was performed for 3 × 45 min- utes, at week two and three for 2 × 45 minutes, at week four to six for 1 × 45 minutes, and week seven and for- ward included one or two sessions per month. At the time of the first interview she had performed flotation for one and half year including approximately 50 sessions in total. At the one-year follow-up, she had floated ap- proximately 75 sessions. 3. Results and Discussion The fourth theme Effects due to the flotation tank treatment (89 MU) comprises the experienced effects of flotation. Her life became better from floating and it made her feel healthy “I feel good and it does not get worse and it just gets better and better… it is hard to explain… but it has helped me so much”. The relaxation extends into her everyday life and includes psychological relaxation as well as relief from earlier painful muscle tensions. Her quality of sleep is good; she has energy and no longer problems with fatigue. She describes being alive, alert and positive, and she no longer feels depres- sive or bothered with negative feelings “I can do more and I am more positive”. The problems with lethargy and dullness are gone and she has the capacity to carry out many things and to be active. Floating has provided feel- ings of security, self-confidence, and the ability to handle setbacks “I have a little more confidence and floating is like an energy boost. I feel like more when I feel better”. Her social abilities have increased, and she is not silent or shy anymore. She is no longer afraid of making mis- takes and about how her behavior might be evaluated. The respondent no longer needs medications and she is happy to avoid the side effects that she previously ex- perienced. The first theme involves The time before floating (in- cluding 52 MU) and contains descriptions of her personal history, setbacks in life (including a case of assault), be- ing on sick-leave and how she was assessed for mental health problems. In social settings, she remained silent most of the time in fear of saying something wrong or behaving clumsy. She described how she used to analyse and reflect upon her thoughts, but could not verbalize what she perceived as frightening. She also described her physical pain including stiffness and tension and how she believed this was the result of the difficult things she had been through. Furthermore, she felt tired and without energy, and everything she did was very slow. She was troubled by frequently recurring depressions and had been prescribed several medicines and had many side effects. The second theme Turning point in her life (including 57 MU) summarizes her first encounter with the flota- tion-REST technique. 3. Results and Discussion At that time, she got a long-term- internship at a company with flotation tanks and since she had heard that it might alleviate headache, which had become a problem since the stimulant therapy, she de- cided to try flotation. Her first floating sessions were not trouble-free since she had problems with finding a good position and was able to relax in the new environment. Her level of stress before entering the tank affected her experiences of floating but she eventually learned that the tensions disappeared when she became relaxed. After three to four times, she began to notice positive effects. She experienced more energy, positive thoughts, deep relaxation and became less tense “I became more posi- tive, had more energy and was no longer as tense”. All this became a big turning point in her life and about ten times she experienced even deeper relaxation. The last theme Perspectives on the method of flota- tion-REST (39 MU) summarizes what flotation means for the respondent personally and her reflections on the method at large. She was surprised by the effect since she did not believe floating would affect her in any signifi- cant way. Her friends and co-workers were also surprised about the positive effects that they saw and they asked her what she had done. She describes floating as a non- demanding process, nothing special is needed in order to float, and it is just a matter of relaxing and let things happen. The effects were gradually reduced and she needed to float regularly for about two times a month, otherwise the lethargy, negative thoughts and social shy- ness would return. Floating made her feel like “a new person” and she came back to life and felt good. She says there are no side effects and prefer floating in front of the drugs she used to take. She believes floating has positive effects for different problems like pain and depression and therefore hopes it gets more attention as a treatment The third theme Experiences in the flotation tank (27 MU) involves experiences during the stay in the tank. A total and profound relaxation was induced in the tank and bodily tensions disappeared. This state of relaxation was appreciated, extended into her everyday life and often accompanied with joy. Copyright © 2013 SciRes. 2.3. The Analysis The Empirical Psychological Phenomenological (EPP) method according to Karlsson [21] was used to analyse the interviews. The EPP-method includes five steps of processing the material; thoroughly reading the tran- scribed text in order to get a comprehensive understand- ing; the text is divided into Meaning Units (MU) based upon the underlying psychological meaning of the text; every meaning unit is transformed from spoken language into abstract language in order to fully recognize the un- derlying psychological phenomenon of the material; the transformed MUs are arranged into categories; these categories are then arranged into clusters of typological structures (themes). In order to control for reliability of Psychopharmacological treatment included central stimulants (Concerta, 54 mg), and anxiolytic medication (Sobril, as needed). She was introduced to pharmacol- ogical stimulant treatment fourteen weeks before flota- OJMP A. KJELLGREN ET AL. 136 the analyses, two of the authors independently sorted MUs into typological structures according to Norlander Credibility Test, NCT (e.g. [21]) and the level of match- ing was found to be very high. Twenty typological struc- tures, based on a total of 264 MUs, were found during the analyses and they have been arranged into five gen- eral themes. Below, these themes are described together with highlighting quotations (in italics) from the respon- dent and discussed with regard to its suggested theoreti- cal and practical implications. She felt completely safe in the tank and had never felt like that before, the feeling of safeness helped her to re- lax “I felt so safe, so I could relax”. Relaxation in the tank induced a “here-and-now” state and the normal thoughts were stopped or changed. Instead she needed not to bother about being evaluated or criticized and she was able to analyse her social phobia and to be herself. She had a feeling of “being” and described altered time perception, “[…]but wow, it has already passed 45 min- utes, I want to be here a little longer because it feels so good”. method. At the follow-up interview 2 additional years later, she is still free from all medications. She still uses the flota- tion-tank about once a month as a stress-reducing tech- nique. As an additional bonus she also had stopped smoking; she had smoked about 15 cigarettes a day for the previous last 9 years, and she attributed the stress reducing effects of the flotation tank as helpful for smoking cessation. Her last sentence during this fol- low-up interview was: “floating is the best thing I have ever tried, no side effects, and it Works!” [7] T. E. Wilens, J. Biederman and E. Mick, “Does ADHD Affect the Course of Substance Abuse? Findings from a Sample of Adults with and without ADHD,” American Journal of Addict, Vol. 7, No. 2, 1998, pp. 156-163. [8] D. J. Nutt, K. Fone, P. Asherson, D. Bramble, P. Hill, K. Matthews, K. A. Morris, P. Santosh, E. Sounga-Barke, E. Taylor, M. Weiss and S. Young, “Evidence-Based Guide- lines for Management of Attention-Deficit/Hyperactivity Disorder in Adolescents in Transition to Adult Services and in Adults: Recommendations from the British Asso- ciation for Psychopharmacology,” Journal of Psycho- pharmacology, Vol. 21, No. 1, 2007, pp. 10-41. doi:10.1177/0269881106073219 p gy doi:10.1177/0269881106073219 From this qualitative single-subject study we learn that floating was associated with beneficial therapeutic ef- fects in terms of quality of life, subjective wellbeing and healthy behavior. The respondent: “feel good well like a new person and so it has made a great difference… it really has and I really want to continue with this because I really need it.” [9] S. J. Rogers and L. A. Vismara, “Evidence-Based Com- prehensive Treatments for Early Autism,” Journal of Cli- nical Child and Adolescent Psychology, Vol. 37, No. 1, 2008, pp. 8-38. doi:10.1080/15374410701817808 [10] T. Spencer, J. Biederman, T. Wilens, R. Doyle, C. Sur- man, J. Prince, E. Mick, M. Aleardi, K. Herzig and S. Faraone, “A Large, Double-Blind, Randomized Clinical Trial of Methylphenidate in the Treatment of Adults with Attention Deficit/Hyperactivity Disorder,” Biological Psy- chiatry, Vol. 57, No. 5, 2005, pp. 456-463. doi:10.1016/j.biopsych.2004.11.043 4. Acknowledgements This project was supported by unrestricted grants from the County Council (Landstinget) of Värmland, LiV, Sweden. [11] J. B. Adams and C. Holloway, “Pilot Study of a Moderate Dose Multivitamin/Mineral Supplement for Children with Autistic Spectrum Disorder,” Journal of Alternative and Complementary Medicine, Vol. 10, No. 6, 2004, pp. 1033- 1039. doi:10.1089/acm.2004.10.1033 3. Results and Discussion It felt as if something “bad” was lifted away from her body during the profound relaxation and sometimes she became so relaxed that she fell asleep. OJMP A. KJELLGREN ET AL. 137 stance Abuse, Vol. 7, No. 3, 1995, pp. 373-378. doi:10.1016/0899-3289(95)90030-6 REFERENCES [12] M. M. Lansbergen, M. van Dongen-Boomsa, J. K. Buite- laar and D. Slaats-Willemse, “ADHD and EEG-Neuro- feedback: A Double-Blind Randomized Placebo-Con- trolled Feasibility Study,” Journal of Neural Transmis- sion, Vol. 118, 2011, pp. 275-284. doi:10.1007/s00702-010-0524-2 [1] T. E. Brown, “ADHD Comorbidities—Handbook of ADHD Complications in Children and Adults,” American Psy- chiatric Publishing, Washington DC, 2009. [2] R. Famularo, T. Fenton, R. Kinscherff and M. Augustyn, “Psychiatric Comorbidity in Childhood Post-Traumatic Stress Disorder,” Child Abuse and Neglect, Vol. 20, No. 10, 1996, pp. 953-961. doi:10.1016/0145-2134(96)00084-1 [13] L. E. Arnold, “Alternative Treatments for Adults with Attention Deficit Hyperactivity Disorder (ADHD),” An- nals of New York Academy of Sciences, Vol. 931, 2001, pp. 310-341. doi:10.1111/j.1749-6632.2001.tb05788.x [3] R. C. Kessler, L. Adler, R. Barkley, J. Biederman, K. C. Conners, O. Demler, S. V. Faraone, L. L. Greenhill, M. J. Howes, K. Secnik, T. Spencer, T. B. Ustun, E. E. Walters and A. M. Zaslavsky, “The Prevalence and Correlates of Adult ADHD in the United Sates: Results from the Na- tional Comorbidity Survey Replication,” American Jour- nal of Psychiatry, Vol. 163, No. 4, 2006, pp. 716-723. doi:10.1176/appi.ajp.163.4.716 [14] S. A. Bood, “Bending and Mending the Neurosignature— Frameworks of Influence by Flotation-REST (Restricted Environmental Stimulation Technique) upon Well-Being in Patients with Stress Related Ailments,” Ph.D. Thesis, Karlstad University, Karlstad, 2007. [15] S. A. Bood, U. Sundequist, A. Kjellgren, T. Norlander, L. Nordstrom, K. Nordenstrom and G. Nordstrom, “Eliciting the Relaxation Response with the Help of Flotation-Rest (Restricted Environmental Stimulation Technique) in Pa- tients with Stress-Related Ailments,” International Jour- nal of Stress Management, Vol. 13, No. 2, 2006, pp. 154- 175. doi:10.1037/1072-5245.13.2.154 [4] [4] T. Wilens, “Attention Deficit Hyperactivity Disorder and Substance Abuse Disorder—The Nature of the Relation- ship, Subtypes at Risk and Treatment Issues. The Nature of the Relationship, Subtypes at Risk and Treatment Is- sues,” Psychiatric Clinics of North America, Vol. 27, No. 2, 2004, pp. 283-301. doi:10 1016/S0193 953X(03)00113 8 Copyright © 2013 SciRes. pp doi:10.1016/S0193-953X(03)00113-8 [16] H. Edebol, S. A. Bood and T. Norlander, “Chronic Whip- lash-Associated Disorders and Their Treatment Using Flo- tation-REST (Restricted Environmental Stimulation Tech- nique),” Qualitative Health Research, Vol. 18, No. 4, 2008, pp. 480-488. doi:10.1177/1049732308315109 [5] R. B. Barkley, “ADHD and Life Expectancy,” ADHD Report, Vol. 4, 1996, pp. 1-4. [6] O. Pomerleau, K. Downey, F. Stelson and C. Pomerleau, “Cigarette Smoking in Adult Patients Diagnosed with At- tention Deficit Hyperactivity Disorder,” Journal of Sub- [17] A. Kjellgren, “The Experience of Flotation-REST (Re- Copyright © 2013 SciRes. OJMP A. KJELLGREN ET AL. 138 [20] World Medical Association Declaration of Helsinki, 2008. http://www.wma.net/en/30publications/10policies/b3/17c. pdf stricted Environmental Stimulation Technique): Conscious- ness, Creativity, Subjective Stress and Pain,” Ph.D. The- sis, Gothenburg University, Gothenburg, 2003. [21] G. Karlsson, “Psychological Qualitative Research from a Phenomenological Perspective,” Almquist & Wiksell In- ternational, Stockholm, 1995. [18] H. Edebol, A. Kjellgren, S. A. Bood and T. Norlander, “Enhanced Independence and Quality of Life with Flota- tion-Restricted Environmental Stimulation Technique of a Patient with Both Attention Deficit Hyperactivity Disor- der and Aspergers Syndrome: A Case Report,” Cases Journal, Vol. 2, 2009, pp. 69-79. doi:10.4076/1757-1626-2-6979 [22] M. Niklasson, I. Niklasson and T. Norlander, “Sensorimotor Therapy: Physical and Psychological Regressions Con- tributes to an Improved Kinesthetic and Vestibular Ca- pacity in Children and Youth with Motor Difficulties and Problems of Concentration,” Social Behavior and Per- sonality, Vol. 38, No. 3, 2010, pp. 327-346. [19] American Psychiatric Association, “Diagnostic and Sta- tistical Manual of Mental Disorders,” 4th Edition, Ameri- can Psychiatric Association, Arlington, 2000. OJMP
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The Teaching of Financial Services Regulation: A Contextual View
International journal of financial research
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1. Introduction This paper seeks to provide a discussion of various themes relating to what, in the business faculties or institutes of many modern tertiary education institutions, and, more particularly, departments focussing on the teaching of financial services, has become a very important academic discipline in its own right: viz the teaching of, and research on, financial services regulation. As with many areas that come under financial services, there is a historic background that had its effect or, even, conceptually non-effect, on the teaching of financial services regulation, and we briefly look at that aspect in both an international and purely local context. In a very broad sense it can be said that financial services institutions were late in fully appreciating that their environment was, so to speak, “being set up” for them by many outside forces: conflicts, politics, society, the law, even the economy itself, not to of course fail to mention the many bank and other financial institution crises. It is therefore correct to see the banks as having for long been (some would say blissfully!) unaware of what was evolving for them as we moved into the post-eighteenth century era. When regulation was, so to speak, “born” – and it is in reality hard to pinpoint when that was the case in many countries – it remained for so long as something happening out there, and perhaps in those times even not something to fret too much over. We consider this aspect in the first part of this paper. Besides examining some of the theories and developments that have had an impact on the teaching of financial services regulation in tertiary education institutions over recent years, and looking at what has impacted on course contents, at what we are doing now, and what we may expect going into the future, we also try to deal with the often intricate, and possibly still undecided, love-hate (or even lukewarm) relationship between the regulatory and legal worlds, and we consider to what extent the teaching of the two disciplines, i.e. bank regulation and law, differs. The tools and methodologies for the teaching of banking regulation are finally emerging as a topic for research and discussion, and this paper tries to make a contribution, albeit small, in that direction. As we shall see it is indeed more than just an issue of printed texts about modernity’s recurrent financial crises. Accepted: November 24, 2015 Accepted: November 24, 2015 ber 24, 2015 Online Published: April 14, 2016 URL: http://dx.doi.org/10.5430/ijfr.v7n2p214 URL: http://dx.doi.org/10.5430/ijfr.v7n2p214 Abstract This paper discusses issues and developments that relate to the teaching of bank regulation in tertiary institutions. It considers how course content, teaching texts, and methodology, can become subject to issues like specific, historical, and jurisdictional, cultures and contexts for the discipline. It considers how economic and political approaches impact such teaching. How banking regulations tools are used, and course structures are built, are matters which impinge on the type of trained personnel who later eventually leave academia and end up working on regulatory or compliance matters. Keywords: regulation, financial services regulation, bank regulation, teaching bank regulation International Journal of Financial Research International Journal of Financial Research Vol. 7, No. 2; 2016 http://ijfr.sciedupress.com Published by Sciedu Press The Teaching of Financial Services Regulation: A Contextual View John. A. Consiglio1 Correspondence: John. A. Consiglio, Department of Banking & Finance, Faculty of Economics, Management & Accountancy, The University of Malta, Malta. Received: October 23, 2015 doi:10.5430/ijfr.v7n2p214 2. Did the Banks Help the Discipline? from the outside of the banks – there was, in term of the single reality of the Rule of Law, an environment that regulated whatever was taught in terms of content, procedures to be followed, etc, and why. So that was the early reality here in Malta. Within the banks ours was an environment that trained bank employees yes, but never really considered outreaching, or interacting, with the evolving greater external environment. (One is led to hypothesise that Barclays Bank, essentially a colonial bank in pure historic terms, had a one-size-fits-all training model for all its territories.) But such environment had and has, as we all know, a life of its own. The economy, the law, the society, the politics of a country, even any bank’s own customers, all of these evolve a dynamic of their own, one that banks ignore at their peril. This dichotomy of institutions being brave, and innovative, amongst Maltese businesses in conceiving and appreciating the value of internal staff training, but then not ever linking or including into their own internal content any awareness of what in the environment was happening to regulate that internal context, must now, a posterior, come to be recognised as an element, one of failure, in the historiography of Maltese banking, even if many gave scant importance to it. The local institutional environment to which I am here referring is that related to the post late-1950s. My research into periods before those times suggests that the terms “financial services regulation” were practically inexistent, and never previously used. For example, even the term “regulation”, on its own, is inexistent in the old legal history text of Judge Dott. Paolo de Bono of 1897. (Note 4) And, closer to our times, even the writings of Malta’s legal historian, Prof. Hugh W. Harding, again do not ever highlight “regulation”, let alone “financial services regulation”, as ever being of specific relevance. Many of us who come to the teaching of bank regulation at some point or other of our lives must, one feels, admit that we do so with a particular luggage and set of biases. Some of us could be practising bankers who, at some stage of our careers would have done work in legal, or internal audit, or even compliance departments. Others may have spent years working full-time with, or consulting for, regulatory agencies or authorities, or even important central banks. 2. Did the Banks Help the Discipline? (Note 3) Because no teaching ever takes place in any environmental vacuum (social, political, cultural, and what not), a good place to start is by acknowledging, especially in the case of the microstate of Malta here often referred to for examples, that it is only by default of the banks themselves that the whole thematic of bank regulation, as a separate study and discipline on its own, one to be taught and absorbed, came to the fore. As part of a 42-year-plus banking career I had the experience of a five-year spell where I was in charge of Barclays Bank’s staff training centre in Malta, and what then (i.e. in the mid-1960s) clearly came down from the top in terms of direction on what course content had to be, well this never ever included the making of bank staff trainees aware of the truth that all course content taught (in tailored-just-for-the-bank’s-needs courses on accounts, forex, bills, credits, advances, branch management, etc) never did these have to consider the contextual reality that from out there – i.e. from the outside of the banks – there was, in term of the single reality of the Rule of Law, an environment that regulated whatever was taught in terms of content, procedures to be followed, etc, and why. Because no teaching ever takes place in any environmental vacuum (social, political, cultural, and what not), a good place to start is by acknowledging, especially in the case of the microstate of Malta here often referred to for examples, that it is only by default of the banks themselves that the whole thematic of bank regulation, as a separate study and discipline on its own, one to be taught and absorbed, came to the fore. As part of a 42-year-plus banking career I had the experience of a five-year spell where I was in charge of Barclays Bank’s staff training centre in Malta, and what then (i.e. in the mid-1960s) clearly came down from the top in terms of direction on what course content had to be, well this never ever included the making of bank staff trainees aware of the truth that all course content taught (in tailored-just-for-the-bank’s-needs courses on accounts, forex, bills, credits, advances, branch management, etc) never did these have to consider the contextual reality that from out there – i.e. Published by Sciedu Press 2. Did the Banks Help the Discipline? Some UK banking historians quote 1866 as being the date when the first recorded bank collapse occurred there. This was the case of Overend Gurney Bank, and, in true contagion or domino fashion, it was a failure that brought down dozens of other banks, and plunged the economy into a crisis. (Note 1) Twentyfour years later, in 1890, Barings Bank suffered a similar meltdown, but most of us are of course knowledgeable about the more notorious 1995 failure ISSN 1923-4023 E-ISSN 1923-4031 214 Published by Sciedu Press International Journal of Financial Research Vol. 7, No. 2; 2016 http://ijfr.sciedupress.com of that same bank, brought about by Nick Leeson’s famous gambles on the SIMEX. Between 1980 and 2003 some 140 countries experienced significant banking sector problems (that is some 75% of the IMF’s membership), and we all know that since then bank failures have certainly not abated. of that same bank, brought about by Nick Leeson’s famous gambles on the SIMEX. Between 1980 and 2003 some 140 countries experienced significant banking sector problems (that is some 75% of the IMF’s membership), and we all know that since then bank failures have certainly not abated. This scenario strikes me as having, over many long years, been the basic underlying background for a lot of the teaching that, in the business faculties and particularly in the banking departments of many tertiary institutions, goes on of financial services sector regulation. When we engage our students into the subject we often, and sometimes inevitably, end up giving them set doses of: ‐ The regulation, deregulation, reregulation, overregulation, under regulation, and regulatory fatigue cocktail (what Eric Gerding’s book presents as the “regularity instability hypothesis”) (Note 2); ‐ The regulation, deregulation, reregulation, overregulation, under regulation, and regulatory fatigue cocktail (what Eric Gerding’s book presents as the “regularity instability hypothesis”) (Note 2); ‐ Current or contemporary materials about laws, directives, regulatory bodies’ own domestic, or international, structures and regulations; ‐ Possibly past, or evolving, notions about soft law; ‐ Regulatory institution change (e.g. the single regulator in a jurisdiction debate), now also at pan-EU inspiration levels; ‐ Licensing, corporate governance, ownership and affiliation, and capital adequacy (with much of and about the Basle process thown in); d, perhaps inevitably, and, perhaps inevitably, ‐ A necessary dose of required reading about the details and development of many individual institutional crises. 3. The Teaching of Regulation and the Legal Profession The nexus between the study of regulation on the one hand, and the legal profession on the other should, speaking idealistically, start from reciprocal respect, and total study and absorption, with each other’s important roles. I posit here some brief considerations that go beyond that. I honestly worry when I hear so many young freshmen moving into the Faculty of Law saying that their only objective for after graduation is to “work in financial services”. Not for them, (as the Italians would say) the necessary “farsi le ossa in tribunale” (Italian), i.e. developing one’s legal backbone in a court of law. Not for them the importance of understanding the law primarily within the larger frameworks of civil or commercial (or criminal) law reasoned litigation, or even of national and/or international financial policy for financial services. Also, with this writing taking place at a time when the European Parliament’s decisions, often sourced in the European Banking Union’s project, is filtering down into member-states’ transposition processes, one even has to query how much – especially in smaller states – such parliaments contain the absolutely needed financial services regulatory sector experience, knowledge, competence, and this even with many lawyers being members of parliaments. Some would hold that the role of lawyers is simply to serve the immediate ends of businesses that are their clients. By contrast (but not necessarily on the contrary) I take the view that some among the legal profession, whether external or in-house lawyers, sometimes submit far too greatly to the will of business executives, or owners, without asserting independent leadership where the long-term interests of the financial institutions, and their shareholders, and their customers, really demand such exercise of independence. The truth is that quite a number of financial lawyers have sometimes allowed themselves to become too much of a service industry, and appear to have abandoned their roles as a source of wise counsel. Indeed “general counsel” has often become “general facilitator” and a service rather than a professional role. And so the teaching of banking regulation should really have as a paramount underlying theme the inculcation in students of an understanding, a particular type of mindset, which is much more concerned with the whys than the whats. 2. Did the Banks Help the Discipline? Others still would simply have been lawyers bitten by the bug, i.e. keenly aware of the importance of ISSN 1923-4023 E-ISSN 1923-4031 ISSN 1923-4023 E-ISSN 1923-4031 215 International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 2; 2016 understanding the law within the larger frameworks of financial policy and social evolution, and wanting to teach all about it. My own time working for financial institutions, and seconded to government corporations, was entirely on the business and strategic side, not within the offices of legal counsel or compliance managers. Perhaps that was a blessing in disguise, because, albeit from a distance, i now tend to think of legal and regulatory practice as being roles that should serve the long term strategic prosperity of society and banking businesses as such, whose long term prosperity necessarily involves both stability and the addition of value for both shareholders and the communities in which they operate. I return to this theme later. Another example of the environmental luggage, or bias, with which the teaching of bank regulation is often saddled is that which Baxter (2014) calls “the problem, or reality, of steady ‘formalisation’ of bank regulation”. In the days before about 1980-1990 – (in the case of the US this was around and even before the S & L (savings and loans) crisis) – bank regulation operated among members of the so-called “club” and according to external beaurocratic discretion, in which the mere hint of regulatory disapproval was generally enough to bring a bank into compliance. With the excesses of the S & L crisis the US Congress reacted angrily and formalised many sanctions. Yet, paradoxically, this led to less cooperation and increased demands for “due process”. At the same time the process had the effect of driving bank regulation into the realm of law. Perhaps it is only in the US that situations such as this, or similar to it, create the perception why the subject is so recent a topic (should we call it a “phenomenon”?) in law schools or faculties. Before events such as these it was as if banking regulation was within the realm of macroeconomists who often seem to me to use language any way they want to and for whom the structures and procedures of the law are inimical. Published by Sciedu Press 3. The Teaching of Regulation and the Legal Profession Such is the situation, for example, in Malta’s College of Arts, Science & Technology (MCAST), where, in their Institute of Business & Commerce, in the second year of their Level 5 Higher Diploma in Financial services course, (a course which actually only started being given in October 2014), this area of studies is not taught at all as a separate credit area of studies, but it is rather thrown in and mixed with other financial services subjects: a situation which inevitably begs the question of “isn’t a little knowledge always a dangerous thing?” (Note 6). In some US universities quite a different situation applies. Some academics hold that it is only in theory that local domestic components of banking regulation courses are easy to build up. In the case of Malta the structuring of this part can be temptingly made to look as a pot pourri of the Malta Financial Services Authorities (MFSA)’s – and other local regulators’ (Note 7) – underpinning laws, and the role, structure, and operations, of local financial intermediaries’ roles and operations, and of other local financial services law generally (Note 8). That would be dangerously close to failing to give students a feel, a realisation, of many other realities that exist even in a local context: themes like e.g. how regulatory capture operates in Malta, the on-site/off-site regulation dichotomy, capital adequacy considerations, and others; and all, yes, again in our own specific cultural context. And so, too, the teaching of banking regulation in separate courses, viz domestic banking regulation and international banking regulation, cannot not be, on one hand, an attractive proposition. Aids and materials can – if one is keen enough to make it a lifetime interest by constantly reviewing taught content – these can be put over sensibly researched, brought together, and delivered successfully to both under and post graduate courses. And, of course on the other hnnd making it absolutely clear to students that they can only become outstanding students in this discipline if they accept the fact that they have to become reading gluttons. Lawrence Baxter, who teaches this discipline at Duke University School of Law in Durham, North Carolina, is one exponent of the opposite position, with regards to this notion of teaching domestic and international banking regulation in separate courses. His is a teaching experience that has gone through several very absorbing facets. 3. The Teaching of Regulation and the Legal Profession When we as teachers of banking regulation spend a number of lectures dissecting and discussing, for example, Michael Moran’s (1986) seminal writings on the public interest, administration, instrumental, and cultural theories of regulation( Note 5), and changes in regulation – with particular reference to the case of financial markets – our hope is that this will encourage students not only to think strategically (some history of the Politics of Regulation, vide e.g. Francis (1993), Haines (2011), and to a lesser extent Stiglitz (2011) helps) but also to recognise and understand both public and private long-term interests. In reality this may very much sound like asking a lot from both banking students and future lawyers, because, soon after some time in their new professions, impatient executives are seldom willing to listen to a sermon on the virtues of constraints which they would very often be trying to avoid or werk around. But if we, as teachers or academics, do not persist in this effort then we might as well consign our students to roles not significantly different to those of marketers, and human resources personnel (with all due respects to colleagues teaching those other specific roles, and indeed the professionals practising them). ISSN 1923-4023 E-ISSN 1923-4031 216 International Journal of Financial Research Vol. 7, No. 2; 2016 http://ijfr.sciedupress.com In Europe, depending on the ECTS spread of courses, different universities are today teaching banking regulation with an eye on both the domestic and international components of the discipline. This is not always the case outside our continent. Colleagues in Hong Kong and Australia tell me that they have more leeway in terms of both choice of content, and time spread available to them for teaching it. So, indeed, a course programme there could even in some institutions extend to years one, two, and three of a full degree course. Compare, inter alia, such teaching situations where the subject is indeed just beginning to find its place. 3. The Teaching of Regulation and the Legal Profession When for example, he structured and started teaching a very focussed course called “Big Bank Regulation” he ran into problems from various fronts. For example: not enough student demand, or demand from certain sources for more specialised courses and which often became hard to change, or some colleagues’ cold reactions because they did not teach courses across both the domestic and international divides, and even examples of snobbing off simply because some courses did not develop business dynamics more than is traditional for law courses. To a certain extent one cannot but agree with him that it is probably artificial to nowadays separate domestic and international banking regulation. As the full impact of the new EU regulatory setup – essentially the Banking Union’s operations through its ESMA, EBA and EIOPA structures (Note 9) – forcibly sinks into the operations of EU markets (including those in smaller states like Cyprus, and Malta), one will come to see that a good component of any course in international banking regulation will, anyway, find its way onto any domestic regulation course. One can here posit a couple of teasing questions: One can here posit a couple of teasing questions: ‐ Firstly how can one possibly teach domestic regulation without recognising that the operations of large banks are in most cases transnational, and, in many other cases indeed global: that in fact such banks are either multinational or international? Basel is indeed integral to domestic bank regulation, while the actions and recommendations of the Financial Stability Board, the G20, and other international institutions, have a greater impact (acknowledged or not) on the shape of domestic regulation, be it through rules or agency decisions ‐ And, secondly, how can any faculty of laws sell as a “Masters course in financial services” a course which would, for example, be built to an extent of, say, some eighty percent of lectures on local law, and only the rest as really having much to do with either international banking or practical in-house financial services work practices. i.e. practical banking (viz the bit done with real life customers and real life banking products)? Shouldn’t such a course really come to be renamed as an “MA in Financial Services Law”? It is interesting to see how, at the University of Wales, the teaching of bank regulation is not essentially focused on the pure discipline per se. Published by Sciedu Press 3. The Teaching of Regulation and the Legal Profession Professor John Ashton teaches a course on “Financial Crises and Regulation”; Professor Sharon Ward focuses very much on compliance in her course on “Financial Services Compliance”; and Professor Bob Souster teaches a course called “Professional Ethics and Regulation”. In all cases therefore specific topics or It is interesting to see how, at the University of Wales, the teaching of bank regulation is not essentially focused on the pure discipline per se. Professor John Ashton teaches a course on “Financial Crises and Regulation”; Professor Sharon Ward focuses very much on compliance in her course on “Financial Services Compliance”; and Professor Bob Souster teaches a course called “Professional Ethics and Regulation”. In all cases therefore specific topics or ISSN 1923-4023 E-ISSN 1923-4031 Published by Sciedu Press 217 International Journal of Financial Research Vol. 7, No. 2; 2016 http://ijfr.sciedupress.com areas (crises, compliance, ethics, etc) are the actual hangers on to which the many basic regulation clothes are hung. In the specific case of Bob Souster’s course the text used is, again, significantly titled “Professionalism, Regulation, and Ethics”, but, as said, regulation is only 25 to 30% of the total module, and certainly not country-specific. The bulk of this course’s content covers in fact ethical and professional behaviour, self-regulation, rules-based vs principles-based regulation, corporate governance, and risk management. It is important to note that in both Bob Souster’s course at Wales, and in Marcel Cassar’s course on “Financial Regulation” in the Faculty of Laws at the University of Malta, it is that part of the market which consists of people who are already graduates in law, accounting, banking, or economics, which support it. Cassar’s course is not focused on the principles of law, or on specific rules and regulations, but much more on considerations pertaining to financial management and behaviour. The emphasis is on a strong message about inherent risks within the banking firm, about corporate governance, politics, and appetite and tolerance of risk, rather than on what is externally, and environmentally, being pushed into, and imposed upon, the firm. This particular financial regulation course is in fact a strong purveyor of the message that the best form of regulation is that which starts within the banking firm itself, and is embedded in the ethos and values of the company. 3. The Teaching of Regulation and the Legal Profession “No amount of laws and rules can ever replace those values of prudence which underpin the trust that the bank must enjoy,” Cassar correctly and emphatically holds. Keen movie watchers could do no worse than quote films like “The Enron Affair”, “Margin Call”, and “Rogue Trader” as ideal illustrations of how it should all not be done. Significantly Cassar uses no particular text-book, but rather sources and materials which are tailored to particular topics or lectures, and, given the limited 30 hours of lecturing that his course covers, he readily recognises that it can only give a small taste for the wider problematic which in fact is banking regulation. (Note 10) By slight analogy, again at the University of Wales there is a compulsory course module in the Chartered MBA programme, called Financial Institutions Risk Management (FIRM), which is run by our colleague Prof. Ted Gardener, and where the pedagogical underscoring focus (especially now after the new Senior Persons Regime in the UK) is, in a way, similar to Cassar’s. Here much emphasis is placed on the integration of ethics, and personal and institutional responsibility and accountability. The main targeted objective is that of integrating bank regulation of risk into a wider macro environment of deregulation of bank structure and conduct rules. (Note 11) The teaching of bank regulation will therefore necessarily differ substantially between institutions and across faculties. How faculty deans look at the importance, or otherwise, of the subject will impact substantially on the output quality of students. Is the area considered of a sine qua non importance, to the point that the subject is given a compulsory, and not elective, area of studies status? And how many hours of teacher-student contact? And what is the predominant method of teaching it? The formal lecture? The case study approach? The rigid familiarity-with-the-law approach? Students’ presentations as a basis for discussion? Or, even, the simulation approach? Insofar as pure legal teaching is concerned the latter is currently being described as a pedagogically valuable and practical tool for teaching modern law curricula, and it is claimed (e.g. Strevens et al (2014) that this form of experiential and problem-based learning enables students to integrate the ‘classroom’ experience with the real world experiences that they will encounter in their professional lives. 3. The Teaching of Regulation and the Legal Profession Answers to questions such as these will determine whether students get a small or larger taste of what in reality is the very wide area falling within this special area of financial studies. And eventual quality will of course also be very much dependent on whether one is teaching young undergraduates, or mature (older aged) students. With a clss of the latter, where many would probably already be graduates in, say, law, accounting, banking, or economics, one can better surmount the problems of lectures probably, and necessarily, only serving to give a mere taste of the subject. With indeed this time factor having already been the case even as much as fifteen years or, say, two decades ago, one can only just imagine how even truer this is the situation nowadays. Inevitably the topic will have to eschew delivery from pure perspectives of law, rules, or regulations, and move instead to a delivery from the angles of financial management and behaviourial considerations. The stronger messages to be imparted would be those concerning risks in the banking firm, the risk management environment, corporate governance, policies and appetite or tolerance of risk. Indeed the good teacher will be emphasising that the best form of regulation is that which starts within the banking firm itself and is embedded in the ethos and values of the company. No amount of laws and rules can ever replace those values of prudence which underpin the trust that every bank must enjoy. (Note 12) Published by Sciedu Press 4. The Problem of Teaching Texts and Contexts Always an important issue when discussing the teaching of banking regulation is the issue of texts. Let me for the moment put aside the fact that academics are these days veritably inundated with constantly being published new texts about what, either the writers or the blurbs hold, are the lessons that we should all be teaching our students to memorise, or at least absorb, from the post-Lehman crisis world. That, I hold, should only be one part of the range of ISSN 1923-4023 E-ISSN 1923-4031 Published by Sciedu Press 218 International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 2; 2016 tools which we as teachers should be using. It is in reality only a small number of writers of such texts who really went to the trouble of updating their casebooks after important legal developments or events, some indeed not necessarily short-lived. In the US Broome and Markham (2010) performed a Herculean task of updating their excellent casebook after the passage of Dodd-Frank. (Note 13) But one is still left somewhat disenchanted with the restrictedly casebook method of teaching financial regulation, particularly now that the field has become so dynamic when compared to what it was twenty years ago when I first started to teach the discipline. The lawyers of course will rightly say that there have been so many decisions, so many well crafted judicial opinions (e.g. should we here in Malta be considering as such the 2014 historical Constitutional Court decision in the National Bank of Malta case?) that regulation students cannot remain aloof of what, the lawyers would hold, is the important mix and interaction of public policy, agency positioning, and industry advocacy. Yes, these all produce important points but still, I would posit, with the passage of time an evanescent level of inflection. Forcing a class to understand the larger evolving picture through the probably purely episodic vignettes, and often procedural contortions, of cases that make their way to and through the courts, seems to me to distort the overall regulatory discipline, or picture, in ways that are not ideal when one is trying one’s best to lay down a long-lasting framework, and the type of mindset to which I have already made reference. In October of 2008 Heidi M. 5. Regulation of Teachers’ Ideals As teachers or lecturers we all however probably realise that texts and course contents are of course motivated with a noble purpose: in general terms that of producing persons who, once they would have left the heights, and excitement, of university life, become valid and welcomed contributors to our countries’ financial services systems, to our countries’ businesses in this sector. Perhaps we try to best encapsulate our efforts in this direction through what we describe as ‘learning outcomes’. In general terms they are described as us aiming that students completing our courses will emerge with a level of understanding of the approaches and basic concepts of financial services regulation, and with a level of knowledge of practical developments in regulatory practices, and how these effect market practitioners in their daily work. One would here be justified in saying that the litmus test of the success, or otherwise, of our daily grind as teachers is to be measured by how much our “products” – i.e. our graduates, even our postgrads and researchers – satisfy the expectations of employers (the financial institutions) out there in the market. And these employers are of course by no means homogeneous in terms of what they do, their internal structures, indeed the terms of their operating licences, not to linger on what they often (fairly or unfairly) often say they want or expect from us. At this point in time the predominantly received vibe is still in the sense that “one of the great things about Malta is its people’s skills....they are perfectly capable of handling the demands of an office” (Mahoney, 2014); or “The financial services sector is one of the most important employers of trained professional staff....here Malta continues to rank as one of the top financial jurisdictions, and is positioned in the top 10 of the World Economic Report Global Conmpetitiveness (WEF) Report *(Lutsch-Emmenegger, 2014). (Note 16) Predominantly employers here, including the major regulator, still seek and manage to employ staff for their regulation needs from the local employment market, and they do it using various methodologies, including local media advertising (vide examples in Appendix Two). In all probability, in then an even higher and more idealistic mindset, the best teachers of banking regulation would tactfully go well beyond this approach. 4. The Problem of Teaching Texts and Contexts Schooner and Michael Taylor produced, again in the US, an excellent text that is full of materials which can be used on both domestic and international regulation courses, and, I feel, in universities probably everywhere. Even as I find myself playing around with this book, alongside those by Haines, and by MacNeil and O’Brien (Note 14), for use of some of their materials, I always feel that these all comfortably lend themselves to my own purely personal framework when teaching this subject to final year banking students. But, that said and done, the inquisitive teacher will still jealously protect his academic liberty to vary lecture content from year to year. My own personal course content is comfortably favoured by the fact that, at this stage of their university undergraduate studies, our students would have become suitably and comfortably familiar with banking in its “modern” forms (Note 15), with the notions of risk in bank financial management, with contagion and moral hazard, with “too big to fail” (or is it “to jail”!?) issues, and other vital thematic. And that all allows one a lot of leeway and flexibility to vary content and topics, with the coverage effectively becoming extensive and, I would add, probably also biased towards the international side of banking. (Vide some of my regular lecture topics in Appendix One to this paper). Published by Sciedu Press 6. Conclusions The above exposition of issues related to the teaching of banking regulation will of course be far away from the minds of our graduates when they move out into the real world of finance. It is worrying that very often the last thing that they would be concerned with should be a comprehensive, let alone dynamic, appreciation of the challenges that regulation is required to address. These challenges are indeed diverse, and they encompass political, social, and even actuarial risks. This diversity demonstrates the limits of studying compliance without considering the goals that infused reforms, and of scrutinising reforms without taking into account whether and how compliance occurs. Regulatory reform cascades from international bodies, from parliaments, from domestic regulatory agencies, and finally makes its presence felt at workplaces. Within the different worksites to which our new graduates go, new infrastructure is often created, routines reconfigured, and records and regularly submitted returns developed (are these factually always needed?..... or in those specific formats?) to demonstrate compliance. In this process they, and we too as their teachers, are thus drawn into the hope that improvements are taking place which could reduce the risk of future disasters. All of this is very much in the vision of Kahn’s (1990) “spiral of progress”. But the disasters keep occurring, and it is easy to become fatalistic. This is wrong, and it is equally wrong to assume that there never have been clear examples of where the lessons of disasters were learnt, leading to enhanced and well-designed regulatory regimes and high levels of compliance. Here “Never again” could ring true. Or could it really?....especially when we consider that our new graduates sent out into the heavily regulated financial services working markets will, in most probability, also be facing that other big problem of uneducated investors. This is a big and hot topic of its own, and has come to the fore very much from 2012 onwards. The former Federal Reserve President, Ben Bernanke, cited its benefits for US economic health, the European Insurance and Occupational Pensions Authority (EIOPA) issued a report decrying the lack of national investor education strategies, and the International Organisation of Securities Commissions (IOSCO) has also argued for a common European framework to investor education and formal evaluation processes. 5. Regulation of Teachers’ Ideals They would be aware that regulation, as a modernist project or topic, involves the development of processes and styles of enforcement that are argued to ensure ever greater and greater levels of compliance which will minimise risk, or avoid a specified harm. Indeed, there are regulatory successes which we as teachers could profitably identify and promote (Note 17). And yet to many (especially in both the media ISSN 1923-4023 E-ISSN 1923-4031 219 International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 2; 2016 and some of the banks themselves) regulation still often continues to appear as not only the solution, but often also the problem. The immense literature on the subject often appears as a never ending saga of teasing apart the technical, the social, and the political elements that seem absolutely inseparable from the discipline. and some of the banks themselves) regulation still often continues to appear as not only the solution, but often also the problem. The immense literature on the subject often appears as a never ending saga of teasing apart the technical, the social, and the political elements that seem absolutely inseparable from the discipline. 6. Conclusions (Note 18) And we here hold that it is wrong to think that it is a problem, or situation, which is very far from, or totally unrelated to the matter of banking regulation education as a thematic of its own. When studying the administrative theories of regulation we are often reminded of Beyer’s (1982) comments about the failure of matching tools to problems. And one of the great problems is that of surveillance either not being supported by the regularity and suitability of the intelligence that it needs (i.e. data, info, regularly submitted returns); or surveillance becoming inflexible once it is bound by routine and, in the process exposing regulators to the mercy of ingenious operators. Much intelligence may indeed be redundant, irrational, or even demanded and gathered only for symbolic purposes. But, on the other hand, many serious problems have been created by the failures of acting efficiently, effectively, or (even more seriously) in timely enough fashion, on information gathered. And so we are, yes, also carrying the responsibility of having to teach our students the problems of coordination and control. The dilemmas which they very quickly come to face at their new workplaces, about making choices between hierarchy, and specialisation, and nitty-gritty technical issues, are often wrongly described as only basic failures. But so far no one has offered an explanation of how we can put together an effective mix of hierarchy and specialisation to tackle several constantly reappearing new technical regulators problems.....thankfully not all of them! Kahn, A. E. (1990). Deregulation: Looking Backward and Looking Forward. Yale Journal on Regulation, 7, 325-54. Schooner, H. M., & Taylor, M. W. (2008). Global Bank Regulation, Principles and Policies. Academic Press, New York. Harding Prof. Hugh W. Notes on the History of Maltese Legislation. Unpublished Notes for Students, Faculty of Law, The University of Malta. Kahn, A. E. (1990). Deregulation: Looking Backward and Looking Forward. Yale Journal on Regulation, 7, 325-54. Notes As in every country they are all jealous of ‘their own patches’, and in this respect, perhaps, we are not dissimilar to the US where the SEC, the FDIC, the Federal Reserve, the Office for Control of the Currency, the General Accounting Office, and other local federal or state bodies, all view with each other in the overall regulatory scenario. Note 7. The Central Bank of Malta, the Malta Stock Exchange, and the Ministry of Finance, must also be considered as part of Malta’s finance sector regulatory setup. As in every country they are all jealous of ‘their own patches’, and in this respect, perhaps, we are not dissimilar to the US where the SEC, the FDIC, the Federal Reserve, the Office for Control of the Currency, the General Accounting Office, and other local federal or state bodies, all view with each other in the overall regulatory scenario. Note 8. Perhaps the major published text in this context is the 2009 “An Introduction to Maltese Financial Services Law” by Ganado & Associates (Allied Publications, Valletta). Note 8. Perhaps the major published text in this context is the 2009 “An Introduction to Maltese Financial Services Law” by Ganado & Associates (Allied Publications, Valletta). Note 9. The European Securities Markets Authority; the European Banking Authority; and the European Insurance and Occupational Pensions Authority. Note 10. Marcel Cassar was First Executive Vice-President and CEO of FIMBank plc, and is now CEO of APS Bank in Malta. APS Bank is a totally Church-owned bank. The conceots here are quoted from his responses on Nov 3, 2014 to this author’s questions on the subject. Note 11. Significantly the text used on this course is Financial Institutions Management: A Risk Management Approach by Anthony Saunders and Marcia Cornett (McGraw-Hill/Irwin). One notes in this context a significant shift at the University of Wales from former MBA (Banking & Finance) courses, where these were simply called Bank Financial Management and made use of the US textbook Bank Financial Management in the Financial Services Industry by Joseph F. Sinkey Jr. (Macmillan Publishing, New York & London). Note 12. The concepts here are sourced in considerations made to the author by Mr. Marcel Cassar (vide Note 14 above) who also lectures on Banking Regulation in the Faculty of Law at the University of Malta. Note 13. Broome L.L. & Markham J.W. References Baxter, L. G. (2014). Capture Nuances in the Contest for Financial Regulation. Wake Forest Law Review, 47, 537-568. Beyer, S. (1982). Regulation and its Reform. Harvard University Press, Cambridge, Mass. Francis, J. (1993). The Politics of Regulation. Blackwell, Masachisetts US & Oxford UK. Gerding, E. (2013). Law, Bubbles, and Financial Regulation. Routledge, London. Haines, F. (2011). The Paradox of Regulation. Edward Elgar, UK. Harding Prof. Hugh W. Notes on the History of Maltese Legislation. Unpublished Notes for Students, Faculty of Law, The University of Malta. Harding Prof. Hugh W. Notes on the History of Maltese Legislation. Unpublished Notes for Students, Faculty of Law, The University of Malta. Kahn, A. E. (1990). Deregulation: Looking Backward and Looking Forward. Yale Journal on Regulation, 7, 325-54. Schooner, H. M., & Taylor, M. W. (2008). Global Bank Regulation, Principles and Policies. Academic Press, New York. ISSN 1923-4023 E-ISSN 1923-4031 Published by Sciedu Press 220 International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 2; 2016 Stiglitz, J. (2001). Principles opf Financial Regulation: A Dynamic Portfolio Approach. The World Bank Research OIbserver, 16(1), 1-18. Stiglitz, J. (2001). Principles opf Financial Regulation: A Dynamic Portfolio Approach. The World Bank Research OIbserver, 16(1), 1-18. Vide e.g. Strevens C. et al. (eds) (2014). Legal Education: Simulation in Theory and Practice. Ashgate Publishing, Abingdon, oxford & US. Vide e.g. Strevens C. et al. (eds) (2014). Legal Education: Simulation in Theory and Practice. Ashgate Publishing, Abingdon, oxford & US. Note 14. Haines F. (2011) – op cit. MacNeil I. & O’Brien J. (2010) – The Future of Financial Regulation – (Hart Publishing, Oxford and Portland, Oregon). Notes Note 1. In 1873 Walter Bagehot, then editor of The Economist, wrote about that crisis and used it as basis for a fierce attack on the Bank of England. Note 2. By 2010 the IMF’s latest estimates had put the total cost of the latest (i.e. post-2008) collapse at some US$ 4 trillions, the vast majority of which could be attributed to systemic failures of corporate, regulatory, and political oversight in the US. Note 3. Students attending the Banking Regulation course at the University of Malta are currently given a case study list of no less than 20 past bank crises to familiarise themselves with. Note 3. Students attending the Banking Regulation course at the University of Malta are currently given a case study list of no less than 20 past bank crises to familiarise themselves with. Note 4. De Bono & Judge Paolo. (1897). Sommario della storia della legislazione in Malta” – Cap XVI, 353-378; Cap XVII, 279-328 – (Tipografia del Malta). This total absence of reference to “regulation” in De Bono’s important work is also confirmed by Prof. Ray Mangion from the Dept of Legal History, Faculty of Law, The University of Malta. Note 5. Vide e.g. Moran M. (1986) – “Theories of Regulation and Changes in Regulation: the Case of Financial Markets” – (Political Studies, Vol. XXXIV, pp 185-201). Moran’s focus is very much based around four major sets of regulatory theories, viz teleogical (or public interest), cultural, instrumental, and administrative. Note 5. Vide e.g. Moran M. (1986) – “Theories of Regulation and Changes in Regulation: the Case of Financial Markets” – (Political Studies, Vol. XXXIV, pp 185-201). Moran’s focus is very much based around four major sets of regulatory theories, viz teleogical (or public interest), cultural, instrumental, and administrative. Note 6. According to Josef Buttigieg from MCAST’s Institute of Business & Commerce there were 18 students on the 2nd Year of this Level 5 Course, and course content generally follows that inspired by the UK’s IFS University College. Note 6. According to Josef Buttigieg from MCAST’s Institute of Business & Commerce there were 18 students on the 2nd Year of this Level 5 Course, and course content generally follows that inspired by the UK’s IFS University College. Note 7. The Central Bank of Malta, the Malta Stock Exchange, and the Ministry of Finance, must also be considered as part of Malta’s finance sector regulatory setup. Notes (2010) – Regulation of Financial Services Activities: Selected Statutes & Regulations – (American Casebook Services, West). But even their updating misses out on e.g. the Libor scandal (JP Morgan et al), Credit Suisse (the derivatives cartel), the whistle-blowing about HSBC’s Latin American money laundering misdemeanours, etc. Note 14. Haines F. (2011) – op cit. MacNeil I. & O’Brien J. (2010) – The Future of Financial Regulation – (Hart Publishing, Oxford and Portland, Oregon). 221 ISSN 1923-4023 E-ISSN 1923-4031 Published by Sciedu Press International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 2; 2016 Note 15. I.e. the “essential”, the “incidental”, and he “closely relateds” of banking. For examples: balance sheet structure and P & L dynamics; how banks can earn (and lose) money; how other sectors fit in; and the conflicting ethics and cultures of modern finance. The issue of “regulation as a modern project” is dealt with by Fiona Haines in The Paradox of Regulation (op cit p.3), and also by Reza Banakar in Law and Regulation in Late Modernity in Law and Society Theory, by Banakar R. & Travers M. (2013) – (Hart Publishing). Note 15. I.e. the “essential”, the “incidental”, and he “closely relateds” of banking. For examples: balance sheet structure and P & L dynamics; how banks can earn (and lose) money; how other sectors fit in; and the conflicting ethics and cultures of modern finance. The issue of “regulation as a modern project” is dealt with by Fiona Haines in The Paradox of Regulation (op cit p.3), and also by Reza Banakar in Law and Regulation in Late Modernity in Law and Society Theory, by Banakar R. & Travers M. (2013) – (Hart Publishing). Note 16. “The Business Observer” – Malta, Oct 23, 2014, pp 11, 12. Note 17. Vide e.g. the experiences of certain jurisdictions such as Australia, Singapore, Canada, and others. The experiences of the Australian regulatory system are particularly important and endlessly fascinating to Australians as well as to those who wonder why the Australian financial system has fared better, on all measures, than most others in developed economies. The statutory language which establishes the mandates of their main relevant agencies (their Australian Securities and Investments Commission (ASIC) and Australian Prudential Regulation Authority (APRA) establishes the key drivers of, for APRA financial safety and systemic stability, and for ASIC market integrity and consumer protection. Note 18. Financial Times, Nov 4, 2012. Appendix One The University of Malta Department of Banking and Finance The University of Malta ISSN 1923-4023 E-ISSN 1923-4031 Recruiting of staff for reggulation duties in Malta Recruiting of staff for reggulation duties in Malta Recruiting Source: Re finance ban of staff for reg ecruitment adv nk The Time gulation duties vert for Regula s of Malta Oct in Malta atory Reportin tober 15th 20 Source: Re finance ban ecruitment adv nk – The Time vert for Regula s of Malta, Oct atory Reporting tober 15th, 201 g Officer by F 4. FIMBank Maltta, Malta’s leaading specialisst trad Apppendix Two Apppendix Two Examples of Bank Regulation Course Topics Examples of Bank Regulation Course Topics ‐ Definition(s) of regulation. ‐ Regulatory theory groups. ‐ Regulation in society and politics (what sectorial mechanisms and structures make bank regulation necessary?). ‐ Regulation in society and politics (what sectorial mechanisms and structures make bank regulation necessary?). ‐ Soft law, and its impact on bank regulation. ‐ The economic history background. ‐ Deregulation; Reregulation; Under-regulation; Over-regulation; Better Regulation; Regulatory Fatigue. ‐ Regulatory capture (the Kane model). ‐ The “competent authority concept”: Malta Financial Services Authority and other regulator ‐ The “competent authority concept”: Malta Financial Services Authority and other regulators. ‐ Regulation and international relations. ‐ Regulation and international relations. ‐ International banking growth and regulation. ‐ Multinationalisation and internationalisation as regulatory backgrounds. ‐ Obstacles and dangers in international banking business. ‐ On consumer protection. ‐ On risks, and corporate governance. ‐ The roles and operations of financial intermediaries. ‐ The EU and its regulatory impact (including the European Systemic Risk Board, Committe ‐ The EU and its regulatory impact (including the European Systemic Risk Board, Committee of European Banking Supervisors, the Single Resolution Mechanism). Banking Supervisors, the Single Resolution Mechanism). Banking Supervisors, the Single Resolution Banking Supervisors, the Single Resolution hanism) Banking Supervisors, the Single Resolution Mechanism). ‐ Home and host country control. ‐ International banking supervision cooperation. ‐ Basle: evolution and impacts. ‐ Key EU Directives and key Malta FS legislation. ISSN 1923-4023 E-ISSN 1923-4031 ISSN 1923-4023 E-ISSN 1923-4031 222 Published by Sciedu Press IInternational Jouurnal of Financiaal Research Vol. 7, No. 2; 2016 http://ijfr.sciiedupress.com Source: Re finance ban ecruitment adv nk – The Time vert for Regula s of Malta, Oct atory Reporting tober 15th, 201 g Officer by F 4. FIMBank Maltta, Malta’s leaading specialisst trade ISSN 1923-40223 E-ISSN 19223-4031 2233 Published byy Sciedu Press IInternational Jouurnal of Financiaal Research Vol. 7, No. 2; 2016 http://ijfr.sciiedupress.com adv lta, N vert by Malta’s November 11ths financial serv h, 2014. vices regulatorr, the Malta FFinancial Serviices Regulator – The Source: Re Sunday Tim ecruitment adv mes of Malta, N vert by Malta’s November 11ths financial serv h, 2014. vices regulatorr, the Malta FFinancial Serviices Regulator – The Source: Re Sunday Tim ecruitment adv mes of Malta, N vert by Malta’s November 11ths financial serv h, 2014. vices regulatorr, the Malta FFinancial Serviices Regulator – The 2244 ISSN 1923-40223 E-ISSN 19223-4031 Published byy Sciedu Press
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An inquiry into the blurring boundaries between professionals and paraprofessionals in Dutch courts and the public prosecution service
International journal of law in context
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Abstract The autonomous position of legal professionals is no longer self-evident. Professionals are under increased pressure to reform. This phenomenon is not only true for legal professionals. A broader trend – which is recognised for the medical profession, academic profession and alike – is that paraprofessionals are gain- ing a more prominent position. In this paper, we focus on the developments in the Dutch public justice system. We conduct a case-study on the role of paraprofessionals in courts and in the public prosecution service – two understudied legal institutions in this regard. By drawing on empirical data unfolding the working routine of the judiciary and the prosecution service, we find two paradigms that define the think- ing about professionalism: a traditional ‘pure professional’ paradigm and a new, more hybrid paradigm that includes (policy-based) managerial thinking. The latter appears to be enhanced by a New Public Management (NPM) approach within these institutions. Although we observe resistance among (para) professionals towards professional changes and ambiguity in the relationships between professionals and paraprofessionals, we also observe that managerialism has changed the work processes and the division of labour between professionals and paraprofessionals. Keywords: professionalism; managerialism; paraprofessionals; judiciary; prosecution office International Journal of Law in Context (2020), 16, 371–389 doi:10.1017/S1744552320000270 An inquiry into the blurring boundaries between professionals and paraprofessionals in Dutch courts and the public prosecution service N.L. Holvast1* and J.M.W. Lindeman2 1Erasmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, The Netherlands *Corresponding author E mail: holvast@law eur nl 1Erasmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, The Netherlands *Corresponding author E mail: holvast@law eur nl versity Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, nds 1Erasmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, The Netherlands *Corresponding author E-mail: holvast@law eur nl asmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht Scho e Netherlands *Corresponding author. E-mail: holvast@law.eur.nl *Corresponding author. E-mail: holvast@law.eur.nl © The Author(s), 2020. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 1The Dutch body of judges that provides the administration of justice (de Rechtspraak) is in an international context com- monly referred to as ‘the judiciary’. The body of public prosecutors (openbaar ministerie) is often referred to as ‘the public prosecution service’. © The Author(s), 2020. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. prosecution service’. 1 Introduction Dutch judges and prosecutors – in the Netherlands, both appointed as ‘judicial civil servants’ – are traditionally portrayed as ‘pure professionals’, as described by Freidson (1994; 2001) and Abbott (1988)1: professionals who establish professional control of work and occupational protection (Noordegraaf, 2007). The adjudication of justice has long been regarded as the outcome of the labour of professional workers (judges and prosecutors). However, over the years, it is increasingly being per- ceived as a service provided by state institutions (the judiciary and the public prosecution service). Similar to many other professional domains (e.g. health care and education), ‘managerialism’ or ‘New Public Management’ (NPM) has had a considerable impact within the administration of justice (Noordegraaf, 2015; Langbroek and Westenberg, 2018; Visser et al., 2019). We notice that this has resulted in a shift from ‘traditional’ professionalism to ‘new’, more hybrid professionalism – which introduces NPM logics into the professionalism-paradigm (see Section 2.1). This shift and the ensuing organisational changes challenge the traditional division of power within these professional institutions (see similarly on the medical profession Bach et al., 2008; Kirkpatrick 1The Dutch body of judges that provides the administration of justice (de Rechtspraak) is in an international context com- monly referred to as ‘the judiciary’. The body of public prosecutors (openbaar ministerie) is often referred to as ‘the public prosecution service’. © The Author(s), 2020. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. NL Holvast and JMW Lindeman 372 et al., 2011). One way in which the power of professionals is challenged is by promoting stratification and by introducing different types, or levels, of professional occupations within the institutions (see Noordegraaf, 2007, pp. 762–763). These processes result in blurring boundaries between ‘full’ profes- sional and varying levels of ‘semi-’, ‘quasi-’ or ‘para-’ professional occupations (for magistrates’ assis- tants in the UK, see e.g. Raine and Willson, 1997; for legal practitioners, see Moorhead et al., 2003; see also Sommerlad et al., forthcoming). 1 Introduction Although it could be argued that both ‘full’ and ‘semi’ profes- sionals have (different) levels of professionalism, in this paper, we refer to the judges and prosecutors as ‘professionals’ and to all others involved – who may meet some, but not all, of the requirements to be a judge or a prosecution officer – as ‘paraprofessionals’.2 The latter group is more diverse in nature, consisting of judicial assistants, staff lawyers and a variety of prosecution assistants. Scholarly observations on the professional shift within the legal domain have predominantly focused on its effect on legal practitioners working at (large) law firms and not on legal professionals working within the administration of justice at state institutions. In this paper, we describe the changes within state institutions in more detail, with specific attention on the changes in the positions of para- professionals. We investigate how new professionalism has changed the formal position of paraprofes- sionals, focusing on the Dutch Judiciary and public prosecution service (PPS) as a case-study. We describe how professionals deal with the changes in what ‘professionalism’ entails and the conse- quences this has for the interaction between professionals and paraprofessionals. 2The latter group’s status is strongly connected to or derived from the ‘full’ professional. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 2.1 Professionalism and NPM Freidson (1970) and Abbott (1988) were among the first authors to introduce the concept of profes- sionalism, which can be contrasted with the concepts of a free market or a bureaucratic logic of orga- nising work. Professional occupations require specific skills and knowledge, which are achieved by both formal education and training as well as informal socialisation into the profession. Lawyers have traditionally been regarded as typical professionals. In their work, they serve public interests, but partly within a commercial setting (the latter being particularly true for legal practitioners; see also the influential work by Abel, 2003). y Recently, however, various authors have noticed a shift in the conditions under which professionals conduct their work. New organisational and managerial principles were introduced into the profes- sions. Evetts (2011) labels this as a transformation from ‘occupational’ professionalism to ‘organisa- tional’ or ‘new’ professionalism. This transformation results in ‘a shift from notions of partnership, collegiality, discretion and trust to increasing levels of managerialism, bureaucracy, standardization, assessment and performance review’ (Evetts, 2011, p. 406). Noordegraaf (2007) similarly refers to this new type of professionalism as ‘controlled professionalism’, considering that the organisations start to gain control over the professionals to some extent. In line with this reasoning, Noordegraaf (2007; 2015) also introduced the concept of ‘hybrid professionalism’. In this concept, organisational and managerial requirements (such as efficiency, budget control and productivity) go together with professional standards (such as quality standards, expertise, autonomy and independence), enhancing rather than weakening each other. g Particularly focusing on the legal profession, Sommerlad (1995) too recognises that the traditional legal profession is under attack by a new paradigm of managerialism. In his inaugural lecture, Moorhead (2014) argues that the legal profession today is precarious and challenged by new legal service providers. Similarly, Kritzer (1999) recognises a movement wherein legal professionalism has changed and ‘formal’ professionals have lost their exclusivity. As a result, ‘services previously provided only by members of formal professions can now be delivered by specialized general professionals or nonprofes- sionals’ (Kritzer, 1999, p. 713). Muzio and Ackroyd (2005) observe that external pressures have mainly resulted in increased internal stratification within law firms. In this context, the term International Journal of Law in Context 373 ‘deprofessionalisation’ has been used. However, we believe (with Paterson, 1996) that this movement can better be recognised as creating an altered form of professionalism rather than resulting in deprofessio- nalisation. 2.1 Professionalism and NPM Professional values still occupy a central place in the execution of the profession but are under pressure by other values and are therefore transformed and relocated within the professional institutions. p y p While the aforementioned literature mainly discusses the position of legal practitioners, there is a fair amount of literature that observes that a worldwide movement to managerialise public-sector insti- tutions has also affected the administration of justice at state institutions (see Freiberg, 2005; Heydebrand and Seron, 1990; Raine and Willson, 1997; Mak, 2008; Hondeghem et al., 2016; Lienhard et al., 2012; Visser et al., 2019). Managerial influences on the administration of justice are, for instance, observed in the US (Heydebrand and Seron, 1990), Australia (Freiberg, 2005; Spigelman, 2001) as well as in various European countries (e.g. for the UK, see Raine and Willson, 1997; McLaughlin et al., 2001; for France, see Vigour, 2015; Mak, 2008; for Switzerland, see Lienhard et al., 2012; for the Netherlands, see Visser et al., 2019; Langbroek and Westenberg, 2018; Mak, 2008). This movement, under the name New Public Management (NPM), stems from organisa- tional theories and focuses on public accountability and the use of best practices from the private sec- tor to improve the quality of services provided by public-service organisations (see e.g. Hood, 1991). Private-sector principles, such as efficiency, effectiveness, transparency and accountability, have become guiding concepts and are widely adhered to in courts and public prosecution offices. This is accompanied by the introduction of private-sector techniques, such as incorporating systems to ‘measure’ court performance and processes and incentives to increase the efficiency of courts (Contini and Carnevali, 2010). While the NPM approach could be regarded as a welcome initiative to help reduce the backlog of cases and improve the quality of justice (Contini and Carnevali, 2010; Jean and Pauliat, 2006), it gen- erates resistance from the professionals working within the system (Vigour, 2006; 2015; Holvast and Doornbos, 2015). This is partly related to the fact that the introduction of NPM principles is often accompanied by – or at least associated with – cuts in the budgets of the organisations. However, pro- fessionals hold more general objections to the movement. Judges particularly fear that NPM principles will impede traditional professional and legal values, such as judicial independence, impartiality and – most importantly – autonomy (Langbroek, 2001; see in relation to legal-aid practice Sommerlad, 1995). 2.1 Professionalism and NPM In fact, it is a much wider grievance that managerialism hinders the autonomous position of profes- sionals. Similar concerns are raised within the health sector and (higher) education (Bezes et al., 2012). g Professional workers are likely to oppose these managerialism influences, for example by creating sets of self-binding conditions for entering the profession and for performing the professional duties. These self-binding conditions (are proclaimed to) accommodate the provision of high-quality services. At the same time, and some authors argue that this is their core purpose, the conditions also function as a means to maintain a professional monopoly of the services offered by the profession (Larson, 1977). Professions will therefore be resilient to changes that challenge the existing system of profes- sional self-organisation and self-control (see e.g. Freidson, 2001). They find it problematic to accept that paraprofessionals can and should perform parts of the professional duties, as it means giving up part of the professional monopoly of providing legal services. However, it has become increasingly clear that this is a lost battle, as the market mechanism forces legal professionals to give up part of this monopoly to become more affordable (Susskind, 2017). This introduces new forms of professionalism and results in paraprofessional positions becoming more professionalised, a notable example being paralegals in law firms (Noordegraaf, 2011; Lively, 2001). ttps://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ownloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 3 The Dutch context: changing position of paraprofessionals in the judiciary and the PPS In the 1990s and 2000s, the Dutch administration of justice has adopted various NPM features (see more in Visser et al., 2019). The introduction of these features affected the formal position of para- professionals in the Dutch judiciary and the PPS in different ways. In both institutions, we observed that paraprofessionals have received a more central position in the work processes. However, the changes in the position of the paraprofessionals in the PPS are more far-reaching and more explicit than in the judiciary. We start this section by providing a brief description of the organisation of both institutions. Subsequently, we successively summarise the changes that occurred regarding the position of paraprofessionals in the judiciary and the PPS. 2.2 Methodology This paper combines a macro-level analysis of regulation and policy on the role of paraprofessionals with micro-level insights from empirical data collected through participant observations and inter- views within the Dutch judiciary and the PPS. For the regulation and policy analysis, we studied the literature on organisational developments within both institutions and conducted a review of NL Holvast and JMW Lindeman 374 policy documents published by the Council for the Judiciary (Raad voor de Rechtspraak), the Board of Prosecutors General (College van Procureurs-Generaal) and the Dutch Association for the Judiciary (Nederlandse Vereniging voor Rechtspraak, the professional body and trade union for judges and pub- lic prosecutors). The previous documents report only marginally about the role and duties of assistants and they provide little insight into the daily practice in the workplace. In order to gain more in-depth informa- tion, empirical data were needed. The empirical data derive from two research projects3: one on the work of public prosecutors in the Dutch PPS and another project on the involvement of judicial assis- tants in judicial decision-making at Dutch district courts. In the first project, Lindeman studied how public prosecutors define and fulfil their duties. He conducted fieldwork for the duration of twelve months within two district offices and one nationwide office of the PPS. He followed the prosecutors in their daily routine and observed their interactions with the PPS staff, police officers, judges, etc. He was able to observe how prosecutors interact with prosecution assistants and to what extent they follow the assistants’ judgments. During his fieldwork, he observed and spoke with numerous prosecution assistants. He also conducted fifteen interviews with prosecutors.4 In the second project, Holvast examined the involvement of judicial assistants in the judicial decision-making in two Dutch district courts (in the criminal-law and administrative-law divisions). She conducted fieldwork for the dur- ation of eight months. Holvast participated in the daily activities of the courts and followed the decision-making process in 137 cases, heard during twenty-seven hearings. She analysed the exchange of views orally and via memos prior to the hearing, observed the hearings and was able to attend and observe the deliberation sessions. She also examined the process of drafting and finalising the judg- ment by judges and assistants. Holvast interviewed over eighty court officials, mainly judges and judi- cial assistants.5 3These two PhD projects by Holvast and Lindeman were published in 2017. 4See more in Lindeman (2017, chapter 2). 5See more in Holvast (2017, chapter 2). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 3These two PhD projects by Holvast and Lindeman were published in 2017. 4See more in Lindeman (2017, chapter 2). 5See more in Holvast (2017, chapter 2). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cam 3.1 Managerial changes in the Dutch judicial and public prosecution organisations This magisterial position is most absolute for judges (as adjudicators) and somewhat less for prosecutors, who have to comply with criminal- policy regulations (Van de Bunt, 1985; Lindeman, 2017). The special position of judges and prosecutors has always been important for the organisational struc- ture of their respective institutions. Traditionally, the judiciary and the PPS were managed by the judges and prosecutors. At all courts, the periodical ‘court meeting’ (gerechtsvergadering), consisting of all judges, used to be an important decision-making body. These local bodies still have some decision-making power, but a more managerial structure was introduced in 2002, with the newly established national Council for the Judiciary at the top. At the court level, an additional management layer consisting of judge team lea- ders was introduced and the courts’ boards were required to include one non-judge board member. Another important change was the introduction of output financing. These changes resulted in a some- what more managerial organisation, in which collective work processes are – to some extent – restricting the autonomy of judges and moving more towards a ‘controlled professionalism’ as described by Noordegraaf (2015). Nonetheless, the autonomous position of judges and their authority in organising their work are still key elements within the judicial organisation. Hence, the NPM values have not replaced the traditional rule-of-law values, but these values co-exist (Mak, 2008; Langbroek and Westenberg, 2018). The organisational structure of the PPS has changed more radically. Significant NPM influences can be recognised in these developments. As within the judiciary, these influences also restrict the autonomy of prosecutors. In order to understand the impact of the changes, a short introduction on the organisation of the PPS is necessary (Zouridis, 2016). The PPS has authority over the police in criminal matters and decides whether to prosecute or dis- miss a case (along the lines of the so-called expediency principle). In the 1970s and 1980s, the Dutch government increasingly regarded criminal policy as a means to proactively fight crime and the PPS was supposed to be the ‘spider in the web’ in this battle. However, in the 1990s, it became clear that the PPS was not up to this task. After long deliberations, legislation implementing rigorous changes in the management structure of the PPS came into force in 1999. 3.1 Managerial changes in the Dutch judicial and public prosecution organisations While we portray judges and prosecution officers as professionals, and thereby compare them to other professionals such as doctors, professors and attorneys, it is important to note that judges and prose- cutors have a special position in society. In the Netherlands, both officials are appointed by law as a so-called ‘judicial civil servant’ (rechterlijk ambtenaar, Art. 1 of the Judicial Organization Act), pro- viding them with a special legal status codified in the Act on the Legal Status of Judicial Civil Servants. This differentiates them from other civil servants and gives them, in some ways, the status of personification of the administration of justice (one of the three branches of state power) (Langbroek and Westenberg, 2018). Especially for judges, this position also comes with various safe- guards regarding their independence, impartiality and integrity: judges are appointed for life (Art. 116 of the Dutch Constitution) and special rules for dismissal apply to ‘judicial civil servants’ International Journal of Law in Context 375 (Bovend’Eert, 2000). These safeguards apply to public prosecutors to a somewhat lesser extent: they are not appointed for life and the Minister of Justice can give directions to the Public Prosecutor’s Office (Van de Bunt and Van Gelder, 2012). Judicial assistants are defined in the Judicial Organization Act as ‘civil servants who work at courts’. Prosecution assistants are regular civil servants and are implicitly referred to as ‘other civil servants working at the prosecutor’s office’.6 The regular terms of appointment and dismissal for Dutch civil servants apply to both types of assistants. The formal status of ‘judicial civil servants’ also resonates in the way judges and prosecutors view their position and how they act. In the Netherlands, this is often referred to as their ‘magisterialness’ (in Dutch magistratelijkheid, which refers to a mindset of making independent decisions as a guardian of the rule of law, thereby considering the interests of all parties involved). For prosecutors, magister- ialness means that they are not merely a party in criminal proceedings seeking to ‘win’ a case: they are expected to oversee a process of establishing the truth that is compatible with the rule of law (Lindeman, 2017, pp. 24–26, 295). Developing and maintaining the attitude of a magistrate is part of the professional training of judges and prosecutors. 6Judicial Organization Act, Art. 126. 7The Judicial Revision Act merged nineteen district offices into ten. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at ww.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge J g , 7The Judicial Revision Act merged nineteen district offices into ten. 3.2 Paraprofessionals in the judiciary The paraprofessional, in the capacity of court assistant (griffier), has a long history in the Dutch judiciary, existing since the early 1800s (De Groot-van Leeuwen, 1991). Other than preparing the court record, which has been a key responsibility since their existence, duties of these assistants have changed drastically over time. From the 1960s to the 1980s, a substantial portion of the assistants consisted of internally trained, originally administrative, staff members. From the 1990s onwards, and particularly after the enact- ment of new legislation, their position was gradually professionalised. The largest change occurred after the introduction of the new management structure with the enactment of the Dutch Judiciary Organization and Management Act (Wet Organisatie en bestuur gerechten) and the Act on the Council for the Judiciary (Wet Raad voor de Rechtspraak), in 2005. The numbers of judicial assistants working at the courts vary somewhat per court type and division but, at district courts and Courts of Appeal, the judicial assistants slightly outnumber the judges working at the courts (see Holvast, 2017, p. 49).8 g y j g g p To implement the output-based financing structure that was introduced in 2005, the judiciary cre- ated a model that specifies how much time (measured in minutes) a judge and a judicial assistant are estimated to work on a specific type of case: the so-called Lamicie-model (see Van der Torre et al., 2007). The model determines what compensation courts subsequently receive for handling the cases, taking into account the supposed time spent on handling those cases by the judicial officers (Van der Torre et al., 2007). The figures are based on time writing surveys and are supposed to reflect the real division of work within the courts. The model revealed the practice that, in most types of cases, judicial assistants spend considerably more time working on the cases than judges. Shortly after the creation of this model, the internal training programme for judicial assistants deteriorated and the minimum requirement for holding a diploma from an institute of higher professional education was introduced (Abram et al., 2011). The latter was primarily a codification of an already existing prac- tice. In fact, many newly hired assistants had (and still have) finished even higher levels of education (an LLM from a Dutch law school). 3.1 Managerial changes in the Dutch judicial and public prosecution organisations A ‘top-down’ hierarchy was introduced, formalising the (until then informal) leadership by the Board of Prosecutors General, but still allowing the Minister of Justice to issue instructions to that board. Also, the practice to shift a part of the pro- secutors’ work to subordinates had a statutory basis that allowed a much broader application. Policy regulations issued by the Board of Prosecutors General became increasingly important instruments. These regulations constrain the discretionary role of individual prosecutors, as they hold instructions regarding the decision to prosecute. g g p Since the formal reorganisation in 1999, the PPS has almost continuously been engaged in revising and adjusting its organisation, for example by increasing the scale of the offices.7 Since the 2008 crisis, NL Holvast and JMW Lindeman 376 budget cuts have been a rule rather than an exception, urging the PPS to work as efficiently as possible, without losing sight of the primary objectives. As of 1 January 2019, the PPS also introduced output financing for a significant part of the organisation. These developments within the PPS display a strong pattern of centralisation of authority and a mandatory accountability within a strict hierarchy, to a certain extent even involving accountability to the Minister of Justice. g y These organisational changes in the institutions also had consequences for the position of parapro- fessionals. We will discuss this position in the next sections. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 8Unfortunately, no historic comparison can be provided; these numbers only recently became available. 3.2 Paraprofessionals in the judiciary The function of judicial assistants in district courts and civil and criminal Courts of Appeal have, since 2007, been divided into five categories: junior judicial staff members, judicial staff members, senior judicial staff members, staff lawyers and senior staff lawyers. The function profiles provide insight into the duties of judicial assistants. These profiles state that (both junior and senior) judicial assistants assist judges in preparing for hearings, during the hearings and in the processing and conceiving of judgments. Staff lawyers are usually specialists of a subfield of law. They are required to possess an LLM from a Dutch law school. Staff lawyers may provide advice on complex legal issues and cases with high social or economic impact. Additionally, they can con- tribute to the creation of new case-law and serve as sounding boards for judges and judicial assistants. The descriptions of duties in the profiles, however, remain abstract and can be interpreted in multiple ways. The function profiles do not indicate the manner in which judicial assistants are supposed to assist prior to the hearings or how to give advice. The profiles also do not mention the assistants’ role in the deliberation room; the research by Holvast (2017) revealed the informal norm that judicial assistants do participate in judicial deliberations. It can be extracted from the aforementioned documents that judicial assistants have attained a more central position within the process of adjudication. Several professional duties that were originally International Journal of Law in Context 377 reserved for judges are now partly conducted by judicial assistants. The transformation of the judicial-assistant position that occurred over time appears to follow from NPM-driven organisational changes, such as the output-based financing structure and efforts to improve the efficiency of work pro- cesses. The required qualifications for the function have become stricter and an assistant’s current role exceeds the more administrative tasks that the role primarily consisted of before. Most judges and assis- tants who worked at the courts during these transitions confirm that the judicial-assistant position has tacitly become more demanding. Examples thereof are the acceleration of the duties to draft judgments and the assistants’ active participation in deliberation sessions. The fact that the position of judicial assistant nowadays requires more legal knowledge and skills is most recently used as leverage in the negotiations of the collective labour agreements of judicial assistants. 3.3.1 Formalised mandate for prosecutor assistants The idea to allow prosecutors to delegate additional duties to assistants developed as early as the 1970s. That is not to say that delegating these duties was immediately a widespread practice. At first, prose- cutors were reluctant to allow the paraprofessionals to perform some of their duties (Van de Bunt, 1985). After all, these assistants did not possess a law degree and, as such, acquired their knowledge ‘on the job’. At the same time, the case-load gave the prosecutors no choice but to allow assistants to at least prepare their cases for them. This practice was facilitated by the prosecution guidelines, support- ing the assistants in their decision-making process. This deployment of assistants increased over the years, without a clear legal basis: the public prosecutor would still formally make the decisions. This changed in 1999 when the legal amendments mentioned above provided a formal mandate to assis- tants within the PPS to perform certain duties normally reserved for public prosecutors.9 Thus, while, in the judiciary, assistants work under the direct authority of a judge, prosecutors’ assistants have been mandated to make individual decisions for the past two decades. As such, in the 1990s and 2000s, NPM principles were more and more incorporated into the PPS organisation. On the one hand, prosecution assistants increasingly were university graduates. On the other hand, the PPS also started employing new types of assistants with lower education levels. Between 2000 and 2015, a broad range of assistants with varying levels of education were employed by the PPS: administrative judicial assistants (senior secondary vocational education)10; junior pros- ecution assistants (higher professional education)11; prosecution assistants (higher professional educa- tion or university education)12; and senior prosecution assistants and (senior) policy officers (mostly university education).13 Nonetheless, even the assistants with the lowest rank (administrative judicial assistants) were mandated to process and decide on (the most simple) cases. 3.2 Paraprofessionals in the judiciary However, as of today, many judicial assistants have temporary contracts and most assistants’ salaries are based on a scale for graduates from Universities of Applied Sciences, even though the vast majority have graduated from regular universities. 9Judicial Organisation Act, Art. 126. 10Administratief Juridisch Medewerkers who received middelbare beroepsopleiding (MBO). 11Junior Parketsecretaris (hoger beroepsonderwijs, HBO). 12Parketsecretarissen, HBO or universitair onderwijs. 13Senior Parketsecretarissen and (senior) beleidsmedewerkers, Universitair geschoold. l d d f htt // b id / IP dd 80 101 62 141 03 M 2021 t 11 00 31 bj t t th C b id C t f il bl t https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 9Judicial Organisation Act, Art. 126. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cam 3.3.2 The role of paraprofessionals in FOO and HIC The work in Public Prosecutor’s Offices can roughly be divided in two work streams: frequently occur- ring offences (FOO) and high impact crimes/organised crimes (HIC). There are significant differences between the positions of the assistants in both divisions. During the period 2000–2015, prosecution assistants were used extensively to process FOO. This was facilitated by an ever-increasing, intricate structure of policy regulations (guidelines and NL Holvast and JMW Lindeman 378 instructions) and ICT systems, which allowed assistants to work independently while still being mon- itored (Van de Bunt and Van Gelder, 2012; Lindeman, 2017).14 Almost 50 per cent of the cases regis- tered by the PPS were dealt with out of court.15 A vast majority of these cases were processed by assistants. This allowed a so-called bifurcation: prosecutors dealt with more serious and/or compli- cated cases, while assistants processed the run-of-the-mill cases. Assistants decided whether cases went to trial or were handled out of court. They decided which offence a suspect would be prosecuted for and (in the case of out-of-court proceedings) on the type and measure of the sentence. Lindeman (2017) observed that there was not much scrutiny of the assis- tants’ work. The FOO departments of the PPS district office virtually operated on a ‘stand-alone’ basis. Prosecutors would only be involved if a case was taken to court because a suspect did not agree to the proposed out-of-court sanction or if an assistant decided to summon the suspect. A highly critical report on the efficiency of the criminal justice chain (Algemene Rekenkamer, 2012) led to the implementation of a new procedure regarding FOO: the so-called ZSM procedure (Jacobs and Van Kampen, 2014).16 In a significant departure from the practice described above, the aim is now that all cases are preliminary decided upon by a prosecutor in an early stage. The prosecu- tor gives an indication as to the processing (a trial or an out-of-court proceeding). Subsequently, a prosecution assistant will execute this decision or, if necessary, prepare the case for trial. The use of prosecution assistants without legal training has diminished. A new function, the ‘assistant-prosecutor’, has been introduced. (Senior) prosecution assistants with a law degree can enter a traineeship to become an assistant-prosecutor. An assistant-prosecutor is always on duty together with a regular prosecutor at the ZSM desk. 3.3.2 The role of paraprofessionals in FOO and HIC Those cases that will be routed towards a trial will, ideally, be processed by this assistant-prosecutor, who will also represent the PPS at the trial. The assistant-prosecutor will also represent the PPS at trial in other simple cases, mostly heard by a single judge. In doing so, the assistant-prosecutor takes over a significant part of the workload tradition- ally assigned to regular prosecutors. Initial research by the Dutch Association for the Judiciary and follow-up research by the Board of Prosecutors General reveal that there are differences between the districts as to the scope of the competence and autonomy of assistant-prosecutors. It is not always clear whether the various manifestations of the assistant-prosecutor are in line with the national policies. While prosecution assistants have increasingly been assigned more demanding duties, their salary grade has remained almost unchanged and they rarely have permanent contracts. Even becoming an assistant-prosecutor is by no means a guarantee to enter the traineeship to become a fully qualified public prosecutor. In the departments where more serious and/or complicated criminal cases are dealt with and where prosecutors oversee the criminal investigations carried out by the police, also known as the HIC departments, (senior) prosecution assistants play a significant role. The role of prosecution assistants within these more complicated criminal cases is more commensurate with the role of judicial assis- tants. The legislation on their mandate does not allow the prosecution assistants to make independent decisions on the use of investigation methods (e.g. wire taps or surveillance orders) and/or coercive measures. However, the assistants do often offer significant assistance to the prosecutors. 14This ICT system called the Decision Support System, Beslissingsondersteuningssysteem – BOS, was used from the start of the reorganisation in 1999 until 2015. 15This figure has been quite consistent over the past decades. 16ZSM stands for ‘zo snel mogelijk’ which translates as ‘as soon as possible’. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 14This ICT system called the Decision Support System, Beslissingsondersteuningssysteem – BOS, was used from the start of the reorganisation in 1999 until 2015. 15This figure has been quite consistent over the past decades. 16ZSM stands for ‘zo snel mogelijk’ which translates as ‘as soon as possible’. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 14This ICT system called the Decision Support System, Beslissingsondersteuningssysteem – BOS, was used fro he reorganisation in 1999 until 2015. 15 4.1 Introduction While further substantiating the professional and paraprofessional collaboration in the workplace, the (para)professionals are guided by shared professional norms and ideals regarding the appropriate pos- ition of paraprofessionals within the process of decision-making. Our fieldwork in the judiciary and PPS reveals that, among professionals and paraprofessionals, there is not one dominant paradigm that clarifies their behaviour. Two different – and on various aspects competing – paradigms co-exist and define the role of paraprofessionals. These professional paradigms are related to the developments in professionalism described in Section 2.1. On the one hand, and that is the first paradigm, (para)professionals conform to a trad- itional (occupational or ‘pure’) definition of professionalism, in which the ‘full’ professional is regarded as holding the single authority over the professional decision-making. In this paradigm, it is regarded as inappropriate for the ‘full’ professionals to rely too strongly on the work of paraprofes- sionals. The professionals claim full jurisdiction over the process of adjudication/prosecution and the position of paraprofessionals is – in some occasions – marginalised. On the other hand, a second para- digm exists, which encompasses that professionals and paraprofessionals accept that their relationships have changed and that managerial indicators regarding productivity and policy influence their work, and they acknowledge that paraprofessionals can – to some extent – be regarded as professionals themselves. Within this paradigm, all officers at the court/PPS are viewed as partners in reaching the goal of delivering justice in an efficient and timely manner. Some (para)professionals have ideas that predominantly fit into one specific paradigm. More often, professionals’ and paraprofessionals’ behaviour appears to fit in both paradigms, opting for different sets of norms in different situations. In the following sections, we provide a detailed account of how we recognised these paradigms in the statements made and actions taken by professionals and paraprofessionals. 3.4 Concluding remarks In the past decades, the (formal) division of duties between professionals and paraprofessionals has been altered. More specifically: further allocation of duties to paraprofessionals has occurred. Within both institutions, these changes can, at least in part, be attributed to the NPM movement. At the PPS, some of the decision-making practices have increasingly been mandated to assistants, International Journal of Law in Context 379 which transferred them into some sort of quasi-prosecutors, culminating in the establishment of assistant-prosecutors. In the judiciary, all decision-making duties are still formally in the hands of the judges, with no official mandates for decision-making by judicial assistants. In both institutions, the official recognition of more demanding paraprofessional functions is still limited: neither the financial compensation nor other forms of appreciation have shown meaningful improvement. Additionally, the official policy provides general guidelines, but leaves a significant mar- gin of discretion as to how the professionals and paraprofessionals should give substance to their collaboration, leaving room for differences in execution. This makes it interesting to investigate which – largely informally defined – standards/norms regarding the division of labour are existent at the courts and PPS, and how these are linked to different concepts of professionalism. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor According to some respondents, this can be explained by the often more timid nature of assistants, related to the fact that they chose to fulfil a subordinate role with no decision-making responsibilities. Observations within the PPS were somewhat different in this regard: especially within the HIC teams, some assis- tants played dominant roles in team meetings. Also, socialising between prosecutors and assistants was customary, which, in the 1980s, would have been unthinkable (Van de Bunt, 1985). Despite prosecutor and assistant positions growing towards each other, periodical ‘prosecutors-only’ meetings were still being held (Lindeman, 2017). While the social boundaries between the groups of professionals and paraprofessionals have faded in the past decades and the interaction has become more informal (on the PPS in the 1980s, see Van de Bunt, 1985; on the judiciary, see Bevers, 2004, pp. 8–9), these boundaries have not disappeared com- pletely. A judge (A1 – resp. 42) explains that, when she started working in the judiciary around the turn of the century, it was explained to her that judges were not supposed to socialise too much with assistants, as that was unworthy of their rank. Within some court divisions, it was still common in 2012–2013 for judges and assistants to have lunch in separate groups. This demonstrates that – to some extent – a social distance still exists. Judicial assistants would, on average, also speak considerably less than judges during general staff meetings that were held in different courts. According to some respondents, this can be explained by the often more timid nature of assistants, related to the fact that they chose to fulfil a subordinate role with no decision-making responsibilities. Observations within the PPS were somewhat different in this regard: especially within the HIC teams, some assis- tants played dominant roles in team meetings. Also, socialising between prosecutors and assistants was customary, which, in the 1980s, would have been unthinkable (Van de Bunt, 1985). Despite prosecutor and assistant positions growing towards each other, periodical ‘prosecutors-only’ meetings were still being held (Lindeman, 2017). The previous paragraphs clearly indicate that both judges and prosecutors believe that there is a certain domain of authority exclusive to their profession. In the judiciary as well as in the PPS, we observed significant differences between individual judges/prosecutors as to the exact dimensions of that domain. 17But see Section 4.3.1. 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor On various occasions, we observed a scepticism towards a movement that would assign more far- reaching adjudicative or prosecutive duties to paraprofessionals. Several times an inherent hierarchy in the relationship between professionals and paraprofessionals was emphasised by respondents. They related this to the fact that the judge/prosecutor is the one appointed by law to adjudicate/pros- ecute. This claim of authority is more obvious with regard to judges (see Section 3.1) and this was indeed a stronger sentiment within the judiciary. However, prosecutors also expressed this claim of authority. The ‘full’ professionals thereby displayed behaviour of marking the boundaries of their pro- fession and being resilient to changes in work processes that might challenge these boundaries – which can be regarded as typical behaviour for ‘pure professionals’. It is interesting that not just professionals, but also several paraprofessionals defend these strict boundaries. 380 NL Holvast and JMW Lindeman Some authors suggest that the sense of responsibility for judgments (and in this context also deci- sions to use coercive measures, prosecute and/or demand a sentence) might be lost when paraprofes- sionals are involved (Fiss, 1983; Hol, 2001). Despite our finding that, indeed, paraprofessionals are involved to a greater extent in the decision-making process, a decline in a sense of responsibility was not generally observed in our research.17 One judge, for example, stated: ‘I make the decision. It is my responsibility, and I have to carry it. And if we can’t decide, I am the one who lies awake at night, not the judicial assistant. He will hear the next day what we’re going to do.’ (A1 – resp. 18) Judges’ responsibilities appear to rest heavily on their shoulders. Relying too strongly on the pro- ducts of judicial assistants is, from this perspective, excluded. Judges draw a clear line at the use of memos produced by judicial assistants. All judges proclaimed that it is inappropriate to prepare for a hearing by exclusively reading the memo prepared by an assistant and not opening the case files. One judge’s (A1 – resp. 38) opinion was more extreme. He said he did not read the memo at all, as he wanted to enter the hearing unbiased. However, this was not the leading opin- ion within the courts. Judges’ responsibilities appear to rest heavily on their shoulders. Relying too strongly on the pro- ducts of judicial assistants is, from this perspective, excluded. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor Judges draw a clear line at the use of memos produced by judicial assistants. All judges proclaimed that it is inappropriate to prepare for a hearing by exclusively reading the memo prepared by an assistant and not opening the case files. One judge’s (A1 – resp. 38) opinion was more extreme. He said he did not read the memo at all, as he wanted to enter the hearing unbiased. However, this was not the leading opin- ion within the courts. In the HIC division of the PPS, a similar claim of authority was displayed. Prosecutors claimed that assistants hardly ever made decisions without prior consent by the prosecutor, especially when it con- cerned decisions on the application of the expediency principle: ‘It’s an equal debate, but at the end of the day, the decision has your name on it, and you will be the one having to discuss the decision with parties concerned. No, I don’t think that decisions leave this office without the involvement of a prosecutor.’ (A2 – resp. 10) ‘It’s an equal debate, but at the end of the day, the decision has your name on it, and you will be the one having to discuss the decision with parties concerned. No, I don’t think that decisions leave this office without the involvement of a prosecutor.’ (A2 – resp. 10) While the social boundaries between the groups of professionals and paraprofessionals have faded in the past decades and the interaction has become more informal (on the PPS in the 1980s, see Van de Bunt, 1985; on the judiciary, see Bevers, 2004, pp. 8–9), these boundaries have not disappeared com- pletely. A judge (A1 – resp. 42) explains that, when she started working in the judiciary around the turn of the century, it was explained to her that judges were not supposed to socialise too much with assistants, as that was unworthy of their rank. Within some court divisions, it was still common in 2012–2013 for judges and assistants to have lunch in separate groups. This demonstrates that – to some extent – a social distance still exists. Judicial assistants would, on average, also speak considerably less than judges during general staff meetings that were held in different courts. 4.3 The ‘new’ professionals paradigm: judges/prosecution officers and assistants as partners in reaching professional goals 4.3.1 (Para)professionals taking into account ‘new professional’ values in their professional collaborations While we observed claims made regarding the hierarchy in the relationship between professionals and paraprofessionals, the equality in and similarities of the positions were just as frequently mentioned. Many judges underlined that they acknowledged judicial assistants for being colleagues with whom they closely collaborate in the process of adjudication and interchange intellectual ideas regarding the merits of cases. Assistants are often also regarded as equal partners by the HIC prosecutors and are welcomed to discuss almost all decisions with the prosecutor. Lindeman (2017) heard a pros- ecutor make the off-the-cuff claim that assistants could perform all the prosecutor’s tasks, except for the trial (A2, p. 105). A judge similarly said: ‘A judge is actually a sort of assistant plus: he should be able to do everything that an assistant can and chair the hearing’ (A1 – resp. 77). Judges and prosecutors regularly work with the same assistants, which can attribute to a certain team spirit. The fact that the majority of recently appointed judicial/prosecution assistants obtained a law degree appears to be important for judges and prosecutors to acknowledge them as discussion partners: ‘I always think: we employ lawyers for a reason …. We should make use of that’ (A1 – resp. 42). Various respondents also highlight the sounding-board function of the assistants and they recognise that the discussion with the assistants affects their considerations regarding cases. One judge explains how she, somewhat to her own surprise, really missed this type of contribution when – due to unforeseen circumstances – she was not accompanied by a knowledgeable assistant: ‘I realized that I really missed a discussion partner then. … I really missed someone to write the judgments. That, too. … But I mainly noticed that what I really missed was the exchange of thoughts. I didn’t expect that to be such an issue.’ (A1 – resp. 42) The discussion-partner role of judicial assistants is also exposed by the informal norm (largely given substance since the 1990s) that judicial assistants are asked to display their views on the case during deliberation sessions.18 Some judges regard this as a duty, emphasising their equal position in the dis- cussion of cases. Others consider this more to be a privilege or a social gesture, which they imagine assistants will appreciate. 18Although, in the interviews, judges widely endorse this informal work practice, they do not always act accordingly during the deliberation sessions (perhaps due to ‘pure’ professional ideas). In just over half of the deliberations observed by Holvast, the assistants were consistently offered the opportunity to speak first. 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor The appearance at the public hearing was generally regarded as one of the key components of the profession of judge or prosecutor, which clearly distinguished these professionals from the paraprofes- sionals. Several assistants mentioned that they actually prefer a position out of the spotlight and believed themselves to be more suitable for such a role. At the courts, a marginal change occurred ttps://doi.org/10.1017/S1744552320000270 .org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at International Journal of Law in Context 381 in which, in administrative-law cases, some judges would allow assistants to ask questions to the par- ties (which in reality rarely happened). At the PPS, a notable shift in this regard is the introduction of the assistant-prosecutor (see Section 3.3). Still, a large portion of the judges and prosecutors believed that performing at the hearing was typ- ical for their profession. This also demarcates their professional position to the general public. These professionals reject the possibility of having assistants involved. When the researcher, for instance, asked a judge about her view on having judicial assistants ask questions, she responded: ‘No, that can- not happen, because the assistant is not present to ask questions during the hearing. If that happens during a hearing where parties are present, then it is wrong. Period’ (A1 – resp. 50). In summary, we observed (para)professionals who (still) adhere to traditional professional values, as described in the original literature on professionalism when it concerns certain parts of their work (see Larson, 1977; Abbott, 1988; Freidson, 1994). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 4.3 The ‘new’ professionals paradigm: judges/prosecution officers and assistants as partners in reaching professional goals In the administrative-law sections of the courts, it is common practice in single-judge cases for the judge and judicial assistant to meet after the hearing to discuss the cases. It is remarkable that these meetings are referred to as ‘deliberation sessions’, while formally the 18Although, in the interviews, judges widely endorse this informal work practice, they do not always act accordingly during the deliberation sessions (perhaps due to ‘pure’ professional ideas). In just over half of the deliberations observed by Holvast, the assistants were consistently offered the opportunity to speak first. NL Holvast and JMW Lindeman 382 judge takes the decision and, thus, actual deliberation is not a necessity. Most of these sessions appeared to occur on equal grounds, with both participants being actively involved in exchanging dif- ferent arguments regarding a case. A similar practice can be found in the various forms of peer ses- sions that prosecutors convene on a regular basis. For example, ‘sentence assessments’19 regularly take place. A prosecutor presents his/her case to other prosecutors with the objective of assessing the demand (regarding the sentence) that fits the crime best. The assistant who worked on the case with the prosecutor is also invited and actively participates in the meeting. Furthermore, judicial and prosecution assistants have another important means of sharing their views on the case: the preparatory memos that they make for judges and prosecution officers to pre- pare for the hearings (see on the importance of these memos in Dutch police judge cases also Van Oorschot, 2014). Some of these memos are mainly summaries of the case files, yet other memos clearly, and deliberately, reveal the views of the assistants on the demands that the prosecutor should present or what the judges’ decision should be. In some administrative-law cases, judicial assistants would write memos in a draft-judgment format, thereby to an extent already anticipating the legal rea- soning. In complicated cases, the prosecution assistant who assisted in conducting and overseeing the investigation and who prepared the case will attend the hearing before the trial such as to be able to assist the prosecutor when last-minute questions arise. Hence, while judges and prosecutors are by law required to act and decide in specific situations, the preparations of an assistant are by many consid- ered indispensable for these actions and decisions. 19Strafmaatoverleggen. 20Dutch law requires these warrants for many investigation methods, such as surveillance orders, requisition of informa- tion (e.g. CCTV footage) and wire taps. 21However, because both our research projects provide an analysis of the institutions at one specific point in time, we do not claim to have proof for a causal relation and/or a gradual development in this regard. It is even likely that both sets of values have always to some extent played a part in defining work relations. 22Lindeman’s (2017) observations on the FOO divisions are representative of the organisation of the processing of FOO in the timeframe prior to the ZSM procedure (roughly from 2000 until 2015). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available a 19Strafmaatoverleggen. 20Dutch law requires these warrants for many investigation methods, such as surveillance orders, requisition of informa- tion (e.g. CCTV footage) and wire taps. 21However, because both our research projects provide an analysis of the institutions at one specific point in time, we do not claim to have proof for a causal relation and/or a gradual development in this regard. It is even likely that both sets of values have always to some extent played a part in defining work relations. 22Li d ’ (2017) b ti th FOO di i i t ti f th i ti f th i f FOO i 4.3 The ‘new’ professionals paradigm: judges/prosecution officers and assistants as partners in reaching professional goals This indicates that these professionals acknowledge that their work is to some extent ‘controlled’ by decisions about work processes – and the role of para- professionals therein – made on an organisational level (see Noordegraaf, 2015). At the HIC departments, when the police need a warrant for a specific investigative method,20 assistants will draw up the warrant (and determine whether there are sufficient grounds that meet the legal requirements). Assistants draft orders for pre-trial detention. Assistants prepare meetings with the police in ongoing investigations, and they even represent the prosecutor in such meetings. Some prosecutors (but certainly not all) sometimes temporarily yield some of their authority and allow assistants to decide on the arrest of persons ‘not caught in the act’, even when there is no legit- imation for such a mandate. The prosecutor will insist on being briefed on such decisions straightaway (Lindeman, 2017). Prosecution assistants also play important roles in the policy-related duties of public prosecutors. They prepare and attend selection consultations with the police, in which the case-load of HIC cases will be prioritised. They also prepare and attend tri-party consultations in which the city mayor, the chief of police and the public prosecutor discuss priorities regarding enforcement of crime and public order within districts. Hence, in this second paradigm, we observe (para)professionals taking into account ‘new profes- sional’ values in their professional collaborations. Thus, we observe that (para)professionals are aware of various values of which the importance was stressed in the NPM movement.21 y p y p g 22Lindeman’s (2017) observations on the FOO divisions are representative of the organisation of the processing of FOO in the timeframe prior to the ZSM procedure (roughly from 2000 until 2015). ww.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge 19Strafmaatoverleggen. 4.3.2 New professionalism in the extreme: the PPS 2000–201522 In the FOO divisions of the PSS, we observed an example of an extreme form of the ‘new professionals’ paradigm in which professionals do not so much work together to reach professional goals, but rather work on reaching these goals separately. y p y p g 22Lindeman’s (2017) observations on the FOO divisions are representative of the organisation of the processing of FOO in the timeframe prior to the ZSM procedure (roughly from 2000 until 2015). International Journal of Law in Context 383 During Lindeman’s fieldwork, assistants in the FOO division would work mostly independently from prosecutors and were formally mandated to independently decide cases. Prosecutors had no individual say as to which assistant processed which cases: management and policy regulations had taken over that part of the process and prosecutors often only came into play once a case ended up before the court. Prosecutors did not have the time or the means to actively interfere in those cases at an earlier stage. The decisions were made at great distance from the prosecutor. Therefore, the prosecutors did not claim authority over the cases: ‘Misdemeanors; I do have them [cases prepared by prosecution assistants] at my hearings, but they do not feel like my cases’ (A2 – resp. 7). At the same time, the prosecutors were not content with this division of duties because they regularly noticed mistakes and/or omissions in cases that would end up in court hearings that they had to prepare. Prosecutors commented on the undesirability of this situation, while at the same time emphasising that it was out of their hands. As a consequence of omissions in the files, they sometimes would ask for an acquittal or a stay of proceedings. One prosecutor (A2 – resp. 7) admitted to being aware that, due to a lack of scrutiny by the responsible assistants, people were paying fines for behaviour that was not necessarily liable for punishment. Still he admitted that he did not make the effort to raise awareness on this topic with the responsible assistants, nor with other prosecutors, nor the leadership of their district office. Another prosecutor (A2 – resp. 1) referred to similar issues as a ‘business risk’, while at the same time admitting that he believed it to be frustrating and that it could affect the credibility of the PPS. 4.3.2 New professionalism in the extreme: the PPS 2000–201522 Thus, the prosecutors did feel that they should have authority over the process, but perceived that – in reality – it was out of their control. They considered this to be primarily a problem for the institution rather than for them as professionals. It is in this regard that we observe how the organisational control strongly affects the division of duties between professionals and paraprofessionals, which leads to a decline in the sense of responsibility (discussed earlier in Section 4.2 above) that we did not observe as strongly in other divisions. In most situations described under this paradigm of ‘new professionals’, we see professionals acting as ‘hybrid professionals’ who combine pursuing professional standards with managerial requirements (see Noordegraaf, 2007). However, the situation at the FOO divisions appears to be one in which the professionals try to meet professional standards but the managerial set-up hinders them from meeting both requirements. This situation has somewhat changed since the introduction of the ZSM procedure (discussed in Section 3.3). One of the aims of this alteration was to increase the commitment of pro- secutors to FOO cases. In practice, however, a lot of cases are still processed by assistants, who have now been promoted to ‘assistant-prosecutors’. p p In the following section, we discuss some consequences of two co-existing, and in some respects conflicting, paradigms regarding the role of paraprofessionals. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 5.1.1 Different levels of delegation of authority in complex and routine cases To anyone with an understanding of the diversity of cases that are handled by the PPS and the judi- ciary, it will not be surprising that different types of cases demand a different paraprofessional con- tribution. Among other aspects, this depends on the type of legislation involved, the procedure for handling the case and the content of the case. In our studies, we found a difference especially between routine, or run-of-the-mill, cases and complex cases.23 In complex cases – dealing with complex legal issues that require a modified process for handling the case, adjusted to the specifics of the case – the collaboration of professionals and paraprofessionals is quite diverse and to a large extent dependent on the specific needs of the professionals. Professionals still have significant control over the work processes and the collaboration is hardly directly affected by NPM requirements such as productivity and efficiency. Different professionals think and act differ- ently regarding which, and how many, content-related tasks are allocated to paraprofessionals (see also Section 4). How much authority judges and prosecutors are prepared to relinquish also depends on the legal knowledge and quality they attribute to the assistants and the trust they have in them. The idea that substantial expertise and specialisation are more important for the quality of one’s work than one’s formal position (being professional or paraprofessional) is consistent with research conducted on the quality of legal practitioners and paralegals (see Moorhead et al., 2003). There are few organisational guidelines regarding the collaboration between professionals and para- professionals in complex cases. In several of such cases, we observed collaboration on an equal basis, with paraprofessionals actively participating in the discussion regarding the merits of a case. However, in other cases, we observed professionals strongly taking the lead and allowing only a small role to be played by paraprofessionals. This was particularly observed at the courts where complex cases are regularly handled by panels of three judges. In that setting, an additional contribution to the judicial discussion by a judicial assistant was, at times, not needed.24 In routine cases – dealing with simple legal issues that can be handled in a rather uniform manner – the procedure for handling the cases is more established and regulated by organisational standards. 5 Analysis of practices in the workplace This section analyses the daily practices that we observed. We demonstrate how the two paradigms that shape the thinking about professional–paraprofessional relations played out at the level of the work- place. When analysing the judiciary and the PPS, first of all, we observe one major collective finding, which is related to the (para)professionals’ juggle between the different paradigms (see similarly Schott et al., 2016). Namely, we observed great variation on a workplace level in the manner in which profes- sionals and paraprofessionals divide tasks and give substance to their collaboration. We thereby observe that the type of collaboration and the level of delegation of authority differ greatly, depending on the type of cases, being either routine cases or complex cases. This – sometimes inexplicable – vari- ation also results in an ambiguity in the relationship between professionals and paraprofessionals. Second, we find that organisational aspects do – sometimes greatly – influence the nature and degree of collaboration and delegation. This influence is particularly visible in the PPS and is more subtly observed in the judiciary. NL Holvast and JMW Lindeman 384 23This is a simplified dichotomy of reality, as different grades of complexity exist. 24This situation has not been observed much at the prosecution’s offices, as it is not very common that multiple prose- cutors deal with one case. See also Lindeman (2017). 5.1 Great variation and ambiguity in the professional–paraprofessional collaboratio On the one end of the spectrum, we observe occasions on which the professional hardly utilises the paraprofessional, other than for purely administrative and secretarial tasks. This occurs mainly in the judiciary. Some judges tend to check all the products of judicial assistants extensively, especially when they consider a judicial assistant to be less meticulous. Other judges simply make little use of the memos and drafts produced by assistants. On the other end of the spectrum, we observe occasions on which the professional utilises the para- professional to the extent that they leave a large part of the initial decision-making up to the parapro- fessional and the professional mainly functions as a controller at the end of the process. The most extreme example of this form of collaborating has been described above regarding the FOO division of the PPS, where prosecutors have given up part of their professional territory. This substantial variation is partly related to the variety in needs with regard to assistance in dif- ferent types of cases; each case requires different legal research to be conducted as well as different clerical assistance. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 23This is a simplified dichotomy of reality, as different grades of complexity exist. 24This situation has not been observed much at the prosecution’s offices, as it is not very common th cutors deal with one case. See also Lindeman (2017). ww.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ed from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge 5.1.2 Ambiguity on the relations between professionals and paraprofessionals Nonetheless, even in cases with very comparable characteristics, the type and amount of involvement of paraprofessionals vary greatly. We believe that this variance is related to the autonomy that the pro- fessionals still are given by their institutions in deciding how to collaborate with paraprofessionals.25 How the professionals give substance to their discretion appears to be dependent on the paradigms they adhere to. The unpredictability of the division of duties also results in a fair amount of ambiguity in the relationship between these officials. In the judiciary, where there is relatively little official policy that defines the role of paraprofessionals, it is unclear which informal norms should be leading when giving substance to the role of paraprofessionals in practice. In the PPS, we observed that prosecutors often were not aware of the specific requirements regarding education and/or experience for assistants: ‘This is not something that was handed out to me when I started working here. I know about it in general, but if you ask me in detail, I won’t know. It’s not an issue that we normally encounter’ (A2 – resp. 8). Furthermore, prosecutors were not always aware of the formal limits to the mandate that most assistants were working under. Although most (para)professionals have certain, sometimes strong, ideas about the appropriate type and amount of division of labour between professionals and parapro- fessionals, they are uncertain about whether and how to propagate these ideas. As a result, judges and prosecutors only rarely talk to paraprofessionals to share their expectations about their work or provide them with feedback.26 Judicial assistants are, for instance, unsure about what is expected of them during deliberation sessions. They are regularly invited to share their views on cases at the start of the discussions in the deliberations room, but are they also expected to get involved in the discussion that follows from this? When judicial assistants have written draft judgments, judges often make adjustments to the drafts and the assistants are asked to revise them. However, the reasons for making the adjustments are hardly ever explained to the assistants. When judges were asked why they rarely shared their views on the quality and usefulness of the work of judi- cial assistants, they explained that this was partly because they were uncertain about the work stan- dards that judicial assistants had to meet. 25Paraprofessionals’ views also play a role in this, but a proactive paraprofessional has to first be accommodated to act as such by the professional; see also Holvast (2017, pp. 161–162). 26The absence of a culture of sharing feedback in the judiciary was also repeatedly observed by a committee that conducted inspections of the courts. See Commissie visitatie gerechten, Rapport visitatie gerechten (2014), pp. 51–55; Rapport Visitatie Gerechten (2018), p. 20. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, availa 5.1.1 Different levels of delegation of authority in complex and routine cases Usually, these cases – which are the bulk of the cases at district courts and the PPS – do not require a lot of deliberations, as the outcome is clear-cut. A relatively large proportion of the paraprofessionals’ duties in these cases therefore consists of administrative duties. However, in this regard, we observe different official views from the institutions regarding the extent to which judicial or prosecutorial duties can be mandated to paraprofessionals. As described above, in the FOO division, paraprofes- sionals are mandated to make prosecution decisions and, more recently, to even function as a International Journal of Law in Context 385 prosecutor in court. While prosecutors are at times sceptical about this delegation of authority, they accept this as something that is out of their control. In the judiciary, where the sentiment that the judge ought to have the final say is much stronger, no such official delegation of authority is observed. Even in routine cases, the judge always is the one who officially decides the cases, even though these cases are at times completely prearranged by judicial assistants. This final responsibility is considered key to many judges. One judge notes: ‘He [the suspect] has the right, which is also captured in the constitution, to get the judge offered to him by law. … So he has a right to see me. Not a judicial assistant, but me!’ (A1 – resp. 38). In short, we observe a rather varied, and in that sense also comparable, situation in courts and the prosecution office regarding complex cases. Regarding routine cases, we observe that – especially the official – delegation of duties goes much further within the prosecution office. 5.2 Organisation and official policy determine the work processes We have already given various examples of situations in which the official policy of the institutions influences the organisation of the work in the workplace. In Lindeman’s study, it became clear that policy (and regulation) played an extensive role in the developments that resulted in the establishment of particular working processes. In fact, the role of policy and regulation was so extensive that prose- cutors felt that they no longer had individual autonomy over the processing of cases. They were not familiar with the assistants who were mandated to decide on the cases and there was no procedure in place to provide them with feedback. This led to a disconnection between the prosecutors and the paraprofessionals. Prosecutors no longer felt that the cases dealt with in the FOO division were ‘their’ cases, even when they ended up having to handle the case on trial. It seems that values trad- itionally associated with the professional (individual prosecutors) have been appropriated by the insti- tution and in turn have been attributed (within a strict framework of policy and regulations) to paraprofessionals. Thus, in this concrete situation, deprofessionalisation of the ‘full’ professional seems to take place. This is accompanied by an increasing professionalisation of the paraprofessional. The PPS has partly reversed these developments with the introduction of the ZSM procedure. However, the introduction of assistant-prosecutors reveals that prosecutors have undeniably lost ter- ritory. The introduction of this new function has met some criticism. It has been touted (by a judge) as being merely a trick with a change of labels, leading to an inflation of the position of prosecutors (Kristen et al., 2017). At the same time, prosecutors, who felt disconnected with these cases anyway, do not always mourn this ‘lost territory’. The previous developments have not been observed at the HIC divisions of the PPS and at the judi- ciary. The professional values and managerial requirements are in those (parts of the) institutions mainly communicating vessels. Still, it cannot be denied that organisational aspects and official policy have also been determinative for the manner in which the professional and paraprofessionals conduct their work. Due to the high workload that is assigned to prosecutors and judges (see Visser et al., 2019, pp. 45–47) and due to centrally introduced work processes introduced to increase efficiency, judges and prosecutors are not able to perform all their professional duties themselves and must rely heavily on assistants. 5.1.2 Ambiguity on the relations between professionals and paraprofessionals Judges were especially unsure about whether they could expect that judicial assistants should function as a discussion partner. Public prosecutors on the one hand appear more at ease about working with assistants. At the same time, they may feel that some assistants have been forced upon them: it is the organisation that decides the scope of the assistants’ mandate. However, even when prosecutors are largely supportive of employing assistants, they do ask themselves what a suitable division of labour would be and what conditions would be necessary to achieve this ideal situation. bridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 m https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core ter 386 NL Holvast and JMW Lindeman In both institutions, this results in a situation in which the conditions for fruitful professional– paraprofessional interaction remain largely unexpressed. Various paraprofessionals mention the uncertainty of this status quo. They are unsure whether they are expected to play an active role in the decision-making process, by challenging the arguments produced by the professionals, or whether they are expected to follow the instructions of professionals without asking critical questions. 5.2 Organisation and official policy determine the work processes This also results in situations in which the professionals are more or less forced to sur- render part of their professional territory. Part of the work that defines the magisterial character of judges and prosecutors (e.g. forming an unbiased opinion without the risk of any prejudice when deciding on a case) has been appropriated by their institutions and attributed to paraprofessionals. While there is room left for discussion about the optimal division of duties, the effects of managerial measurements have made it difficult for judges and prosecutors to arrange significant changes in order to reclaim territory in situations that they believe need this. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cam 6 Conclusion In this paper, we described how, in the past decades, the traditionally professional-oriented organisa- tion of the Dutch judiciary and the PPS changed, under influences such as the NPM movement. In our analysis of official documents, we find that these changes also altered the position of the judges, pro- secutors and their assistants. Similarly to many other public-sector institutions as well as to legal prac- tice, the employment of assistants has increased significantly in the judiciary and the PPS. There also has been a considerable amount of professionalisation among these assistants, to the point at which they can be referred to as paraprofessionals. Our empirical research reveals that these paraprofessionals International Journal of Law in Context 387 play a significant role in the day-to-day work at the judiciary and PPS. Especially at the PPS, parapro- fessionals have been given relatively large autonomy, by way of a mandate construction. However, individual judges, prosecutors and paraprofessionals have different attitudes towards the appropriate division of duties. In these attitudes, we recognise attributes from the traditionally leading ‘pure pro- fessionalism’ (which emphasises the superior status and autonomy of the professional) as well as the ‘new professionalism’ (which emphasises the collaborative aspect of the professional and paraprofes- sional in reaching professional goals). We have defined these as two competing paradigms. In practice, we observe that these paradigms are visible in different forms and variations in both institutions. The views and practices of professionals in the judiciary fit more dominantly within the first paradigm and the views and practices within the PPS (particularly the divisions dealing with FOO) fit more dom- inantly within the second paradigm. It has become clear that, while professionals have continued to occupy an important position within their institutions, the managerialism of the organisational structures of both institutions has had a considerable impact on the division of duties between professionals and paraprofessionals. The new professional perspective, however, has not replaced the traditional perspective on the alloca- tion of duties, but has set a new, more hybrid perspective in action. The co-existence of these two para- digms and the fact that both institutions have little regulation that concretely defines the territory of the (para)professionals result in a wide variation in collaboration between professionals and parapro- fessionals in practice as well as in ambiguity regarding the position of paraprofessionals. 6 Conclusion Such a diver- sified landscape in daily practices in the workplace is not unique for our research setting. It can be distinguished in other professional settings as well (see e.g. Maynard-Moody and Musheno, 2003). In fact, many (legal) professionals are likely to struggle to define their relationship to paraprofes- sionals. Nonetheless, empirical evidence of such a struggle within the public justice system has been scarce up until now. We believe that some discretion in defining the territory of professionals and the collaboration between professionals and paraprofessionals is essential, as different situations may require different professional and paraprofessional attention. At the same time, not discussing the ideals behind differ- ent practices can result in incomprehensible or arbitrary differences in the allocation of duties in the workplace. It can even result in a vacuum in which no one feels responsible anymore. A possible answer to the, sometimes inexplicable, variation in work practices and the uncertainty about the appropriate division of labour among (para)professionals might be the creation of professional stan- dards for the employment of paraprofessionals. In order to have an actual effect on the daily work practices, we believe that it is key that not just managers, but also professionals and paraprofessionals, are involved in the creation of these standards. Additionally, if significant duties in the adjudication process are delegated to paraprofessionals, this should also be reflected in the safeguards relating to their function and in the financial compensation. In recent publications, Noordegraaf (2015) argued that the perspective of professionalism ‘beyond hybridity’ may counter these issues. Professionals will have to take up responsibilities regarding the organisation of their work, so that they are not only treating cases based on parameters set out by managers (Noordegraaf, 2015; Noordegraaf and Siderius, 2016). ‘Organising professionalism’ means that professional workers also share a responsibility for organising the structures around their work, such as acquiring and maintaining expertise, co-operating with others (e.g. in teams), prioritis- ing and selecting the work (bearing in mind strategic and budgetary considerations), detecting and preventing risks and failures, and accounting for their actions. We believe that this might work not only for the relationship of professionals and managers, but also for professionals and paraprofessionals. Conflicts of Interest. None Conflicts of Interest. None Acknowledgements. 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Programa de sensibilización legal para reducir la violencia de género familiar en la ciudad de Guayaquil 2020
Ciencia latina
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Programa de sensibilización legal para reducir la violencia de género familiar en la ciudad de Guayaquil 2020 Laura Estela Chacón Chacón ab.laurachacon@hotmail.com Escuela de posgrado: Programa Académico Doctorado en Gestión Pública y Gobernabilidad Universidad Cesar Vallejo Piura - Perú RESUMEN La presente investigación abordó el tema de la violencia de género familiar que va aumentando con el pasar de los días, inclusive, en medio de esta situación pandémica que nos toca vivir, violencia que, al interior del grupo familiar, la mujer es mayormente la afectada, este trabajo tuvo como objetivo proponer un Programa de Sensibilización Legal para Reducir La violencia de género familiar en la ciudad de Guayaquil. La metodología tuvo un propósito como Aplicado, enfoque mixto y un alcance Descriptivo y Explicativo, donde el diseño fue no experimental de corte transversal, se obtuvo una muestra de 282 personas, conformada por las familias de Guayaquil en el año 2020. Los resultados mostraron que el programa de sensibilización social tiene amparo legal en función a sus normativas, mientras que la atención que se viene brindando como soporte asistencial a la comunidad es casi nulo, por lo que consideran importante la aplicación de talleres prácticos. Concluyendo que un Programa de Sensibilización Legal reduciría la Violencia de Género Familiar en la ciudad de Guayaquil, y que, de acuerdo con la opinión de especialistas sobre esta propuesta, arrojaron un Coeficiente de validez de contenido es 0.941 por lo que se recomienda la pertinencia de implementar y aplicar el protocolo. Palabras Clave: programa; sensibilización legal; violencia de género; familias; mujeres afectadas. Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7472 Chacón Chacón Legal awareness program to reduce family gender violence in the city of Guayaquil 2020 ABSTRACT This research addressed the issue of family gender violence that increases with the passing of days, including, in the midst of this pandemic situation that we have to live, violence that, within the family group, women are mostly affected The objective of this work was to propose a Legal Awareness Program to Reduce Family Gender Violence in the city of Guayaquil. The methodology had a purpose such as Applied, mixed approach and a Descriptive and Explanatory scope, where the design was non-experimental, crosssectional, a sample of 282 people was obtained, made up of families from Guayaquil in 2020. The results showed that The social awareness program has legal protection based on its regulations, while the care that has been provided as assistance to the community is almost nil, which is why they consider it important to apply practical workshops. Concluding that a Legal Awareness Program would reduce Family Gender Violence in the city of Guayaquil, and that, according to the opinion of specialists on this proposal, they yielded a content validity coefficient is 0.941, so the relevance of implement and apply the protocol. KeyWords: program; legal awareness; gender violence; families; affected women Artículo recibido: 10 Agosto. 2021 Aceptado para publicación: 07. Setiembre. 2021 Correspondencia: ab.laurachacon@hotmail.com Conflictos de Interés: Ninguna que declarar Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7473 Programa de Sensibilización I. INTRODUCCIÓN Según Arisis y Cortés (20202), manifestaron que los latinoamericanos tienen en frente actualmente dos emergencias: en el exterior de su domicilio el coronavirus y en su interior sus agresores. El enclaustro y la necesidad de acoplarse a rutinas diarias a entornos digitales, la pérdida de trabajos, el alternar dentro del mismo hogar con las obligaciones laborales y las actividades domésticas, ente otros elementos, han incrementado el riesgo de violencia dentro de la familia. Los grupos más perjudicados son los adolescentes y menores, las mujeres, las personas adultas y con discapacidad. Es así que, en la región andina, algunos datos oficiales muestran un incremento de actos violentos dentro del hogar durante el aislamiento. Actualmente la violencia dentro de la familia es un problema que está afectando a la mayoría de los territorios desde tiempo atrás y en la actual situación de la pandemia pone a prueba la capacidad de réplica de los gobiernos. Podemos observar que estas ideas prácticas nos pueden ayudar en el cambio: 1.Con la colaboración público-privada. Podemos enfrentar la violencia doméstica requiriendo acciones colectivas. 2. Utilizando tecnología adecuada al nuevo contexto, ya que es sin duda un mecanismo esencial para prevenir, atender y mitigar la violencia doméstica. Sin embargo, también puede ser usada por los agresores. Por eso, debe fortalecerse el trabajo entre autoridades y redes de vecinos, habilitar contenidos y herramientas para facilitar el acceso a la oferta y mecanismos de denuncia pública para personas con discapacidad visual y/o auditiva, campañas para menores y adolescentes usando redes sociales, e incorporar nuevas tecnologías o reconocimiento facial.(párr1-2) Basados en la publicación de Duque (2017), en donde atribuye que está un escenario que Ecuador lamenta la presencia de actos violentos en el seno de la familia. Según las tasas emitidas por el Ministerio del Interior, se recibieron 24376 denuncias por violencia producida en el seno familiar hasta junio de 2017, siendo 2923 de hombres y 21453 fueron de mujeres los afectados; lo que significa que el 88% de mujeres reciben maltrato en sus domicilios caudado por los esposos. Este problema fue afrontado bajo la temática de: “Violencia familiar: prevención e intervención”, por lo que, al conversar sobre violencia dentro del hogar, nace el término “la casa del abuso”, que se refiere al control y poder de las víctimas, referenciándose que en la mayoría de casos se trata de violencia conyugal. (párr. 1-4). Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7474 Chacón Chacón Dada la situación actual Cortés (2020), nos brinda herramientas para poder contrarrestar la violencia familiar se debe implantar reglas claras con la familia para tener una buena convivencia. Es básico establecer reglas que contengan deberes y derechos a quienes viven en un mismo hogar, ya que éstas ayudan a definir límites y formas de comportarse, las mismas que tienes que ser de fácil entendimiento y aceptadas por todos los que viven dentro del hogar. Además, es Vigorizar los conductos de comunicación al interno de la familia. Se debe escuchar de manera activa, no enjuiciar los comportamientos, no criticar, colocarse en posición del otro, demuéstrales respecto e interés por los sentimientos del resto de los miembros de la familia, con la finalidad de generar un ambiente de confianza para mantener la comunicación en el hogar. Recordando que lo que se dice, debe ser coherente con lo que se hace. La plática y la conciliación deberían ser formas alternativas a usarse para dar fin a los conflictos que aparezcan. Definir el costo emocional que originan los conflictos es tener una ventaja para resolverlos. Por medio de los acuerdos, el ser neutral y el ser reservado es la forma de mediación en el hogar que ayuda a originar ambientes de cooperación y negociación, buscando que una persona ajena intervenga en resolver la situación para que no se incremente un conflicto. Se debe divulgar el afecto que se tiene para con todos los miembros del hogar. Igual apartar el orgullo y la timidez permite que se expresen de manera más fácil los sentimientos, se sonría, se romper el hielo, realizando actividades de manera conjunta siendo amables y ofreciendo disculpas, para señalar lo importante que es cada miembro que compone una familia. (párr. 1-7) Es de apreciarse que esta situación que evidenciamos nos genera mucha preocupación y a la vez una serie de preguntas y que en algunos casos no encontramos respuestas, en ese sentido es que se plantea la siguiente pregunta de investigación: ¿Cómo reducir la violencia de genero familiar en la ciudad de Guayaquil. 2020?, este problema nos permite tomar conciencia de aquello que está aquejando a las familias de Guayaquil, por lo que creo que la investigación se justifica, partiendo del hecho que a través de los años esta situación se ha hecho muy recurrente, a pesar que se vienen instruyendo a la población a defenderse de muchas formas o maneras, no incitando a la violencia, sino por el contrario a tener la calma y madurez necesaria que permita que las familias de Guayaquil puedan vivir en armonía, superando las diferencias que muchas veces son el remedio para esta situación por la cual mayormente se agrede a la mujer por su condición de tal, a quien se le pretende atribuir derechos limitativos, cuando realmente no lo son, la constitución del Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7475 Programa de Sensibilización Ecuador defiende tanto al hombre como a la mujer en sus derechos básicos, pero debido a los excesos de maltrato a la mujer por justificaciones de contextos machistas, el estado ha generado las normativas legales para protegerlas de aquellos individuos que no respetan el estado de derecho de las personas. En el campo del conocimiento, esta investigación se justifica en lo teórico, dado que el conocer a fondo la problemática existente , se puede entender las causas, orígenes, tipos y características típicas en que manifiestan, desde la vertiente del conocimiento se desea profundizar en el conocimiento de esta realidad y poder sentar las bases para la realización de mayores trabajos de investigación que aborden la temática y sus causas, con la finalidad de poder plantear alternativas de solución desde el campo del conocimiento. Justifica en lo práctico, pues debido a que es una situación que amerita atención oportuna, es que el conocimiento contribuye a plantear soluciones con el debido sustento y que fortalece o respalda cada conocimiento aplicado en pos de una solución viable y pertinente, las diversas alternativas son diseñadas, elaboradas, analizadas en su magnitud de solución y finalmente son evaluadas para medir el impacto en la solución de problemas que afectan a las familias y en especial a la mujer guayaquileña. Y en lo social, esta investigación generará nuevas condiciones de vida para aquellas mujeres afectadas por algún tipo de violencia de género, con lo cual se le estaría protegiendo, tanto a ella como al grupo familiar que no es ajena a cualquier tipo de violencia que se genera al interior del hogar. Lo mencionado anteriormente nos permite plantear como Objetivo General  Proponer un Programa de Sensibilización Legal para Reducir La violencia de género familiar en la ciudad de Guayaquil. 2020. Teniendo además como Objetivos Específicos 1. Diagnosticar el estado actual de La violencia de género familiar en la ciudad de Guayaquil. 2020 2. Identificar los factores influyentes en La violencia de género familiar en la ciudad de Guayaquil. 2020, 3. Diseñar Programa de Sensibilización Legal para Reducir La violencia de género familiar la ciudad de Guayaquil. 2020 y Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7476 Chacón Chacón 4. Estimar los resultados que generará la implementación de Programa de Sensibilización Legal en La violencia de género familiar en la ciudad de Guayaquil. 2020. El análisis de lo descrito nos lleva a proponer la siguiente Hipótesis  La misma que versa de la siguiente forma: La implementación de un Programa de Sensibilización Legal reduciría la violencia de género familiar en la ciudad de Guayaquil. 2020. II. METODOLOGÍA 2.1. Tipo y diseño de investigación. Según el propósito es aplicada, dado que permite incrementar y ahondar sobre el conocimiento de nuestro entorno, de forma tal que este saber que se intenta erigir sea uno científico, pues el fin es alcanzar generalidades cada vez mayores. (Arboleda, 2013), según el enfoque es Mixta, porque representan un conglomerado de procedimientos ordenados prácticos y críticos de investigación, conllevando a recolectar y analizar datos cuantitativos y cualitativos, permitiendo hacer inferencias de toda la información lograda, asimismo según el alcance es Descriptiva, porque buscan detallar rasgos sobre alguna particularidad, característica, así como el perfil de personas conformantes de un determinado grupo y que todo ello sea plausible de análisis. Su fin es poder realizar mediciones o recogida de información en forma independiente sobre aquello que es el interés en la investigación. (García, 2011) y Explicativa, porque busca conocer cuáles son aquellas causan que generan o propician tal efecto. así mismo en cuanto al Diseño de investigación fue no experimental, ha sido elegida debido que tiene los caracteres de no acudir a la manipulación de las variables con alguna intención, se observa la anomalía tal cual ocurre, el investigador recurre a simplemente observar, la investigación con la que se gestionó este diseño fue de corte transversal, para la obtención de información en un momento especifico, así lograr una interpretación de cuanto influye y así lograr relacionar a grupos o subgrupos, (Hernández-Sampieri, Fernández y Baptista, 2010, p. 120). 2.2. Variables y operacionalización. Variable Independiente: Programa Legal de Sensibilización Social El programa de sensibilización es en parte el comienzo del proceso de transformación, por esto se requieren otras actividades e iniciativas para darle seguimiento y asesoría a las personas Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7477 Programa de Sensibilización que pasan por distintos niveles en el proceso de aceptación de la transformación y que garanticen su eficacia en su implementación. (Scott, 2008, p. 90) Variable Dependiente: La Violencia de Genero Familiar La violencia hacia la mujer se da por la condición de sexo, el abusador presupone tener derecho de maltratar a la víctima, estos sucesos afectan de forma significativa la salud mental y física, bienestar social, sexualidad y libertad reproductiva de una persona. (Raffino, 2020). 2.3. Población, muestra y muestreo. Población. Ventura-León (2017) define a la población como el conjunto de unidades relacionados por cierto tipo de características que son de interés para el estudio. En tal sentido, respecto a la población frente a la muestra, vemos la existencia de una relación inductiva, con la cual se espera que la porción sometida a la observación tenga el componente representativo de la realidad y con ello asegurar las conclusiones. En el análisis, el enfoque de estudio se ha determinado como población a las familias de Guayaquil en el año 2020, pues existen diversas formas de violencia de género, por ello se ha incluido a todas las familias en mención. Muestra Muestra es cualquier porción o subconjunto de una población que, sometida al análisis de la ciencia estadística, pueden ser determinadas a través del muestreo probabilístico o no probabilístico. En cuanto al Muestreo esta es una técnica que nos ayudó a calcular el número de familias componentes de la muestra de estudio. En ese sentido el estudio al enfoque de análisis, se le aplico un muestreo probabilístico, se logró articular la muestra con la que elaboro el estudio. La fórmula establecida para este procedimiento tanto para obtener la muestra de docentes como de estudiantes fue la siguiente. Calculo para la muestra. 𝑍2𝑃𝑄𝑁 𝑛 = 𝐸2(𝑁 − 1) + 𝑍2𝑃𝑄 Donde: Z = 1.96 Valor al 95% de confianza PQ = 0.5 * 0.5 = 0.25 Proporción máxima que puede afectar a la muestra Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7478 Chacón Chacón E = 5.83 % = 0.0583 Error máximo permisible N = 958 965 (datos obtenidos del INEC) n = 282 2.4. Técnicas e Instrumentos de recolección de datos. De acuerdo con la RAE (2021) precisa que la encuesta es un conglomerado de preguntas elaboradas minuciosamente con la finalidad de dirigirlas para su aplicación a una muestra que represente a un determinado grupo social, y con ello indagar que opinan o conocen sobre puntos que los afectan. En relación a los instrumentos estos han sido un medio para sobre guardar los datos obtenidos de la información, (Redacción, 2021) enfatiza que el cuestionario es una forma organizada y práctica a través de la cual podemos realizar preguntas y obtener respuestas. Adicionalmente a ello se debe validar y confiabilidad el instrumento, a través de os programas específicos para dicha tarea. 2.5. Procedimientos. Toda investigación sigue un proceso ordenado y en lo concerniente al recojo de datos estos deben de realizarse teniendo en cuenta las recomendaciones sobre ciertas particularidades que pueda presentar el instrumento, luego de completado este trabajo, se tabulan los resultados para que a través de los diferentes programas o softwares se procesen y nos permitieron contar con resultados que se analizaron y nos llevaron a obtener argumentos válidos en nuestra investigación. La encuesta se aplicó mediante aplicaciones digitales, los cuestionarios de Google drive. Que nos brindó la información requerida sobre las variables estudiadas. 2.6. Método de análisis de datos. Obtenidos los datos a través de las aplicaciones mencionadas, se continuo a la revisión exhaustiva de las respuestas obtenidas, procesadas mediante el programa estadístico SPSS, así como del Excel. 2.7. Aspectos éticos. Esquema de la ética y sus principios tiene el principal objetivo de velar por la dignidad e integridad de cada individuo que participa en el proceso de investigación es por lo que durante todo el desarrollo de este trabajo se tiene en cuenta lo siguiente: Para empezar las personas que participan de esta investigación los hacen desde su libre voluntad y libertad para ellos entre un consentimiento informado en donde expresa de manera clara y abierta que está brindando su autorización y participar dentro del proceso Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7479 Programa de Sensibilización de evaluación (Parveen y Showkat, 2017, pp. 4-6). Otro aspecto a tener en cuenta es el de la confidencialidad, no se hace una divulgación de los datos obtenidos del evaluado, por eso es que en las encuestas se omite el tema de nombres y apellidos. Finalmente se pone de manifiesto de manera sencilla clara por qué se está haciendo la evaluación evitando así el engaño al evaluador cómo plantear consignas falsas (Weinbaum et al., 2019, pp. 5-7). IV. RESULTADOS Y DISCUSIÓN. Tabla 1 El programa de sensibilización social tiene amparo legal en función a sus normativas Descripción fi % D 98 34.75 NO 55 19.50 A 77 27.30 TA 52 18.44 Total 282 99.99 Fuente: El Autor Con respecto a los resultados de la tabla 1, sobre si El programa de sensibilización social tiene amparo legal en función a sus normativas, al respecto un 34.75 % solo refieren estar en Desacuerdo con el enunciado, asimismo un 19.5 % optaron No Opinar, mientras que un 27.3 % ante dicha afirmación refieren estar de Acuerdo y finalmente el 18.44 % están Totalmente de Acuerdo con lo manifestado. Esto se compara con lo manifestado por Rivera (2021) en su investigación hallaron que, por un lado, las normas fijadas para la ejecución del programa cumplen con los requerimientos que sirva para que se genere valor público, por otro lado, se muestra que existen intervalos de control político y operativo que son plausibles de mejorarse en la gestión pública gubernamental. (p. 75), también González (2017). menciona que este aporte investigativo tiene como fin planes de educativo-legal, la observación de los resultados emitidos permite darnos cuenta que el foco de estudio no es consciente de los mecanismos del gobierno que permite garantizar los derechos de los seres humanos. No obstante, se debe seguir trabajando en fomentar el estudio de las leyes para que los ciudadanos seamos garantes y conocedores de nuestros derechos, así las instituciones encargadas en velar la efectiva aplicación de las mismas se vean fortalecidas. (p. 16). Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7480 Chacón Chacón Tabla 2 El programa de sensibilización social viene brindando soporte asistencial a la comunidad Descripción fi % TD 95 33.69 D 90 31.91 NO 41 14.54 A 56 19.86 Total 282 100.00 Fuente: el Autor Con respecto a los resultados de la Tabla 2, sobre si , , al respecto un 33.69 % manifiestan estar Totalmente en Desacuerdo y un 31.91 % solo refieren estar en Desacuerdo con el enunciado, asimismo un 14.54 % optaron No Opinar, mientras que un 19.86 % ante dicha afirmación refieren estar de Acuerdo. Esto se compara con lo manifestado por CastilloSepúlveda, Winkler y Berroeta (2019) trabajo donde se propone que se cambie de punto de vista de acción frente a la comunidad, empleando como perspectiva analítica lo establecido en la teoría del actor-red. Partiendo de la representación de las relaciones entre prácticas y organizaciones técnicas mostradas por los profesionales, diseñadores, y usuarios de tres programas sociales en la nación chilena, mostrando cómo estos se pueden entender como ensamblajes heterogéneos compuestos por las nuevas capacidades de los actores involucrados. En tal ruta, en lugar de ser un proceso totalmente social, se muestra el empoderamiento como una agencia que emerge en procesos de mediación técnica y social, dicho proceso se ha formulado en base a relaciones entre humanos y no humanos podría ser entendido como prehensión. (p. 1), asimismo, Oddone y Chernobilsky (2019) analizan a las organizaciones sociales que tienen a personas mayores que viven en Buenos Aires en relación a los programas sociales que se destinan a este grupo de edad. Los resultados obtenidos parcialmente de un estudio mayor que se planteó como objetivo recabar información sobre la imagen, el uso, el rol de las redes sociales y las formas de supervivencia de las personas adulto mayor en correlación con los ya mencionados programas sociales. (p. 122) Tabla 3 Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7481 Programa de Sensibilización Los talleres prácticos vienen siendo un soporte clave dentro de los programas de sensibilización Descripción fi % D 26 9.22 NO 19 6.74 A 127 45.04 TA 110 39.01 Total 282 100.01 Fuente: el Autor Con respecto a los resultados de la tabla 3, sobre si Los talleres prácticos vienen siendo un soporte clave dentro de los programas de sensibilización, al respecto un 9.22 % solo refieren estar en Desacuerdo con el enunciado, asimismo un 6.74 % optaron No Opinar, mientras que un 45.04 % ante dicha afirmación refieren estar de Acuerdo y finalmente el 39.01 % están Totalmente de Acuerdo con lo manifestado. Esto se compara con lo manifestado por Hengen (2020) quien argumentó que, si enseñamos justicia social en el marco dictado por los compromisos tradicionales de educación superior, lo más seguro es que podamos equivocarnos. Pero podemos cumplir con la promesa de justicia social si nuestros cursos y programas están (1) centrados en un proyecto (2) involucra la acción colectiva y (3) explícitamente no sea neutral. A medida que nuestros estudiantes aprenden a través de talleres y capacitaciones pueden identificar la injusticia, hablar de ella con otros y promulgar estrategias para el cambio, están logrando los resultados de aprendizaje del curso mientras mejoran la vida de muchos. (p. 67), también Blanco et al. (2019). Refieren que Uruguay está implementando (UT) el cual es un plan colectivo-laboral donde puedan ser partícipes las personas de tercera edad que no tienen trabajo y que forman parte de familias disfuncionales. Tomando como base las percepciones de hábitos de Bourdieu y de individualización de las coberturas de Castel, lo complejo de la vida diaria; la no existencia de vínculos entre participantes y dentistas y la casi inexistencia de tiempo de seguimiento, para sostener el transcurso de socialización son puntos que deben ser considerados para comprender lo abandonado que se tiene en este derecho y que es transitorio para otorgar la asistencia (p. 1), asimismo, Bermúdez y Arcidiácono (2018) puntualizan que hay programas que logran transformar y articular las diferentes instancias de gobierno, retomando las exigencias de las prestaciones por medio de cooperativas Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7482 Chacón Chacón creadas por el gobierno y usa las capacitaciones para que se fortalezca el capital humano de las entidades. Se establecerá el problema alrededor de la diferencia entre elaborar políticas “para mujeres” y con una mirada transversal a partir del género. (p. 1) Tabla 4 Las lesiones físicas como parte de un maltrato están incrementando la violencia de genero familiar Descripción fi % D 41 14.54 NO 92 32.62 A 39 13.83 TA 110 39.01 Total 282 100.00 Fuente: el Autor Con respecto a los resultados de la tabla 4, sobre si Las lesiones físicas como parte de un maltrato están incrementando la violencia de genero familiar, al respecto un 14.54 % solo refieren estar en Desacuerdo con el enunciado, asimismo un 32.62 % optaron No Opinar, mientras que un 13.83 % ante dicha afirmación refieren estar de Acuerdo y finalmente el 39.01 % están Totalmente de Acuerdo con lo manifestado. Esto se compara con lo manifestado por Chanamé et al. (2017) donde evidencia que el 62 % de las personas que fueron encuestadas alcanzó un bajo nivel de conocimiento en violencia física por negligencia; el 34 % en violencia psicológica y el 52 % en violencia sexual. Desde otro punto de vista, se consideró el puntaje total sobre la noción de violencia, obteniendo el 45 % un nivel bajo. Como conclusión se tiene que los cuidadores o los padres tienen una noción deficiente de los conceptos de violencia y sus tipologías. (p. 30), mientras que Pérez-Rey et al. (2019) refieren que se hallaron ideologías sobre la violencia de género hacen que se influencie para que se continúen incrementando los casos, concluyendo que los hombres tienen una mayor creencia en la violencia de género que las mujeres, no dimensionando el origen de la violencia de género, ni el impacto perjudicial que causa a nivel personal, social, laboral, económico, entre otros, ya que en ciertos momentos se puede tomar de forma normal la interior de las relaciones de pareja. (p. 547), también Rivera et al. (2020). se hallaron elevados porcentajes de violencia en la familia y de acciones agresivas en el contexto educativo, así como escasos índices educativos acerca Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. ISN 2707-2207 / ISSN 2707-2215 (en línea), setiembre-octubre, 2021, Volumen 5, Número 5. https://doi.org/10.37811/cl_rcm.v5i5.859 p.7483 Programa de Sensibilización la temática que tiene relación con la violencia hacia la mujer. En el caso de la violencia física se ejerció con mayor frecuencia en los adolescentes y niños varones, en el rango de 15 a 19 años, y en los municipios de El Carmen de Atrato y Bahía Solano. En cambio, las violaciones sexuales fueron practicada a las niñas, de 9 hasta 11 años en los municipios de Pivijay y de Uribía. En definitiva: las leyes enfocadas al género y las perspectivas colectivas imputadas a los individuos según su sexo presentan a niños, niñas y adolescentes a diferente tipo de maltratos que son diferenciadas; sumándose a ellos su ubicación geográfica y su edad. (p. 2) IV. CONCLUSIONES 1) La presente investigación concluye con una propuesta sobre la implementación de un Programa de Sensibilización Legal para reducir la Violencia de Género Familiar en la ciudad de Guayaquil. 2020, programa requerido para enfrentar la creciente tasa de violencia de género que se viene percibiendo en la sociedad guayaquileña. 2) Se lograron identificar elementos importantes que se vienen manifestando en el diario vivir de las víctimas de violencia de género, manifestaciones crecientes como lesiones, miedos, temores, gastos en tratamientos, traumas entre otros pero con un denominador en común: la mujer, la misma que está viviendo tiempos muy complicados. 3) Se analizaron factores que resaltaron y se corroboraron como el maltrato físico el mismo que se traduce propiciar a las víctimas no solo lesiones físicas, sino también incapacidad temporal y en algunos casos incapacidad permanente; el maltrato psicológico el cual trae consigo, traumas que sin el tratamiento adecuado es perjudicial para la víctima de la violencia, vidas inseguras y el perjuicio económico, traducido en gastos para tratamientos, para acciones legales y la carencia de ingresos. 4) Se elaboró un programa acorde a la situación a abordar, la misma que tendrá un sustento legal con normas y regulaciones pertinentes, una estructura asistencial que genere un acompañamiento procesal como un soporte comunitario y un eje educativo, por el cual se capacite desde las diferentes necesidades identificadas. 5) Los resultados del juicio de expertos, demuestran la importancia y necesidad de aplicar este programa, el mismo que se revisó y evaluó por especialistas, profesionales del ámbito de la gestión pública, análisis que arrojaron un Coeficiente Ciencia Latina Revista Científica Multidisciplinar, Ciudad de México, México. 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A 3D SEDIMENT TRANSPORT MODEL FOR COMBINED WAVE-CURRENT FLOWS
Proceedings of Conference on Coastal Engineering/Proceedings of ... Conference on Coastal Engineering
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138602 2 Parsons Laboratory, Massachusetts Institute of Technology, Room 48-216C, 15 Vassar Street, Cambridge, MA, 02139, USA A 3D SEDIMENT TRANSPORT MODEL FOR COMBINED WAVE-CURRENT FLOWS Peifeng Ma1 and Ole Secher Madsen2 Peifeng Ma1 and Ole Secher Madsen2 Accurate prediction of current velocity and bottom shear stress, which both can be significantly influenced by wind waves, is essential for sediment transport predictions in the coastal environment. Consequently wind-wave effects must be taken into account in a numerical sediment transport model for application in coastal waters. In the present study, elements of a large-scale 3D numerical coastal circulation and sediment transport model are developed to predict net, i.e. the wave-period-averaged, sediment transport rates. The sediment transport components considered are (i) bed-load transport; (ii) mean suspended load sediment transport within the wave boundary layer, which is obtained from an analytical solution; and (iii) suspended load sediment transport above the wave bottom boundary layer, which is obtained from a numerical model. In all model components wind wave effects are accounted for through simple analytical models. Thus, the roughness prescribed for the hydrodynamic part of the numerical coastal circulation model is the apparent roughness, i.e. the roughness experienced by a slowly varying current in the presence of waves. Similarly, the reference concentration specified for the sediment transport part of the numerical model is obtained from analytical solutions for suspended sediment concentrations within the combined wave-current bottom boundary layer. Stratification effects caused by suspended sediment are included in the large-scale numerical sediment transport model. Results of idealized tests suggest that wind wave effects can be pronounced, e.g. in some typical coastal scenarios sediment can only be mobilized when wind waves are present and accounted for. It is also shown that stratification can significantly affect suspended sediment transport rates of fine sediments. Keywords: sediment transport; bottom boundary layer; wave-current interaction; sediment-induced stratification 1 Singapore-MIT Alliance for Research and Technology, 1 CREATE way, #09-03 CREATE Tower, Singapore, 138602 1. INTRODUCTION As illustrated in Figure 1, a typical coastal environment consists of wind waves, slowly varying currents, such as tide- and wind-driven flows, and sediment in the bottom and water column. Sediment can be moved as bed-load transport on the seabed or stirred up into the water column by bed shear stress and then transported by a current as suspended load transport. It is well-known that wind waves can intensify the near bottom turbulence significantly due to the limited thickness of the wave boundary layer. As a result, the bottom shear stress and turbulent mixing in the water column can be markedly enhanced by the presence of wind waves resulting in more sediment being mobilized and moved to upper layers of the water column where it is then made available for transport by currents. Consequently, wind wave effects must be included in numerical coastal circulation and sediment transport models. Since coastal circulation models cannot resolve the short time-scales governing the wave bottom boundary layer, the hydrodynamic as well as sediment transport processes within the combined wave-current bottom boundary layer must be calculated separately to produce physically realistic, slowly varying boundary conditions that are passed on to the numerical coastal circulation and sediment transport model. Figure 1 Illustration of a typical coastal environment WAVES CURRENTS SHEAR STRESS SUSPENSION Figure 1 Illustration of a typical coastal environment Many numerical sediment transport studies have been conducted in the past few decades (e.g. Li and Amos, 2001; Lesser et al., 2004; Warner et al., 2008). Wind wave effects have been taken into 1 COASTAL ENGINEERING 2012 2 account in some models, e.g. some wave-associated coefficients are introduced by Lesser et al. (2004) to account for wave effects on sediment transport. Wave-current interaction models (e.g. Madsen, 1994) are introduced by Li and Amos (2001) and Warner et al. (2008) to predict combined wave-current shear stresses for sediment transport predictions. However, most numerical sediment transport models treat the wave boundary layer as a portion of numerical domain, regardless of the much different physics within the layer which usually cannot be resolved by coastal circulation models. Density changes caused by suspended sediment have been included in some sediment transport models (e.g. Lesser et al., 2004; Warner et al., 2008), but hardly any information has been reported on the importance of this sediment stratification effect. 1. INTRODUCTION The objective of the present study is to develop a three dimensional numerical sediment transport model for combined wave-current flows. A bottom boundary layer model is introduced to account for wind wave effects on current flow and sediment transport. The mean suspended sediment transport within the boundary layer is calculated separately and analytically. The sediment stratification effect is studied. The paper is organized as follows. A model description, including flow model, bottom boundary layer model and sediment transport model, is presented in Section 2. In Section 3, numerical experiments are conducted to test the model. Finally conclusions are provided in Section 4. 2. MODEL DESCRIPTION In coastal waters, sediment is usually stirred up by the bed shear stress and then transported by a current. Bed shear stress determines the amount of sediment being mobilized and flow velocity determines how far the sediment can be transported. In the present model, a bottom boundary layer model is introduced to predict bed shear stress in combined wave-current flows, including the total and sediment transport shear stresses. A numerical coastal circulation model is used to compute eddy viscosity and velocity profiles for the slowly varying currents above the bottom boundary layer, for which wave effects are incorporated through the specification of an enhanced apparent bottom roughness, i.e. the roughness experienced by a current in the presence of wind waves. The bed-load transport rate and the reference concentration for suspended sediment concentration are computed from the so-called skin friction or sediment transport shear stress. The mean suspended load transport within the bottom boundary layer is solved analytically. The suspended load sediment transport rate above the bottom boundary layer is computed by a numerical sediment transport model that solves the unsteady, i.e. slowly varying, advection-diffusion equation to obtain the suspended sediment concentration corresponding to a specified reference concentration predicted by the analytical bottom boundary layer model that includes the effects of wind waves. The present model is formulated in a terrain following coordinate system (x, y, σ) with x and y indicating the two directions in the horizontal plane and σ representing the scaled vertical direction. The velocities in the three directions are denoted by (u, v, w). The still water depth is h and surface elevation is denoted by η. The water density and molecular viscosity are taken to be ρ = 1,025kg/m3 and ν = 1.3x10-6m2/s, respectively. To be realistic, we assume the presence of random wind waves which have a root mean square (rms) wave height Hr and a representative wave period Tr or angular frequency ωr = 2π /Tr and assumed described by linear wave theory. Non-cohesive sediment with median diameter d and density ρs = 2,650kg/m3 is considered in the present study. 2.1 Hydrodynamic Model The Princeton Ocean Model (POM), Blumberg and Mellor (1987), is used to predict 3D flow velocities (u, v, w) in the x, y and σ directions, respectively. POM is a primitive equation ocean model based on hydrostatic and Bousinesq assumptions, in which the turbulence closure scheme of Mellor and Yamada (1982), hereafter referred to as MY, is incorporated to estimate eddy viscosity KM and eddy diffusivity for heat, KH. In the present study, we assume the suspended sediment diffuses in the same way as the heat and therefore take the sediment eddy diffusivity KS = KH. As pointed out by Warner et al. (2005), the MY scheme predicts a substantially smaller eddy viscosity than analytical solutions and other turbulence closure schemes. This under-prediction may have significant influence on suspended sediment concentrations causing it to decay too rapidly with distance from the bed. To avoid the under- prediction, the wall proximity function with open channel correction proposed by Blumberg et al. (1992) is used to replace the original wall function in the MY scheme. Since POM cannot resolve the small scale wave motion, only net or wave-period averaged quantities are computed in the present study, e.g. the net bed-load transport rate and mean concentrations. COASTAL ENGINEERING 2012 3 2.2 Bottom Boundary Layer Model A wave-current interaction model (Grant and Madsen, 1979; Madsen, 1994; Humbyrd and Madsen, 2010) is incorporated into POM to account for wind wave effects. The model is based on a bilinear time-invariant eddy viscosity model that is proportional to the combined maximum wave- current shear velocity *m u within and to the enhanced current shear velocity *c u above the bottom boundary layer, i.e. * * , ( ) , m cw M c cw u z z K z u z z κ δ κ δ ≤  =  ≥  (1) (1) The model is able to predict the physical, i.e. movable, bottom roughness kn, the apparent bottom roughness, i.e. the enhanced bottom roughness experienced by a current in the presence of waves, kna, the wave boundary layer thickness δcw, the total current and wave shear velocities, * * and c wm u u , and the current and wave skin friction, or sediment transport, shear velocities * * and cs wms u u . The information required to implement the wave-current interaction model include: water depth h; rms wave height Hr and period Tr or angular frequency ωr; current bottom shear velocity u*c or a reference current velocity ur at z = zr; and sediment diameter d. The solution procedure for the wave- current interaction can be divided into three steps. 2.2.1 Prediction of physical bottom roughness kn based on wave and sediment specification It is assumed that wind waves dominate fluid-sediment interaction and therefore determine the bed condition, i.e. flat without or with sediment motion or rippled, so that the physical roughness can be predicted solely from wave information. To do this, the maximum wave bottom orbital velocity Ubm or the excursion amplitude Abm = Ubm / ωr is computed from rms wave height, period and water depth. Then the skin friction Shields Parameter ' ψ d is obtained from [ ] [ ] ' ' ' 2 / ( 1) 0.5 / ( 1) d wm w bm s gd f U s gd ψ τ ρ = − = − (2) (2) where g = 9.81m2/s is the acceleration due to gravity and / 2.59 s s ρ ρ = = is the relative density of sediment. The wave skin friction factor ' wf is computed based on a bottom roughness equal to the median sediment diameter, i.e. 0.078 2 ' 0.109 2 exp 7.02 8.82 , / 10 exp 5.61 7.30 , / 10 − −    − = <    =    − = ≥     bm w bm X X A d f X X A d (3) (3) There will be no sediment motion if the skin friction Shields Parameter ' ψ d given by (2) is smaller than the critical value, cr ψ , which can be estimated by the formula proposed by Herrmann and Madsen (2007) ( ) 2/3 3/4 * * 0.095 0.056 1 exp / 20 cr S S ψ −   = + − −   (4) (4) where ( ) * 1 / 4 S d s gd ν = − . The bed will be flat with no transport if ' ψ ψ < d cr , rippled if ' 0.35 ψ ψ ≤ ≤ cr d and in sheet flow condition if ' 0.35 ψ > d . The threshold Shields Parameter for sheet flow condition is taken to be 0.35, rather than the usual value of 0.7, because the Shields Parameter computed using the significant wave orbital velocity, ' ' 2 ψ ψ = s d , has been found to describe onset of sheet flow for random waves. 2.2.2 Prediction of total shear velocities, boundary layer thickness and apparent roughn With the physical bottom roughness kn obtained as outlined in Section 2.2.1, and the current shear velocity *c u , a wave-current interaction analysis can be performed to yield the maximum wave shear velocity, *wm u . The general procedure is ( ) 1/2 2 2 2 * * 1 2 cos with / cw c wm C u u µ φ µ µ µ = + + = (7a) 0.078 2 0.109 2 exp 7.02 8.82 , / 10 exp 5.61 7.30 , / 10 bm n cw bm n C X X C A k f C X X C A k µ µ µ µ µ µ µ µ − −    − = <    =    − = ≥     (7b) * / 2 wm cw bm u f U = (7c) (7c) where cw c w φ φ φ = − is the angle between currents and waves, with cφ and w φ denoting the current and wave directions with respect to the x axis. In this wave-current interaction procedure, the current shear stress or shear velocity *c u is kept unchanged since it would be slowly varying. But this does not mean the current shear stress is fixed for the entire simulation, since it varies slowly with time as predicted by the numerical circulation model and/or slowly changing wind wave conditions. Hence, the bottom boundary layer model and the flow model are fully coupled. 2.2.1 Prediction of physical bottom roughness kn based on wave and sediment specification The threshold Shields Parameter for initiation of motion is still taken as cr ψ in order to be consistent with the critical value used in the calculation of bed-load transport rate and reference concentration for sediment suspension in combined wave-current flows. The physical roughness for different bed conditions can therefore be computed from ' ' ' (no transport) 0.35(rippled bed) 0.35 (sheet flow) d cr cr d d ψ ψ ψ ψ ψ < ≤ ≤ > (5) ( ) ' ' ' , (no transport) , / , 0.35(rippled bed) max( , ), 0.35 (sheet flow) d cr n n bm n cr d n d d k k d A k d A d ψ ψ ψ ψ ψ  <  = ≤ ≤   >  (5) (5) COASTAL ENGINEERING 2012 4 4 COASTAL ENGINEERING 2012 where An is a function of ' ψ d suggested to be 15 ' ψ d by Madsen (2002) based on very limited data. where An is a function of ' ψ d suggested to be 15 ' ψ d by Madsen (2002) based on very limited data. For rippled bed conditions, the physical roughness in (5) can be calculated iteratively through the empirical relationship for the energy dissipation factor established by Humbyrd and Madsen (2010) ( ) 0.25 ' * 0.14 exp 4.94ψ = − e d f S (6a) ( ) 0.25 ' * 0.14 exp 4.94ψ = − e d f S (6a) This energy dissipation factor is a function of bottom roughness This energy dissipation factor is a function of bottom roughness ( ) ( ) { } 10 cos / 60 11 2log / e w bm n f f A k π = −     (6b) (6b) where wf is computed from (3) with / bm n X A k = . Therefore, the energy dissipation factor ef can be calculated by (6a) based on the skin friction Shields Parameter obtained from (2). Having ef , the physical roughness can be computed iteratively from (6b). where wf is computed from (3) with / bm n X A k = . Therefore, the energy dissipation factor ef can be calculated by (6a) based on the skin friction Shields Parameter obtained from (2). Having ef , the physical roughness can be computed iteratively from (6b). 2.2.1 Prediction of physical bottom roughness kn based on wave and sediment specification 2.2.2 Prediction of total shear velocities, boundary layer thickness and apparent roughness ' max( ,15 ) ns d k d d ψ = (13) Unfortunately, no experimental data are available to support a specific choice of the reference level for the reference current velocity to be used in the computation of the sediment transport shear stresses for combined wave-current flows. Thus we choose, somewhat arbitrarily but physically and computationally pleasing, the upper boundary of the wave bottom boundary layer as the reference level at which the reference current velocity is specified, i.e. we have from (9) ( ) * / ln30 / at r c cw na r cw u u k z κ δ δ = = (14) (14) Hence the sediment transport shear velocities * * and cs wms u u can be computed from the following iterative procedure Hence the sediment transport shear velocities * * and cs wms u u can be computed from the following iterative procedure ( ) 1/2 2 2 2 * * 1 2 cos with / µ φ µ µ µ = + + = s cw s s s cs wms C u u (15a) 0.078 2 0.109 2 exp 7.02 8.82 , / 10 exp 5.61 7.30 , / 10 µ µ µ µ − −    − = <    =    − = ≥     s s s s bm ns cws s s s s bm ns C X X C A k f C X X C A k (15b) 2 2 * 0.5 wms cws bm u f U = (15c) * * ms s wms u C u µ = (15d) 0 * * 2 0 * ln( / ) ln( / ) 1 1 ln( / ) 2 4 ln( / ) r cws r cws s cs ms cws s ms r cws z u z u u z u z δ κ δ δ δ   = − + +       (15e) where 0 / 30 s ns z k = and the thickness cws δ is computed in the same way as cw δ in (8a-c) but with the skin friction parameters * , , and ns s s ms k C u µ µ . 2.2.2 Prediction of total shear velocities, boundary layer thickness and apparent roughn Once the iteration procedure in (7a-c) converges, we can obtain * * and c wm u u and the combined maximum shear velocity * * m wm u C u µ = , from which we can calculate the bottom boundary layer thickness * / cw m r PA u µ δ κ ω = (8a) * / cw m r PA u µ δ κ ω = (8a) where ( ) 0.071 exp 2.96 / 1.45 bm n A C A k µ µ −   = −     (8b) ( ) ( ) / 1 / 16.3 C C P C µ µ µ µ µ − = (8c) (8b) (8c) The current velocity within and above the boundary layer can now be computed from ( ) ( ) 2 * * * / ln 30 / , ( ) / ln 30 / , c m n cw c c na cw u u z k z u z u z k z κ δ κ δ  ⋅ ≤  =  ⋅ ≥  (9) (9) where the apparent roughness, kna, is obtained by matching the velocity at cw z δ = ( ) * * / 30 30 / c m u u na cw cw n k k δ δ − = (10) (10) The apparent roughness is the roughness experienced by the current in the presence of wind waves, which is used to compute the quadratic bottom friction factor The apparent roughness is the roughness experienced by the current in the presence of wind waves, which is used to compute the quadratic bottom friction factor COASTAL ENGINEERING 2012 5 ( ) 2 2 1 / ln 30 / κ =     D na C z k ( ) 2 2 1 / ln 30 / κ =     D na C z k (11) (11) where 1z is the first interior velocity grid level above the bottom in POM simulations . A logarithmic velocity distribution near the bottom is assumed to obtain (11). Through this bottom friction factor, CD, the numerical circulation model captures the enhancement of bottom resistance induced by wind waves. 2.3 Prediction of sediment transport shear stress To compute sediment transport rates, we need to evaluate the shear stresses or shear velocities responsible for moving and entraining sediment. For sheet flow conditions, the shear stresses obtained as outlined above are the sediment transport shear stresses. For rippled bed conditions, the total shear stresses obtained in the preceding section consist of skin friction and form drag, and of these only the skin friction shear stress is responsible for sediment motion. Thus, when the bed is rippled, the skin friction shear stress needs to be separated from the total shear stress. To do this, another wave-current interaction analysis is performed based on a reference current velocity and a skin friction roughness. For rippled bed conditions, a reasonable skin friction roughness should be related to the flow intensity rather than a constant multiple of the grain size. In order to make a smooth transition from rippled bed to sheet flow conditions, we adopt the roughness for sheet flow condition in (5) to calculate skin friction bottom roughness, i.e. we take ' max( ,15 ) ns d k d d ψ = (13) 2.2.2 Prediction of total shear velocities, boundary layer thickness and apparent roughn The apparent roughness is the only parameter passing from the bottom boundary layer model to the flow model, which in turn will provide an updated current shear stress to the bottom boundary layer model. The current bottom shear stress τbc in POM is computed from the friction factor and the horizontal velocity U1 at the level 1 = z z 2 1 = bc D C U τ ρ (12) 2 1 = bc D C U τ ρ (12) (12) ' max( ,15 ) ns d k d d ψ = After the iteration procedure in (15a-e) converges, we can obtain the sediment transport shear stresses for the current, 2 * cs cs u τ ρ = , and for the waves, 2 * wms wms u τ ρ = . The total sediment transport shear stress can then be written as COASTAL ENGINEERING 2012 6 ( ) ( ), ( ) bs bsx bsy t t t τ τ τ φ   =     ( ) ( ), ( ) cos cos cos , cos sin sin bs bsx bsy wms r w cs c wms r w cs c t t t t t τ τ τ τ ω φ τ φ τ ω φ τ φ   =   = + +      (16) (16) 2.3 Sediment Transport Model The net bed-load transport rate can be computed by averaging (17) over a wave period as B q ( ) 0 1 = ∫  T B q t dt T (18) (18) The instantaneous bed-load transport rate for a pure sinusoidal wave condition is ( ) cos cos τ ω τ ω ∝ ⋅  B wms r wms r q t t t , which obviously leads to a period-averaged bed-load transport of zero. Thus, wind waves can only give rise to a non-zero net bed-load transport when they are acting together with a current and/or on a sloping bottom. For example, if we assume wave shear stress to be dominant and the bed slope to be small, the net bed-load transport was shown by Madsen (2002) to be approximately given by ( ) 2 2 * * * 9 tan / 2tan B q wms cs wms m u u u β ϕ ∝ − . The instantaneous bed-load transport rate for a pure sinusoidal wave condition is ( ) cos cos τ ω τ ω ∝ ⋅  B wms r wms r q t t t , which obviously leads to a period-averaged bed-load transport of zero. Thus, wind waves can only give rise to a non-zero net bed-load transport when they are acting together with a current and/or on a sloping bottom. For example, if we assume wave shear stress to be dominant and the bed slope to be small, the net bed-load transport was shown by Madsen (2002) to be approximately given by ( ) 2 2 * * * 9 tan / 2tan B q wms cs wms m u u u β ϕ ∝ − . To predict suspended load transport rate above the bed-load transport layer, i.e. z > 7d, a reference concentration at z = 7d, related to the excess sediment transport shear stress, was introduced by Madsen (2002) ( ) 0 , ( ) 0.0022 max ( ) / 1 ,0 at 7 β ρ τ τ   = − =    R s bs cr C t c t z d (19) (19) where 0 0.65 = c is the volume concentration of sediment in the bed. 2.3 Sediment Transport Model p Instantaneous sediment transport rates are averaged over a wind wave period to yield mean or net sediment transport rates. Three categories of sediment transport are considered: (i) Net bed load transport, B q , which takes place below the reference level for mean concentration specification for the suspended load, z = 7d; (ii) Mean suspended load transport within the wave boundary layer, 7d < z < δcw, S1 q , which is obtained from analytical solutions for current velocity and mean sediment concentration within the wave boundary layer, since POM does not resolve wave boundary layer scales; and (iii) Mean suspended load transport above the wave boundary layer, z > δcw, S2 q , which is obtained by solving an advection-diffusion equation for the mean sediment concentration and the current velocity using POM. g The instantaneous bed-load mass transport rate is computed by the model proposed by Madsen (1991). { } ( ) , , 1.5 8 ( ) ( ) ,0 tan tan ( 1) β β β τ α τ ρ τ τ τ ϕ β τ ρ − = − ⋅ ⋅ ⋅ − −      bs cr s bs B bs cr m bs q t Max t s g (17) where β is the bottom slope in the direction of the instantaneous sediment transport shear stress and considered positive when sloping up in the shear stress direction. In large scale coastal simulations, the bed slope is usually very small, i.e. tan sin 1 β β β ≈ ≈ << . The critical shear stress with slope effect is given by ( ) , 1 tan / tan cr cr s β τ τ β ϕ = + with [ ] ( 1) τ ψ ρ = − cr cr s gd and cr ψ computed by (4). s ϕ and m ϕ are static and dynamic friction angles for sediment which have values of 30o and 50o, respectively. Thus, the parameter ( ) ( ) tan tan / tan tan 0.7 m s β α ϕ β ϕ β = + + ≈ . 2.3 Sediment Transport Model Therefore the net suspended load transport rate in the wave bottom boundary layer is 7 cw c d u Cdz δ =∫  S1 q (23) 7 cw c d u Cdz δ =∫  S1 q (23) (23) where the current velocity vector ( ) c u z  is given by (9). This integration of (23) can be performed analytically (e.g. Madsen, 2002) or numerically by discretizing the boundary layer into a large number of layers. In the present study, the latter method is adopted. The hydrodynamic and sediment transport processes above the boundary layer are far more complex than their counterparts within the bottom boundary layer and are solved numerically in the present study. The slowly varying current velocity is computed from the numerical circulation model, POM, whereas the mass concentration C(z) is obtained by numerically solving the advection-diffusion equation for suspended sediment, ( ) ( ) ( ) ( ) ( ) f S H H DC DuC DvC w w C t x y K C C C A D A D D x x y y σ σ σ ∂ ∂ ∂ ∂  + + + − =   ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂  + +   ∂ ∂ ∂ ∂ ∂ ∂   (24) (24) where D h η = + is the total water depth with η the free surface elevation predicted by POM, AH is the horizontal eddy diffusivity which is assumed the same as the horizontal eddy viscosity and predicted by POM, and the vertical diffusivity for sediment, KS, is assumed identical to the heat diffusivity predicted by MY turbulence closure scheme in POM. Boundary conditions need to be specified in order to solve (24). At land boundaries, a no flux condition is applied, i.e. / 0 C n ∂ ∂= with n denoting the outward normal direction at the boundary. At open boundaries, concentration is specified for inflow condition, i.e. if 0 in n C C u = < with n u the flow velocity in the outward normal direction, n, and an advection condition is applied for outflow, i.e. / / 0 if 0 n n C t u C n u ∂ ∂+ ∂ ∂= > . 2.3 Sediment Transport Model Again, only the mean reference concentration is considered here, which is 0 1 ( ) T R R C C t dt T = ∫ (20) 0 1 ( ) T R R C C t dt T = ∫ (20) It can be shown that, unlike the net bed-load transport, linear wind waves alone can produce a significant mean reference concentration, e.g. the mean concentration for pure wave conditions can be expressed by ( ) 0 0.0044 / / 1 R s wms cr C c πρ τ τ = − which obviously does not vanish. Therefore, wave- induced bottom shear stress can play a very important role in predicting suspended load transport and 7 COASTAL ENGINEERING 2012 should not be neglected, especially for wave dominated conditions which are common in most coastal waters. Since the large scale numerical model does not resolve wave bottom boundary layer scales, the mean concentration in this layer is computed analytically based on an eddy diffusivity KS which is assumed identical to the eddy viscosity, KM, given by (1). To obtain the analytical concentration solution, we assume the time scale of vertical diffusion in the boundary layer is much smaller than the scale of the slow current flow changes so that equilibrium can be assumed. This assumption should be reasonable, since the wave boundary layer is thin and the turbulent mixing is very strong. For equilibrium, the vertical diffusion and the sediment settling effects are balanced to yield an analytical concentration solution within the wave boundary layer ( ) * / ( ) / 7 f m w u R C z C z d κ − = (21) (21) where wf is the sediment fall velocity. It is calculated from the formula proposed by Jiménez and Madsen (2003) sediment fall velocity. It is calculated from the formula proposed by Jiménez and ( ) ( ) * 1 / 0.95 5.1/ f n n w s gd S = − + (22) (22) where / 0.9 n d d ≈ and ( ) * 1 / 4 n n n S d s gd ν = − . where / 0.9 n d d ≈ and ( ) * 1 / 4 n n n S d s gd ν = − . 2.3 Sediment Transport Model At the free surface, zero net sediment flux is assured by setting / 0 S f K C Dw C σ ∂ ∂ + = at z = h +η , whereas a sediment concentration, the reference concentration, is specified as b C C = at bottom. To obtain the reference concentration, Cb, we first compute the mean concentration at the outer edge of the wave boundary layer, i.e. at z = δcw, from the analytical wave boundary layer solution, (21), ( ) * / ( ) /7 f m w u a cw R cw C C z C d κ δ δ − = = = ⋅ (25) ( ) * / ( ) /7 f m w u a cw R cw C C z C d κ δ δ − = = = ⋅ (25) Physically, the bottom level in POM simulations is defined at the outer edge of the wave boundary layer, i.e. cw z δ = . However, due to the staggered grid system used in POM, the bottom level for Physically, the bottom level in POM simulations is defined at the outer edge of the wave boundary layer, i.e. cw z δ = . However, due to the staggered grid system used in POM, the bottom level for COASTAL ENGINEERING 2012 8 COASTAL ENGINEERING 2012 8 8 concentration is not at cw z δ = but a half grid spacing above it, i.e. at / 2 b cw b z z z δ = = + ∆ with bz ∆ the spacing for the interior grid cell immediately above the bottom. Assuming equilibrium conditions to apply within the bottom half of the first grid, the concentration Cb can be obtained analytically based on the reference concentration Ca at cw z δ = and the linear eddy diffusivity above the boundary layer given by (1). 2.3 Sediment Transport Model In this manner, the reference concentration for the large scale numerical sediment transport model clearly accounts for wind wave effects and it becomes ( ) * / 1 / 2 at f c w u b a b cw b C C z z z κ δ − = + ∆ = (26) (26) The eddy diffusivity predicted by POM approaches zero at the bottom level which is actually at the outer edge of the wave boundary layer where the eddy diffusivity according to the wave boundary layer model should be *c cw u κ δ . This relatively small eddy diffusivity may produce a large difference in sediment concentration, since suspended sediment concentration is very sensitive to the diffusivity near the bottom. For example, if we assume a typical condition with 1 / f w cm s = , * 2.5 / c u cm s = , 5 cw cm δ = , 10 bz cm ∆ = and 3 1 / a C kg m = , we obtain 3 0.5 / b C kg m = from (26), and the concentration at the upper boundary of the first grid cell, cw b z z δ = + ∆ is roughly 0.33kg/m3. If directly using the eddy diffusivity predicted by POM, the concentration at cw b z z δ = + ∆ predicted by the present model can be estimated analytically to be 3 0.25 / kg m . This underestimates the concentration by about 24%. To avoid this under-predictions and be more physically consistent, we add the constant eddy diffusivity, *c cw u κ δ , to the POM predicted eddy diffusivity SPOM K over the entire water depth * S SPOM c cw K K u κ δ = + (27) (27) * S SPOM c cw K K u κ δ = + This adjustment may lead to some minor error in eddy diffusivity near the surface as it does not approach zero. However, this error should have very little influence on the predicted sediment concentration as it is usually quite small near the free surface. 2.3 Sediment Transport Model y q Having the flow velocity and the concentration, the suspended load transport rate above the wave boundary layer is computed by cw h uCdz η δ + =∫  S2 q (28) cw h uCdz η δ + =∫  S2 q (28) Suspended sediment will change the water-sediment mixture density and therefore produce stratification, which in turn will affect the predictions of flow velocity and sediment concentration. The density of the water-sediment mixture can be computed from ( )(1 1/ ) mix C z s ρ ρ = + − (29) ( )(1 1/ ) mix C z s ρ ρ = + − (29) A stable stratification, which would generally be the case for sediment suspended in the water column since the concentration and therefore the mixture density would decrease with distance above the bottom, suppresses the turbulence and therefore weakens the turbulent mixing. This would result in an increase in velocity and a reduction in sediment concentration. In contrast, an unstable stratification intensifies the flow turbulence and therefore results in more sediment transport. Due to the sediment settling effect, positive stratification should be more common. Unstable stratification may, however, occur in rapidly varying flows when the near-bed concentration, which responds nearly instantaneously to the reference concentration, decreases so rapidly that high sediment concentrations in upper layers have insufficient time to settle out of suspension. 3. MODEL TESTS In this section, a few idealized tests are performed to examine the model’s ability to predict wind wave effects and sediment transport in combined wave-current flows. The computational domain is an open channel of 100km length, 10km width and 10m depth. In the numerical simulations, uniform horizontal grid cells of ∆x = ∆y = 2km and 40 non-uniform sigma layers with finer spacing near the bed are used. The external and internal time steps are 6sec and 120sec, respectively. In the tests, Coriolis force is neglected. A steady open channel flow is considered which is driven by a prescribed constant volume discharge at both open boundaries, equivalent to applying a depth- COASTAL ENGINEERING 2012 9 averaged velocity ( ) 0.5 / 1 / U h η = + m/s at the boundaries. This leads to U = 0.5m/s at the center of the domain where the surface elevation 0 η = after a steady state is established. The flow is uniform in the cross-channel direction. A random wind wave condition with a rms height of Hr = 1m and a representative period of Tr = 8sec is specified. The waves are assumed to propagate in the same direction as the current flow, i.e. this is a co-directional combined wave-current flow case. Uniform sediment with a median grain size of d = 0.1mm or 0.2mm is assumed. This corresponds to a critical shear stress of 2 2 0.19 / or 0.20 / cr N m N m τ = or a critical shear velocity of * 1.36 / cr u cm s = or 1.40 / cm s as computed from (4) and a fall velocity of 0.72 / or 2.2 / = f w cm s cm s from (22). For the inflow boundary condition, the sediment concentration is assumed zero. This creates a transition region near the upstream boundary. The length of this transition region can be estimated approximately from 2 / S L Uh K = 2 0.5 10 / 0.01 × ∼ 5km = . Evidently, this is a very short distance compared to the domain length of 100km, most of which will therefore be unaffected by transition effects. The simulations are run for 3 days. To avoid simulation crash, the forcing is imposed gradually with a linear ramp up during the first 12hours. 3. MODEL TESTS The results show that steady state is established within a few hours after the ramp up period. All the results shown in this Section are obtained at the center of the domain where the surface elevation is close to zero at a time corresponding to the end of the simulation, t = 72 hours. For the 0.1mm sediment, three simulations are performed: (1) Pure current case; (2) Combined wave-current case without stratification; (3) Combined wave-current case with stratification. For the 0.2mm sediment, only simulations (2) and (3) are performed. Some predicted parameters for simulations (2) and (3) are listed in Table 1, including physical bottom roughness, apparent roughness, boundary layer thickness, wave and current shear velocities and reference concentrations. Sediment transport rates are listed in Table 2 for the different simulations. 3.1 Effects of Wind Waves The vertical profiles of eddy viscosity and horizontal velocity from simulations (1) and (2) for 0.1mm sediment are shown in Figure 2. For the pure current simulation, the physical roughness is chosen to be the larger one of the sediment diameter d and 3.3ν/u*c, i.e. the simulation starts with kn = d by assuming a rough turbulent flow, then checks and switches to smooth turbulent flow with kn = 3.3ν/u*c if d < 3.3ν/u*c. The flow model predicts a bottom shear velocity of 1.5cm/s, indicating a smooth turbulent flow condition and therefore the roughness is kn = 3.3ν/u*c = 0.28mm. When wind waves are present, a rippled bed is obtained, leading to a much larger physical roughness of kn = 1.66cm and an apparent roughness of kna =11.1cm (non-stratified case in Table 1) and a bottom shear velocity of 2.72cm/s. As a result, the maximum eddy viscosity (seen in Figure 2a) almost doubles from 0.01m2/s to 0.02m2/s due to the presence of wind waves. The current velocity profiles in Figure 2b show that the presence of wind waves leads to a lower current velocity near the bed due to the larger flow resistance. The velocity at the first interior grid point is decreased from about 0.3m/s for the pure current condition to 0.15m/s in the presence of wind waves. Table 1. Parameters predicted by the model for different sediment in stratified and non-stratified flows. Diameter 0.1mm 0.2mm Stratified Parameters No Yes No Yes kn (cm) 1.66 1.66 12.3 12.3 kna (cm) 11.1 14.1 51.8 53.1 δcw (cm) 1.82 1.91 4.31 4.35 u*c (cm/s) 2.72 2.24 3.43 3.34 u*cs (cm/s) 1.24 1.00 0.88 0.85 u*wm (cm/s) 5.30 5.20 8.14 8.12 u*wms (cm/s) 2.82 2.76 2.80 2.80 R C (kg/m3) 7.23 6.75 6.39 6.34 Ca (kg/m3) 2.72 2.38 0.75 0.73 COASTAL ENGINEERING 2012 10 0 2 4 6 8 10 0.000 0.007 0.014 0.021 KM (m 2/s) z (m) Eddy Viscosity @x=50km, t=72th hour No Wave Hr = 1m (a) 0 2 4 6 8 10 0.2 0.3 0.4 0.5 0 2 4 6 8 10 0.000 0.007 0.014 0.021 KM (m 2/s) z (m) Eddy Viscosity @x=50km, t=72th hour No Wave Hr = 1m (a) Velocity @x=50km, t=72th hour No wave Hr = 1m u (m/s) z (m) (b) Figure 2. 3.1 Effects of Wind Waves Predicted vertical profiles of (a) eddy viscosity and (b) current velocity for pure current and combined wave-current cases at the center of domain. Sediment size is 0.1mm. 0 2 4 6 8 10 0.2 0.3 0.4 0.5 Velocity @x=50km, t=72th hour No wave Hr = 1m u (m/s) z (m) (b) Figure 2. Predicted vertical profiles of (a) eddy viscosity and (b) current velocity for pure current and combined wave-current cases at the center of domain. Sediment size is 0.1mm. Due to the flat bed condition, the sediment transport shear velocity for the pure current condition is equal to the total value of 1.5cm/s, which is very close to the critical shear velocity of 1.36cm/s for the 0.1mm sediment. This means that there is essentially no sediment transport for the pure current condition. In the presence of wind waves the sediment transport shear velocities are calculated to be 1.24 cm/s and 2.82cm/s for current and waves, respectively, and are based on a reference current velocity of ur = 11.1cm/s at zr = 1.82cm and a skin friction roughness of kns = 0.45mm. Hence, the sediment transport shear stress in this co-directional flow case is ( ) ( ) 0.8cos 0.15,0 τ ω = +  bs t t N/m2. This gives a maximum instantaneous value of 0.95 N/m2, which is much larger than the critical shear stress of 0.19N/m2. The rather dramatic effect of the presence of wind waves is seen in the reference concentrations, 7.23kg/m3 and 2.72kg/m3 (Table 1), at the lower and upper boundaries of the wave boundary layer. As shown in Table 2, the predicted transport rates are 0.01kg/m/s, 0.0051kg/m/s and 0.5kg/m/s for the net bed-load transport, the mean suspended load transport within and above the boundary layer, respectively. It can be seen that suspended load transport in the water column is dominant as it is about 30 times the combined bed-and suspended load transport rate within the wave boundary layer. This is mainly caused by the small fall velocity of the fine sediment and strong turbulent mixing enhanced by wind waves. Due to the thin boundary layer and fine sediment, the mean suspended load transport rate within the boundary layer is much smaller than that in the overlying water column, but it is still comparable to the net bed-load transport rate. Table 2. Transport rates (kg/m/s) of net bed-load, suspended load within and above the w.b.b.l. 3.1 Effects of Wind Waves for two types of sediment. The total transport rate T q is T B S S q q q q 1 2 = + + = + + = + + = + + . B q 1 S q 2 S q T q Stratified Diameter d No Yes No Yes No Yes No Yes 0.1mm 1.0x10-2 7.3x10-3 5.1x10-3 3.5x10-3 5.0x10-1 1.2x10-1 5.2x10-1 1.3x10-1 0.2mm 6.2x10-3 5.9x10-3 3.1x10-3 2.9x10-3 1.6x10-2 1.3x10-2 2.5x10-2 2.2x10-2 Table 2. Transport rates (kg/m/s) of net bed-load, suspended load within and above the w.b.b.l. for two types of sediment. The total transport rate T q is T B S S q q q q 1 2 = + + = + + = + + = + + . 3.2 Effects of Sediment-Induced Self-stratification for 0.1mm Sediments Sediment stratification effects are accounted for in the present study by considering the stratification effects associated with the varying density of the water-sediment mixture as expressed in (29). The routine available in POM to account for stratification due to heat and/or salinity gradient is used to account for sediment stratification effects. In general, the stratification effect is incorporated by relating a turbulent kinetic energy (TKE) production to the vertical density gradient. This leads to an enhanced turbulence intensity by a positive vertical density gradient and vice versa. In this steady flow case, the concentration decreases all the way from the bed to surface, leading to stably stratified conditions which mean that the turbulence intensity will be reduced and therefore turbulent mixing will be weakened. This is confirmed by the decreased current shear velocity of 2.24cm/s predicted by POM and listed in Table 1 for 0.1mm sediment when stratification is accounted for compared to 2.72cm/s for COASTAL ENGINEERING 2012 11 the non-stratified case. As a result, the current velocity within the boundary layer (seen in Figure 3a) shows quite a significant reduction, e.g. the velocity at the outer edge of the boundary layer is decreased from 11cm/s to 8cm/s. It should be recalled that stratification effects within the wave boundary layer are not accounted for, and this is the reason why the wave shear velocity, as seen in Table 1, hardly changes (it goes from 5.3cm/s to 5.2cm/s). The reduced current shear velocity, in addition to the reduced mixing associated with stable stratification, makes the eddy diffusivity above the wave boundary layer decrease substantially as shown in Figure 4a, reducing the maximum value from about 0.027m2/s to 0.015m2/s. 0.000 0.005 0.010 0.015 0.020 2 3 4 5 6 7 0.000 0.005 0.010 0.015 0.020 0.00 0.02 0.04 0.06 0.08 0.10 u (m/s) z (m) Velocity @x=50km, t=72th hour Non-stratified Stratified (a) Concentration (kg/m 3) z (m) Concentration @x=50km, t=72th hour Non-stratified Stratified (b) Figure 3 Analytical solutions of vertical profiles of (a) current velocity and (b) sediment concentration within the wave boundary layer at the center of domain. Sediment size is 0.1mm. 3.2 Effects of Sediment-Induced Self-stratification for 0.1mm Sediments 0.000 0.005 0.010 0.015 0.020 0.00 0.02 0.04 0.06 0.08 0.10 u (m/s) z (m) Velocity @x=50km, t=72th hour Non-stratified Stratified (a) 0.000 0.005 0.010 0.015 0.020 2 3 4 5 6 7 Concentration (kg/m 3) z (m) Concentration @x=50km, t=72th hour Non-stratified Stratified (b) Figure 3 Analytical solutions of vertical profiles of (a) current velocity and (b) sediment concentration withi the wave boundary layer at the center of domain. Sediment size is 0.1mm. 0 2 4 6 8 10 0.1 0.2 0.3 0.4 0.5 0.6 0 2 4 6 8 10 0.000 0.007 0.014 0.021 0.028 KS (m 2/s) z (m) Eddy Diffusivity @x=50km, t=72th hour Non-stratified Stratified (a) Velocity @x=50km, t=72th hour Non-stratified Stratified u (m/s) z (m) (b) 0 2 4 6 8 10 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 Concentration @x=50km, t=72th hour Non-stratified Stratified Concentration (kg/m 3) z (m) (c) Figure 4 Predicted vertical profiles of (a) eddy viscosity; (b) Velocity; (c) Concentration above the wave boundary layer at the center of domain. Sediment size is 0.1mm. 0 2 4 6 8 10 0.000 0.007 0.014 0.021 0.028 KS (m 2/s) z (m) Eddy Diffusivity @x=50km, t=72th hour Non-stratified Stratified (a) 0 2 4 6 8 10 0.1 0.2 0.3 0.4 0.5 0.6 Velocity @x=50km, t=72th hour Non-stratified Stratified u (m/s) z (m) (b) 0 2 4 6 8 10 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 Concentration @x=50km, t=72th hour Non-stratified Stratified Concentration (kg/m 3) z (m) (c) Concentration (kg/m 3) Figure 4 Predicted vertical profiles of (a) eddy viscosity; (b) Velocity; (c) Concentration above the wave boundary layer at the center of domain. Sediment size is 0.1mm. 12 COASTAL ENGINEERING 2012 Due to the reduced current shear velocity, the velocity gradient near the bottom decreases, but this tendency is counteracted by the reduced mixing due to stable stratification which would tend to increase the velocity gradient. The combined effect of these processes is a decrease of velocity near the bottom and, since the depth-averaged velocity has to be maintained at 0.5m/s, a slight increase in the upper layers (seen in Figure 4b). For example, calculations suggest that the velocity for stratified case is reduced by 27% (from 14.8cm/s to 10.9cm/s) at bottom to about 1% at 2m above the bed and the difference is less than about 1.2% in the rest portion of the water column. 3.2 Effects of Sediment-Induced Self-stratification for 0.1mm Sediments Since a reduction in shear velocity leads to a smaller reference concentration and stable stratification reduces upward mixing of sediments into the water column, the two effects mentioned above both result in a decrease in suspended sediment concentration. One can therefore expect a significant reduction in the sediment concentration and transport rates due to self-stratification effects. The reference concentrations in Table 1 show that these are reduced by about 7% from 7.23kg/m3 to 6.75kg/m3 at z = 7d. This very minor reduction is, of course, associated with the virtually unchanged wave shear velocities, which dominate in the creation of the mean reference concentration. Consequently, the concentration within the boundary layer does not show significant reduction as seen in Figure 3b. In contrast, the concentration above the wave boundary layer (shown in Figure 4c) is significantly decreased by the stratification effect, e.g. the concentration at 1m above the bed is reduced by about 68% from 0.28kg/m3 to 0.09kg/m3, although the difference between non-stratified and stratified cases is only 13% at bottom. y The transport rates in Table 2 reveal that the net bed-load and mean suspended load transport rates within the boundary layer decrease only by roughly 30%. The relatively small reduction of net bed-load transport rate is because it depends only on the sediment transport shear stress which, as seen from Table 1, does not change appreciably. The reason for the slight reduction of mean suspended load transport rate within the boundary layer reflects primarily the reduction in current velocity (shown in Figure 3a), whereas the concentration (Figure 3b), predicted without considering stratification, hardly changes. However, the suspended load transport rate above the boundary layer is significantly reduced as a result of stratification. The stratified value of 0.12kg/m/s is only 24% of the 0.5kg/m/s predicted when stratification effects are neglected. This significant influence of stratification effects above the boundary layer is mainly caused by the reduction of turbulent mixing associated with the stable stratification and, to a lesser extent, by the reduced current shear stress. 4. CONCLUSIONS In this paper, a three dimensional sediment transport model is proposed for the computation of sediment transport in combined wave-current flows in coastal waters. The model calculates three contributions to the total sediment transport rate: (i) net bed-load transport in the bed-load layer z < 7d; (ii) mean suspended load transport within the bottom boundary layer 7d < z < δcw; and (iii) suspended load transport above the wave boundary layer z > δcw. Wind wave effects on flow and sediment transport are accounted for through an analytical bottom boundary layer model. For the hydrodynamics, the presence of wind waves enhances the flow resistance by increasing the roughness, the so-called apparent roughness, experienced by a current in the presence of waves. For the sediment transport processes, wind waves increase the bottom shear stress and the near-bed turbulence and therefore mobilize and suspend more sediment than would be the case if the waves were absent. In addition, the wave enhanced turbulent mixing in the water column above the wave boundary layer diffuse more sediment up into the water column. In the water column above the wave boundary layer hydrodynamics and sediment transport processes are solved numerically using POM and stratification effects induced by suspended sediment are taken into account. Idealized tests were performed to examine the model’s ability to predict sediment transport in combined wave-current flows. The test results reveal that wind waves have a pronounced effect on hydrodynamics and suspended sediment transport for typical conditions of coastal waters, e.g. sediment may only be mobilized and transported when wind waves are present. The sediment transport rate within the wave boundary layer, which has been neglected in most previous studies, is more important for coarser sediment, e.g. it is about 20% the suspended load transport above the boundary layer for 0.2mm sediment compared to 1% for 0.1mm sediment case in the present study’s computational example. Therefore, wind wave effects should never to be neglected in sediment transport modeling in coastal waters. The numerical tests suggest that the self-stratification effect is more significant for fine sediment as it causes 75% drop in total transport rate for the 0.1mm sediment case, but becomes less significant for coarser sediment as the reduction of total transport rate is only 12% for 0.2mm sediment. COASTAL ENGINEERING 2012 COASTAL ENGINEERING 2012 13 ACKNOWLEDGMENTS The research described in this paper was funded in whole or in part by the Singapore National Research Foundation (NRF) through the Singapore-MIT Alliance for Research and Technology’s (SMART) Center for Environmental Sensing and Modeling (CENSAM). 4. CONCLUSIONS Considering the significant influence of self-stratification on suspended transport above the boundary layer, it would be prudent to examine the effect of stratification within the wave boundary layer, or in the extrapolation of the mean concentration from the outer edge of the wave boundary layer to the numerical model’s grid point where a reference concentration is specified. We are currently pursuing extensions of the sediment transport model along these lines as well as examining its performance in more realistic scenarios. 4. CONCLUSIONS Essentially, the stratification effect can be very significant when concentration gradients are large, which usually happens for relatively fine sediment and strong flow conditions. In such circumstances, our results show that the self-stratification effect should be accounted for when modeling sediment transport in coastal waters. It is noted that the present model tests were performed for a steady co-directional wave-current flow. Further tests must be conducted to test the model’s capabilities in more realistic flows, e.g. in unsteady flows and in the cases where waves and current are in different directions. For unsteady, slowly varying flows, the applicability of the concentration bottom boundary condition, which assumes equilibrium conditions, needs to be tested. To more accurately predict the sediment transport in combined wave-current flows, the wave-associated suspended load transport within the wave boundary layer, 1 7 cw S d q uCdz δ = ∫    , with ( ) ( ) and u t C t   denoting the time-varying wave-associated velocity and concentration within the wave boundary layer, should be included. Considering the significant influence of self-stratification on suspended transport above the boundary layer, it would be prudent to examine the effect of stratification within the wave boundary layer, or in the extrapolation of the mean concentration from the outer edge of the wave boundary layer to the numerical model’s grid point where a reference concentration is specified. We are currently pursuing extensions of the sediment transport model along these lines as well as examining its performance in more realistic scenarios. It is noted that the present model tests were performed for a steady co-directional wave-current flow. Further tests must be conducted to test the model’s capabilities in more realistic flows, e.g. in unsteady flows and in the cases where waves and current are in different directions. For unsteady, slowly varying flows, the applicability of the concentration bottom boundary condition, which assumes equilibrium conditions, needs to be tested. To more accurately predict the sediment transport in combined wave-current flows, the wave-associated suspended load transport within the wave boundary layer, 1 7 cw S d q uCdz δ = ∫    , with ( ) ( ) and u t C t   denoting the time-varying wave-associated velocity and concentration within the wave boundary layer, should be included. Blumberg, A.F., B. Galperin, and D.J. O’Connor. 1992. Modeling vertical structure of open-channel flows, Journal of Hydraulic Engineering, 118(H8), 1119-1134. Blumberg, A.F., and G.L. Mellor. 1987. A description of a three-dimensional coastal ocean circulation model, Three Dimensional Coastal Ocean Models, N. Heaps, Ed., Coastal Estuarine Science, Vol. 4, Amer. Geophys. Union, 1–16. Blumberg, A.F., B. Galperin, and D.J. O’Connor. 1992. Modeling vertical structure of open-channel flows, Journal of Hydraulic Engineering, 118(H8), 1119-1134. Blumberg, A.F., and G.L. Mellor. 1987. A description of a three-dimensional coastal ocean circulation model, Three Dimensional Coastal Ocean Models, N. Heaps, Ed., Coastal Estuarine Science, Vol. 4, Amer. Geophys. Union, 1–16. 3.3 Effects of Sediment Diameter In the preceding sections, we discussed wind wave and stratification effects based on numerical results for 0.1mm sediment. To investigate the influence of sediment size on sediment transport, we perform two simulations, one with and the other without stratification effects, for sediment of 0.2mm diameter. The same flow and wave conditions as those for the 0.1mm sediment are used, and results are listed in Tables 1 and 2. As shown in Table 1, the physical and apparent roughness are about 12cm and 52cm. These are significantly larger than those for the 0.1mm sediment, indicating larger ripples and therefore smaller ratios between skin friction shear velocities and total shear velocities. This is confirmed by the results in Table 1, e.g. the ratio of skin friction and total current shear stress for non-stratified case is 0.26 for 0.2mm sediment compared to 0.46 for 0.1mm sediment. The reference concentrations at z = 7d are quite similar for the 0.1mm and 0.2mm sediments; however, due to its much larger fall velocity, the mean concentration at the outer edge of the bottom boundary layer, 0.75kg/m3for 0.2mm sediment, is only 28% of the value for 0.1mm sediment. Due to the near-identical critical shear stresses and much different fall velocities for the two sediments, the difference of net bed-load transport rates between the two sediments is smaller than that of suspended load transport rates. As shown in Table 2, the difference of net bed-load transport rates for non-stratified case is about 38%, whereas the difference between suspended transport rates above the boundary layer is as large as 97%. As for the stratification effect, the results in Table 2 show that the stratification only results in a 12% reduction of total sediment transport rate for 0.2mm sediment, i.e. much less than the 75% reduction found for the 0.1mm sediment. The primary reason for this is that the concentration above the boundary layer is significantly reduced for the 0.2mm sediment due to its much larger fall velocity, 2.2cm/s vs. 0.72cm/s, and therefore generates less stratification effect. REFERENCES Blumberg, A.F., and G.L. Mellor. 1987. A description of a three-dimensional coastal ocean circulation model, Three Dimensional Coastal Ocean Models, N. Heaps, Ed., Coastal Estuarine Science, Vol. 4, Amer. Geophys. Union, 1–16. 14 COASTAL ENGINEERING 2012 Grant, W.D., and O.S. Madsen. 1979. Combined wave and current interaction with a rough bottom Journal of Geophysical Research, 84(C4), 1797-1808. Herrmann, M.J., and O.S. Madsen. 2007. Effect of stratification due to suspended sand on velocity and concentration distribution in unidirectional flows, Journal of Geophysical Research, 112, C02006, doi:10. 1029/2006JC003569. Humbyrd, C.J., and O.S. Madsen. 2010. Predicting movable bed roughness in coastal waters, Proceedings of the International Conference on Coastal Engineering. No. 32(2010), Shanghai, China. Paper #: sediment.6. Retrievable from http://journals.tdl.org/ICCE/ g f f g g China. Paper #: sediment.6. Retrievable from http://journals.tdl.org/ICCE/ Jiménez, J.A. and O.S. Madsen. 2003. A simple formula to estimate settling velocity of natura sediment, Journal of waterway, port, coastal and ocean engineering, 129, No.2, 70-78. Lesser, G.R., J.A. Roelvink, J.A.T.M. van Kester, and G.S. Stelling. 2004. Development and validation of a three-dimensional morphological model, Coastal Engineering, 51, 883-915. Li, M.Z. and C.L. Amos. 2001. SEDTRANS96: the upgraded and better calibrated sediment-transpor model for continental shelves, Computers & Geosciences, 27(6), 619-645. p ( ) Madsen, O.S. (1991) Mechanics of cohesionless sediment transport in coastal waters. Proceedings of Coastal Sediments '91, ASCE, Seattle, USA. 1:15-27 Madsen, O.S. 1994. Spectral wave-current bottom boundary layer flows. Proceedings of 24t International Conference on Coastal Engineering, ASCE, 384-398. Madsen, O.S. 2002. Sediment Transport Outside the Surf Zone. In: Walton, T. (editor), Coastal Engineering Manual, Part III, Coastal Processes, Chapter III-6, Engineer Manual 1110-2-1100, U.S. Army Corps of Engineers, Washington, DC. y p g g Mellor, G.L., and T. Yamada. 1982. Development of a turbulence closure model for geophysical fluid problems, Review of Geophysics and Space Physics, 20, 851–875. Warner, J.C., C.R. Sherwood, H.G. Arango, and R.P. Signell. 2005. Performance of four turbulence closure models implemented using a generic length scale method, Ocean Modelling, 8, 81-113. Warner, J.C., C.R. Sherwood, R.P. Signell, C.K. Harris, and H.G. Arango. 2008. Development of a three-dimensional, regional, coupled wave, current and sediment-transport model, Computers & Geosciences, 34, 1284-1306.
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Effects of Annular-finned Tube Deformation on Thermal Stresses in a Heat Exchanger
Nigerian Journal of Technological Development
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298 298 NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 19, NO.4, DECEMBER 2022 Effects of Annular-finned Tube Deformation on Thermal Stresses in a Heat Exchanger M. Kashani, A. Hatami*, M. Hosseini Faculty of Mathematics, University of Sistan and Baluchestan, Zahedan, Iran ABSTRACT: Nowadays, along with the developments in the industry, many machines and structures are exposed to very high-temperature environments, which has caused various types of thermal load in them. These conditions have led to focused studies on thermal stresses in structures. Heat exchangers are among the structures in which the temperature difference causes thermal stress. In this paper, “finite volume” and “finite element” methods are used to solve flow equations and thermal stress equations in solids, respectively. In addition, the effect of changes in the shape of the annular finned pipe on the thermal stresses in the heat exchanger is investigated. The study results on changing the pipe shape from circular to elliptical show that by changing the pipe shape, the thermal stress in the fin can be significantly reduced; for instance, in the cases reviewed in this paper, the thermal stress has decreased by 9%. Furthermore, the results of thermal stress show that the Maximum Effective Stress point, both before and after changing the shape, is still located at the fin’s base. So, according to the results of this study, examining the effect of pipe shape changing on thermal stress is an imperative measure when designing heat exchangers. KEYWORDS: Fluid-thermal-structural analysis, Annular fin, Thermal stress, Turbulent flow, Heat e uid-thermal-structural analysis, Annular fin, Thermal stress, Turbulent flow, Heat exchanger [Received May 19, 2022; Revised Aug. 11, 2022; Accepted Aug. 22, 2022] Received May 19, 2022; Revised Aug. 11, 2022; Accepted Aug. 22, 2022] Print ISSN: 0189-9 [Received May 19, 2022; Revised Aug. 11, 2022; Accepted Aug. 22, 2022] doi: http://dx.doi.org/10.4314/njtd.v19i4.2 I. INTRODUCTION They proposed a two-dimensional analytical solution for a fin with a constant base temperature and a locally invariant convective heat transfer coefficient around the fin. (Sharma, et al., 2019) calculated temperature and thermal stress gradients in an annular fin. They presented their results for different aspect ratios (ratio of inner to outer radii) by changing the annular fin inner radius. They obtained a second-order nonlinear differential equation as the governing equation for all parameters. (Hatami & Ganji, 2013) used the fourth-order Runge–Kutta method to examine heat transfer from the surface of annular fins with rectangular, triangular, convex, and power- law cross sections. (Kundu & Das, 2007) used a semi-analytical method to study elliptical fins. Their study showed that an optimal elliptical fin could dissipate heat at a much higher rate than a circular fin. The study recommended considering the fin volume or heat dissipation rate as the constant parameter in optimizing the fin geometry. (Jang & Yang, 1998) also published a research on elliptical fins. Their experimental– numerical study addressed flow and heat transfer in a four-row assembly of elliptical-finned tubes with alternating and linear arrangements. They also studied a circular-finned tube with an alternating arrangement. Their experimental results showed that the heat transfer coefficients of elliptical-finned tubes were 35–50% of that of circular-finned tubes of the same perimeter, whereas the total pressure drop in the assembly was only 25– 30% of that in the circular-finned tubes. Furthermore, (Nagarani, et al., 2014) also discussed the advantages of elliptical fins and tubes compared to other geometries. These advantages can be listed as follows: They considered aluminum, copper, and silicon nitride fins and showed that the fin made of silicon nitride with a power- law cross-section maximized heat transfer. (Nagarani, et al., 2014) reviewed previous studies on bringing extended surfaces into heat transfer problems in heat exchangers. Their results help select extended surfaces with different geometries based on available space and costs. In an experimental–numerical study, (Jang, et al., 1998) investigated the performance of an annular tube bundle comprising four rows of annular-finned tubes in dry and wet conditions for inlet velocities in the range of 1–6 m/s. They showed that using a one-dimensional model with increasing flow velocity causes an increase in fin efficiency estimation errors. I. INTRODUCTION They showed that the heat transfer coefficient of an annular-finned tube is 20– 50% higher than that of an elliptical-finned tube with identical tube (geometry). However, circular tubes were found to experience a higher pressure drop than elliptical ones. stresses. Roy and Ghosal (Roy & Ghosal, 2017) used the Homotopy Perturbation Method (HPM) to investigate temperature distribution across an annular fin and the effect of temperature-induced variations of thermal conductivity coefficient on fin’s efficiency and temperature distribution. Peng and Chen (Peng & Chen, 2011) also studied temperature distribution across the annular fin using hybrid differential transforms and finite-difference methods. (Roozbahani, et al., 2017) used the Homotopy Analysis Method (HAM) to find the temperature and stress distributions across an annular disc. p g p p p (Hu & Jacobi, 1993) carried out numerical and experimental investigations of heat transfer in a four-row annular tube bundle. It was shown that in the Re = 3300–12000 range, a considerable change is observed in convective mass transfer and mass transfer behavior even for an infinitesimal angle of attack. (Mon, 2003) studied the effects of geometrical parameters, including fin thickness, fin height, tube diameter, fin pitch, and the annular-finned tube bundle arrangement on the heat transfer coefficient. It was shown that increasing the fin-spacing-to-fin-height ratio increases the heat transfer coefficient and reduces pressure drop in a linear arrangement. However, doing the same in a staggered arrangement initially increases the heat transfer coefficient before reducing it. (Shokouhmand, et al., 2014) optimized an annular-finned tube through structural design aiming to find the optimal geometry that maximized heat exchange. It was shown that the optimal geometry depends on flow conditions. In a numerical study, (Nemati & Moghimi, 2014) investigated flow in finned tube bundles of heat exchangers with various types of turbulence. Their results indicated that the Transition SST model is consistent with experimental results. Moreover, (Nemati & Samivand, 2016) used Transition SST and investigated flow and heat transfer in annular and elliptical-finned tubes. Other works include that of (Aziz, 1993), who investigated the two-dimensional conductivity of a rectangular fin addressing the effect of thermal source, non-uniform fin base (inner radius) temperature, and changes in convective heat transfer coefficient. (Lau & Tan, 1973) focused on errors in one-dimensional heat transfer analysis of straight annular fins. I. INTRODUCTION *Corresponding author: ahatami@math.usb.ac.ir NOMENCLATURE English symbols cp Specific heat at constant pressure, j/k E Modulus of elasticity, Pa H Convective heat transfer coefficient, kf Thermal conductivity, W/mk kt turbulent thermal conductivity, W/m R Radius, m rb Inner radius of the fin, m re Outer radius of the fin, m Srr Radial stress S Tangential stress rS  Shear tress T Temperature, k Ta Fluid temperature, k Tb Temperature at the fin base, k u Displacement, m wt Fin thickness, m Greek symbols  Surface absorptivity  Thermal expansion coefficient  Surface emissivity rr   Radial strain    Tangential strain  Poisson’s ratio ρ Density, kg/m3  Dynamic viscosity, kg / m.s t  Turbulent viscosity, kg / m.s Engineers strive to improve heat transfer from mechanical devices and researchers from various engineering backgrounds have proposed numerous techniques for this purpose. Using fins is considered a versatile straightforward solution. Thanks to their simple structure and low cost, fins are abundantly used in various applications including aviation, automotive, electronics, and other industries. Fins improve heat transfer by increasing the surface-to-volume ratio. However, the resulting temperature gradient brings about thermal stresses, and thus damages the fin structure. This has prompted researchers to carry out extensive studies on fin temperature distribution and thermal stresses. Some of these works on annular fins are reviewed in what follows. Using the inverse Laplace transform and Simpson's rule, Shang-Sheng (Wu, 1997) investigated transient thermal stresses in a uniform, isotropic annular fin, assuming one- dimensional heat transfer. Yu and Chen (Yu & Chen, 1999) used a hybrid one-dimensional method to study steady-state and transient stresses and heat transfer in an isotropic annular fin under convection, radiation, and convection–radiation boundary conditions. They compared the resulting temperature distributions and stresses using various boundary conditions. Chio and Chen (Chiu & Chen, 2002) used decomposition to study temperature and thermal stress distribution across an isotropic annular fin. Their results showed that even small temperature perturbations have a significant impact on thermal 299 HATAMI et al: EFFECTS OF ANNULAR-FINNED TUBE DEFORMATION ON THERMAL STRESSES with inlet velocities in the range of 2–7 m/s. They showed that the heat transfer coefficient of an annular-finned tube is 20– 50% higher than that of an elliptical-finned tube with identical tube (geometry). However, circular tubes were found to experience a higher pressure drop than elliptical ones. with inlet velocities in the range of 2–7 m/s. I. INTRODUCTION (Mon & Gross, 2004) adopted a numerical method to study the effects of fin spacing on heat transfer, pressure drop, and flow behavior in a four-row annular-finned tube bundle under turbulent conditions. They showed that boundary layer development around the fin and tube surface primarily depends on the ratio of fin spacing to fin height.  By using elliptical fins, the pressure drop across circular tubes can be reduced. Further, their results showed that, increasing this ratio in a linear arrangement increases the heat transfer coefficient while reducing pressure drop. Drawing on (Mon & Gross, 2004) works, (Bilirgen, et al., 2013) addressed the case of one row of annular-finned tubes under turbulent flow conditions and carried out on a detailed investigation of the effect of Reynolds number (Re), fin spacing, fin height, fin thickness, and fin material, on heat transfer and pressure drop. They showed that fin thickness has a much less significant effect on heat transfer and pressure drop than fin height and spacing. (Jang & Yang, 1998) studied numerically and experimentally pressure drop and heat transfer in elliptical and annular-finned tube bundles  Space limitation in one side and sufficient space in the vertical direction improves the heat transfer rate.  Oblong tubes with a principal axis parallel to the flow direction generally enhance heat transfer.  Furthermore, this design allows extension of the external surface, improving overall heat transfer. This prompted several studies on heat transfer and hydrodynamics of non- circular tubes and tube (Hatami, et al., 2020) assemblies.  A non-circular geometry reduces scaling due to the small surface area of the front portion of the tubes and their smaller separation zone. NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 19, NO.4, DECEMBER 2022 300 Figure 2: Tube deformation. Figure 2: Tube deformation.  There is increasing power density with in a given volume and envelope shape  There is increasing power density with in a given volume and envelope shape A review of previous studies on annular fins showed that the extensive applications of fins across different industries had inspired several experimental, numerical, or analytical studies. Some researchers have focused on the effect of temperature on annular fins thermal stresses and strains (Wu, 1997; Chiu & Chen, 2002; Bidzard, et al., 2017; Hatami, et al., 2020). I. INTRODUCTION The present study relied on the fluid-thermal-structural analysis to investigate the deformations of the annular-finned tube and its effects on thermal stresses in heat exchangers. This study draws on research by (Hosseini, et al., 2020; Hosseini, et al., 2020), who investigated the effects of the deformation of tubes in the first row of fin bundles. The aim was to investigate the effects of tube deformation in other rows of fin bundles. Figure 2: Tube deformation. Table 1: Geometrical specifications. a = 10 mm Distance from inlet b = 10 mm Distance from outlet h = 14 mm Solution domain height c = 140 mm Solution domain width wt = 4 mm Fin thickness rb = 20 mm Base radius re = 60 mm Tip radius A. Problem Description Figures 1 and 2 illustrate the problem geometry. The turbulent flow of air runs at 0.002 kg/s around the fin with an initial surface temperature of Tsur = 300 K. At Tb = 600 K, the tube wall temperature was also assumed to be constant and equal to the base (inner radius) temperature. Tables 1 and 2 present geometrical conditions and fin material, respectively. This study investigated thermal stresses deforming the tube into an elliptical shape as depicted in Figure 2. Since the sharpest temperature gradient in the heat exchanger develops during the initial moments, all discussions in this study are limited to the instant after 10 seconds. Table 2: Fin material properties. Density Young’s modulus Poisson’s ration Specific heat Coefficient of thermal expansion 280 MPa Tensile yield strength 310 MPa Tensile ultimate strength 280 MPa Compressive yield strength s 3 2700 kg / m  s E 71000 MPa  s 0.33  s p c 925J / KgK  *s 5 1 2.3 10 C      Figure 1: Three-dimensional annular fin geometry. B. Governing Equations and Boundary Conditions B. Governing Equations and Boundary Conditions 1) Fluid domain 1) Fluid domain Equations governing heat transfer and flow under transient, turbulent, three-dimensional, and incompressible conditions are (Nemati & Samivand, 2016). A Continuity equation: (1) y q i i ( u ) 0 t x        (1) B- Momentum equation: B- Momentum equation: In Eqn. (2), μ is the dynamic viscosity, μr is the turbulent viscosity, and k is the turbulent kinetic energy. Figure 1: Three-dimensional annular fin geometry. (2) HATAMI et al: EFFECTS OF ANNULAR-FINNED TUBE DEFORMATION ON THERMAL STRESSES 301 HATAMI et al: EFFECTS OF ANNULAR-FINNED TUBE DEFORMATION ON THERMAL STRESSES 301 C- Energy equation: * rr rr 2 * rr 2 r r E [ (1 ) T] 1 E [ (1 ) T] 1 E 1                     (3) C- Energy equation: i t i i i ( E p) T (u ( E p)) (k k ) t x x x                   In Eqn. (3), E is the total energy. D-Boundary conditions: i Inlet and outlet boundary conditions: * rr rr 2 * rr 2 r r E [ (1 ) T] 1 E [ (1 ) T] 1 E 1                     (7) (3 gy q i t i i i ( E p) T (u ( E p)) (k k ) t x x x                   (7) In Eqn. (3), E is the total energy. In Eqn. (3), E is the total energy. ii. Constitutive equations: (5) * rr rr 2 * rr 2 r r E [ (1 ) T] 1 E [ (1 ) T] 1 E 1                     (9) iii. The no-slip condition defined for the tube and the fin is: (9) (6) f i u 0  f i u 0  (6) (6) In Eqns. (5) and (6), f iu is the component i of the fluid velocity. i. Equilibrium equations: In the absence of body forces, the two equilibrium equations are as follows: (4) (4) (4) r rr rr r r ( ) 1 0 r r r 2 1 0 r r r                     (8) (8) ii. The symmetry boundary condition for the surfaces shown in Figs. 1 and 2 are as follows: where σrr and σθθ are the radial and tangential stresses, respectively. where σrr and σθθ are the radial and tangential stresses, respectively. (5) f f i i 2 3 f f 2 3 u u 0, 0 x x T T 0, 0 x x             ii. Constitutive equations: D-Boundary conditions: i. Inlet and outlet boundary conditions: In Eqn. (7), s T is the temperature, s is the density, ks is the thermal conductivity, and s pc is the special heat capacity of the fin. Periodic boundary conditions for the inlet and outlet (Patankar, et al., 1977) are as follows: i. Equilibrium equations: i. Equilibrium equations: ii. Solid-Fluid Interface Domain ii. Solid-Fluid Interface Domain ii. Solid-Fluid Interface Domain ii. Solid-Fluid Interface Domain The following boundary condition holds over the fin– fluid interface. (14) (14) where n is the direction normal to the surface. where n is the direction normal to the surface. Figure 4: The magnified mesh for the rectangular region. A. Grid The grid types used in the solution domain for the fins and the fluid are illustrated in Figures 3 and 4, respectively. Figure 3: Structured mesh for the fin. Figure 4: The magnified mesh for the rectangular region. 2) Solid domain (fin) where E denotes the elastic modulus,  is Poisson’s ratio, and   is the thermal expansion coefficient. where E denotes the elastic modulus,  is Poisson’s ratio, and   is the thermal expansion coefficient. The equations governing the solid domain include energy, equilibrium, constitutive, and strain–displacement equations. An important assumption here is to consider the energy equation to be independent of the equilibrium, constitutive, and strain–displacement equations. In other words, the energy equation is solved first using the thermal boundary conditions. The resulting temperature is used subsequently in solving the other three equations. iii. Strain–displacement equations: iii. Strain–displacement equations: (10) r r r rr r u u u u u u 1 1 1 , , ( ) r r r 2 r r r                     (10) Moreover, the fins physical properties were assumed to be uniform and isotropic. Given the small fin thickness, the equations governing the solid domain were solved using the plane-stress assumption. A constant temperature thermal boundary condition was used at the fin base and a coupled boundary condition between the fin and the fluid to solve the energy equation. The zero-radial-stress boundary condition was used to solve the equilibrium, constitutive, and strain– displacement equations for the fin base and the outer radius. Moreover, the axial stress was assumed to be zero normal to the fin (in the z direction). The solid domain equations are presented as follows. iv. Boundary conditions for the equilibrium, constitutive, and strain–displacement equations: iv. Boundary conditions for the equilibrium, constitutive, and strain–displacement equations: (11) (11) where ur, uθ, and uz are displacements along r, θ, and z, respectively. where ur, uθ, and uz are displacements along r, θ, and z, respectively. where ur, uθ, and uz are displacements along r, θ, and z, respectively. p y Srr and Sθθ are defined as given in Eq. (12) and (13) respectively. i. Energy equation and its boundary conditions: 302 NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 19, NO.4, DECEMBER 2022 Figure 4: The magnified mesh for the rectangular region. (12) (13) (12) (13) (13) 2) Fluid domain 2) Fluid domain For the fluid domain, validation was performed by comparing the results of the present numerical solution with the experimental (Verein Deutscher Ingenieure, 2000) and numerical (Mon, 2003) results. All conditions are similar to the i1 case in page 152 from Mon,s thesis (Mon, 2003). 1 0.6 0.15 3 t A Nu 0.22Re Pr ( ) , Maximum deviation 25%, A    (15) 1 0.6 0.15 3 t A Nu 0.22Re Pr ( ) , Maximum deviation 25%, A    (15) Chiu and Chen [3] Present Work (15) IV. RESULTS AND DISCUSSION This section discusses the effect of changes in rh on the temperature and effective stress. Figure 7 shows the directions of the angles. temperature and effective stress. Figure 7 shows the directions of the angles. Figure 6. Comparison of Tangential stresses. Table 3: Comparison of temperature values (Kelvin) at specific Table 3: Comparison of temperature values (Kelvin) at specific Solution method Roy & Ghosal, 2017) Present work 578.52 578.45 566.28 566.35 560.19 560.19 558.3 558.36 Table 3: Comparison of temperature values (Kelvin) at specific locations (insulated boundary condition for the fin tip and convection boundary condition for the fin’s top and bottom surfaces). p p p locations (insulated boundary condition for the fin tip and convection boundary condition for the fin’s top and bottom surfaces). Solution method Roy & Ghosal, 2017) Present work 578.52 578.45 566.28 566.35 560.19 560.19 558.3 558.36 Table 4: Comparison of temperature values (Kelvin) at specific locations (convection boundary condition for the fin tip and its top and bottom surfaces). Solution method (Chiu & Chen, 2002) 557.070 563.724 556.613 554.022 (Roy & Ghosal, 2017) 557.826 565.053 558.482 556.513 (Peng & Chen, 2011) 576.99 563.88 556.86 554.31 Present work 576.98 563.90 556.86 554.33 Table 4: Comparison of temperature values (Kelvin) at specific locations (convection boundary condition for the fin tip and its top and bottom surfaces). Figure 6: Comparison of tangential stresses. B. Validation 1) Solid domain In order to validate the solid-domain results, the radial and tangential stresses and the annular fin temperature were compared against similar reports from the literature (Chiu & Chen, 2002; Roy & Ghosal, 2017; Peng & Chen, 2011). As evident from Fig. 5, the radial stress results are consistent with the results of (Chiu & Chen, 2002) with a 0.3% relative error. Also, reasonable accuracy was observed in the tangential stress as shown in Fig. 6. The temperature distribution results are also compared in Tables 3, 4, 5, 6 and 7 with corresponding results from the literature (Chiu & Chen, 2002; Roy & Ghosal, 2017; Peng & Chen, 2011). According to (Chiu & Chen, 2002; Roy & Ghosal, 2017; Peng & Chen, 2011), b e b r r r r    and b r r   . h for the fin. 1 1.5 2 2.5 3 -6 -5 -4 -3 -2 -1 0 RadialStresses, S  Present Work Chiu and Chen [3] Without Fluid Flow h = 50 W/mK 2   rr  Figure 5: Comparison of radial stresses. b r r   . Figure 3: Structured mesh for the fin. 1 1.5 2 2.5 3 -6 -5 -4 -3 -2 -1 0 RadialStresses, S  Present Work Chiu and Chen [3] Without Fluid Flow h = 50 W/mK 2   rr  Figure 5: Comparison of radial stresses. Present Work Chiu and Chen [3] Figure 5: Comparison of radial stresses. 303 HATAMI et al: EFFECTS OF ANNULAR-FINNED TUBE DEFORMATION ON THERMAL STRESSES Figure 6. Comparison of Tangential stresses. 1 1.5 2 2.5 3 -40 -30 -20 -10 0 10 Tangential Stresses, S Chiu and Chen [3] Present Work Without Fluid Flow h = 50 W/mK       Figure 6 Comparison of Tangential stresses 1 1.5 2 2.5 3 -40 -30 -20 -10 0 10 Tangential Stresses, S Chiu and Chen [3] Present Work Without Fluid Flow h = 50 W/mK       A. Temperature Variations Figure 8 shows temperature contours in the fin for circular and elliptical tubes with respect to rh. These contours show that increasing rh reduced temperature gradient in the radial direction. The reduction was even more substantial at 0°, which corresponds to the maximum effective stress (Hosseini, et al., 2020). It is also observed that the temperature gradient intensified in the  direction as tube deformed. Table 5: Comparison of radial stresses values at specific locations (insulated boundary condition for the fin tip and convection boundary condition for the fin’s top and bottom surfaces). Table 5: Comparison of radial stresses values at specific locations (insulated boundary condition for the fin tip and convection boundary condition for the fin’s top and bottom surfaces). Solution method (Chiu & Chen, 2002) 0 -6.20 -4.60 -1.95 0 Present work 0 -6.21 -4.60 -1.95 0 Solution method (Chiu & Chen, 2002) -34.62 -6.49 4.90 9.79 10.41 Present work -34.62 -6.50 4.90 9.79 10.41 Table 6: Comparison of Tangential stresses values at specific locations (insulated boundary condition for the fin tip and convection boundary condition for the fin’s top and bottom surfaces). Nu (Verein Deutscher Ingenieure, 2000) Nu (Mon, 2003) Nupresent work Errwith Experimental Errwith Numerical 34.00 34.40 32.21 5.26% 6.37% Table 7: Fluid-domain validation results. Table 6: Comparison of Tangential stresses values at specific locations (insulated boundary conditio for the fin tip and convection boundary condition for the fin’s top and bottom surfaces). Table 6: Comparison of Tangential stresses values at specific locations (insulated boundary condition for the fin tip and convection boundary condition for the fin’s top and bottom surfaces). Solution method (Chiu & Chen, 2002) -34.62 -6.49 4.90 9.79 10.41 Present work -34.62 -6.50 4.90 9.79 10.41 p y p ) Nu (Verein Deutscher Ingenieure, 2000) Nu (Mon, 2003) Nupresent work Errwith Experimental Errwith Numerical 34.00 34.40 32.21 5.26% 6.37% Table 7: Fluid-domain validation results. NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 19, NO.4, DECEMBER 2022 304 = 20mm hr mm 22 = hr Figure 8: Temperature contours of the fin. mm 24 = hr mm 26 = hr = 20mm hr mm 22 = hr Figure 8: Temperature contours of the fin. mm 24 = hr mm 26 = hr Figure 8: Temperature contours of the fin. It is observed that the peak radial stress occurred in the same areas with the steepest radial temperature gradient. A. Temperature Variations Figure 10 indicate that the largest absolute values of the tangential stress before and after deformation occurred at the fin base, where it is attached to the tube. Furthermore, the results show that the tangential stresses are predominantly affected by temperature. B. Radial, Tangential and Shear Stress Variations B. Radial, Tangential and Shear Stress Variations B. Radial, Tangential and Shear Stress Variations Figures 9–11 depict radial, tangential, and shear stress contours. These contours show that radial and tangential stresses decreased as the initially circular tube deformed into an elliptical shape and the shear stress increased due to the temperature gradient being intensified along . = 20mm hr mm 22 = hr = 20mm hr mm 22 = hr HATAMI et al: EFFECTS OF ANNULAR-FINNED TUBE DEFORMATION ON THERMAL STRESSES 305 Figure 10: Tangential stress contours. mm 24 = hr mm 26 = hr Figure 9: Radial stress contours. mm 24 = hr mm 26 = hr = 20mm hr mm 22 = hr Figure 9: Radial stress contours. mm 24 = hr mm 26 = hr Figure 9: Radial stress contours. mm 24 = hr mm 24 = hr Figure 9: Radial stress contours. = 20mm hr mm 22 = hr Figure 10: Tangential stress contours. mm 24 = hr mm 26 = hr Figure 10: Tangential stress contours. NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 19, NO.4, DECEMBER 2022 306 NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 19, NO.4, DECEMBER 2022 = 20mm hr mm 22 = hr mm 24 = hr mm 26 = hr Figure 11: Shear stress contours. Figure 11: Shear stress contours. C. Effective Strain and Strain Variations Figure 12 shows that the tube deformation from a circular to an elliptical shape reduced the effective stress by 9%. The maximum effective stress occurred at the fin base. It was also found that the tube deformation changed the distribution of effective stress in the fin along  due to an increase in the shear stress. The effective thermal stress serves as the criterion for determining the critical points in terms of strength and deformation. Figure 12 depicts effective stress contours. The effective stress equation used in this study is presented in Eqn. 16. (16) mm = 22 hr mm = 20 hr (16) mm = 22 hr mm = 20 hr HATAMI et al: EFFECTS OF ANNULAR-FINNED TUBE DEFORMATION ON THERMAL STRESSES 307 mm = 24 hr Figure 12: Effective stress contours. mm = 26 hr mm = 24 hr Figure 12: Effective stress contours. mm = 24 hr Figure 12: Effective stress contours. Figure 12: Effective stress contours. fins with temperature-dependent thermal conductivity. Acta Mechanica, 157, 147-158. fins with temperature-dependent thermal conductivity. Acta Mechanica, 157, 147-158. AUTHOR CONTRIBUTIONS Study concept and design: M. Kashani, and A. Hatami; analysis and interpretation of data: M. Kashani, and M. Hosseini; drafting of the manuscript: M. Kashani; critical revision of the manuscript for important intellectual content: M. Kashani, A. Hatami, and M. Hosseini; statistical analysis: M. Kashani. Jang, J. Y. and Yang, J.-. Y. (1998). Experimental and 3-D numerical analysis of the thermal-hydraulic characteristics of elliptic finned-tube heat exchangers. Heat Transfer Engineering, 19(4), 55-67. Jang, J. Y.; J. T. Lai and L. C. Liu. (1998).The thermal-hydraulic characteristics of staggered circular finned- tube heat exchangers under dry and dehumidifying conditions. International journal of heat and mass transfer, 41(21), 3321- 3337. M. Kashani, A. Hatami, and M. Hosseini; statistical analysis: M. Kashani. M. Kashani. V. CONCLUSION Hatami, A.; P. Samira and M. Hosseini. (2020). Numerical analysis of thermal stresses and strains of annular finned tube bundle in turbulent flow regime. Mechanics & Industry, 21(6), 601. The effects of deformation of an annular finned tube from a circular to an elliptical shape on its thermal stresses were investigated and critical points of the fin in terms of thermal stress were determined. The finite-volume method and Transition SST model were coupled with the SIMPLE algorithm to solve flow equations, while the thermal stress equations were solved in the solid domain by the finite-element method. The results can be summarized as follows: Hatami, M. and Ganji, D. D. (2013). Thermal performance of circular convective–radiative porous fins with different section shapes and materials. Energy Conversion and Management, Volume 76, 185-193. Management, Volume 76, 185-193.  Overall, it is concluded that using elliptical-finned tubes significantly reduces the effective stress. Hosseini, M.; A. Hatami and S. Payan. (2020). Comparison of the effect of laminar and turbulent flow regimes on thermal stresses and strains in an annular fin. Journal of Mechanical Science and Technology, 34(1), 413-424.  The maximum effective stress before and after deformation consistently remained at the base of the fin. echanical Science and Technology, 34(1), 413-424  Tube deformations reduced radial and tangential stresses while increasing shear stress. Hosseini, M.; A. Hatami and S. Payan. (2020). Impact of Flow around Annular Fins on their Thermal Stresses and Strains. Amirkabir J. Mech. Eng., 52(1), 51-54.  The deformation caused the temperature gradient to decrease along r but intensified it along . Hu, X. and Jacobi, A. M. (1993). Local Heat Transfer Behavior and Its Impact on a Single-Row, Annularly Finned Tube Heat Exchanger. ASME. J. Heat Transfer, 115(1), 66-74. REFERENCES Aziz, A., (1993). The effects of internal heat generation, anisotropy, and base temperature nonuniformity on heat transfer from a two-dimensional rectangular fin. Heat Transfer Engineering, 14(2), 63-70. Aziz, A., (1993). The effects of internal heat generation, anisotropy, and base temperature nonuniformity on heat transfer from a two-dimensional rectangular fin. Heat Transfer Engineering, 14(2), 63-70. Kundu, B. and Das, P. K. (2007). Performance analysis and optimization of elliptic fins circumscribing a circular tube. International journal of heat and mass transfer, 50(1-2), 173- 180. Bidzard, A.; M. Mahzoon and M. Yaghoubi. (2017). Analytical Solution for Temperature, Stress and Displacement Fields for a Hollow Cylinder Subjected to Asymmetric and Time Dependent Heat Flux. Amirkabir J. Mech. Eng., Volume 49, 253-260. Lau, W. and Tan, C. W. (1973). Errors in One- Dimensional Heat Transfer Analysis in Straight and Annular Fins. ASME. J. Heat Transfer, 95(4), 549–551. Mon, M. S. (2003). Numerical investigation of air-side heat transfer and pressure drop in circular finned-tube heat exchangers, s.l.: Freiberg. Bilirgen, H.; S. Dunbar and E. K. Levy. (2013). Numerical modeling of finned heat exchangers. Applied Thermal Engineering, 61(2), 278-288. Mon, M. S. and Gross, U. (2004). Numerical study of fin-spacing effects in annular-finned tube heat exchangers. Chiu, C. H. and Chen, C. K. 2002. Application of the decomposition method to thermal stresses in isotropic circular 308 NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 19, NO.4, DECEMBER 2022 International journal of heat and mass transfer, 47(8-9), 1953- 1964. International journal of heat and mass transfer, 47(8-9), 1953- 1964. quadratic temperature-dependent thermal conductivity. Journal of Thermal Stresses, 40(17), 828-845. Nagarani, N.; K. Mayilsamy; A. Murugesan and G. S. Kumar. (2014).Review of utilization of extended surfaces in heat transfer problems. Renewable and Sustainable Energy Reviews, 29, 604-613. Roy, R. and Ghosal, S. (2017). Homotopy Perturbation Method for the Analysis of Heat Transfer in an Annular Fin with Temperature-Dependent Thermal Conductivity. ASME. J. Heat Transfer, 139(2), 022001. Nemati, H. and Moghimi, M. (2014). Numerical study of flow over annular-finned tube heat exchangers by different Nemati, H. and Moghimi, M. (2014). Numerical study of flow over annular-finned tube heat exchangers by different turbulent models. CFD Letters, 6(3), 101-112. Sharma, R.; L. Sondhi; V. K. Gaba and S. Bhowmick. (2019). A Study of Thermo-structural Behavior of Annular Fin. In: Innovative Design, Analysis and Development Practices in Aerospace and Automotive Engineering. Singapore: Springer, 381-387. Nemati, H. and Samivand, S. (2016). REFERENCES Numerical study of flow over annular elliptical finned tube heat exchangers. Arabian Journal for Science and Engineering, 41(11), 4625- 4634. Shokouhmand, H.; S. Mahjoub and M. R. Salimpour. (2014).Constructal design of finned tubes used in air-cooled heat exchangers. Journal of Mechanical Science and Technology, 28(6), 2385-2391. Patankar, S. V.; H. Cheng; H. L. Liu and M. S. Ephraim. (1977).Fully developed flow and heat transfer in ducts having streamwise-periodic variations of cross-sectional area. J. Heat Transfer, 99(2), 180-186. Verein Deutscher Ingenieure. (2000). VDI- Wärmeübergang, Berechnungsblätter fürden Wärmeübergang. 8 ed. Berlin: Springer. Peng, H. -S. and Chen, C. -L. (2011). Hybrid differential transformation and finite difference method to annular fin with temperature-dependent thermal conductivity. International Journal of Heat and Mass Transfer, 54((11-12)), 2427-2433. Wu, S. -S., (1997). Analysis on transient thermal stresses in an annular fin. Journal of thermal stresses, 20(6), 591-615. Yu, L. & Chen, C., (1999). Application of the Hybrid Method to the Transient Thermal Stresses Response in Isotropic Annular Fins. ASME. J. Appl. Mech., 66(2), 340– 346. Roozbahani, M. M.; H. Razzaghi; M. Baghani; M. Baniassadi and M. Layeghi. (2017).Temperature and stress distribution in hollow annular disk of uniform thickness with
https://openalex.org/W4319746568
https://journals.us.edu.pl/index.php/PLS/article/download/13140/11228
Polish
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Macedoński fenomen wzrostu liczby tłumaczeń z języka polskiego w latach 1990-2020
Przekłady Literatur Słowiańskich
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cc-by-sa
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Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. ABSTRACT  |  This article presents the rise of translated Polish literature to the Mac- edonian language in the thirty years period since Macedonia gained independence from Yugoslavia and transformed from a socialist to a capitalist system. The rise of private publishing houses automatically meant the rise of translating from other languages; in addition, the rise of foreign funds and especially the financial support by the EU in culture was a relevant factor for developing literary translations from Polish to the Macedonian language. The main part of this article is about the works which have been translated, their importance and the methods used in making the choices on what to translate. KEYWORDS  |  Polish literature, translating, publishing, Macedonian language, re­ ception artykuły i rozprawy Ostatnie trzydzieści lat w przestrzeni macedońskiej było punktem zwrotnym dla wielu działań społecznych, w tym również dla humanistyki. Wraz z dojściem do głosu kapitalizmu po rozpadzie Jugosławii powstało wiele prywatnych i małych wydawnictw o różnych sympatiach i zainteresowaniach. Odzyskanie niepod- ległości przyczyniło się do zwiększenia liczby książek wydawanych w języku macedońskim, co znacząco wypełniło lukę, jaka powstała w okresie powojen- nym, kiedy to brak literatury pięknej w języku macedońskim rekompensowano literaturą w przekładzie na jeden z pozostałych języków społeczności jugosło- wiańskiej. Niezależność państwa dała szansę na powiększenie się reprezentacji literatury polskiej na macedońskim rynku wydawniczym. Znaczący wzrost liczby polskich dzieł tłumaczonych na język macedoński zaobserwowano po raz pierwszy pod koniec 2013 roku. Przeprowadzono wówczas przegląd lite- ratury tłumaczonej z języka polskiego. Badania wykazały, że w ciągu zaledwie pięciu lat, czyli od 2009 do grudnia 2013 roku1, ukazało się dwadzieścia pięć dzieł literackich tłumaczonych z języka polskiego. Porównując badane pięć lat z okresem je poprzedzającym, zauważamy, że w Republice Macedonii w ciągu dwudziestu lat, tj. od 1988 do 2008 roku, wydano w sumie dwadzieścia pięć przekładów dzieł autorów polskich. Oznacza to wzrost o prawie 400 procent rocznie. A to, trzeba przyznać, imponująca liczba przykładów literatury polskiej w macedońskiej przestrzeni literackiej. Przekłady literatury polskiej wydane w latach: 2009—2013 (25 dzieł) 1988—2008 (25 dzieł) 1. Кучок Војќех: Ѓубре — Македонска реч, 2009 1. Милош Чеслав: Заробениот ум. — Наша книга, 1988 2. Грохола Катажина: Никогаш повеќе — Македонска реч, 2009 2. Лем Станислав: Соларис — Детска радост, 1989 3. Милош Чеслав: Родната Европа — Микена, 2009 3. Косидовски Зенон: Кога сонцето беше бог — Наша книга, 1989 4. Сјенкјевич Хенрик: Избрани раскази — Микена, 2010 4. 1 Л. Танушевска, 2014: 25/5 — Книжевниот превод од полски јазик како позитивен феномен во преведувачката дејност во поново време. W: Годишен зборник бр. 39—40, Универзитет „Св. Кирил и Методиј” / Филолошки факултет „Блаже Конески”, Skopje, s. 655—659. Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. Мочарски Казимјеж: Разговори со џелатот — Култура, 1991 5. Сјенкјевич Хенрик: Крстоносци — Бегемот, 2010 5. Анджејевски Јежи: Апелација — Култура, 1995 6. Пјонтковска Моника: Валкани приказни — Македонска реч, 2010 6. Современа полска поезија — Скопје 1996 PLS.2022.12.01.14  |  s. 2 z 11 Lidija Tanuševska  |  Macedoński fenomen wzrostu liczby… 7. Сјенкјевич Хенрик: Скици нацртани со јаглен — Бегемот, 2011 7. Шимборска Вислава: Крај и почеток  — Детска радост, 1996 8. Сјенкјевич Хенрик: Без догма — Или- или, 2011 8. Вавжкјевич Марек: Доцна попладне. Избор — Скопје 1998 9. Виткјевич Стањислав: Збогување со есента — Македонска реч, 2011 9. Гжејшчак Маријан: Струга во светлината. Избор — СВП, 1998 10. Шулц Бруно: Продавниците со боја на цимет / Санаториумот во знакот на клепсидрата — Бегемот, 2011 10. Мицкјевич Адам: Лирски напеви — Струшки вечери на поезијата, 1998 11. Рејмонт Стањислав: Селани — Конгресен центар, Микена, Макавеј, 2012 11. Херберт Збигњев: Избор — Наше писмо, 1998 12. Гомбрович Витолд: Дневник 1953— 1958 — Конгресен центар, Микена, Макавеј, 2012 12. Гомбрович Витолд: Порнографија — ЕИН-СОФ, 2000 13. Барт Анджеј: Фабрика на смртта — Македонска реч, 2012 13. Твардовски Јан: Љубовта љубов бара — Скопје 2000 14. Токарчук Олга: Талкачи — Или-или, 2013 14. Адамчевски Јан: Легенди за стариот Краков — СС на Флф „Блаже Конески”, 2002 15. Дукај Јацек: Мраз — Бегемот, 2013 15. Кјешловски К.; Пјешевич К.: Декалог — Аз-Буки, 2002 16. Крајевски Марек: Смрт во Бреслау — Бегемот, 2013 16. Колаковски Лешек: 13 Сказни од кралството Лајлонија за големи и мали и други сказни — Лист, 2002 17. Гутовска-Адамчик Малгожата:  110 улици — Македонска реч, 2013 17. Шулц Бруно: Продавници со боја на цимет — Темплум, 2004 18. Батор Јоана: Песочна планина — Македонска реч, 2013 18. Херберт Збигњев: Патот до вообразбата —Макавеј, 2005 19. Милош Чеслав: Заробениот ум — Три, 2013 19. Билски Ришард: Пожари над Тетово — Култура, 2006 20. Сјенкјевич Хенрик: Quo vadis — Три, 2013 20. Токарчук О., Хвин С., Сташук А.: Сон за недофатливото место. Фрагменти од современата полска проза — СС на Флф „Блаже Конески”, 2006 21. Варга Кшиштоф: Булеварот на Независноста — Антолог, 2013 21. Херлинг-Груѓински Густав: Бела ноќ на љубовта —Или—или, 2006 22. Гомбрович Витолд: Космос — Бегемот, 2013 22. Дубоа Јацек: И сè заради фараонот — Детска радост, 2007 PLS.2022.12.01.14  |  s. 3 z 11 artykuły i rozprawy 23. Норвид Ципријан: Цивилизација — Бегемот, 2013 23. Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. Сјенкјевич Хенрик: Quo vadis — Матица македонска, 2007 24. Гомбрович Витолд: Транс- Атлантик — Бегемот, 2013 24. Токарчук Олга: Правек и другите времиња —Македонска реч, 2007 25. Грабињски Стефан: Мистични приказни — Бегемот, 2013 25. Гутовска-Адамчик Малгожата:  220 линии — Детска радост, 2008 Jednym z powodów takiego wzrostu zainteresowania wydawaniem literatury polskiej jest wsparcie finansowe polskiego Ministerstwa Spraw Zagranicznych, w ramach którego działa Instytut Książki, a także wsparcie Programu Kul- turalnego Unii Europejskiej czy rządowe projekty przekładu literatury obcej: Нобеловци (Nobliści) i Ѕвезди на светската книжевност (Gwiazdy Literatury Światowej). Wzrost ten przypisuje się przede wszystkim działalności prywat- nych wydawnictw zainteresowanych literaturą polską, zwłaszcza wydawnictwa Begemot, którego właściciel jest tłumaczem z polskiego na macedoński i wywo- dzi się ze szkoły polonistycznej Wydziału Filologicznego Uniwersytetu Świę- tych Cyryla i Metodego w Skopju. Oczywiście publikowaniem dzieł polskich autorów zajmują się również inne oficyny, takie jak: Makedonska recz, Ili-ili, Antolog, a ostatnio także Wostok, Bata Press, Tri, Goten, Slavika Libris i in. W ciągu kolejnych sześciu lat (do 2020 roku) liczba przekładów literackich z języka polskiego wzrosła jeszcze bardziej, ponad dwukrotnie od ostatniego spisu w 2014 roku, a więc w ciągu sześciu lat ukazało się około sześćdziesięciu dzieł polskich autorów. To w sumie setki przekładów na język polski wydanych przez trzydzieści lat, co niewątpliwie świadczy o rewolucyjnym zwrocie w tłu- maczeniu. Wynika to także z ogromnego zaangażowania tłumaczy w realizację całego procesu. Mówi się, że tłumaczenie to proces, który wzbogaca nasz język i przedłuża jego życie, więc tłumacze niewątpliwie mają w tym swój nieoceniony udział. O ile w okresie przed odzyskaniem niepodległości aktywnie działało tylko kilku tłumaczy z języka polskiego — głównie osoby, które ukończyły edu- kację w Polsce podczas ich (lub ich rodziców) emigracji z egejskiej części Grecji, jak np. Petre Nakovski i Grigori Poposki, o tyle w ostatnich latach pojawiło się wielu młodszych tłumaczy. Należy w tym miejscu wspomnieć o polonistach starszego pokolenia, takich jak Zdravko Stamatovski, Aneta Stamatovska, Dejan Gegovski, Milica Mirkulovska i Lidija Tanuševska. Działa już także solidna grupa młodych tłumaczy, którzy stopniowo stają się doświadczonymi profe- sjonalistami, z powodzeniem mierzącymi się z translatorskimi wyzwaniami. Większość z nich to absolwenci skopijskiej polonistyki Wydziału Filologicz- nego im. Blaże Koneskiego: Aneta Todevska, Filip Dimevski, Zvonko Dimoski, Ilija Trajkovski, Slobodan Krstevski i in., którzy uczyli się swego rzemiosła od obecnej kadry naukowo-dydaktycznej polonistyki: Milicy Mirkulovskiej i Lidiji PLS.2022.12.01.14  |  s. 4 z 11 Lidija Tanuševska  |  Macedoński fenomen wzrostu liczby… Tanuševskiej. Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. Niektórzy z tych tłumaczy nie kontynuują swojej przekładowej działalności, kończąc na jednej, dwóch lub trzech książkach, natomiast inni są już rozpoznawalni jako wysokiej klasy tłumacze. Są też tacy, którzy próbowali wykorzystać swoją dwujęzyczność mimo innej specjalizacji: Mihail Barakov- ski (przełożył Fabrykę muchołapek Andrzeja Barta) czy Maciej Kaczorowski (przełożył 13 bajek z królestwa Lailonii dla dużych i małych i inne bajki Leszka Kołakowskiego). W  ciągu ostatnich trzydziestu lat wypełniono lukę brakujących dzieł z kanonu literatury polskiej, ale tłumaczono także autorów nowych i popu- larnych na polskiej scenie literackiej. Sienkiewicz był obecny na rynku mace- dońskim tylko ze starszym tłumaczeniem Quo vadis, ale do 2014 roku na język macedoński zostało przetłumaczonych pięć innych jego dzieł. Jego realistyczne powieści i nowele — przełożone przez dwoje tłumaczy — stanowią interesu- jący materiał do analizy z punktu widzenia teorii przekładu. Są to Wybrane opowiadania wydane w ramach rządowego projektu Nobliści oraz zbiór Szkice węglem — przetłumaczone przez Milicę Mirkulovską oraz Filipa Dimevskiego. Ze słynnych powieści historycznych Sienkiewicza po raz pierwszy na język macedoński została przełożona powieść Krzyżacy (tłum. Filip Dimevski), a po raz trzeci ukazało się Quo vadis w przekładzie Slobodana Krstevskiego. Jedna z dwóch „współczesnych” powieści noblisty — Bez dogmatu — w tłumaczeniu Lidiji Tanuševskiej została wydana w Macedonii po raz pierwszy. Biorąc pod uwagę, że 2014 rok był Rokiem Henryka Sienkiewicza, można powiedzieć, że tłumacze oddali hołd temu wielkiemu polskiemu, ale też światowemu pisarzowi. W 2018 roku nakładem wydawnictwa Begemot ukazało się kapitalne wydanie Ogniem i mieczem w tłumaczeniu Filipa Dimevskiego. W 2014 roku po raz pierwszy opublikowano utwór innego polskiego noblisty z początku XX wieku — Władysława Reymonta, tj. powieść Chłopi w tłumacze- niu Petre Nakovskiego. Dzieło to realistycznie przedstawia ówczesne życie pol- skiej wsi, a jego język jest specyficzny ze względu na użycie — nie tylko w dia- logach, lecz także w narracji — gwary, z której Reymont stworzył uniwersalny język polszczyzny chłopów, co jest niewątpliwie dużym wyzwaniem dla tłu- macza. W kolejnych latach przetłumaczone zostały także: Komediantka (2015), Bunt (2017), Ziemia obiecana (2019) i Wampir (2019). Do 2017 roku dzieła Stefana Żeromskiego, Elizy Orzeszkowej czy Gabrieli Zapolskiej nie funkcjono- wały w języku macedońskim. Dzięki młodemu tłumaczowi, Iliji Trajkovskiemu, Ludzie bezdomni Żeromskiego, Ostatnia miłość Orzeszkowej i Menażeria ludzka Zapolskiej ukazały się w pięknie stylizowanym języku macedońskim. Ponadto wydany został przekład opowiadań Stefana Grabińskiego z lat 20. ubiegłego wieku, zbiór nosi tytuł Opowieści mistyczne. PLS.2022.12.01.14  |  s. 5 z 11 Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. 6 z 11 Lidija Tanuševska  |  Macedoński fenomen wzrostu liczby… W 2014 również po raz pierwszy w języku macedońskim ukazały się repor- taże Ryszarda Kapuścińskiego. Wydano Imperium, następnie Jeszcze dzień życia. Za przekład Imperium Fundacja im. Ryszarda Kapuścińskiego przyznała nagrodę tłumaczce Lidiji Tanuševskiej. Tym samym język macedoński stał się jednym z 39 języków, na jakie do tej pory przetłumaczono twórczość Kapuścińskiego. Wraz z pierwszym przekładem tekstu Kapuścińskiego w Macedonii zaczęto się interesować polskim reportażem literackim. Przetłumaczono książki współ- czesnych polskich autorów prozy non-fiction: Narzeczona Schulza i Oskarżona: Wiera Gran Agaty Tuszyńskiej, Gottland i Zrób sobie raj Mariusza Szczygła, a także Wszystkie dzieci Louisa Kamila Bałuka. Wiele z dzieł przetłumaczonych na macedoński we wspomnianym trzydzie- stoletnim okresie w Polsce zostało wydanych po 1989 roku, czyli po upadku komunizmu. Literatura — wolna od cenzury i psychologicznej presji wyraża- nia czegoś bądź nie — traci wspólny język i autorytety literackie, a wszystko w niej staje się prywatne, intymne. Każdy może pisać, o czym tylko chce, co prowadzi do eksplozji różnych zainteresowań i stylów literackich. Częstym kryterium wydawców w Macedonii przy wyborze polskich autorów i dzieł do tłumaczenia są zdobyte nagrody europejskie i polskie. Spośród polskich laurów najistotniejsza jest Nagroda Literacka Nike. Tak więc na początku XXI wieku jednym z najważniejszych wydarzeń przekładowych była powieść Gnój Wojcie- cha Kuczoka, przetłumaczona na język macedoński przez Filipa Dimevskiego i Natalię Łukomską. Niektórzy autorzy podejmują się wielkich osobistych pisar- skich wyzwań, jak na przykład Jacek Dukaj, który w obszernej powieści Lód tworzy alternatywną wersję historii początku XX wieku. Przekładu tego fascy- nującego dzieła na język macedoński podjął się Filip Dimevski. W ostatnich latach popularna staje się powieść kryminalna, w Polsce szcze- gólnie znana jako sposób opisu przeszłości. Dobrym przykładem są powieści Marka Krajewskiego, przedstawiające przedwojenny świat, za którym wiele osób odczuwa nostalgię. Filip Dimevski przetłumaczył Śmierć w Breslau i Koniec świata w Breslau. Jest także autorem przekładu Ofiary Polikseny Marty Guzow- skiej i Uwikłania Zygmunta Miłoszewskiego, a kontynuację tego ostatniego dzieła, Ziarno prawdy, przetłumaczyła Lidija Tanuševska. W  literaturze najnowszej na szczególną uwagę zasługuje fascynująca kobieca fala, a Olga Tokarczuk przebija się jako jej najważniejsza przedstawi- cielka. Powieści Prawiek i inne czasy, Bieguni i E.E. przełożyła Lidija Tanuševska. Pomimo zainteresowania wydawnictw twórczością Olgi Tokarczuk uzyskanie dofinansowania z Instytutu Książki stało się problematyczne po zmianie kie- rownictwa tej instytucji w 2016 roku. Ponadto, po przyznaniu autorce Nagrody Nobla zdobycie praw autorskich stało się trudniejsze dla wydawców w regionie. Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. Przetłumaczono także wybór prozy Cypriana Kamila Norwida oraz opowiadania Sławo- PLS.2022.12.01.14  |  s. 5 z 11 artykuły i rozprawy mira Mrożka. Żaden z tych autorów nie był wcześniej tłumaczony na język macedoński, z  wyjątkiem niektórych sztuk Mrożka na potrzeby spektakli teatralnych. W ciągu tych trzydziestu lat intensywnie tłumaczono utwory polskich przed- stawicieli awangardy, będących krzewicielami nowego literackiego wyrazu, form pisarskich, stylów i idei obowiązujących do dziś. Do drugiej dekady XXI wieku zostały przetłumaczone tylko fragmenty esejów i powieści Stanisława Ignacego Witkiewicza, ale w 2011 roku po raz pierwszy ukazała się w języku macedoń- skim powieść Pożegnanie jesieni w przekładzie Anety Todevskiej. W 2016 roku nakładem wydawnictwa Begemot opublikowano w dwóch tomach Nienasycenie w przekładzie Filipa Dimevskiego, a także, po raz pierwszy, kompletne wydanie zbiorów opowiadań Brunona Schulza Sklepy cynamonowe i Sanatorium pod klepsydrą oraz eseje, listy i grafiki Schulza zatytułowane Mityzacja rzeczywistości (tłum. Lidija Tanuševska). Dzieła Witolda Gombrowicza, do tej pory tłumaczone jedynie we frag- mentach w czasopismach literackich, w ciągu minionych trzydziestu lat były intensywnie przekładane. Ostatnio przetłumaczono całą prozaiczną twórczość literacką tego pisarza, głównie dzięki staraniom tłumacza Filipa Dimevskiego i wydawnictwa Begemot. Powieść Pornografia w tłumaczeniu Dejana Gegov- skiego ukazała się już w 1991 roku. Dziennik przełożył Petre Nakovski, a utwór został wydany w ramach wspomnianego już ministerialnego projektu Gwiazdy Literatury Światowej. Następnie w macedońskiej wersji językowej ukazały się powieści Kosmos, Trans-Atlantyk, Ferdydurke oraz zbiór opowiadań Bakakaj (tłum. Filip Dimevski). Poszukiwaniem tożsamości, a dokładniej tożsamości wschodnioeuropejskiej jako szczególnego zjawiska historycznego i społecznego w dziele Rodzinna Europa zajął się Czesław Miłosz, a tłumaczenia tej prozy podjęła się Milica Mirkulovska. Na macedoński został przełożony również zbiór esejów noblis­ty — Zniewolony umysł — jest to prawdopodobnie pierwszy przekład bezpośrednio z języka polskiego tych kultowych filozoficzno-politycznych tekstów opisują- cych stosunki w społeczeństwie polskim w okresie stalinizmu. Autorem prze- kładu jest Filip Dimevski. W 2016 roku z inicjatywy wydawnictwa Antolog i tłumaczki Milicy Mir- kulovskiej rozpoczął się projekt „100 lat od narodzin Stanisława Lema”, który miał swój finał w 2020 roku. W tym stosunkowo krótkim okresie ukazało się pięć przekładów dzieł Lema. Powstało nowe tłumaczenie Solaris, a Cyberiada, Eden i Niezwyciężony (prawdopodobnie pierwsze tłumaczenie bezpośrednio z języka polskiego) zostały wydane po raz pierwszy. Projekt przekładu dzieł Lema to ogromny wkład w promocję polskiej literatury w Macedonii i ważne wydarzenie na macedońskiej scenie literackiej. PLS.2022.12.01.14  |  s. PLS.2022.12.01.14  |  s. 7 z 11 Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. Mimo wszystko, choć z  wielkim trudem okupionym biurokratycznymi PLS.2022.12.01.14  |  s. 7 z 11 artykuły i rozprawy zmaganiami, przekłady dzieł Tokarczuk ukazały się po okresie będącym przed- miotem niniejszej analizy. zmaganiami, przekłady dzieł Tokarczuk ukazały się po okresie będącym przed- miotem niniejszej analizy. Ostatnio popularna staje się również pisarka Joanna Bator, a jej powieści Piaskowa góra i Ciemno, prawie noc wydano w przekładzie Anety Todevskiej- -Sapety. Powrotu do tematu Holokaustu, do skomplikowanych relacji polsko- -żydowskich w bardzo oryginalny sposób dokonują Andrzej Bart w powieści Fabryka muchołapek (przetłumaczonej jako Fabryka śmierci), a także Piotr Paziński w powieści Pensjonat — te dzieła również zostały wydane po macedoń- sku w omawianym czasie. Kwestia żydowska poruszona jest także we wspo- mnianych już przełożonych dziełach Agaty Tuszyńskiej. Niektórzy z  przetłumaczonych autorów w  najbardziej brutalny sposób oddają w swej twórczości trudną współczesność, ukazując życie takie, jakie jest naprawdę: rozkład, beznadziejność, zgnilizna i samotność, które towa- rzyszą człowiekowi do końca. Taka jest Aleja Niepodległości Krzysztofa Vargi. W  Nikczemnych historiach Moniki Piątkowskiej w  ten sam brutalny spo- sób w dwunastu odrębnych opowieściach opisane jest zło, nicość, brud człowie­- ka i poszukiwanie przyczyn, które mogą, ale nie muszą, usprawiedliwiać zbrod- nie. O upadku wartości moralnych piszą też Jerzy Pilch w Innych rozkoszach, Daniel Odija w Szklanej hucie i Dorota Masłowska w Wojnie polsko-ruskiej pod flagą biało-czerwoną. l Śmiałych przekładowych strategii podjęli się młodzi tłumacze w translacjach Lubiewa Michała Witkowskiego (tłum. Zvonko Dimoski), Morfiny Szczepana Twardocha (tłum. Ilija Trajkovski) i Sońki Ignacego Karpowicza (tłum. Filip Dimevski). W ostatnim czasie macedońskim wydawcom przyznano wiele grantów na literaturę dla dzieci, co wpłynęło na wyraźniejszą obecność tego gatunku litera- tury polskiej w Macedonii. Wydane w tych latach tłumaczenia to: 110 ulic Mał- gorzaty Gutowskej-Adamczyk, Czarownica piętro niżej Marcina Szczygielskiego, Humory Hipolita Kabla Roksany Jędrzejewskiej-Wróbel, Miś Uszatek Czesława Janczarskiego, Córka czarownic Doroty Terakowskiej i in. Nagrody literackie zawsze stanowią zachętę do przetłumaczenia większej liczby dzieł danego autora. Laureatem macedońskiej nagrody Złoty Wieniec podczas renomowanych Strużańskich Wieczorów Poezji w 2018 roku został Adam Zagajewski. Przyznanie jej przyczyniło się do opublikowania praktycz- nie całej twórczości polskiego poety. Organizatorzy Strużańskich Wieczorów Poezji we współpracy z europejską platformą poetycką Versopolis odpowiadają również za coroczne publikacje przekładów innych polskich poetów, takich jak m.in. Grzegorz Kwiatkowski czy Krystyna Dąbrowska. Odnośnie do tłuma- czeń polskiej poezji warto zauważyć, że Treny Jana Kochanowskiego zostały po PLS.2022.12.01.14  |  s. Data zgłoszenia: 24.01.2022 r.  |  Data akceptacji: 5.07.2022 r. 8 z 11 Lidija Tanuševska  |  Macedoński fenomen wzrostu liczby… raz pierwszy wydane w Antologii poezji światowej w 2015 roku w przekładzie Zdravka Stamatovskiego. g Zaprezentowany krótki przegląd przekładów literatury polskiej w Macedonii nie obejmuje wszystkich tłumaczeń dokonanych w ciągu ostatnich trzydziestu lat. Celem tekstu było bowiem zobrazowanie, jak bardzo wzrosła aktywność translatorska i o ile większe jest zainteresowanie literaturą polską w Macedo- nii, dzięki czemu obecność i widoczność polskiej twórczości na macedońskim rynku wydawniczym są coraz wyraźniejsze. Przetłumaczyła z macedońskiego Natalia Łukomska Przetłumaczyła z macedońskiego Natalia Łukomska Lidija Tanuševska Lidija Tanuševska Македонскиот феномен на подем на преведувачката дејност од полски јазик во периодот 1990—2020 г. Македонскиот феномен на подем на преведувачката дејност од полски јазик во периодот 1990—2020 г. Literatura Танушевска Л., 2009: Преводи на литературни дела  — библиографија. W: Педесет години на полонистиката во Македонија 1959—2009. Уни- верзитет „Св. Кирил и Методиј” / Филолошки факултет „Блаже Конески”, Skopje, s. 119—126. Танушевска Л., 2014: 25 / 5 — Книжевниот превод од полски јазик како пози- тивен феномен во преведувачката дејност во поново време. W: Годишен зборник бр. 39—40. Универзитет „Св. Кирил и Методиј” / Филолошки факултет „Блаже Конески”, Skopje, s. 655—659. Танушевска Л., 2017: Јазичната норма во книжевниот превод од полски на македонски јазик. W: Г. Цветановски, ред.: Македонистиката меѓу традицијата и современите предизвици. Зборник на трудови од Меѓуна- родната научна конференција одржана на 21 и 22 ноември 2013 година во Скопје. Институт за македонски јазик „Крсте Мисирков”, Skopje, s. 529—536. Македонскиот феномен на подем на преведувачката дејност од полски јазик во периодот 1990—2020 г. Резиме  |  Оваа статија е за рецепцијата на полската книжевност во Македонија и за промените во преведувачката политика во последните триесет години. Авторка та се обидува да ги издвои најважните библиографски единици од преведената литература и да даде една слика на тоа што и зошто се преведувало од полски јазик во овој период. Но, исто така, дава и еден кус преглед на мотивите и факторите кои влијаеле на овој неверојатен подем на преведувањето од полски јазик. Се разбира, PLS.2022.12.01.14  |  s. 9 z 11 artykuły i rozprawy разликите со претходниот период се очигледни, но исто така е важно да се нагласи дека полската литература во поново време станува значително позабележлива на книжевната сцена, што во голем дел е заслуга на амбициите на одделни издавачки куќи и преведувачи. КЛУЧНИ ЗБОРОВИ  |  полска литература, превод, македонски јазик, рецепција Lidija Tanuševska Macedonian Phenomenon of Rise of Translation Works from Polish Language in the Period Between Years 1990—2020 SUMMARY  |  This article deals with the summarization of the translated Polish litera- ture in the period of the past thirty years. The motive is the phenomenal rise of translated books caused by political but also financial and human factors. This text shows the most significant translated books, the innovations in the selection of the literature, the interests and eagerness of the translators and the comparison between this kind of activity nowa- days and the same one thirty years ago. Its goal is also to give an individual outlay of the reception by the author, who is a translator too. Many of the works presented here were translated for the first time long after their originals were published for the first time, and the reception is expected to differ a lot. This article also presents the new tendencies in Macedonian publishing of Polish literature. KEYWORDS  |  Polish literature, translation, publishing houses, reception, Macedonian language LIDIJA TANUŠEVSKA LIDIJA TANUŠEVSKA  |  prof. dr hab., pracuje w Katedrze Slawistyki na Wydziale Filologicznym im. Blaże Koneskiego Uniwersytetu Świętych Cyryla i Metodego w Skopju w Macedonii; prowadzi zajęcia z literatury polskiej, prak- tyki przekładu z języka polskiego na język macedoński oraz z przekładoznawstwa. Jej zainteresowania badawcze koncentrują się głównie na zagadnieniach prze- kładoznawstwa, ale również językoznawstwa porównawczego i literaturoznaw- stwa. Jest wybitną tłumaczką literatury polskiej na język macedoński. Została wyróżniona nagrodami translatorskimi za przekłady Prawieku i innych czasów Olgi Tokarczuk oraz opowiadań Brunona Schulza, a także Nagrodą im. Ryszarda Kapuścińskiego za przekład Imperium na nowy język (2015). W 2017 roku wydała monografię Przyczynek do gramatyki konfrontatywnej języka macedoń- skiego i języka polskiego. Przełożyła książki: Bieguni, Prowadź swój pług przez kości umarłych Olgi Tokarczuk, Bez dogmatu Henryka Sienkiewicza, Gottland Mariusza Szczygła, a także opowiadania i nowele Cypriana Kamila Norwida, Stefana Grabińskiego, Mikołaja Grynberga i in. PLS.2022.12.01.14  |  s. 10 z 11 Lidija Tanuševska  |  Macedoński fenomen wzrostu liczby… NATALIA ŁUKOMSKA  |  dr nauk humanistycznych w zakresie językoznaw- stwa, polonistka i slawistka, tłumaczka z języka macedońskiego, lektorka języka polskiego na Wydziale Filologicznym Uniwersytetu Świętych Cyryla i Metodego w Skopju. PLS.2022.12.01.14  |  s. 11 z 11
https://openalex.org/W4254471524
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0119209&type=printable
English
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Correction: Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice
PloS one
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CORRECTION OPEN ACCESS Citation: The PLOS ONE Staff (2015) Correction: Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice. PLoS ONE 10(3): e0119209. doi:10.1371/journal. pone.0119209 In Fig. 1B, phase contrast and GFP fluorescence images of 967B2, 957F1 and 979F1 are incorrect. Please view the correct Fig. 1 here. CORRECTION Correction: Generation and Characterization of Induced Pluripotent Stem Cells from Aid- Deficient Mice The PLOS ONE Staff In Fig. 1B, phase contrast and GFP fluorescence images of 967B2, 957F1 and 979F1 are incorrect. Please view the correct Fig. 1 here. Published: March 18, 2015 Copyright: © 2015 The PLOS ONE Staff. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 / 3 PLOS ONE | DOI:10.1371/journal.pone.0119209 March 18, 2015 PLOS ONE | DOI:10.1371/journal.pone.0119209 March 18, 2015 column) images are shown. Scale bars; 200 μm. (C, D) The number of GFP-positive colonies from Aid+/+, Aid+/and Aid/MEFs induced by 4Fs (C) and 3Fs (D). For each genotype, three different lots of MEFs were used in each experiment, and the experiments were repeated four times. Colonies were counted 25 (4Fs) and 30 (3Fs) days after the induction. (E) The number of total colonies from Aid+/+, Aid+/−and Aid−/−MEFs subjected to transduction of the 3Fs with or without Aid. (F) The proportion of GFP-positive colonies out of the total colonies from Aid+/+, Aid+/−and Aid−/−MEFs induced by 3Fs with or without Aid. (G) The number of GFP-positive colonies from Aid+/+ and Aid−/−primary B cells induced with 4Fs. Experiments were repeated five times. doi:10.1371/journal.pone.0119209.g001 PLOS ONE | DOI:10.1371/journal.pone.0119209 March 18, 2015 1. Shimamoto R, Amano N, Ichisaka T, Watanabe A, Yamanaka S, Keisuke O, et al. (2014) Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice. PLoS ONE 9(4): e94735. doi:10.1371/journal.pone.0094735 PMID: 24718089 Fig 1. Generation of iPS cells from Aid−/−mice. (A) The relative expression of Aid and Gapdh. Total RNA was isolated from three E B6ES and MG1.19), three Aid+/+ iPS cell clones (967B2, 967C1 and 979B1), three Aid−/−iPS cell clones (957F1, 979F1 and 979E1), and Aid−/−MEF clones and primary B cells (pB cells), and was used for the quantitative RT-PCR analysis. The data are shown as the morphology of Aid+/+ and Aid−/−iPS colonies 25 days after the introduction of 4Fs into MEFs. Phase contrast (left column) and GFP f Fig 1. Generation of iPS cells from Aid−/−mice. (A) The relative expression of Aid and Gapdh. Total RNA was isolated from three ES cell clones (RF8, B6ES and MG1.19), three Aid+/+ iPS cell clones (967B2, 967C1 and 979B1), three Aid−/−iPS cell clones (957F1, 979F1 and 979E1), three parental Aid+/+ and Aid−/−MEF clones and primary B cells (pB cells), and was used for the quantitative RT-PCR analysis. The data are shown as the average ± SD. (B) The morphology of Aid+/+ and Aid−/−iPS colonies 25 days after the introduction of 4Fs into MEFs. Phase contrast (left column) and GFP fluorescence (right Fig 1. Generation of iPS cells from Aid/mice. (A) The relative expression of Aid and Gapdh. Total RNA was isolated from three ES cell clones (RF8, B6ES and MG1.19), three Aid+/+ iPS cell clones (967B2, 967C1 and 979B1), three Aid−/−iPS cell clones (957F1, 979F1 and 979E1), three parental Aid+/+ and Aid−/−MEF clones and primary B cells (pB cells), and was used for the quantitative RT-PCR analysis. The data are shown as the average ± SD. (B) The morphology of Aid+/+ and Aid−/−iPS colonies 25 days after the introduction of 4Fs into MEFs. Phase contrast (left column) and GFP fluorescence (right PLOS ONE | DOI:10.1371/journal.pone.0119209 March 18, 2015 2 / 3 Reference 3 / 3
https://openalex.org/W3184567430
https://inserm.hal.science/inserm-03312491/file/s12916-021-02027-z.pdf
English
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Variability of multi-omics profiles in a population-based child cohort
BMC medicine
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To cite this version: Marta Gallego-Paüls, Carles Hernández-Ferrer, Mariona Bustamante, Xavier Basagaña, Jose Barrera- Gómez, et al.. Variability of multi-omics profiles in a population-based child cohort. BMC Medicine, 2021, 19 (1), pp.166. ￿10.1186/s12916-021-02027-z￿. ￿inserm-03312491￿ Variability of multi-omics profiles in a population-based child cohort Marta Gallego-Paüls, Carles Hernández-Ferrer, Mariona Bustamante, Xavier Basagaña, Jose Barrera-Gómez, Chung-Ho E Lau, Alexandros P Siskos, Marta Vives-Usano, Carlos Ruiz-Arenas, John Wright, et al. Variability of multi-omics profiles in a population-based child cohort Marta Gallego-Paüls1,2,3, Carles Hernández-Ferrer1,2,3, Mariona Bustamante1,2,3,4, Xavier Basagaña1,2,3, Jose Barrera-Gómez1,2,3, Chung-Ho E. Lau5,6, Alexandros P. Siskos7, Marta Vives-Usano1,2,3,4, Carlos Ruiz-Arenas1,2,3, John Wright8, Remy Slama9, Barbara Heude10, Maribel Casas1,2,3, Regina Grazuleviciene11, Leda Chatzi12, Eva Borràs2,4, Eduard Sabidó2,4, Ángel Carracedo13,14, Xavier Estivill4, Jose Urquiza1,2,3, Muireann Coen6,15, Hector C. Keun7, Juan R. González1,2,3, Martine Vrijheid1,2,3 and Léa Maitre1,2,3* © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Multiple omics technologies are increasingly applied to detect early, subtle molecular responses to environmental stressors for future disease risk prevention. However, there is an urgent need for further evaluation of stability and variability of omics profiles in healthy individuals, especially during childhood. Methods: We aimed to estimate intra-, inter-individual and cohort variability of multi-omics profiles (blood DNA methylation, gene expression, miRNA, proteins and serum and urine metabolites) measured 6 months apart in 156 healthy children from five European countries. We further performed a multi-omics network analysis to establish clusters of co-varying omics features and assessed the contribution of key variables (including biological traits and sample collection parameters) to omics variability. Results: All omics displayed a large range of intra- and inter-individual variability depending on each omics feature, although all presented a highest median intra-individual variability. DNA methylation was the most stable profile (median 37.6% inter-individual variability) while gene expression was the least stable (6.6%). Among the least stable features, we identified 1% cross-omics co-variation between CpGs and metabolites (e.g. glucose and CpGs related to obesity and type 2 diabetes). Explanatory variables, including age and body mass index (BMI), explained up to 9% of serum metabolite variability. Conclusions: Methylation and targeted serum metabolomics are the most reliable omics to implement in single time-point measurements in large cross-sectional studies. In the case of metabolomics, sample collection and individual traits (e.g. BMI) are important parameters to control for improved comparability, at the study design or analysis stage. This study will be valuable for the design and interpretation of epidemiological studies that aim to link omics signatures to disease, environmental exposures, or both. Keywords: Multi-omics, Exposome, Variability, Population study, Metabolomics, DNA methylation, Cross-omics, mRNA, miRNA, Children * Correspondence: lea.maitre@isglobal.org 1ISGlobal, Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain Full list of author information is available at the end of the article HAL Id: inserm-03312491 https://inserm.hal.science/inserm-03312491v1 Submitted on 2 Aug 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Gallego-Paüls et al. BMC Medicine (2021) 19:166 https://doi.org/10.1186/s12916-021-02027-z * Correspondence: lea.maitre@isglobal.org 1ISGlobal, Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain Full list of author information is available at the end of the article Background due to genetics, although some proteins are highly in- fluenced by body mass composition and acute inflam- mation [28]. g Characterizing early indicators of health and disease tra- jectories during pregnancy and childhood is at the core of the life course approach [1–4]. Early life englobes the most critical/sensitive periods for organ development, which makes it especially vulnerable to the effects of en- vironmental exposures [5, 6]. The integration of multiple omics data—such as epigenomics, transcriptomics, pro- teomics and metabolomics—is increasingly applied to detect early, subtle molecular responses to environmen- tal exposures because it employs a holistic view on all cellular processes [7–10]. However, there is an urgent need for further evaluation of stability and variability of omics profiles, between and within healthy children. Epi- demiological studies that incorporate omics profiles to monitor healthy individuals over time need to be in- formed of technical and biological variability in order to interpret changes in omics profiles, even if they are small. Omics variability may be determined by factors hindering subtle biological changes of interest, such as seasonality, individual characteristics (age and BMI), stage of life (i.e. hormones might vary between pre- puberty and adulthood stages) as well as by technical variability (due to measurement error and limited preci- sion of analytic tools), which therefore must be con- trolled at the design of the study [2, 5, 6]. To date, multi-omics platforms have mainly been used in studies with a small sample size that focused on diet- ary or physical activity interventions rather than follow- ing up healthy people from the general population [2, 29, 30]. They agreed that inter-individual variation, ra- ther than intra-individual variation, was the main ex- planatory factor for all omics measurements. However, previous multi-omics profile studies have not considered changes related to short and medium term. Especially, there is a lack of evidence regarding children from the general population, nor single or multi-omics studies, and the contribution of several factors such as age, sex and BMI. In the present study, we estimated intra- and inter- individual variability in multi-omics profiles (blood DNA methylation, gene expression, miRNA, proteins and serum and urine metabolites) in 156 children from five European countries at two time points with a 6-month interval. We further aimed to assess interrelationships between the variability in different omics layers by performing a cross- omics correlation network analysis. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 2 of 16 Page 2 of 16 Background Finally, we aimed to decompose the variance in multi-omics profiles according to (1) inter-individual characteristics: sex, ancestry, age, maternal education, Mediterranean diet quality index (KIDMED score) and zBMI (which do not change in a 6- month period); and (2) intra-individual characteristics: hours of fasting before the visit for blood/ urine sampling, heavy exercise practice the same day or the day before sampling, having a cold at the moment of the visit, hour of sampling, day of the week and season (Fig. 1). g y Temporal variability in omics profiles has been de- scribed previously to assess the reliability of single time-point measurements in cross-sectional studies and to understand ageing and disease processes [11, 12]. In this paper, we define “intra-individual variabil- ity” as the variability estimated within individuals over time and “inter-individual variability” as the between- individuals variability. We also define the “short-term” as a time span of hours or days, the “medium-term” as a time span of months, and the “long-term” when considering years. For example, in the short term, the metabolome in urine and blood is assumed to be more dynamic than other omics as it is the down- stream result of in vivo substances and environmental factor influence [4]. On the other hand, DNA methy- lation is generally considered to be the most stable omics profile over short periods of time [13] and could provide more valuable information for environ- mental epidemiological purposes than other omics, as some methylomics signatures (e.g. smoking signatures) have been shown to persist over time even when the exposure no longer exists [2, 13–19]. Previous studies have also shown low levels of intra-individual variabil- ity in > 95% of the gene expression profile and in 25.5% of the miRNAs analysed, which are proposed as good biomarkers for many human diseases [20– 27]. Overall, the blood proteome is considered quite stable over time with strong inter-individual variability Summary of laboratory processing of omics signatures, quality control and normalization We performed in-depth omics profiling at two time points ~ 6 months apart for all 156 children. Because only 87 children had complete data of all omics analyses at both visits, we decided to analyse each omics profile independ- ently (i.e. all paired samples available for each specific omics layer). The final sample size for each omics layer was 149, 105, 100, 149, 154 and 154 children for DNA methylation, blood gene expression, miRNA expression, proteins, serum metabolites and urine metabolites, re- spectively. Details on laboratory and data processing methods are available in Additional file 1 - Supplementary Methods [39–48]. While DNA methylation, gene and miRNA expression screenings were based on genome- wide arrays, the other methods were targeted or semi- targeted. From now on, we use the term “features” to refer to the omics variables in our study: CpGs, gene and miRNA transcripts, proteins and metabolites. Because our study did not have technical replicates (biological samples systematically aliquoted in two replicates before sample preparation), which would be the ideal way to measure technical variability, potential technical variability was fil- tered out as much as possible in each omics layers, before fitting the variance partition models. Concretely, the panel study consisted of two visits (A and B) where data on exposures, individual behaviours, phenotypes and omics profiles were collected [38]. The mean difference between the two visits (A and B) was 6.11 months (standard deviation (SD): 2.18 months). In order to characterize in depth the variability of the omics measurements, the study population was further restricted to children with complete information for both visits (A and B) for at least one of the omics (N= 156). Prior to the start of data collection, national ethics committees had granted all the required permissions that allowed cohort participant recruitment and follow- up visits. Additionally, all the participants were asked to sign a HELIX specific informed consent. g p All samples were randomized in the arrays by sex and cohort, and in addition, the samples from the same indi- vidual in the microarray-based platforms were paired in the same plate/array (see Supplementary Methods). In the methylation, gene and miRNA expression data, we corrected remaining technical batch effects and blood cell composition by calculating surrogate variables (SVs) while protecting for cohort, sex and age with the SVA and SmartSVA methods [44, 49]. Study design and population y g p p The HELIX (Human Early Life Exposome) study is a collaborative project of six-population based cohorts in different European Countries: UK (BiB: Born in Brad- ford) [31], France (EDEN: Étude des Déterminants pré et postnatals du développement et de la santé de l’Én- fant) [32], Spain (INMA: Infancia y Medio Ambiente) [33], Lithuania (KANC: Kaunus cohort) [34], Norway (MoBa: Norwegian Mother and Child Cohort Study) [35] and Greece (RHEA: Mother Child Cohort study in Crete) [6, 36]. The aim of the HELIX study was to meas- ure and describe multiple environmental exposures dur- ing early life (pregnancy and childhood) in a prospective cohort and associate these exposures with molecular omics signatures and child health outcomes. From the six existing European longitudinal population-based birth cohorts studies participating in HELIX, a subcohort of 1301 mother-child pairs was se- lected to be fully characterized for a broad suite of Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 3 of 16 Gallego-Paüls et al. BMC Medicine Fig. 1 Study workflow Fig. 1 Study workflow Page 4 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 extraction, red cells and a buffy coat for DNA extraction. After processing, these samples were frozen at −80 °C under optimized and standardized procedures [6]. environmental exposures and omics data, to be clinic- ally examined, and to have biological samples col- lected [6]. From this subcohort, 156 children were selected to be part of the panel study: 28 from BiB (UK), 28 from EDEN (France), 40 from INMA Saba- dell (Spain), 30 from KANC (Lithuania) and 30 from RHEA (Greece) [37]. Children from MoBa (Norwe- gian Mother and Child Cohort Study; Norway) were not included in this panel study. The Child Panel Study had the same inclusion criteria as the HELIX subcohort: (a) age 6–11 years at the time of the visit, with a preference for ages 7–9 years if possible; (b) sufficient stored pregnancy blood and urine samples; (c) complete address history available; and (d) no ser- ious health problem. Summary of laboratory processing of omics signatures, quality control and normalization We used residuals from the correction process to analyse blood DNA methylation, gene and miRNA expression. We excluded probes for CpGs that did not reach a 62.5% interclass correlation coefficient (ICC) to minimize technical vari- ability, based on a previous analysis with technical repli- cates [46]. Gene and miRNA expression were filtered out based on call rate or other omics based on other technical parameters (see Table 1). All omics measure- ments were normalized and log2 transformed except for DNA methylation. Sample collection Biological samples were collected using the same stan- dardized protocols across all five cohorts. Urine samples were collected twice daily (first morning void and bed- time sample) in high-quality polypropylene tubes. The two urine samples were brought by the participants to the centre in cool packs and stored at −4 °C until pro- cessing. After aliquoting, the urine samples were frozen at −80 °C under optimized and standardized procedures. A pooled sample of both the morning and the night urine samples was used for the analysis when available (94.9% of individuals in the first visit and 95.5% in the second visit). In visit A, 7 children only had a morning sample available, and 1 child only had night sample. For visit B, this happened in 4 and 3 children for morning and night samples, respectively. Eighteen milliliters of blood was collected at the end of the clinical examination of the child, ensuring an approxi- mate 3-h fasting time since the last meal (visit A mean: 3: 34 h (SD: 1:11 h); visit B mean: 2:35 h (SD: 1:31 h)). Blood samples were collected using a ‘butterfly’ vacuum clip and local anaesthetic and processed into a variety of sample matrices for serum, plasma, whole blood for RNA Linear mixed effect models biological traits and sample collection parameters that were obtained through questionnaires. All omics, except the urine metabolome, were corrected for omics platform technical variables; therefore, we only included the run order as a covariate in the urine metabolome model. Time to last meal and hour of sample collection were not in- cluded in the urine metabolome model because we used pooled samples (morning and night). Variability present in the different omics layers was cal- culated with the variancePartition R package [47]. Briefly, it fits a linear mixed effect model to partition the variance attributed to multiple variables in the data. As this analytical process uses a multiple regression model, the effect of each variable is assessed while correcting for the others. Therefore, variancePartition assessed the contribution of each meta-data variable to variation in each feature. Before running the models, we ensured the absence of collinearity between the explanatory variables by obtain- ing a collinearity score: if this score were to be > 0.99, the variance partition estimates would produce mislead- ing results and overestimate the contribution of variables modelled as fixed effects [47]. No variables were elimi- nated due to collinearity. We considered two mixed effect models: (1) a model to estimate the proportion of variance attributable to intra-individual, inter-individual and cohort variability and (2) the same model adjusting for several explanatory variables (inter- and intra-individual variability-related variables, see list below) to determine the proportion of variance they accounted for. Individual IDs were entered in the models to account for inter-individual variability, whereas we took residuals as a measure of intra- individual variability. In the case of the methylome, we aimed to assess the amount of variance attributed to differences in the im- munological cell type composition. For this purpose, an extra model was performed with a dataset corrected for batch effect using the ComBat method [52] instead of using SVA method, in order to keep the effect of cell type composition. Statistical analyses All statistical analyses were performed using R version 3.6.3 [50]. Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 5 of 16 Table 1 Omics data description and technical variability management Omics profile Matrix Sample size (omics available for both visits) Number of features Laboratory processing Batch correction Criteria for feature exclusion DNA methylation Blood leukocytes 149 91601 Randomized by cohort and sex, and panel samples paired in plate and array Residuals of SVs protecting for cohort, sex and age. Cell type composition also corrected with SVs. < 98% call rate and < 62.5% ICC Gene expression Whole blood 105 45438 Randomized by cohort and sex, and panel samples paired in plate. Residuals of SVs protecting for cohort, sex and age. Cell type composition also corrected with SVs. < 25% call rate miRNA expression Whole blood 100 453 Randomized by cohort and sex, and panel samples paired in plate and array. Residuals of SVs protecting for cohort, sex and age. Cell type composition also corrected with SVs. < 25% call rate Proteins Plasma 149 36 Randomized by cohort Overall protein average minus plate specific protein average subtracted for each individual and each protein < 30% measurements in the linear range (LIN) Serum metabolites Serum 154 177 Fully randomized - > 30% CV and > 30% BLD + zeros Urine metabolites Urine 154 44 Fully randomized - > 30% CV Definitions. Call rate (for DNA methylome): proportion of detection of a given CpG among samples; Call rate (for miRNA and gene expression): proportion of detection of gene or miRNA among samples. Abbreviations. SV surrogate variables, ICC interclass correlation coefficient [35], CV coefficient of variation, BLD below limit of detection Table 1 Omics data description and technical variability management Definitions. Call rate (for DNA methylome): proportion of detection of a given CpG among samples; Call rate (for miRNA and gene expression): proportion of detection of gene or miRNA among samples. Abbreviations. SV surrogate variables, ICC interclass correlation coefficient [35], CV coefficient of variation, BLD below li i f d i Definitions. Call rate (for DNA methylome): proportion of detection of a given CpG among samples; Call rate (for miRNA and gene expression): proportion of detection of gene or miRNA among samples. Abbreviations. SV surrogate variables, ICC interclass correlation coefficient [35], CV coefficient of variation, BLD below limit of detection DNA methylation A total of 156 children from five cohorts across Europe (BIB in the UK, EDEN in France, KANC in Lithuania, RHEA in Greece and INMA in Spain) were followed up for this study, with demographic data detailed in Table 2. Time span between both visits (A and B) was 6.1 months (2.2 SD). Children were on average 7.8 (1.7 SD) years old in visit A. Most of the participants of the study (71.2%) were in the healthy BMI range and remained in the same category from visit A to visit B. Samples were collected on average 3:36 h (1:12 SD) after the last meal during visit A, and after 2:36 h (1:30 SD) during visit B. The hour of sam- ple collection at both visits was almost the same, being 16: 54 h (2:54 SD) and 16:18 h (3:06 SD) for visits A and B, re- spectively. Table 2 shows the description of other explana- tory variables measured in the study. Variation in blood DNA methylation was mainly due to intra-individual variability with a median of 62.2% across all CpG sites and a median inter-individual variability of 37.6% (Q1: 4.1%; Q3: 65.8%). We note however a large heterogeneity between the CpGs with the lower and upper quartiles ranging from 33.9 to 95.6% for intra- individual variability and from 4.1 to 65.8%, for inter. This heterogeneity was partially expected due to the intra-experimental variation in Illumina BeadChip data. Indeed, intra-individual variability of each CpG site in our study was associated to previously reported ICCs, described in the same tissue and the same array, but in adults [46] (Additional file 1 - Figure S1). CpG sites with the highest inter-individual variability were enriched for CpG shores, whereas CpG sites with high intra-individual variability were enriched in open sea areas. We further aimed to evaluate the importance of blood cellular composition in blood DNA methylation variability. For this, we applied the same model for vari- ance partition but without residualizing the effect of blood cell proportions (Additional file 1 - Figure S2). Linear mixed effect models The data matrix contained 139 samples × 13,167 features (13,103 CpGs, 9 proteins, 44 serum me- tabolites and 11 urine metabolites). We computed GGMs using the ggm.estimator.pcor function from the R package “GeneNet” [48]. This function estimates pair- wise partial correlation coefficients conditioned against all remaining variables, allowing to filter out indirect as- sociations that may appear in omics data [48, 54]. We considered significant partial correlations between fea- tures those with p values below the false discovery rate (FDR) threshold of 0.05 (p < 1.28 × 10−7). To construct and visualize the resulting GGM networks, we used Cytoscape 3.8.2 [59]. Edges connecting the nodes repre- sent significant partial correlations and they were weighted using partial correlation coefficients (PCCs). The opacity of each node is based on its connectivity de- gree (number of edges connecting a particular feature). omics features presented little variability due to cohort (median variability of features ranging from 0% for the methylome to 15.7% for the proteome). Linear mixed effect models The following explanatory variables were added to the model as a measure of (1) inter-individual variation: sex, ancestry of the child, age, maternal education—as a gen- eral measure of socio-economic status—KIDMED score as a measure of healthy diet pattern [51] and zBMI, as we did not observe significant changes in the 6-month period; and of (2) intra-individual variation: time to last meal (hours of fasting), heavy exercise practice the same day or the day before the sample collection, having a cold at the moment of sampling, hour of sample collection, run order for the urine metabolome model, day of the week and sea- son at which the samples were collected. These were Additionally for the methylome, we checked whether CpGs located in the 4th quartile for intra- and inter- individual variability were enriched for CpG island rela- tive position (island, shore, shelf, open sea) and for over- lap with CpGs associated with exposures/traits in the EWAS Atlas [53]. Gaussian graphical modelling (GGM)—network analysis A GGM was used to assess direct associations between changes in omics features and elucidate biologically Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Page 6 of 16 Table 2 Population description (N=156) Start of the study Sex Male 89 Female 67 Ancestry European ancestry 145 Pakistani 10 Other 1 Cohort BIB, UK 28 EDEN, France 28 KANC, Lithuania 30 RHEA, Greece 30 INMA, Spain 40 Age (years); mean (SD) Total 7.8 (1.7) BIB 6.7 (0.2) EDEN 10.8 (0.5) KANC 6.7 (0.5) RHEA 6.3 (0.12) INMA 8.6 (0.5) zBMI; mean (SD) 0.4 (1.2) zBMI categories Thinness (zBMI < −2) 1 Healthy (−2 ≤zBMI < 1) 111 Overweight (1 ≤zBMI ≤2) 27 Obese (zBMI > 2) 17 Table 2 Population description (N=156) relevant associations [54–58]. GGMs were built on the delta matrix calculated as the change in omics features between visits (i.e. the change in feature X for a child be- tween time points is correlated with the change in Y, where the correlation is again calculated across all chil- dren). The omics features included: CpG sites with 100% intra-individual variability and proteins, serum and urine metabolites in the highest quartile for intra-individual variability. Gene expression and miRNAs were excluded because the number of participants with complete data including these layers was significantly lower (n=87) and would penalize the identification of biologically relevant associations. Serum and urine metabolites On average, for all the omics, intra-individual variabil- ity was negligibly affected by the inclusion of the ex- planatory variables. However, these variables explained a large percentage of variance in some particular features (Fig. 5) as described further below. Considering all the omics features as a whole, we identified that age, zBMI and hour of sample collection had a major effect on fea- ture variability. The serum metabolome presented, in average, the high- est inter-individual variability (median: 43.4%; Q1: 31.1%; Q3: 53.7%), and the lowest intra-individual vari- ability (median: 50.7%; Q1: 41.3%; Q3: 60.3%). Urine me- tabolites also presented a relatively high median inter- individual variability (median: 28.82%; Q1: 16%; Q3: 40.2%), compared to other omics. Proteins Proteins presented large heterogeneity as well (median cohort variability median: 15.7%; Q1: 4.5%; Q3: 35.3%; median intra-individual variability: 60%; Q1: 48.8%; Q3: 66.3%;). For instance, the variability of C-reactive protein (CRP) was largely explained by intra-individual variabil- ity (87.2%), while the variability of the epidermal growth factor protein (EGF) was attributed to cohort by 55%. Variance partition analysis shows large heterogeneity between and within omics A large heterogeneity in terms of the proportion of vari- ance explained by cohort, inter- and intra-individual variability between omics layers and within the same omics layer was found (Fig. 2 and Table S1). Overall, Page 7 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Fig. 2 Variance partition analysis of omics data. Total variance was apportioned between cohort, inter-individual and intra-individual effects. A The heatmap colour (yellow to red) indicates the variance of features at each coordinate. B The violin plot describes the statistics of the variance explained by each component Fig. 2 Variance partition analysis of omics data. Total variance was apportioned between cohort, inter-individual and intra-individual effects. A The heatmap colour (yellow to red) indicates the variance of features at each coordinate. B The violin plot describes the statistics of the variance explained by each component Fig. 2 Variance partition analysis of omics data. Total variance was apportioned between cohort, inter-individual and intra-individual effects. A The heatmap colour (yellow to red) indicates the variance of features at each coordinate. B The violin plot describes the statistics of the variance explained by each component Page 8 of 16 Page 8 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 When these were added as explanatory variables, differ- ences in immune cell type composition explained a me- dian of 14.0% of the intra-individual variance (Q1: 6.0%; Q3: 34.8%). model (P=9) did not show any significant partial correlation to other features. Among the connected components com- posed by features from different omics layers, all consisted of one serum or urine metabolite directly correlated to a group of CpGs, indicating CpG-metabolite change dependencies. These metabolites included trimethylamine oxide (TMAO), carnitine C3-DC (C4-OH), PC ae C38:1, glucose and citrate (Fig. 4A–E, respectively). CpGs cg16076587 and cg08510264 present in the same connected component as glucose are an- notated to INPP5A (Inositol polyphosphate-5-phosphatase A) and IRS2 (Insulin receptor substrate 2) genes, respectively. Gene expression Intra-individual variability explained the majority of the variance in most of the transcript clusters—groups of probes that define the expression of a gene (median: 93%; Q1: 78%; Q3: 100%). Biological traits can help to interpret some of the omics variability, especially in the serum metabolome MiRNAs presented a variance partition pattern similar to the gene expression, although not as pronounced. Intra-individual variability was predominant for most of the miRNAs (median: 65.2%; Q1: 51.8%; Q1: 81.4%). y y We further aimed to evaluate the association of omics variability with several anthropometric and dietary traits. Overall, the inclusion of explanatory variables accounted for up to 9% of the serum metabolite inter-individual variability (change from a median of 43% to 34% with additional explanatory variables) and up to 3–4% (me- dian change) of the intra-individual variability in the gene expression, miRNA and proteins (Additional file 1 - Table S1). Percentage of omics features explained by each explanatory variable per omics dataset, considering three different thresholds: ≥1%, ≥2% and ≥5% of vari- ance explained are also provided (Additional file 1 – Table S2). Gaussian graphical model networks identify few CpG- metabolite change dependencies Generally, DNA methylation was poorly explained by the explanatory variables: only 3% of the CpGs had more than 2% of the variance explained by individual traits; the KIDMED score explained the most variability (≥2% of the variance in 9.6% of CpGs). Our multi-omics study design also allowed us to analyse co- dependencies in the variability of biological features across the different molecular layers. After applying GGM on the delta matrix (e.g. correlations on the change in omics fea- tures between visits, see the “Methods” section [54–58]), we found 70 connected components and a total of 755 nodes and 1781 undirected edges (FDR < 0.1, Fig. 3). Edges were weighted using partial correlation coefficients [PCCs ranged from 0.003 to 0.007, p values ranged from 2.22 × 10−16 to 1.28 × 10−7]. The largest connected component contained 409 nodes mainly formed by CpG sites (99%), plus 69 smaller connected components that contained from 26 to 2 nodes. Most connected components (88.5%) were formed by features from the same omics layer, including three formed exclusively by serum metabolites as follow: (1) amino-acids (Arg, Phe, Trp, Met, Met.SO, Tyr and His), (2) carnitines and (3) phosphatidylcholines (PCs). Proteins included in the In gene expression, age, maternal education, KIDMED score, week day and hour of sample collection were re- sponsible for at least 2% of the variance in more than 5% of the transcripts each, with the KIDMED score showing the largest influence. MiRNAs were mainly influenced by the hour of sample collection, age, ancestry, and KIDMED score. Concretely, ancestry and hour of sample collection explained more than 5% of the variance in 4.6% and 8.7% of the miRNAs, respectively. Further- more, the KIDMED score explained more than 2% of variance in 10.6 % of miRNAs. Proteins were mostly influenced by zBMI, ancestry, and age, where each trait explained more than 5% of the variance in 22.2%, 16.7% and 16.7% of the Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 9 of 16 Gallego-Paüls et al. BMC Medicine Fig. 3 Network representation of the Gaussian graphical model (GGM) of the DNA methylome, proteins, serum and urine metabolites with high intra-individual variability measured in 157 children from five European countries. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). Gaussian graphical model networks identify few CpG- metabolite change dependencies The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM Fig. 3 Network representation of the Gaussian graphical model (GGM) of the DNA methylome, proteins, serum and urine metabolites with high intra-individual variability measured in 157 children from five European countries. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM Serum metabolites were mostly affected by ancestry and age, explaining more than 5% of the variance in 24.3% and 16.4% of the metabolites, respectively. By contrast, ancestry and age explained more than 5% of variance in just 2.3% and 9.1% of the urine metabo- lites, respectively. Our results showed that time to last meal explained more than 2% of the variance in only a 10.7% of the serum metabolites. Among the metab- olites most influenced by sex, BMI and hours of fast- ing, we found sphingomyelin C18:1 and tyrosine for proteins, respectively. Among the proteins largely ex- plained by zBMI were leptin and insulin. Similarly, these same proteins showed sex differences, found in higher concentrations in females. Ancestry explained 25–12% of the variability in these proteins (in order of magnitude): interleukin (IL)8, TNF alfa, BAFF, in- sulin and HGF (see Additional file 2). Insulin ap- peared to be the only protein significatively influenced by the hours of fasting and CRP was asso- ciated with having a cold. proteins, respectively. Among the proteins largely ex- plained by zBMI were leptin and insulin. Similarly, these same proteins showed sex differences, found in higher concentrations in females. Ancestry explained 25–12% of the variability in these proteins (in order of magnitude): interleukin (IL)8, TNF alfa, BAFF, in- sulin and HGF (see Additional file 2). Insulin ap- peared to be the only protein significatively influenced by the hours of fasting and CRP was asso- ciated with having a cold. Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Page 10 of 16 irect associations between features from Fig. 4 Main connected components of the Gaussian graphical model (GGM) network that involve direct associations between features from different omics layers. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. Gaussian graphical model networks identify few CpG- metabolite change dependencies The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM Fig. 4 Main connected components of the Gaussian graphical model (GGM) network that involve direct associations between features from different omics layers. Blue nodes represent CpG sites. Red and yellow nodes represent serum and urine metabolites, respectively. The opacity of the nodes is dependent on their degree (number of edges connecting a particular feature). The edge thickness was weighted based on the partial correlation coefficients (PCCs) obtained from the GGM sex; glucose and 4-deoxyerythronic acid for BMI z- score; and alanine for hours of fasting. metabolites were somewhat in the middle, with strong heterogeneity between features. Consequently, DNA methylation and serum metabolites (targeted assay) will better inform epidemiological studies that rely on single measurements to compare individuals in the search of biomarkers, whereas less stable omics profiles such as gene expression will give more reliable information to studies that assess individual trajectories over time (mul- tiple time point measurements). We evidenced that vari- ability of omics features comes from several sources. Besides technical or analytical variability, which we tried to control for, we identified physiological patterns in intra-individual variability through inter-omics network analysis. In all omics profiles, features with high inter- individual variability were identified, which can be as- cribed to biological (between individuals) variability. While it is always preferable to adhere to standard sam- ple collection conditions, this is not always possible, and omics features with substantial biological variation are potentially robust enough to yield meaningful findings in Discussion The current study offers a multi-omics perspective of medium-term omics variability in childhood. We mod- elled the variability of 6 different types of omics data (blood DNA methylation, gene expression, miRNA, proteome, serum and urine metabolomes) for 156 chil- dren from five European countries at two time points with a 6-month interval, and found a large range of intra- and inter-individual variability between and within each omics profiles. We pointed out that overall intra- individual variation accounted for the largest part of the total variation in all omics. While DNA methylation and serum metabolites exhibited stronger stability over time for many features (median inter-individual variability: 37.6 and 43.4%, respectively), gene expression was the less stable omics profile in average (median inter- individual variability: 6.6%) and proteins and urinary Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine Page 11 of 16 Fig. 5 Violin plots showing multi-omics variability decomposed by biological traits and sample collection parameters measured in the study. Labels correspond to omics features mostly explained by each variable. Abbreviations. Proteome: IL: interleukin; Apo A1: apolipoprotein A1; RA: receptor antagonist; CRP: c-reactive protein. Serum metabolome: C: acylcarnitine; SM: sphingomyelin; PC: phosphatidylcholine; lysoPC: lysophosphatidylcholine Fig. 5 Violin plots showing multi-omics variability decomposed by biological traits and sample collection parameters measured in the study. Labels correspond to omics features mostly explained by each variable. Abbreviations. Proteome: IL: interleukin; Apo A1: apolipoprotein A1; RA: receptor antagonist; CRP: c-reactive protein. Serum metabolome: C: acylcarnitine; SM: sphingomyelin; PC: phosphatidylcholine; lysoPC: lysophosphatidylcholine which are regions 0–2 kb from CpG islands (CpG rich regions) [33]. On the other hand, CpG sites with high intra-individual variability were enriched in open sea areas, which are isolated CpG sites in the genome that have been linked to chromosomal instability and loss of imprinting [33, 34]. Recent studies point out that pheno- typically relevant CpGs tend to be located in CpGs shores [13, 16]. These CpGs with high inter-individual variability are especially relevant for large-scale epi- demiological studies since these probes could be used as reliable biomarker candidates [16]. Our results reinforce that strong methylation differences between individuals spite of collection inconsistencies. The small proportions of variability attributed to cohort demonstrated that standardized sample collection protocols can produce robust results in large-scale omics studies across differ- ent countries. Discussion Our results, in contrast, attributed the majority of the variation to intra- individual effects, potentially due to RNA quality that might differ between visits A and B. Effort has to be put on the initial sample preparation (DNA, RNA extraction and quality) and its harmonization across different cen- tres or time point collection, since it strongly determines the quality of omics measurements and might hinder real biological response. To our knowledge, only two previous studies have estimated miRNA variability in terms of intra- and inter-individual effects, but these studied a longer time span (5 years) [26] or a daily time span (48 h) in cerebrospinal fluid [27]. Despite these dif- ferences in study design, both studies agree that there is diversity within the microRNome in terms of stability of its features. Previous proteome studies in healthy human volunteers have demonstrated moderate inter-individual variability (CV ranging from 30% to 50%) [35, 36], simi- lar to the urine proteome in seven adult donors [37]. A study that estimated intra-individual variation of plasma adipokines concluded that they may be useful bio- markers of inflammation in population-based studies of obesity-related disease due to their stability over time [38]. These corroborate our results for IL-1β, IL-6, IL-8, leptin, adiponectin, hepatocyte growth factor (HGF) and CRP which presented the highest stability in our study. Finally, metabolomics studies comparing serum and urine metabolomes corroborate in children and adult populations strongly corroborate our findings that the serum metabolome is more stable and captures more inter-individual specific variance, compared to the urine metabolome [39–43]. A few recent studies have reported changes in multiple omics profiles in clinical settings and after dietary well- ness coaching intervention or physical activity [30, 66, 67], revealing omics signatures that may serve as poten- tial diagnostic markers. However, variability of omics profiles in healthy population and “normal” living condi- tions remains under-studied. Large observational studies would benefit enormously from this information, be- cause it allows the interpretation of changes that do not match a pattern. This is especially important for early- life studies that focus on the origin of diseases in chil- dren because omics profiles are able to capture very early and subtle molecular responses, even before physiological manifestations appear. Discussion Biological traits and sample collection var- iables, easy to collect in cohort studies, might help to account for the unwanted variability, in particular for metabolomics. We found, in the case of the methylome, that the most stable CpG sites were enriched in functional methylation regions of the genome. CpG sites with the highest inter- individual variability were enriched for CpG shores, Page 12 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 12 of 16 Gallego-Paüls et al. BMC Medicine (2021) 19:166 already appear in childhood as previously reported [18], even in our population of similar European ancestry children. On the other hand, gene expression may re- quire repeat sampling to account for intra-individual and technical variability, in order to generate stable enough markers to be deployed in epidemiological stud- ies. Previous studies in healthy individuals show that gene expression profiles are mostly stable and repeatable in the short/medium term (< 5% of transcripts with high intra-individual variability) [20–22, 24]. Our results, in contrast, attributed the majority of the variation to intra- individual effects, potentially due to RNA quality that might differ between visits A and B. Effort has to be put on the initial sample preparation (DNA, RNA extraction and quality) and its harmonization across different cen- tres or time point collection, since it strongly determines the quality of omics measurements and might hinder real biological response. To our knowledge, only two previous studies have estimated miRNA variability in terms of intra- and inter-individual effects, but these studied a longer time span (5 years) [26] or a daily time span (48 h) in cerebrospinal fluid [27]. Despite these dif- ferences in study design, both studies agree that there is diversity within the microRNome in terms of stability of its features. Previous proteome studies in healthy human volunteers have demonstrated moderate inter-individual variability (CV ranging from 30% to 50%) [35, 36], simi- lar to the urine proteome in seven adult donors [37]. A study that estimated intra-individual variation of plasma adipokines concluded that they may be useful bio- markers of inflammation in population-based studies of obesity-related disease due to their stability over time [38]. These corroborate our results for IL-1β, IL-6, IL-8, leptin, adiponectin, hepatocyte growth factor (HGF) and CRP which presented the highest stability in our study. Discussion Finally, metabolomics studies comparing serum and urine metabolomes corroborate in children and adult populations strongly corroborate our findings that the serum metabolome is more stable and captures more inter-individual specific variance, compared to the urine metabolome [39–43]. intake and fat storage [61] and the pro-inflammatory cy- tokines IL-6 and IL-1 beta, both known to be elevated in subjects with obesity or with serum lipid concentrations abnormalities, leading to a state of chronic inflammation [62]. Similarly, these same proteins showed sex differ- ences, in particular leptin and IL-1 beta, known to be higher in females and resulting in a higher risk to de- velop obesity than males [63]. Interestingly, circulating IL-8, a pro-inflammatory cytokine, was strongly influ- enced by ancestry (6% of children had Pakistani ances- try) in our cohorts (25% of variance explained). IL-8 was previously found to be influenced by genetic polymor- phisms in an eastern Indian population, potentially driv- ing individual variations in the host’s immune response, in particular to infectious diseases [64]. Intra-individual factors such as hours of fasting and having a cold gave the expected results: insulin appeared to be the only pro- tein significatively influenced by the hours of fasting and CRP was associated with having a cold. This is in line with a previous study that considered the fasting/post- prandial state of the samples and revealed that, on aver- age, it explained less than 2% of the total variance [41]. Associations of serum and urine metabolites with sex, BMI and hours of fasting (only for serum) was also pre- viously reported in the cross-sectional study of 1300 HELIX children [39]. Interestingly, high levels of 4- deoxyerythronic acid in children, found correlated to higher BMI in our study, have been previously related to early-onset type I diabetes although further understand- ing of its metabolism is required [65]. already appear in childhood as previously reported [18], even in our population of similar European ancestry children. On the other hand, gene expression may re- quire repeat sampling to account for intra-individual and technical variability, in order to generate stable enough markers to be deployed in epidemiological stud- ies. Previous studies in healthy individuals show that gene expression profiles are mostly stable and repeatable in the short/medium term (< 5% of transcripts with high intra-individual variability) [20–22, 24]. Discussion Variations in omics profiles due to factors such as BMI, age, physical activity, fasting time and sampling conditions need to be well characterized in order to interpret subtle changes related to environmental exposures such as air pollution or endocrine-disrupting chemicals, the ultimate goal of re- cent exposome initiatives [60]. Omics measurements in our study are of great clinical relevance as they provide the basis for the discovery of new biomarkers of multiple medical conditions such as cancer, heart disease, neuro- logical disorders or inflammatory diseases [61–64]. Here, Biological traits, such as body weight, sex and age, accounted for inter-individual variability in gene expres- sion, similar to results obtained by Hughes et al. (2015) [25], where roughly 2% of total gene expression variation was explained by these traits in placenta samples. Age and hour of sample collection have also been found to be significant sources of variation in blood gene expres- sion patterns of healthy individuals [60]. Previous studies on miRNA variability identified the strong influence of age and sample storage time [15, 23], but the time of the day or dietary intake has not been previously studied to our knowledge. Among the proteins largely explained by zBMI were leptin and insulin, hormones related to food Gallego-Paüls et al. BMC Medicine (2021) 19:166 Page 13 of 16 Page 13 of 16 approach provided reduced biological coverage, we are confident that this insured reliable annotation and quan- tification of markers. In contrast, we report high intra- individual effects in a large proportion of genome-wide omics features that are potentially due to technical vari- ability. Despite considering quality control parameters (e.g. filtering for call rate) in order to minimize this ef- fect, we note that precision in the intra-individual vari- ability apportionments can be strengthened by adding technical replicates. This, together with increasing the number of individuals, would be the ideal way to strengthen the biological signal. Moreover, despite hav- ing measured many biological traits in the children under study, there are relevant missing variables, such as recent dietary intake in the last 24 h, which is expected to influence the variability of omics such as the urine metabolome. It would be interesting to study if and how other factors account for variability within omics. Our study could also benefit from a more exhaustive collec- tion of repeated samples across short periods of time. Discussion For example, having morning and night samples in the same individual at different time points, as done previ- ously for metabolomics [77, 78] or miRNAs [27]. we provide insight into which omics features are stable within individuals and demonstrate sufficient inter- individual variation in order to reduce chance findings when conducting epidemiological studies with follow-up for disease outcomes [16]. We emphasize the fact that children from five Euro- pean countries took part in the study. The multiple loca- tions of our participants allow to generalize the influence of factors such as lifestyle habits, seasonal in- fluences and maternal education, which vary greatly be- tween countries/cultures and not many studies consider. A major strength of our study lies in the cross-omics ap- proach. We created a network of interacting omics fea- tures of four different omics layers (DNA methylome, proteome, serum and urine metabolomes) using Gaussian graphical models (GGMs). GGMs circumvent the selec- tion of indirect associations that usually appear between omics measurements, as Pearson correlations are generally high in these data. GGMs are based on partial correlation coefficients and provide an estimate of conditional de- pendencies between variables, elucidating direct associa- tions [54]. This enabled us to obtain a holistic view of the biological significance of our results. By studying the inter- action of multiple omics features, we exploited the data to their full potential for further disease prediction and pre- vention studies [56]. Moreover, it allowed us to go a step further by identifying interactions of features across omics layers, which is the main goal of the recent discipline in- teractome within systems biology. Despite not finding many dependencies across omics, a few metabolites were related to groups of CpGs. These included TMAO, a com- pound generated by the gut microbiota from diet-derived components, hence with high variability and strongly de- termined by diet, gut microbial flora and drug administra- tion [65]. Elevated levels of TMAO positively correlate to cardiovascular disease through the development of athero- sclerosis in previous studies [68–73]. Variation in four CpGs was related to glucose in urine (pool morning and night urine), which level depends on lifestyle factors like diet and exercise behaviours and its dysregulation related to obesity and type 2 diabetes (T2D) in children [74]. Interestingly, two of these CpGs are within genes related to obesity and T2D [53]. Discussion This supports previous studies on GGMs that show their ability to reconstruct metabolic pathways [54, 75, 76], including in this case across omics. Conclusions d We assessed omics profiles variability over the medium- term in child cohorts from the general population, using a multi-omics approach. We found large heterogeneity within and between omics profiles. Intra-individual vari- ability presented the highest median variability in all cases. The cross-omics analysis we performed provides global insights into how different omics features vary over time, within and between individuals and among cohorts from different countries. This study thereby pro- vides a valuable framework for future epidemiological studies that aim to detect omics signatures linked to disease, environmental exposures or both. Abbreviations BMI: Body mass index; CRP: C-reactive protein; CV: Coefficient of variation; FDR: False discovery rate; GGM: Gaussian graphical model; HELIX: Human Early Life Exposome; ICC: Interclass correlation coefficient; IL: Interleukin; KIDMED score: Mediterranean diet quality index; miRNA: MicroRNA; PC: Phosphatidylcholine; PCCs: Partial correlation coefficients; Q1, 2, 3, 4: Quartile 1, 2, 3, 4; QC: Quality control; SD: Standard deviation; SVA: Surrogate variable analysis; TC: Transcript cluster; TMAO: Trimethylamine oxide Our study had some limitations. In the first place, we studied omics variability in 156 children and a larger population size would have provided greater statistical power to our model. However, this sample size allowed us to analyse 6 different molecular layers in the same in- dividual and at the same time-point, twice, data rarely obtained in the past. Our study used targeted ap- proaches to measure the proteome and the serum me- tabolome profiles (semi-targeted for urine). While this References 1. Karczewski KJ, Snyder MP. Integrative omics for health and disease. Nat. Rev. Genet. 2018;19:299–310. https://doi.org/10.1038/nrg.2018.4. 1. Karczewski KJ, Snyder MP. Integrative omics for health and disease. Nat. Rev. Genet. 2018;19:299–310. https://doi.org/10.1038/nrg.2018.4. 2. Everson TM, Marsit CJ. Integrating -omics approaches into human population-based studies of prenatal and early-life exposures. Curr. Environ. Heal. 2018;5(3):328–37. https://doi.org/10.1007/s40572-018-0204-1. 2. Everson TM, Marsit CJ. Integrating -omics approaches into human population-based studies of prenatal and early-life exposures. Curr. Environ. Heal. 2018;5(3):328–37. https://doi.org/10.1007/s40572-018-0204-1. 3. Pinu FR, Beale DJ, Paten AM, Kouremenos K, Swarup S, Schirra HJ, et al. 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Acknowledgements e would like to thank all the families for their generous contributio We would like to thank all the families for their generous contribution. Availability of data and materials The summarized results generated during the current study are available in supplementary material. The raw data supporting the current study are available from the corresponding author on request subject to ethical and legislative review. 5. Barker DJP. The origins of the developmental origins theory. J Intern Med. 2007;261(5):412–7. https://doi.org/10.1111/j.1365-2796.2007.01809.x. 5. Barker DJP. The origins of the developmental origins theory. J Intern Med. 2007;261(5):412–7. https://doi.org/10.1111/j.1365-2796.2007.01809.x. Declarations 6. Maitre L, de Bont J, Casas M, Robinson O, Aasvang GM, Agier L, et al. Human Early Life Exposome (HELIX) study: a European population-based exposome cohort. BMJ Open. 2018;8:e021311. 6. Maitre L, de Bont J, Casas M, Robinson O, Aasvang GM, Agier L, et al. Human Early Life Exposome (HELIX) study: a European population-based exposome cohort. BMJ Open. 2018;8:e021311. Authors’ contributions B RS C RG Authors contributions LM, MB, JW, RS, MC, JRG, HK and MV designed the omics study in HELIX. The following authors participated in omics data acquisition and quality control: AC, CR-A, MB (DNA methylation), XE, MV-U (transcriptomics), ES, EB (proteo- mics), CEL, APS, LM, HK, MC (metabolomics). JW, RS, BH, RG, MV and LCh are the PIs of the cohorts or participated in sample and data acquisition. CH-F, LM, MB, JU, JRG, XB and JB, performed statistical analyses and JRG functional enrichment analyses. The project was coordinated by MV. MG-P wrote the original draft of the paper and LM, MB, MV, JRG, CH-F and HK contributed to reviewing and editing the manuscript. All authors read and approved the manuscript. Funding Th d The study has received funding from the European Community’s Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project) and the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project). y g p y Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project) and the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project). Additionally, BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/ N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Rhea project was finan- cially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV- 1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in pre- school children, in Heraklion district, Crete, Greece: 2011- 2014; “Rhea Plus”: Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012-15). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) and it is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I+D+i 2013-2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. MV-U and CR-A were supported by a FI fellowship from the Catalan Govern- ment (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (MS16/00128). LM is funded by a Juan de la Cierva-Incorporación fellowship (IJC2018-035394-I) awarded by the Spanish Ministerio de Economía, Industria y Competitividad. Received: 30 March 2021 Accepted: 8 June 2021 Received: 30 March 2021 Accepted: 8 June 2021 Consent for publication Not applicable. Consent for publication Not applicable. Competing interests Th h d l h The authors declare that they have no competing interests. MC is currently affiliated to AstraZeneca but the company had no role in the design, conduct, or analysis of the H2020-EU funded project. Author details 1 1ISGlobal, Barcelona, Spain. 2Universitat Pompeu Fabra (UPF), Barcelona, Spain. 3Consorcio de Investigacion Biomedica en Red de Epidemiologia y Salud Publica (CIBERESP), Madrid, Spain. 4Center for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain. 5MRC Centre for Environment and Health, School of Public Health, Imperial College London, London, UK. 6Division of Systems Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College London, South Kensington, London, UK. 7Cancer Metabolism & Systems Toxicology Group, Division of Cancer, Department of Surgery & Cancer and Division of Systems Medicine, Department of Metabolism, Digestion & Reproduction, Imperial College London, London, UK. 8Bradford Institute for Health Research, Bradford Teaching Hospitals NHS Foundation Trust, Bradford, UK. 9Team of Environmental Epidemiology applied to Reproduction and Respiratory Health, Institute for Advanced Biosciences (IAB), Inserm, CNRS, Université Grenoble Alpes, Grenoble, France. 10Université de Paris, Centre for Research in Epidemiology and Statistics (CRESS), INSERM, INRAE, F-75004 Paris, France. 11Department of Environmental Sciences, Vytautas Magnus University, Kaunas, Lithuania. 12Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. 13Medicine Genomics Group, Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER), University of Santiago de Compostela, CEGEN-PRB3, Santiago de Compostela, Spain. 14Galician Foundation of Genomic Medicine, Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Servicio Gallego de Salud (SERGAS), Santiago de Compostela, Galicia, Spain. 15Oncology Safety, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, UK. Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12916-021-02027-z. The online version contains supplementary material available at https://doi. org/10.1186/s12916-021-02027-z. Additional file 1. Supplementary tables, figures and R code. This file contains supplementary methods, tables and figures summarizing the results of the variance partition models. It also contains the code used in R to perform variance partition analyses and Gaussian graphical models. Page 14 of 16 Page 14 of 16 Gallego-Paüls et al. BMC Medicine Gallego-Paüls et al. BMC Medicine (2021) 19:166 forskningsetikk, Rhea: Ethical committee of the general university hospital of Heraklion, Crete. Informed consent was obtained from a parent and/or legal guardian of all participants in the study. forskningsetikk, Rhea: Ethical committee of the general university hospital of Heraklion, Crete. 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Freshening of the Mediterranean Salt Giant: controversies and certainties around the terminal (Upper Gypsum and Lago-Mare) phases of the Messinian Salinity Crisis
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Andreetto, F., Aloisi, G., Raad, F., Heida, H., Flecker, R. M., Agiadi, K., & Lofi, J. (2021). Freshening of the Mediterranean Salt Giant: controversies and certainties around the terminal (Upper Gypsum and Lago-Mare) phases of the Messinian Salinity Crisis. Earth-Science Reviews, 216, Article 103577. https://doi.org/10.1016/j.earscirev.2021.103577 Andreetto, F., Aloisi, G., Raad, F., Heida, H., Flecker, R. M., Agiadi, K., & Lofi, J. (2021). Freshening of the Mediterranean Salt Giant: controversies and certainties around the terminal (Upper Gypsum and Lago-Mare) phases of the Messinian Salinity Crisis. Earth-Science Reviews, 216, Article 103577. https://doi.org/10.1016/j.earscirev.2021.103577 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1016/j.earscirev.2021.103577 Link to publication record on the Bristol Research Por PDF-document Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1016/j.earscirev.2021.103577 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via Elsevier at 10.1016/j.earscirev.2021.103577. Please refer to any applicable terms of use of the publisher. Freshening of the Mediterranean Salt Giant: controversies and certainties around the terminal (Upper Gypsum and Lago-Mare) phases of the Messinian Salinity Crisis Littoral Cˆote d’Opale, UMR 8187, LOG, Laboratoire d’Oc´eanologie et de G´eosciences, F 59000, Lille, France l Geological Survey of Israel, Jerusalem 95501, Israel m Institute of Earth Sciences, The Hebrew University of Jerusalem, Israel n CASP, West Building, Madingley Rise, Madingley Road, Cambridge CB3 0UD, United Kingdom o Senckenberg Research Biodiversity and Climate Research Centre, Senckenberganlage 25, 60325 F n CASP, West Building, Madingley Rise, Madingley Road, Cambridge CB3 0UD, United Kingdom n CASP, West Building, Madingley Rise, Madingley Road, Cambridge CB3 0UD, United Kingdom o Senckenberg Research Biodiversity and Climate Research Centre, Senckenberganlage 25, 60325 F , g, g y , g y , g , g o Senckenberg Research Biodiversity and Climate Research Centre, Senckenberganlage 25, 60325 Frankfurt am Main, Germany o Senckenberg Research Biodiversity and Climate Research Centre, Senckenberganlage 25, 60325 Frankfurt am Main, Germany * Corresponding author. E-mail address: f.andreetto@uu.nl (F. Andreetto). Contents lists available at ScienceDirect Contents lists available at ScienceDirect University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Earth-Science Reviews 216 (2021) 103577 Available online 3 March 2021 0012-8252/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Corresponding author. E-mail address: f.andreetto@uu.nl (F. Andreetto). https://doi.org/10.1016/j.earscirev.2021.103577 Received 1 November 2020; Received in revised form 4 February 2021; Accepted 25 February 2021 Freshening of the Mediterranean Salt Giant: controversies and certainties around the terminal (Upper Gypsum and Lago-Mare) phases of the Messinian Salinity Crisis F. Andreetto a,*, G. Aloisi b, F. Raad c, H. Heida d, R. Flecker e, K. Agiadi f, J. Lofi c, S. h i j d F. Andreetto a,*, G. Aloisi b, F. Raad c, H. Heida d, R. Flecker e, K. Agiadi f, J. Lofi c, S. Blondel g, F. Bulian h, A. Camerlenghi g, A. Caruso i, R. Ebner j, D. Garcia-Castellanos d, V. Gaullier k, L. Guibourdenche b, Z. Gvirtzman l,m, T.M. Hoyle a,n, P.T. Meijer j, J. Moneron l,m, F.J. Sierro h, G. Travan k, A. Tzevahirtzian i, I. Vasiliev o, W. Krijgsman a a Paleomagnetic Laboratory “Fort Hoofddijk”, Dept. of Earth Sciences, Utrecht University, Budapestlaan 17, 3584 CD Utrecht, The Netherlands b Universit´e de Paris, Institut de physique du globe de Paris, CNRS, F-75005 Paris, France c G´eosciences Montpellier, CNRS, Universit´e de Montpellier, Montpellier, France d Geosciences Barcelona, GEO3BCN, CSIC, Sol´e i Sabarís s/n, Barcelona, Spain e BRIDGE, School of Geographical Sciences and Cabot Institute, University of Bristol, University Road, Bristol BS8 1SS, United Kingdom f Department of Palaeontology, University of Vienna, Althanstraße 14 (UZA II), 1090 Vienna, Austria g Istituto Nazionale di Oceanografia e di Geofisica Sperimentale (OGS), Trieste, Italy h Department of Geology, University of Salamanca, Salamanca, Spain i Dipartimento di Scienze della Terra e del Mare, Universit`a degli studi di Palermo, via Archirafi 20-22, 90123 Palermo, Italy j Department of Earth Sciences, Utrecht University, Utrecht, The Netherlands k Univ. Lille, CNRS, Univ. Littoral Cˆote d’Opale, UMR 8187, LOG, Laboratoire d’Oc´eanologie et de G´eosciences, F 59000, Lille, France l Geological Survey of Israel, Jerusalem 95501, Israel m Institute of Earth Sciences, The Hebrew University of Jerusalem, Israel n CASP West Building Madingley Rise Madingley Road Cambridge CB3 0UD United Kingdom k Univ. Lille, CNRS, Univ. 1. Introduction hyposaline (substage 3.2; 5.42-5.33 Ma) ponds which only received water from local streams and were colonized by Black Sea organisms carried by aquatic migratory birds (Fig. 1b; e.g. Ruggieri, 1967; Decima and Sprovieri, 1973; Decima and Wezel, 1971, 1973; Cita et al., 1978; Müller et al., 1990; Benson and Rakic-El Bied, 1991; Benson et al., 1991; Müller and Mueller, 1991; Butler et al., 1995; Orszag-Sperber et al., 2000; Rouchy et al., 2001; Kartveit et al., 2019; Madof et al., 2019; Camerlenghi et al., 2019; Caruso et al., 2020; Raad et al., 2021). As morphological and seismic reflection studies at the Strait of Gibraltar documented a ~400 km long erosional trough connecting the Gulf of Cadiz (Atlantic Ocean) to the Mediterranean Sea, this scenario of a lowered Mediterranean Sea was promptly linked to the termination of the MSC (McKenzie, 1999; Blanc, 2002; Garcia-Castellanos et al., 2009, 2020). This conclusion has recently been reinforced by the discovery of vast chaotic deposits sitting at the claimed Miocene/Pliocene transition in the area of the Malta Escarpment-Ionian Abyssal Plain (Micallef et al., 2018, 2019; Spatola et al., 2020). At the end of the Miocene, orbital and tectonic drivers combined to alter the amount of water delivered to the Mediterranean Basin by the Atlantic Ocean from the west, the brackish Eastern Paratethys (i.e. Euxinic-Caspian Basin system) from the east and the major peri- Mediterranean freshwater drainage systems (e.g. African rivers and Rhˆone; Griffin, 2002; Gladstone et al., 2007; Van der Laan et al., 2006; Hilgen et al., 2007; Ryan, 2009; Flecker et al., 2015; Marzocchi et al., 2015, 2016, 2019; Simon et al., 2017; Krijgsman et al., 2018; Capella et al., 2020). The changes in extra and intrabasinal connectivity resulted in unprecedented paleoceanographic and paleohydrological budget changes that led to a relatively short-lived environmental and ecological crisis (approx. 660 kyr; 5.97-5.33 Ma), for which the term Messinian Salinity Crisis (MSC) was coined (Selli, 1954, 1960). Most conspicuous was the rapid accumulation of several kilometers of halite (i.e. ~1 million km3) on the Mediterranean abyssal plains (e.g. Hsü, 1972; Ryan, 1973; Montadert et al., 1978; Haq et al., 2020). This happened within 50 kyr, from 5.60-5.55 Ma, according to Roveri et al. (2014a) and Manzi et al. (2018), or in >300 kyr, when starting at 5.97 Ma, as put forward by Meilijson et al. (2018, 2019). 1. Introduction In more recent years, the desiccated basin model was challenged by the observation of deposits that are uniform in terms of sedimentology and stratigraphic architecture (Roveri et al., 2008a), ostracod content (Gliozzi et al., 2007; Stoica et al., 2016) and geochemistry (McCulloch and De Deckker, 1989; García-Veigas et al., 2018; Andreetto et al., 2021) throughout the Mediterranean marginal belt and of δDn-alkanes and δDalkenones sharing similarities with the coeval Atlantic Ocean and Black Sea, respectively (Vasiliev et al., 2017). A model of a (relatively) full Mediterranean Sea developed (Fig. 1c), where the debate mainly con­ cerns the provenance of the hydrological fluxes and the resultant hydrochemical composition of the water mass. In this scenario, the Mediterranean was first, during substage 3.1, transformed into a new gypsum-precipitating basin filled with marine and continent-derived During the ~200 kyr lapse (i.e. MSC Stage 3 following Roveri et al., 2014a; Fig. 1a) between the end of salt precipitation (5.55 Ma) and the restoration of the still enduring marine conditions (5.33 Ma), the Med­ iterranean underwent a sequence of paleohydrological and base-level changes that are the topic of intense and long-standing debates. The initial and still widely endorsed hypothesis was that the Mediterranean Sea, following the major drawdown event that led to halite deposition (i. e. Stage 2), maintained the isolated, deeply-desiccated geography con­ taining a series of hypersaline (substage 3.1; 5.55-5.42 Ma) and Fig. 1. (a) Consensus chronostratigraphic model for the MSC events (Roveri et al., 2014a). Stage 3, here of interest, spans between 5.55 Ma and 5.332 Ma, the astronomical ages of the base of the Upper Gypsum Unit (following Manzi et al., 2009) and Trubi Formation (Van Couvering et al., 2000) in the Sicilian Eraclea Minoa section, respectively. CdB: Calcare di Base; PLG: Primary Lower Gypsum; RLG: Resedimented Lower Gypsum; UG: Upper Gypsum. (b), (c) Map of the Mediterranean region showing the two extreme and mutually exclusive paleoenvironmental scenarios proposed to have featured the Mediterranean during Stage 3 (see discussion in Chapter 7; modified after Krijgsman et al., 2018). Fig. 1. (a) Consensus chronostratigraphic model for the MSC events (Roveri et al., 2014a). Stage 3, here of interest, spans between 5.55 Ma and 5.332 Ma, the astronomical ages of the base of the Upper Gypsum Unit (following Manzi et al., 2009) and Trubi Formation (Van Couvering et al., 2000) in the Sicilian Eraclea Minoa section, respectively. A B S T R A C T Keywords: Messinian Salinity Crisis Mediterranean stratigraphy Connectivity proxies Paleogeography Paratethys Lago-Mare The late Miocene evolution of the Mediterranean Basin is characterized by major changes in connectivity, climate and tectonic activity resulting in unprecedented environmental and ecological disruptions. During the Messinian Salinity Crisis (MSC, 5.97-5.33 Ma) this culminated in most scenarios first in the precipitation of gypsum around the Mediterranean margins (Stage 1, 5.97-5.60 Ma) and subsequently > 2 km of halite on the basin floor, which formed the so-called Mediterranean Salt Giant (Stage 2, 5.60-5.55 Ma). The final MSC Stage 3, however, was characterized by a "low-salinity crisis", when a second calcium-sulfate unit (Upper Gypsum; substage 3.1, 5.55- 5.42 Ma) showing (bio)geochemical evidence of substantial brine dilution and brackish biota-bearing terrigenous sediments (substage 3.2 or Lago-Mare phase, 5.42-5.33 Ma) deposited in a Mediterranean that received relatively large amounts of riverine and Paratethys-derived low-salinity waters. The transition from hypersaline evaporitic (halite) to brackish facies implies a major change in the Mediterranean’s hydrological regime. However, even after nearly 50 years of research, causes and modalities are poorly understood and the original scientific debate between a largely isolated and (partly) desiccated Mediterranean or a fully connected and filled basin is still vibrant. Here we present a comprehensive overview that brings together (chrono)stratigraphic, sedimentological, paleontological, geochemical and seismic data from all over the Mediterranean. We summarize the paleo­ environmental, paleohydrological and paleoconnectivity scenarios that arose from this cross-disciplinary dataset and we discuss arguments in favour of and against each scenario. F. Andreetto et al. Earth-Science Reviews 216 (2021) 103577 1. Introduction CdB: Calcare di Base; PLG: Primary Lower Gypsum; RLG: Resedimented Lower Gypsum; UG: Upper Gypsum. (b), (c) Map of the Mediterranean region showing the two extreme and mutually exclusive paleoenvironmental scenarios proposed to have featured the Mediterranean during Stage 3 (see discussion in Chapter 7; modified after Krijgsman et al., 2018). 2 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Fig. 2. Map of the Mediterranean Basin (modified from Lofi, 2018) showing: a) the location of the key intermediate and deep basins as well as physical thresholds that influenced the connectivity history of the Mediterranean; b) the onshore (i.e. basins and/or sections) and offshore (DSDP/ODP/Industrial drill sites) localities where deposits attributed to MSC Stage 3 have been studied. Mixed assemblages of Paratethyan-like ostracods and foraminifera are known from all mentioned onshore localities and some offshore locations (see text). The present-day spatial extent of the MSC seismic units, except for the Lower Unit, is also shown. The paleogeography of the (Eastern and Central) Paratethys and of the North Aegean domain is contoured after Van Baak et al. (2017) and Krijgsman et al. (2020a), respectively. W-E onshore localities: 1-6 Betic Cordillera (SE Spain): 1-Marbella and 2-Malaga basins (Guerra-Merch´an et al., 2010); 3-Sorbas Basin (Roveri et al., 2009, 2019a); 4-Nijar Basin (Fortuin and Krijgsman, 2003); 5-Vera Basin (Fortuin et al., 1995); 6-Bajo Segura Basin (Soria et al., 2005, 2008a, 2008b); 7-Mallorca (Mas and Forn´os, 2020); 8-Melilla Basin (Rouchy et al., 2003); 9-Boudinar Basin (Merzeraud et al., 2019); 10-Chelif Basin (Rouchy et al., 2007); 11-Sahel area (Frigui et al., 2016); 12-Al´eria Basin and 13-Rhˆone Valley (Carbonnel, 1978); 14-Piedmont Basin (Dela Pierre et al., 2011, 2016); 15-Po Plain (Ghielmi et al., 2010, 2013; Amadori et al., 2018); 16-Fine Basin (Cava Serredi section; Carnevale et al., 2006a, 2008). 1. Introduction 17-21 Apennine system: Romagna sections (17, Roveri et al., 1998), Trave section (18, Iaccarino et al., 2008), Maccarone section (19, Bertini, 2006, Grossi et al., 2008; Sampalmieri et al., 2010; Pellen et al., 2017), Colle di Votta (20)-Fonte dei Pulcini (21)-Stingeti (22) sections (Cosentino et al., 2005, 2012, 2013, 2018), Mondragone 1 well (23, Cosentino et al., 2006), Crotone Basin (24, Roveri et al., 2008a); 25-27 Sicily: Villafranca Tirrena (25) and Licodia Eubea (26) sections (Sciuto et al., 2018), Caltanissetta Basin (27, Manzi et al., 2009); 28-Corfu (Pierre et al., 2006); 29-Zakinthos (Karakitsios et al., 2017b); 30-Crete (Cosentino et al., 2007); 31-Cyprus (Rouchy et al., 2001; Manzi et al., 2016a); 32-Adana Basin (Radeff et al., 2016). Fig. 2. Map of the Mediterranean Basin (modified from Lofi, 2018) showing: a) the location of the key intermediate and deep basins as well as physical thresholds that influenced the connectivity history of the Mediterranean; b) the onshore (i.e. basins and/or sections) and offshore (DSDP/ODP/Industrial drill sites) localities where deposits attributed to MSC Stage 3 have been studied. Mixed assemblages of Paratethyan-like ostracods and foraminifera are known from all mentioned onshore localities and some offshore locations (see text). The present-day spatial extent of the MSC seismic units, except for the Lower Unit, is also shown. The paleogeography of the (Eastern and Central) Paratethys and of the North Aegean domain is contoured after Van Baak et al. (2017) and Krijgsman et al. (2020a), respectively. W-E onshore localities: 1-6 Betic Cordillera (SE Spain): 1-Marbella and 2-Malaga basins (Guerra-Merch´an et al., 2010); 3-Sorbas Basin (Roveri et al., 2009, 2019a); 4-Nijar Basin (Fortuin and Krijgsman, 2003); 5-Vera Basin (Fortuin et al., 1995); 6-Bajo Segura Basin (Soria et al., 2005, 2008a, 2008b); 7-Mallorca (Mas and Forn´os, 2020); 8-Melilla Basin (Rouchy et al., 2003); 9-Boudinar Basin (Merzeraud et al., 2019); 10-Chelif Basin (Rouchy et al., 2007); 11-Sahel area (Frigui et al., 2016); 12-Al´eria Basin and 13-Rhˆone Valley (Carbonnel, 1978); 14-Piedmont Basin (Dela Pierre et al., 2011, 2016); 15-Po Plain (Ghielmi et al., 2010, 2013; Amadori et al., 2018); 16-Fine Basin (Cava Serredi section; Carnevale et al., 2006a, 2008). 1. Introduction Then, a uniform fauna populated this immense lac-mer which was divided […] into Pannonian basin, […] Dacique Basin, and Euxin and Caspian basin […]” (Gillet, 1932). “[…] During the Volhynien (Sarmatique inferior) there was a lac-mer of uniform fauna that extended through all the eastern Europe. […] and the fauna of the eastern regions of that huge lac-mer was completely differ­ enciated [sic] from the one in the western regions. […]” (Gillet, 1933). “[…] The Pontien fauna is not anymore a fauna characteristic of an internal saline sea, as in the Sarmantien, but is a fauna of a “desalinated lagoon”, a lac-mer, as the Russian geologists named it. […]” (Gignoux, 1936b). In the late 19th (Capellini, 1880) and 20th century (Ogniben, 1955; Ruggieri, 1962, 1967; Decima, 1964), late Messinian ostracod- and mollusk-bearing deposits in the Mediterranean were described at several Italian localities. Initially, the expressions “Congeria beds” (Capellini, 1880) and "Melanopsis beds" (Ruggieri, 1962) were used. Later on, Ruggieri (1967) pointed out the affinity of these faunal elements with those of the Pontian of the Paratethys. Consequently, he speculated on a feasible Paratethys-like paleoenvironmental configuration for the Med­ iterranean in the latest Messinian and he coined the Italian translation (i. e. "Lago-Mare") from the French “Lac-Mer” in reference to the shallow- water lakes claimed to be widely distributed across the Mediterranean. Progress in the 1970s in onshore and offshore exploration highlighted the temporally well-constrained distribution of the Paratethyan organ­ isms in the Mediterranean (Carbonnel, 1978). On this premise, Hsü et al. (1978a) proposed to use "Lago-Mare" to "designate the latest Messinian oligohaline environment, postdating evaporite deposition and predating Pliocene marine sedimentation […] in order to distinguish it from "lac mer" which, strictly speaking, was a Paratethyan environment". Notwithstanding the new definition, in various parts of the text they used "Lago-Mare" to refer to the Paratethyan lakes (pp. 1071-1072: "[…] The upper Messinian Mediterranean was floored by a series of desert basins, some with salt lakes, prior to inundation by the Lago-Mare."), thus giving rise to the confusion on how to use the term properly. After nearly 50 years of research on both onshore and offshore lo­ calities (Fig. 2), the observations backing up the competing desiccated and full-basin Mediterranean models remain extremely difficult to reconcile. 1. Introduction 17-21 Apennine system: Romagna sections (17, Roveri et al., 1998), Trave section (18, Iaccarino et al., 2008), Maccarone section (19, Bertini, 2006, Grossi et al., 2008; Sampalmieri et al., 2010; Pellen et al., 2017), Colle di Votta (20)-Fonte dei Pulcini (21)-Stingeti (22) sections (Cosentino et al., 2005, 2012, 2013, 2018), Mondragone 1 well (23, Cosentino et al., 2006), Crotone Basin (24, Roveri et al., 2008a); 25-27 Sicily: Villafranca Tirrena (25) and Licodia Eubea (26) sections (Sciuto et al., 2018), Caltanissetta Basin (27, Manzi et al., 2009); 28-Corfu (Pierre et al., 2006); 29-Zakinthos (Karakitsios et al., 2017b); 30-Crete (Cosentino et al., 2007); 31-Cyprus (Rouchy et al., 2001; Manzi et al., 2016a); 32-Adana Basin (Radeff et al., 2016). Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. waters (e.g. Manzi et al., 2009; Roveri et al., 2014c; Flecker et al., 2015; Vasiliev et al., 2017; García-Veigas et al., 2018; Grothe et al., 2020). Then, during substage 3.2, it became a brackish lake-sea comparable to the present-day Black Sea or Caspian Sea (Roveri et al., 2008a; Stoica et al., 2016; Andreetto et al., 2021), depending on whether a marine connection with the Atlantic was active (Manzi et al., 2009; Roveri et al., 2014b, 2014c; Flecker et al., 2015; Marzocchi et al., 2016; Vasiliev et al., 2017; García-Veigas et al., 2018) or not (e.g. McCulloch and De Deckker, 1989; Roveri et al., 2008a), and with a base-level fluctuating by hun­ dreds of meters with precessional periodicity (Fortuin and Krijgsman, 2003; Ben Moshe et al., 2020; Andreetto et al., 2021). In the relatively full scenario, the revival of marine conditions is ascribed to either connectivity changes (Marzocchi et al., 2016) or to a moderate sea-level rise (Andreetto et al., 2021). In contrast, Carnevale et al. (2006a, 2006b, 2008, 2018) and Grunert et al. (2016), based on the recovery of fish remains ascribed to marine species, proposed that fully marine condi­ tions were in force in the Mediterranean already at the end of substage 3.1. geologists Suzette Gillet (Gillet, 1932, 1933) and Maurice Gignoux (Gignoux, 1936a) we can state with relative certainty that the original meaning of the word “Lago-Mare” (here reported with the French counterpart “Lac-Mer”) had its provenance in the Russian literature: “[…] An isolation of the basin, that became a brackish, isolated basin. 1. Introduction Uncertainties regarding the chronostratigraphic framework of Stage 3 deposits, the origin and migration of its characteristic biota, the meaning of the data derived from the applied geochemical techniques and the relationship between the Mediterranean and its surrounding water bodies (i.e. Atlantic Ocean, Indian Ocean and Paratethys) all inhibit a clear understanding of the Mediterranean base-level and its hydrochemical structure. In this paper we attempt to summarize all the existing, but heavily scattered, data resulting from ~50 years of cross-disciplinary studies with the aim of providing a comprehensive overview of the stratigraphic arrangement of Stage 3 onshore and offshore deposits, as well as of their sedimentological, paleontological, geochemical and seismic properties. Subsequently, we assemble the observations favoring both end-member scenarios of a relatively desiccated and relatively full Mediterranean. Finally, we focus on novel future analytical techniques and approaches that have the potential to constrain Mediterranean base-level during MSC Stage 3 as well as the changing hydrological fluxes and connec­ tivity phases between the intra-Mediterranean basins and the neigh­ boring Atlantic Ocean and Paratethyan domains as a mean of reconstructing the state of the art of the complex history of this enig­ matic period of the Mediterranean history once and for all. In the most recent stratigraphic overview of the MSC (Fig. 1a; Roveri et al., 2014a), the terminal MSC stage is called Stage 3, which is in turn subdivided into substages 3.1 and 3.2 (also termed Lago-Mare). Beside such a chronostratigraphic definition, the term “Lago-Mare” has also been used for a typical biofacies of the late Messinian Mediterranean (e. g. Fortuin et al., 1995; Gliozzi, 1999; Gliozzi and Grossi, 2008; Sciuto et al., 2018), for the pelitic beds encasing the Paratethyan-related fauna (i.e. a lithofacies; e.g. Fortuin and Krijgsman, 2003; Sciuto et al., 2018), as the name of an informal lithostratigraphic unit (usually distinguished by its fossil content) sandwiched between the Sicilian Upper Gypsum and the Arenazzolo Fm. (Fig. 4b; Clauzon et al., 2005; Londeix et al., 2007; Popescu et al., 2009; Bache et al., 2012) and to denote multiple (3 to 4) spilling events of the Paratethys into the Mediterranean (Clauzon et al., 2005, 2015; Popescu et al., 2007, 2009, 2015; Suc et al., 2011; Bache et al., 2012; Do Couto et al., 2014; Frigui et al., 2016; Mas and Forn´os, 2020). i 2.2. Development of a chronostratigraphic framework Issues of the timing and duration of the MSC only began to be tackled in the 1990s, in parallel with discussion concerning the nature of its extreme paleoenvironments (Schmalz, 1969; Hsü et al., 1973a, 1973b, 1973c, Hsü et al., 1978a, 1978b; Nesteroff, 1973; De Benedetti, 1982). While published models (Butler et al., 1995; Clauzon et al., 1996; Krijgsman et al., 1999a; Rouchy and Caruso, 2006) mostly converged on the (astronomical) age of the marine replenishment at the beginning of the Pliocene (5.332 Ma; Van Couvering et al., 2000), there were dis­ agreements about the age of the onset of the MSC (synchronous vs diachronous) and of specific events within it (see discussion in Roveri et al., 2014a). Among these, the work of Krijgsman et al. (1999a) has obtained wide consensus. Their cyclostratigraphic tuning and correla­ tion of continuous and bio-magnetostratigraphically constrained pre- evaporitic sections in Spain (Sorbas), Sicily (Gibliscemi/Falconara) and Greece (Metochia) resulted in a synchronous age of 5.96±0.02 Ma for the MSC onset (later refined to 5.97 Ma by Manzi et al., 2013). The astronomical ages for the onset (Krijgsman et al., 1999a) and termina­ tion (Van Couvering et al., 2000) of the MSC are not contentious since the characteristic sedimentary cyclicity and sediments’ properties (e.g. color of the lithologies and biota content) of the pre- and post-MSC successions fit robustly with the insolation curve (see also Van der Laan et al., 2006 and Topper and Meijer, 2015). The age of the base of Stage 3 is largely determined by correlating the sedimentary cycles of the Upper Gypsum unit (UG) at Eraclea Minoa (Sicily) with the astronomical curve La2004 (Laskar et al., 2004). The UG sedimentary cyclicity consists of alternating gypsum and mudstone beds of variable thickness (Figs. 5g-i; see subsection 3.8). Precessional variation of the Mediterranean freshwater budget tied tightly to the African monsoon and Atlantic storms are the drivers interpreted to lie behind the gypsum-mudstone cycles (e.g. Marzocchi et al., 2015, 2019; Simon et al., 2017). Variations of the freshwater discharge cause the pycnocline to shift vertically, resulting in brine concentration and gyp­ sum precipitation during to the arid/dry phases of the precession cycles (precession maxima-insolation minima) and brine dilution and mudstone deposition during the humid/wet phases (precession minima- insolation maxima) (Van der Laan et al., 2006; Manzi et al., 2009). 2.1. Historic overview of nomenclature and concepts The final phase of the MSC (i.e. substage 3.2), also known as “Lago- Mare”, finds its sedimentary expression in cyclically-arranged terrige­ nous and evaporitic sediments hosting unique faunal assemblages of ostracods, mollusks and dinoflagellate cysts (dinocysts). They are related, at species level, to those inhabiting, during the Miocene, the brackish basins of the Paratethys realm (e.g. Gliozzi et al., 2007; Stoica et al., 2016). But what exactly is the “Lago-Mare”? This widely employed expression in the MSC literature encompasses a variety of meanings that make its application doubtful and misleading. The root of the wording “Lago-Mare” is to be found in the Russian scientific litera­ ture of the late 1800s. Nikolai Andrusov (1890) used the corresponding Russian term with a geographical and chronological connotation in reference to the series of central-eastern European basins that during the Miocene turned from marine settings to desalinized semi-isolated lakes with an endemic fresh-brackish water biota association (e.g. Popov et al., 2006 and references therein). The original monograph of Andrusov (1890) was not widely available outside Russia, but his attendance of international conferences allowed his research to spread outside the Russian borders. From the publications of the French This being a review, we use the widely employed definition of the model of Roveri et al., 2014a) (Fig. 1a) and regard the Lago-Mare as a “phase of massive biota migration from the Paratethys realm, cyclo­ stratigraphically constrained between 5.42 Ma and 5.332 Ma (Roveri et al., 2008a; Grossi et al., 2011), during which the Mediterranean sedimentary environments underwent an impressive freshening”. Nevertheless, we call for caution in the use of this definition of “Lago- Mare” in future studies, since 5.42 Ma as the (astronomical) age of the first entrance of Paratethyan organisms into the Mediterranean is likely 4 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. to be incorrect (see subsection 5.1) and evidence of ‘impressive fresh­ ening’ are already present much earlier (e.g. at Eraclea Minoa; Vasiliev et al., 2017; García-Veigas et al., 2018). gain precise dates for events within the MSC (e.g. Hilgen et al., 1995; Vai, 1997; Krijgsman et al., 1999b, 2001), bypassing the challenge posed by the unsuitability of the classic biomagnetostratigraphic tools for the MSC successions. Characteristic interference patterns of eccentricity and precession have been tentatively recognized in the Sicilian Eraclea Minoa section (see subsection 3.8; Van der Laan et al., 2006). 2.1. Historic overview of nomenclature and concepts However, clear orbital signals are typically poorly expressed in MSC records and, when they are present, like in Sicily, they are not (vertically) repeated with sufficient frequency to establish clear phase relations with the as­ tronomical cyclicity. For this reason, the simple counting of cycles with no analysis of cyclostratigraphic pattern in proxy records has mostly been employed as a correlation method (Roveri et al., 2008a; Manzi et al., 2009; Manzi et al., 2016a; Cosentino et al., 2013). 2.2. Development of a chronostratigraphic framework (2006) tentatively recognized sedimentary pat­ terns that they correlated with the astronomical curves by using the same phase relationships between the sedimentary cycles and the astronomical cycles as are seen in Plio-Pleistocene sapropel-bearing marine successions of the Mediterranean (Hilgen, 1991). The four closely spaced gypsum beds III to VI were regarded as a cluster, i.e. the sedimentary expression of a 100 kyr eccentricity maximum (Hilgen, 1991; Strasser et al., 2006), whereas the preceding and following evaporite-free marly interval were attributed to a phase of low-amplitude precession oscillations caused by a 100 kyr eccen­ tricity minimum (Fig. 3a). Tuning downward from the conformable Miocene/Pliocene boundary (Fig. 6d) and arguing that the preces­ sion peak at ~5.38 Ma, which has an extremely low amplitude, is not expressed in the sedimentary record, Van der Laan et al. (2006) correlated gypsums III to VI with the four successive precession/ insolation peaks of the 100 kyr eccentricity maximum dated around 5.44 Ma and the overlying and underlying gypsum-free marly in­ terval fell within 100 kyr eccentricity minimum cycles (Fig. 3a, right log). This tuning resulted in an astronomical age of ~5.51 Ma for the first gypsum bed in their log (i.e. gypsum II in the log of Manzi et al., 2009), and an approximate duration of 175 kyr for Stage 3 as whole. Although the substage 3.1/3.2 transition is linked to a major Mediterranean-scale hydrological re-organization possibly coinciding with the migration of the Paratethyan biota (Roveri et al., 2008a; Grossi et al., 2011), the facies change used for its definition is hardly recog­ nizable elsewhere (see Chapter 3). As such, other tools have been used to equip fragmentary and/or lithological cyclicity-lacking sections with an age model: the (highly controversial) ostracod biozonation (see sub­ section 5.1; e.g. Stoica et al., 2016; Karakitsios et al., 2017a; Cosentino et al., 2018; Caruso et al., 2020) and the astronomical tuning of mag­ netic susceptibility records (e.g. Fonte dei Pulcini section, Central Apennines; Cosentino et al., 2012). Comparison of Atlantic oxygen isotope records (Van der Laan et al., 2005, 2006) and the chronostratigraphy of Roveri et al. (2014a) revealed that Stage 3 sedimentation started during a prominent global eustatic lowstand associated with oxygen isotope (glacial) stage TG12, followed by a latest Messinian deglacial interval which comprised multiple obliquity- and possibly precession-forced global eustatic pha­ ses. As documented by Hodell et al. 3.1. The Albor´an region The westernmost outcrops of Stage 3 deposits in the Mediterranean are located in the Albor´an region, close to the present-day Strait of Gibraltar (Fig. 2b). MSC deposits on the margins of this region are poorly developed, possibly because of a late Tortonian uplift that raised the margins above the Mediterranean water level (L´opez-Garrido and Sanz de Galdeano, 1999). Near Malaga, however, two facies associations consisting of m-thick conglomerate-sandstone beds alternating with laminated pelites are documented in the Rio Mendelín section (infor­ mally referred to as “LM unit”; Guerra-Merch´an et al., 2010) and attributed to (part of) the Lago-Mare phase (Fig. 4a) based on their paleontological content. These sediments are squeezed between the Paleozoic basement units, with an erosive contact and associated angular unconformity, and the Pliocene, from which they are separated by another erosional surface draped by conglomeratic accumulations (Fig. 6a). A well-preserved and diverse in situ Paratethyan-type ostracod and molluskan fauna (i.e. Lymnocardiinae and Dreissenidae) typical of shallow waterbodies (up to 100 m deep; Grossi et al., 2008; Gliozzi and Grossi, 2008) with low salinities (5-18‰) is reported from the pelitic units (Guerra-Merch´an et al., 2010). The overlying Pliocene in the deeper depocenters starts with 30 m-thick littoral conglomerates with marine mollusks passing progressively upwards into deeper water facies, while fan deltas developed at the basin margins (L´opez-Garrido and Sanz de Galdeano, 1999; Guerra-Merch´an et al., 2010, 2014). Notably, the overall thickness of the Pliocene deposits reaches 600 m. The detailed regional studies by L´opez-Garrido and Sanz de Galdeano (1999) and Guerra-Merch´an et al. (2014) concluded that accommodation space was created during (Zanclean) sedimentation by local fault-driven subsi­ dence, and that movement on these faults only reversed at the end of the Zanclean causing uplift. An age of 5.53 Ma for the first gypsum bed was also obtained by the astronomical tuning of the Upper Gypsum in Cyprus (Manzi et al., 2016a), but there the tuning is performed just by following the recog­ nition, from the base up, of 6 gypsum beds just like in Sicily and therefore arguing for a bed-to-bed correlation with the Sicilian gypsums I-VI. In the consensus model of Roveri et al. (2014a) the base of Stage 3 coincides with the base of the Sicilian UG, placed by Manzi et al. (2009) at 5.55 Ma (Fig. 1A). However, in the model of Manzi et al. 2.2. Development of a chronostratigraphic framework (2006) correlated gypsums III to VI with the four successive precession/ insolation peaks of the 100 kyr eccentricity maximum dated around 5.44 Ma and the overlying and underlying gypsum-free marly in­ terval fell within 100 kyr eccentricity minimum cycles (Fig. 3a, right log). This tuning resulted in an astronomical age of ~5.51 Ma for the first gypsum bed in their log (i.e. gypsum II in the log of Manzi et al., 2009), and an approximate duration of 175 kyr for Stage 3 as whole. 2. An alternative tuning by Manzi et al., 2009; Fig. 3a, left log) argued that every precessional/insolation peak must have an expression in the rock record. Manzi et al. (2009) agreed with the solution of Van der Laan et al. (2006) on the sedimentary inexpressiveness of the (barely visible) insolation minima peak at ~5.38 Ma. However, these authors considered the insolation minima peaks immediately above and below of too low amplitude to promote the conditions required for gypsum precipitation, but also too high not to have some sedi­ mentary expression. They therefore identified sandstone horizons VI’ and VI" as the sedimentary response to these weak insolation/pre­ cession signals. The addition of two precessional cycles (i.e. a total of 9) resulted in an astronomical solution that was adjusted one pre­ cessional cycle lower than that of Van der Laan et al. (2006), trans­ lating into an age of 5.53 Ma for the base of the UG and a total duration of ~200 kyr for Stage 3. But the more conspicuous differ­ ence between the two astronomical solutions discussed lies in the timing at which gypsum precipitation occurred, restricted to the 100 kyr eccentricity maxima according to Van der Laan et al. (2006), extended to the 100 kyr eccentricity minima by Manzi et al. (2009). rather than the amplitude of the peaks responsible for the formation of the other facies. This approach resulted in an age of 5.42 Ma for the first conglomerate (i.e. the substage 3.1/3.2 transition; Fig. 5g) and an approximate duration of 90 kyr for substage 3.2 (the Lago-Mare phase). The same astronomical age is obtained by tuning the Upper Member of the Feos Formation in the Nijar Basin (Omodeo-Sal´e et al., 2012), where four pelite-conglomerate cycles plus one sandstone capped by the Miocene/Pliocene boundary mark the interval attributed to Stage 3.2 (Fortuin and Krijgsman, 2003). 1. Van der Laan et al. 2.2. Development of a chronostratigraphic framework (2001) (later revised by Drury et al., 2018), Van der Laan et al. (2006) and Roveri et al. (2014a), the marine replenishment of the Mediterranean did not coincide with any major deglaciation, so non-eustatic causes of the Zanclean megaflood hy­ pothesis are required. 2. An alternative tuning by Manzi et al., 2009; Fig. 3a, left log) argued that every precessional/insolation peak must have an expression in the rock record. Manzi et al. (2009) agreed with the solution of Van der Laan et al. (2006) on the sedimentary inexpressiveness of the (barely visible) insolation minima peak at ~5.38 Ma. However, these authors considered the insolation minima peaks immediately above and below of too low amplitude to promote the conditions required for gypsum precipitation, but also too high not to have some sedi­ mentary expression. They therefore identified sandstone horizons VI’ and VI" as the sedimentary response to these weak insolation/pre­ cession signals. The addition of two precessional cycles (i.e. a total of 9) resulted in an astronomical solution that was adjusted one pre­ cessional cycle lower than that of Van der Laan et al. (2006), trans­ lating into an age of 5.53 Ma for the base of the UG and a total duration of ~200 kyr for Stage 3. But the more conspicuous differ­ ence between the two astronomical solutions discussed lies in the timing at which gypsum precipitation occurred, restricted to the 100 kyr eccentricity maxima according to Van der Laan et al. (2006), extended to the 100 kyr eccentricity minima by Manzi et al. (2009). 2. 2.2. Development of a chronostratigraphic framework Two different tuning options exist in literature (Van der Laan et al., 2006 versus Manzi et al., 2009; Fig. 3a): The cyclic arrangement of the MSC sediments (Fig. 3a) led scientists to interpret that the same cyclostratigraphic approach could be used to Fig. 3. (a), (b) Available astronomical tunings to astronomic curves of climatic precession (P), 100 kyr eccentricity (E) and 65◦N insolation curve (I) of Laskar et al. (2004) of the lithological cyclicity of onshore Stage 3sections (a) and of the seismic cycles and/or well logs (gamma ray and resistivity) of the MU in the Levant Basin (b). Tunings of onshore sections in (a) are carried out downward from the M/P boundary (conformable in all sections). Astronomically-tuned glacial (even numbers) and interglacial (odd numbers) stages (i.e. TG) as defined by Hodell et al. (1994) are also indicated. Fig. 3. (a), (b) Available astronomical tunings to astronomic curves of climatic precession (P), 100 kyr eccentricity (E) and 65◦N insolation curve (I) of Laskar et al. (2004) of the lithological cyclicity of onshore Stage 3sections (a) and of the seismic cycles and/or well logs (gamma ray and resistivity) of the MU in the Levant Basin (b). Tunings of onshore sections in (a) are carried out downward from the M/P boundary (conformable in all sections). Astronomically-tuned glacial (even numbers) and interglacial (odd numbers) stages (i.e. TG) as defined by Hodell et al. (1994) are also indicated. 5 5 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. 1. Van der Laan et al. (2006) tentatively recognized sedimentary pat­ terns that they correlated with the astronomical curves by using the same phase relationships between the sedimentary cycles and the astronomical cycles as are seen in Plio-Pleistocene sapropel-bearing marine successions of the Mediterranean (Hilgen, 1991). The four closely spaced gypsum beds III to VI were regarded as a cluster, i.e. the sedimentary expression of a 100 kyr eccentricity maximum (Hilgen, 1991; Strasser et al., 2006), whereas the preceding and following evaporite-free marly interval were attributed to a phase of low-amplitude precession oscillations caused by a 100 kyr eccen­ tricity minimum (Fig. 3a). Tuning downward from the conformable Miocene/Pliocene boundary (Fig. 6d) and arguing that the preces­ sion peak at ~5.38 Ma, which has an extremely low amplitude, is not expressed in the sedimentary record, Van der Laan et al. 3.1. The Albor´an region Note the large controversies in timing, duration and chronostratigraphic position of the main erosion phase between models in (a) and (b). Models in (a) follow the recently established MSC chronostratigraphic model of Roveri et al. (2014a), according to which the Mediterranean base-level dropped and halite deposited on sea floor during Stage 2 and the Upper Gypsum/Upper Evaporites-Lago-Mare sequence followed. Models in (b) were proposed following the alternative scenario of Clauzon et al. (1996, 2005), which envisaged two Lago-Mare episodes (LM1 and LM3) that occurred before and after the main Mediterranean drawdown event, during which LM2 was deposited in the deep desiccated basins (Do Couto et al., 2014; Popescu et al., 2015; see Roveri et al., 2008c and Grothe et al., 2018 for further explanations). Note, in (b), the shifting of the position of the main erosional phase in Sicily through time as well as the time of the marine replenishment in the Apennines. An alternative scenario, based on the finding of (a few) specimens of the nannofossil Ceratolithus acutus, ascribed the LM unit of Guerra- Merch´an et al. (2010) to the earliest Zanclean (Fig. 4b; Do Couto et al., 2014). Miocene up until MSC Stage 1 (Fortuin and Krijgsman, 2003), which is represented by the gypsiferous Yesares Member (e.g. Lu, 2006). How­ ever, facies differences are prominent in the Stage 3 formations ac­ cording to the chronostratigraphic frameworks of Roveri et al. (2009) for the Sorbas Basin and Omodeo-Sal´e et al. (2012) for the Nijar Basin (Fig. 4a). Lithostratigraphically, two members are discerned between the Yesares Member and the basal Zanclean: the Sorbas and Zorreras members in the Sorbas Basin (Figs. 4a, 5a) and the lower and upper members of the Feos Fm. in Nijar (Figs. 3a, 4a; Roep et al., 1998; Krijgsman et al., 2001; Fortuin and Krijgsman, 2003; Braga et al., 2006; Roveri et al., 2009, 2019a; Omodeo-Sal´e et al., 2012). On the southern Albor´an margin in Morocco, latest Messinian de­ posits are reported from the Boudinar and Melilla basins (Fig. 2b). Up to 100 m-thick chaotic deposits containing selenite gypsum fragments, azoic conglomerates, sandstones yielding planktic foraminifera and nannofossils and lacustrine limestones are capped by early Pliocene marine marls (Rouchy et al., 2003; Azdimousa et al., 2006; Corn´ee et al., 2016; Merzeraud et al., 2019). 3.1. The Albor´an region (2009) this age is attributed to a cumulate gypsum horizon interpreted as laterally equivalent of the Halite (i.e. Stage 2), and therefore implying the kickoff of Stage 3 at 5.53 Ma (Fig. 3a). The post-evaporitic successions of the Romagna (Cusercoli and Sapigno sections; Roveri et al., 1998) and Marche (e.g. Trave and Maccarone sections; Iaccarino et al., 2008; Cosentino et al., 2013) areas provided evidence that led to the splitting of Stage 3 into substage 3.1 and 3.2. In the resulting composite section (Roveri et al., 2008a), a shift in the sedimentary facies and stacking pattern is observed (see description in subsection 3.7). Correlation of the sedimentary cyclicity in Romagna was from the (conformable) base of the Pliocene down­ wards (or from an U-Pb-dated ash layer upward; Cosentino et al., 2013) and linked three fluvial conglomerates and two black mudstone layers of unknown sedimentological significance to the arid phases of the pre­ cession cycles (Fig. 3a; Roveri et al., 2008a). The greater thickness of the oldest conglomerate was possibly assumed to be evocative of an oscil­ lation of the amplitude of the corresponding precession minima peak 6 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Fig. 4. Schematic overview of different chronostratigraphic models for some of the Messinian successions presented in Chapter 3. Note the large controversies in timing, duration and chronostratigraphic position of the main erosion phase between models in (a) and (b). Models in (a) follow the recently established MSC chronostratigraphic model of Roveri et al. (2014a), according to which the Mediterranean base-level dropped and halite deposited on sea floor during Stage 2 and the Upper Gypsum/Upper Evaporites-Lago-Mare sequence followed. Models in (b) were proposed following the alternative scenario of Clauzon et al. (1996, 2005), which envisaged two Lago-Mare episodes (LM1 and LM3) that occurred before and after the main Mediterranean drawdown event, during which LM2 was deposited in the deep desiccated basins (Do Couto et al., 2014; Popescu et al., 2015; see Roveri et al., 2008c and Grothe et al., 2018 for further explanations). Note, in (b), the shifting of the position of the main erosional phase in Sicily through time as well as the time of the marine replenishment in the Apennines. Fig. 4. Schematic overview of different chronostratigraphic models for some of the Messinian successions presented in Chapter 3. 3.2. Algeria The Chelif Basin in Algeria (Fig. 2b) displays the typical marginal Messinian succession comprising Tortonian to lower Messinian blue marls, diatomite-bearing sediments (Tripoli unit), cyclically-arranged primary evaporites (13 couplets), ostracod-rich post-evaporitic de­ posits and Zanclean foraminiferal marls (Rouchy et al., 2007). The post- evaporitic sediments show a great lateral variability in both thickness (from few meters up to 125 m) and facies. They are mainly dominated by terrigenous clastic lithologies, associated in the marginal areas with sandy carbonates and stromatolitic limestones. A mixed faunal assem­ blage of non-marine (Paratethyan-like ostracods) and marine (benthic and planktic foraminifera) organisms is present, showing an increase in ostracod species diversity from the bottom to the top (Rouchy et al., 2007). 3.1. The Albor´an region Due to their stratigraphic position, these continental to lacustrine deposits are interpreted as the local expression of the Lago-Mare phase (Corn´ee et al., 2016) or alternatively as Zanclean successions (Azdimousa et al., 2006). The Sorbas Member (see Roep et al., 1998 and Aufgebauer and McCann, 2010 for a more detailed sedimentological description) con­ sists of three overlapping coarsening-upward depositional sequences made of offshore clays and marls passing upward into shelf muds and coastal sandstone bodies. Still unclear is the chemistry of the subaqueous environment during the formation of the Sorbas Member and the provenance of the water fluxes. These shallow-water deposits are conformably replaced upward by the Zorreras Member that comprises alternations of reddish siltstones and sandstones (Fig. 5a) organized in five (or eight) lithological cycles expressing continental environments (Martín-Su´arez et al., 2000; Aufgebauer and McCann, 2010). Up to four lenticular white limestone beds bearing brackish Paratethyan-like os­ tracods (Cyprideis, Loxocorniculina djafarovi and freshwater species of the family Limnocytheridae), bivalves and Chara oogonia (Roep and Harten, 1979; Aufgebauer and McCann, 2010) are found interrupting the fluviatile sequence (Fig. 5a) and are linked to either episodic flooding by local rivers (Braga et al., 2006; Aufgebauer and McCann, 2010) or episodic Mediterranean ingressions (Fortuin and Krijgsman, 2003; Andreetto et al., 2021). A correct interpretation of the paleo­ depositional environment of these limestone beds is crucial for the dis­ cussion concerning the Mediterranean base-level position during the Lago-Mare phase. In fact, if the Sorbas Basin was relatively shallow during Zorreras deposition (50-100 m; Roveri et al., 2019a, 2020), repeated and sudden Mediterranean incursions would indicate that the Mediterranean Basin was relatively full and that its base level was oscillating, possibly with precessional periodicity (Andreetto et al., 2021). The contact between the Zorreras Mb. and the overlying near­ shore Pliocene (<50 m depositional paleodepth; Roveri et al., 2019a) in the Sorbas Basin is conformable and expressed differently around the 3.3. Neogene basins of the Eastern Betics (Spain) The external Neogene basins (Sorbas, Nijar, Vera and Bajo Segura) of the eastern Betic Cordillera (SE Spain; Fig. 2b) represent an important laboratory for understanding Messinian events. In particular, the Sorbas and Nijar basins preserve two allegedly continuous successions spanning the entire MSC (e.g. Roveri et al., 2009; Omodeo-Sal´e et al., 2012). The two basins are similar in many respects. Their stratigraphic organiza­ tion, for example, suggests they were connected for much of the late 7 F. Andreetto et al. Earth-Science Reviews 216 (2021) 103577 ntary expression of Stage 3 from selected onshore Mediterranean localities. (a) Photograph from the Sorbas Basin showing Zorreras member with intercalated white limestones (white arrows; from Andreetto et al., 2021). The conformable resting of the nd underneath the Gochar Fm. of Pliocene age is also appreciable. Car for scale. (b) One typical lithological (and precessional) cy in the Nijar Basin, here constituted by an ostracod-bearing, white and laminated mudstone bed overlain by an azoic fluvial sa c) Panoramic view of the Cuevas del Almanzora section (from Andreetto et al., 2021). Red rectangle indicates the position of th M)/Zanclean (Z) transition and studied by Fortuin et al. (1995), Stoica et al. (2016), Caruso et al. (2020) and Andreetto et al. ( ub-unit a of the Piedmont Basin composed of azoic grey mudstones grading into yellowish, mammal-rich overbank deposits. (e Plain showing incised valleys filled during Stage 3 by suggested clastic deposits and sealed by deep-water turbidites in the Zanc 2018). (f) Typical aspect of the di Tetto/San Donato Formation in the Northern Apennines composed by grey mudstones (det dstone bodies (white arrows). The picture is taken from the Maccarone section. (g) The di Tetto Fm.-Colombacci Fm. transition na, Fig. 2b), defined by the facies change underlined by the appearance of a fluvial conglomerate. This lithostratigraphic bound 3.2 boundary of Roveri et al. (2014a). (h), (i), (l) Lithological cycles of the Upper Gypsum Unit in Eraclea Minoa (h), Siculiana M l l thi k d i il d b b d f i lt ti ith d t b i P t th Fig. 5. Sedimentary expression of Stage 3 from selected onshore Mediterranean localities. (a) Photograph from the Sorbas Basin showing the red continental sediments of the Zorreras member with intercalated white limestones (white arrows; from Andreetto et al., 2021). The conformable resting of the Zorreras Mb. above the Sorbas Mb. and underneath the Gochar Fm. 3.3. Neogene basins of the Eastern Betics (Spain) These clays contain a well-preserved and diversified in situ fauna of Paratethyan-like ostracod and shallow-water, benthic foraminifera mixed with physically reworked (mostly from the lower Messinian Abad marls) planktic and deep-water benthic forami­ nifera (Fortuin et al., 1995; Stoica et al., 2016; Caruso et al., 2020). The marly clays are assigned by Stoica et al. (2016) and Caruso et al. (2020) to (roughly) the whole late Messinian Lago-Mare phase (Fig. 4a) based on the ostracod biozonation of Grossi et al. (2011) and are considered to represent either sedimentation in an isolated lake subject to base-level and salinity fluctuations (Caruso et al., 2020) or deposition in a coastal lagoon that was connected to the water mass filling the open Mediterranean (Stoica et al., 2016; Andreetto et al., 2021). Similar to Malaga, these sediments are topped by an erosive surface draped by a conglomeratic accumulation which is overlain by the open marine fauna-rich sediments of the basal Zanclean (Fortuin et al., 1995; Caruso et al., 2020). This erosion feature likely indicates that the Miocene/ Pliocene transition followed a base-level lowstand in the Vera Basin. i In the Nijar Basin (Fig. 2b), the latest Messinian Feos Formation is bracketed at the base and top by an erosional surface along the basin margins and its correlative conformity in the deeper parts (Fig. 3a; Fortuin and Krijgsman, 2003; Aguirre and S´anchez-Almazo, 2004; Omodeo-Sal´e et al., 2012). The Lower Feos Member consists of azoic, graded and locally slumped siliciclastic-carbonate beds alternating with gypsarenites and gypsiltites and including a laterally continuous Mn- rich bed (Fortuin and Krijgsman, 2003; Omodeo-Sal´e et al., 2012). In the basin center (e.g. Barranco de los Castellones section; Fig. 3a) the Upper Feos member comprises four complete lithological cycles of m- thick conglomerate to sandstone beds alternating with laminated pelites (Fig. 5b), and one incomplete cycle, which only consists of a sandstone horizon conformably capped by the Pliocene Cuevas Fm. (Fig. 6b; For­ tuin and Krijgsman, 2003). A rich fauna of mixed brackish ostracods and marine foraminifera is found in all four pelitic beds (Bassetti et al., 2006). Its origin is questionable. These ostracods were regarded as endemic to the Mediterranean and inhabiting endorheic lakes by Bas­ setti et al. (2006). However, later they were shown to have been mis­ identified and were instead considered Paratethys-derived by Stoica et al. (2016; see subsection 5.1). 3.3. Neogene basins of the Eastern Betics (Spain) and the Zanclean biocalcarenites of the Cuevas Fm. in the Barranco de los Castellones section, Nijar Basin (hammer for scale; modified from Andreetto et al., 2021). (c) The Messinian/Zanclean boundary in the Pollenzo section (Piedmont Basin) marked by a characteristic black layer interbedded between Paratethyan ostracods-rich mudstones and marine foraminifera-rich marls (modified from Dela Pierre et al., 2016). (d) Uppermost segment of the Eraclea Minoa section (Caltanissetta Basin, Sicily) displaying the (non erosive) contact between the Pliocene Trubi Formation above and the sandy Arenazzolo Formation below. The inset is a close view of the transition, which occurs above a ~50 cm-thick burrowed mudstone horizon rich in Paratethyan ostracods and marine foraminifera. (e) Lago-Mare sediments in the Kalamaki section (Zakynthos) unconformable, through an erosional surface (i.e. the Messinian Erosional Surface, MES), over the PLG unit and also unconformable beneath the Trubi Fm. (modified from Karakitsios et al., 2017b). (f) Close view of the M/P boundary in the Pissouri Basin, where the foraminifera-rich Trubi marls lie above a black layer (paleosol according to Rouchy et al., 2001). (2003), Bassetti et al. (2006) and Omodeo-Sal´e et al. (2012), in place by Aguirre and S´anchez-Almazo (2004). basin, ranging from a bivalves-rich bed overlain by a yellow, fossilif­ erous calcarenite floored by a gravelly lag deposit (Mather et al., 2001) to a grey marl horizon with marine foraminifera assemblages followed by a second shell-rich bed (Roveri et al., 2019a). Similar to the situation in Malaga, the rare identification of Ceratolithus acutus in sediments of the continental Zorreras Mb. led Clauzon et al. (2015) to put forward an alternative chronostratigraphic and paleoenvironmental interpretation for the Sorbas MSC succession, shifting the Zorreras Mb. into the Plio­ cene (Fig. 4b) and thus associating the presence of brackish Paratethyan- like ostracods with exchanges between the Mediterranean and Para­ tethys following the Mediterranean re-filling, at high sea level. In the Vera Basin (Fig. 2b), in situ gypsum deposits are missing because of widespread erosion or non-deposition and MSC deposits are only represented by ~12 m of laminated varicolored marly clays (Unit 2 Fig. 4a), which are best exposed in the Cuevas del Almanzora section (Fortuin et al., 1995; Fig. 5c). 3.3. Neogene basins of the Eastern Betics (Spain) of Pliocene age is also appreciable. Car for scale. (b) One typical lithological (and precessional) cycle of the Upper Mb. of the Feos Fm. in the Nijar Basin, here constituted by an ostracod-bearing, white and laminated mudstone bed overlain by an azoic fluvial sandstone (courtesy of Anne Fortuin). (c) Panoramic view of the Cuevas del Almanzora section (from Andreetto et al., 2021). Red rectangle indicates the position of the section straddling the Messinian (M)/Zanclean (Z) transition and studied by Fortuin et al. (1995), Stoica et al. (2016), Caruso et al. (2020) and Andreetto et al. (2021). Buildings for scale. (d) The sub-unit a of the Piedmont Basin composed of azoic grey mudstones grading into yellowish, mammal-rich overbank deposits. (e) WNW-ESE seismic profile in the Po Plain showing incised valleys filled during Stage 3 by suggested clastic deposits and sealed by deep-water turbidites in the Zanclean (modified from Amadori et al., 2018). (f) Typical aspect of the di Tetto/San Donato Formation in the Northern Apennines composed by grey mudstones (detail in the inset) with interbedded sandstone bodies (white arrows). The picture is taken from the Maccarone section. (g) The di Tetto Fm.-Colombacci Fm. transition in the Cusercoli area (Eastern Romagna, Fig. 2b), defined by the facies change underlined by the appearance of a fluvial conglomerate. This lithostratigraphic boundary also corresponds to substage 3.1/3.2 boundary of Roveri et al. (2014a). (h), (i), (l) Lithological cycles of the Upper Gypsum Unit in Eraclea Minoa (h), Siculiana Marina (i) and Polemi (l) sections. Cycles are several m-thick and primarily composed by beds of primary gypsum alternating with mudstones bearing Paratethyan ostracods (at least in Eraclea Minoa). Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Fig. 6. Photographs of the Miocene/Pliocene boundary (yellow lines) from selected onshore Mediterranean localities. (a) Erosive M/P transition in the Mendelín section (Malaga Basin). Note the conglomeratic lag draping the erosional surface and sharply overlain by foraminifera-rich marls. (b) Conformable stratigraphic contact between the uppermost Messinian sandstone of the Feos Fm. and the Zanclean biocalcarenites of the Cuevas Fm. in the Barranco de los Castellones section, Nijar Basin (hammer for scale; modified from Andreetto et al., 2021). (c) The Messinian/Zanclean boundary in the Pollenzo section (Piedmont Basin) marked by a characteristic black layer interbedded between Paratethyan ostracods-rich mudstones and marine foraminifera-rich marls (modified from Dela Pierre et al., 2016). 3.3. Neogene basins of the Eastern Betics (Spain) (d) Uppermost segment of the Eraclea Minoa section (Caltanissetta Basin, Sicily) displaying the (non erosive) contact between the Pliocene Trubi Formation above and the sandy Arenazzolo Formation below. The inset is a close view of the transition, which occurs above a ~50 cm-thick burrowed mudstone horizon rich in Paratethyan ostracods and marine foraminifera. (e) Lago-Mare sediments in the Kalamaki section (Zakynthos) unconformable, through an erosional surface (i.e. the Messinian Erosional Surface, MES), over the PLG unit and also unconformable beneath the Trubi Fm. (modified from Karakitsios et al., 2017b). (f) Close view of the M/P boundary in the Pissouri Basin, where the foraminifera-rich Trubi marls lie above a black layer (paleosol according to Rouchy et al., 2001). Fig. 6. Photographs of the Miocene/Pliocene boundary (yellow lines) from selected onshore Mediterranean localities. (a) Erosive M/P transition in the Mendelín section (Malaga Basin). Note the conglomeratic lag draping the erosional surface and sharply overlain by foraminifera-rich marls. (b) Conformable stratigraphic contact between the uppermost Messinian sandstone of the Feos Fm. and the Zanclean biocalcarenites of the Cuevas Fm. in the Barranco de los Castellones section, Nijar Basin (hammer for scale; modified from Andreetto et al., 2021). (c) The Messinian/Zanclean boundary in the Pollenzo section (Piedmont Basin) marked by a characteristic black layer interbedded between Paratethyan ostracods-rich mudstones and marine foraminifera-rich marls (modified from Dela Pierre et al., 2016). (d) Uppermost segment of the Eraclea Minoa section (Caltanissetta Basin, Sicily) displaying the (non erosive) contact between the Pliocene Trubi Formation above and the sandy Arenazzolo Formation below. The inset is a close view of the transition, which occurs above a ~50 cm-thick burrowed mudstone horizon rich in Paratethyan ostracods and marine foraminifera. (e) Lago-Mare sediments in the Kalamaki section (Zakynthos) unconformable, through an erosional surface (i.e. the Messinian Erosional Surface, MES), over the PLG unit and also unconformable beneath the Trubi Fm. (modified from Karakitsios et al., 2017b). (f) Close view of the M/P boundary in the Pissouri Basin, where the foraminifera-rich Trubi marls lie above a black layer (paleosol according to Rouchy et al., 2001). Fig. 6. Photographs of the Miocene/Pliocene boundary (yellow lines) from selected onshore Mediterranean localities. (a) Erosive M/P transition in the Mendelín section (Malaga Basin). Note the conglomeratic lag draping the erosional surface and sharply overlain by foraminifera-rich marls. (b) Conformable stratigraphic contact between the uppermost Messinian sandstone of the Feos Fm. Pierre et al., 2011). euryhaline, shallow-water benthic foraminifera (Ammonia beccarii, Elphidium granosum, Elphidium macellum, Haynesina germanica and Quinqueloculina laevigata). Planktic foraminifera are also observed and for a long time were considered to be physically reworked (Soria et al., 2005, 2008b). However, some stratigraphic levels contain dwarf tests of long-ranging taxa such as Globoturborotalita decoraperta, Globigerina bulloides, and Neogloboquadrina spp. which recently have been inter­ preted as being in-situ mostly due to the absence of notable signs of reworking (Corbí and Soria, 2016). Among these dwarf taxa is Neo­ globoquadrina acostaensis (dextral; Corbí and Soria, 2016). Since this group is mainly dextral in the latest Messinian Atlantic successions (e.g. Sierro et al., 1993; Bassetti et al., 2006), this may indicate that Atlantic inflow to the Mediterranean occurred during the late Messinian and the base level of the Mediterranean was high enough to reach the marginal Bajo Segura Basin. The Miocene/Pliocene boundary is, once again, marked by an erosional surface which outlines up to 200 m deep pale­ ovalleys engraved down into the pre-MSC sediments and filled with conglomerates and sandstones of claimed coastal and shallow marine environments (Soria et al., 2005, 2008b; García-García et al., 2011; Corbí et al., 2016). The Cassano Spinola Conglomerates is splitted in two sub-units by Dela Pierre et al. (2016). Sub-unit a consists of azoic grey mudstones turning to yellowish silty mudstones (Fig. 5d) typified by in situ root traces, paleosols and mud cracks and including three/four intercalated lens-shaped, cross-bedded conglomeratic layers (Ghibaudo et al., 1985; Dela Pierre et al., 2011, 2016). Abundant land plant leaves and a diverse terrestrial vertebrate fauna are found in the yellowish siltstones, which have been interpreted as overbank deposits (Harzhauser et al., 2015; Colombero et al., 2017 and references therein). In this continental in­ terval, a low-diversity fish fauna consisting of otoliths of marine and Paratethyan species is found (Grunert et al., 2016; Carnevale et al., 2018; Schwarzhans et al., 2020). These otoliths were Sr-dated to the early-middle Miocene (Grunert et al., 2016). Nevertheless, they were concluded not to be physically reworked, but rather to have been transported by large marine predators, therefore implying a Piedmont Basin-(marine) Mediterranean connection was in force (Grunert et al., 2016; see subsection 5.6). Sub-unit b (i.e. 3.4. Mallorca Mallorca, which constitutes an emerged segment of the Balearic Promontory (Fig. 2), does not expose the classical MSC evaporite sequence. Instead, two main MSC-related units are found above late Tortonian-Messinian reefal carbonates (Reef Complex Unit) and beneath the Pliocene: the Santanyí limestones and the Ses Olles Formation (Mas and Forn´os, 2020 and references therein). The Santanyí limestones are microbialites and oolite-dominated sediments in which a baleen whale neurocranium has been found (Mas et al., 2018a). This unit was inter­ preted either as a Terminal Carbonate Complex (TCC) laterally equiva­ lent to the Primary Lower Gypsum (PLG) which has been drilled in the deeper parts of the bay of Palma (Mas and Forn´os, 2020) or as time- equivalent to the Reef Complex Unit (e.g. Arenas and Pomar, 2010; Su´arez-Gonz´alez et al., 2019). The Ses Olles Formation consists of marls, sandy-marls and marly-calcareous lacustrine deposits rich in in-situ freshwater Chara spp., brackish water Paratethyan-like mollusks and ostracods and littoral benthic foraminifera (Elphidium sp., Ammonia sp.). The upper contact of the Ses Olles Formation with the Pliocene corresponds to an erosional ravinement surface draped by a trans­ gressive lag of coastal deposits usually containing coquinas and/or conglomerates (Mas, 2013, 2015; Mas and Forn´os, 2020). The lower contact of the Ses Olles Formation with the Santanyí limestones is sporadically marked by a well-developed reddish paleosol (Mas, 2013, 2015; Mas and Forn´os, 2020), which indicates that a (unquantified) period of subaerial exposure occurred before the emplacement of the Ses Olles Fm. However, in their more recent study, Mas and Forn´os (2020) surprisingly conclude that the Ses Olles Formation has a conformable contact with the Santanyí limestones, ascribed to part of Stage 1. This led Mas and Forn´os (2020) to conclude that the emplacement of the Ses Olles Fm. pre-dated the MSC peak and that the erosional surface marking the Miocene/Pliocene boundary is associated with a 270 kyr hiatus linked to the main MSC base-level drawdown (Fig. 4b). This conclusion is, however, in disagreement with the unconformity at the base of the Ses Olles Fm., which instead points to the deposition of the Ses Olles Fm. (and therefore to the arrival of the Paratethyan fauna in Mallorca) at some point during Stage 3 of Roveri et al. (2014a). Mallorca, which constitutes an emerged segment of the Balearic Promontory (Fig. 2), does not expose the classical MSC evaporite sequence. 3.6. Po Plain The upper contact of the Ses Olles Formation with the Pliocene corresponds to an erosional ravinement surface draped by a trans­ gressive lag of coastal deposits usually containing coquinas and/or conglomerates (Mas, 2013, 2015; Mas and Forn´os, 2020). The lower contact of the Ses Olles Formation with the Santanyí limestones is sporadically marked by a well-developed reddish paleosol (Mas, 2013, 2015; Mas and Forn´os, 2020), which indicates that a (unquantified) period of subaerial exposure occurred before the emplacement of the Ses Olles Fm. However, in their more recent study, Mas and Forn´os (2020) To the east, the Messinian sediments in the Piedmont Basin disappear beneath the km-thick Plio-Quaternary succession of the Po Plain- Adriatic Foredeep (PPAF; Fig. 2a). By definition of Ghielmi et al. (2010) and Amadori et al. (2018), the PPAF includes two main elon­ gated depocenters enclosed within the northern Apennines to the South and the Southern Alps to the North: the easternmost portion of the Po Plain and the whole present-day northern Adriatic Sea. Here, for simplicity, we include in the definition of PPAF also its westernmost depocenters of the Western Po Plain Foredeep. The Messinian-Pleistocene sedimentary sequence, studied through the integration of seismic and borehole observations, is mostly repre­ sented by thick sequences of turbidite deposits in the foreland depo­ center passing, towards the margins, to fluvial and deltaic systems related to the proximity of the marginal thrust-fold-belts (Cipollari et al., 1999; Ghielmi et al., 2010, 2013; Rossi et al., 2015a; Rossi, 2017). During MSC Stage 1, primary evaporites and dolomicrites were depos­ ited in some shallow-water settings, while evaporitic deposition was inhibited in the deep-water settings, where it was replaced by deposition of anoxic mudstones (Ghielmi et al., 2010). Instead, the post-evaporitic deposits consist of large thicknesses (up to 1 km) and volumes of coarse- grained clastics (LM1 and LM2 of Rossi and Rogledi, 1988; ME3 or Fusignano Fm. of Ghielmi et al., 2010; ME4 of Ghielmi et al., 2013; ME3b and possibly ME3a of Rossi et al., 2015a). Several authors (Ghielmi et al., 2010, 2013; Rossi et al., 2015a; Amadori et al., 2018; Cazzini et al., 2020) showed that these post-evaporitic sediments are the infilling of ca. N-S and NW-SE trending, V-shaped valleys (Fig. 5e). Pierre et al., 2011). Strati a Congeria sensuSturani, 1973) is made of grey mudstones bearing a mixture of in-situ brackish water mollusks (Sturani, 1973; Esu, 2007) and ostracods (Trenkwalder et al., 2008) of Paratethyan affinity along with physically reworked foraminifera and calcareous nannofossils (Trenkwalder et al., 2008; Violanti et al., 2009). The transition to the Pliocene Argille Azzurre Fm. is sharp above a characteristic black and azoic sandy layer (Fig. 6c) rich in terrigenous and intrabasinal (i.e., glaucony and phosphates) grains and disarticulated valves of both brackish-water and continental bi­ valves, but barren of in-situ fossils (Trenkwalder et al., 2008). The occurrence, at its top and directly below the Argille Azzurre Fm., of abundant Thalassinoides trace fossils filled with Pliocene sediments led Trenkwalder et al. (2008) and Dela Pierre et al. (2016) to interpret the top surface of this layer as an omission surface. This surface indicates a period of basin starvation (and therefore a hiatus) due to a sudden in­ crease in water-depth, ascribed by Trenkwalder et al. (2008) to the Zanclean reflooding. This hiatus may have lasted for only part of the late Messinian (Violanti et al., 2009; Dela Pierre et al., 2016) or may have endured into the Pliocene (Trenkwalder et al., 2008). 3.6. Po Plain These valleys were carved at least as far as 50 km into the Alps, to a surprisingly conclude that the Ses Olles Formation has a conformable contact with the Santanyí limestones, ascribed to part of Stage 1. This led Mas and Forn´os (2020) to conclude that the emplacement of the Ses Olles Fm. pre-dated the MSC peak and that the erosional surface marking the Miocene/Pliocene boundary is associated with a 270 kyr hiatus linked to the main MSC base-level drawdown (Fig. 4b). This conclusion is, however, in disagreement with the unconformity at the base of the Ses Olles Fm., which instead points to the deposition of the Ses Olles Fm. (and therefore to the arrival of the Paratethyan fauna in Mallorca) at some point during Stage 3 of Roveri et al. (2014a). 3.4. Mallorca Instead, two main MSC-related units are found above late Tortonian-Messinian reefal carbonates (Reef Complex Unit) and beneath the Pliocene: the Santanyí limestones and the Ses Olles Formation (Mas and Forn´os, 2020 and references therein). The Santanyí limestones are microbialites and oolite-dominated sediments in which a baleen whale neurocranium has been found (Mas et al., 2018a). This unit was inter­ preted either as a Terminal Carbonate Complex (TCC) laterally equiva­ lent to the Primary Lower Gypsum (PLG) which has been drilled in the deeper parts of the bay of Palma (Mas and Forn´os, 2020) or as time- equivalent to the Reef Complex Unit (e.g. Arenas and Pomar, 2010; Su´arez-Gonz´alez et al., 2019). The Ses Olles Formation consists of marls, sandy-marls and marly-calcareous lacustrine deposits rich in in-situ freshwater Chara spp., brackish water Paratethyan-like mollusks and ostracods and littoral benthic foraminifera (Elphidium sp., Ammonia sp.). 3.3. Neogene basins of the Eastern Betics (Spain) Planktonic and deep-water benthic foraminifera are widely considered reworked by Fortuin and Krijgsman Stage 3 deposits (Garrucha Fm.) in the easternmost basin of the Betic Cordillera, the Bajo Segura Basin (Fig. 2b), are bounded below and above by two erosional surfaces related to lowered Mediterranean base- levels and discontinuously present due to the widespread fluvial erosion that occurred at the Miocene/Pliocene boundary (Soria et al., 2005, 2008a, 2008b). The Garrucha Fm. shows a maximum thickness of 100 m in its type section (Soria et al., 2007, 2008b). It consists of 20-50 cm thick sandstone bodies interrupting a dominantly marly succession deposited in a subaqueous environment inhabited by Cyprideis sp. and 9 9 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. 3.7. Apennine system The Messinian deposits resurface to the south of the PPAF sector and extensive sections are found in several basins on both the foreland domain (Adriatic side of the partially uplifted Apennine chain), sub­ jected to compressional tectonics during the late Messinian, and the back-arc domain (Tyrrhenian side), contemporaneously affected by extension (Fig. 2b; Cipollari et al., 1999; Schildgen et al., 2014; Cosen­ tino et al., 2018). Overall, the MSC record of the Apennines is subdivided into an evaporitic and post-evaporitic interval squeezed in between two marine units (Messinian Euxinic Shales Fm. at the base and Zanclean Argille Azzurre Fm. atop; Fig. 4a). Different vertical motions related to ongoing Apenninic tectonics resulted in the deposition of Stage 3 sedi­ ments with highly variable sedimentary expression and stratigraphic resolution from basin to basin. The post-evaporitic deposits are alter­ natively found resting unconformably, with an erosional contact asso­ ciated to an angular unconformity, above the alternations of the Gessoso Solfifera Fm./PLG, or conformably above evaporitic-free cycles lateral equivalent of the marginal PLG (Fig. 4a; e.g. Roveri et al., 1998, 2008a). This led to the conclusion that both shallow and deep-water successions are present in the Apennine foredeep system (Roveri et al., 2001). Stage 3 sediments are poorly exposed on the Tyrrhenian Sea onshore side of Italy (Fig. 2b). The best known succession crops out in the Cava Serredi quarry in the Fine Basin (Tuscany; Bossio et al., 1978, 1993; Carnevale et al., 2006b, 2008). Here the MSC has a thickness of ~150 m, of which only the uppermost ~100 m are attributed, without clear ar­ guments, to Stage 3 by Carnevale et al. (2006b). The lowermost ~40 m of the Stage 3 unit consists of mudstone with alternating sandstone bodies which have been attributed to Roveri et al. (1998)’s p-ev1 allounit, while the uppermost ~60 m form the p-ev2 allounit and include two prominent conglomerate bodies alternating with mudstones inter­ bedded with sandstone horizons and black, organic-rich layers (Carne­ vale et al., 2006b). A few and more fragmented sections are also described on the Tyrrhenian Sea side of Italy by Cipollari et al. (1999). p p p y The physical-stratigraphic model developed for the post-evaporitic interval in the Romagna area (i.e. Northern Apennines) and applied to the whole Apennine domain was subdivided into two allounits (named p-ev1 and p-ev2) based on a basin-wide shift in facies, overall stacking patterns and depositional trends (i.e. 3.5. Piedmont Basin The Piedmont Basin (NW Italy) contains the northernmost record of the MSC (Fig. 2b). The terminal MSC sediments (i.e. the Cassano Spinola Conglomerates Fm.) overlay pre-MSC units, the PLG deposits (Gessoso Solfifera Fm.) or reworked evaporites (Valle Versa chaotic complex, VVC) and underly the Zanclean marls of the Argille Azzurre Fm. (Dela 10 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. colored mudstone beds alternating with fine-grained mudstones/clays with intercalated three micritic limestones (known in literature as Colombacci limestones; Figs. 3a, 5g; Bassetti et al., 2004). By contrast, an incomplete Colombacci Fm. deposited in the shallower thrust-top basins (e.g. Vena del Gesso Basin and Molise sections; Pellen et al., 2017; Cosentino et al., 2018). The p-ev2 cycles have been interpreted as reflecting the alternation of wet (mudstones and Colombacci limestones in Eastern Romagna) and dry (coarse-grained facies and Colombacci limestones in the Maccarone section) phases controlled by Milankovitch-driven climatic factors (Fig. 3a; Roveri et al., 2008a; Cosentino et al., 2013) and, as such, they have been used for the as­ tronomical tuning of the Colombacci Fm. to the Lago-Mare phase (Figs. 3a, 4a; see subsection 2.2). By contrast, Clauzon et al. (2005) and Popescu et al. (2007) moved the Colombacci Fm. into the Pliocene (Fig. 4b). However, this conclusion has been proven to rely on wrong stratigraphic and paleontological arguments (see Roveri et al., 2008c, Grothe et al., 2018 and subsection 5.5). Substage 3.2 records in the Apennines do not always contain the three prominent conglomeratic facies as in Romagna, but only laminated to massive clays with sandy intercalations equivalent to the ones typifying substage 3.1 records (e.g. Maccarone section; Sampalmieri et al., 2010; Cosentino et al., 2013; Fig. 5f). The absence of a lithological cyclicity that clearly mimics an orbital signal largely hampered the astronomical tuning of these clay- dominated sections, although an attempt has been made with the Maccarone section (Cosentino et al., 2013). The only exception is rep­ resented by the Fonte dei Pulcini section, which has been equipped with an age framework by astronomical tuning of the magnetic susceptibility record (Cosentino et al., 2012). Despite the lack of outstanding litho­ logical changes these sections are often provided with a lithostrati­ graphic subdivision using the same nomenclature as in the Romagna area. When applied, the di Tetto Fm.-Colombacci Fm. boundary is placed high in the sections, i.e. 3.5. Piedmont Basin few tens of meters underneath the Miocene/Pliocene boundary, resulting in a much different thickness of the formations compared to the Romagna area. depth up to 1 km into the pre- and syn-evaporitic Messinian deposits and nicely shape the present-day river network of the southern Alps (Ama­ dori et al., 2018). Different mechanisms for the incision have been proposed, with major implications for the desiccated vs full Mediterranean controversy (Figs. 1b-c). Ghielmi et al. (2010, 2013), Rossi et al. (2015a), Amadori et al. (2018) and Cazzini et al. (2020) ascribed the valley incision along the PPAF northern margin to fluvial erosion, whose basinward shifting was triggered by the Stage 2 Mediterranean drawdown, estimated to have been around 800-900 m (Ghielmi et al., 2013; Amadori et al., 2018). In this case, Stage 3 deposition in the PPAF occurred in endorheic lakes fed by the Alpine rivers and kept isolated until the Zanclean, when the sudden sea-level rise following the Zanclean reflooding was enough to bypass morphological highs (e.g. Gargano-Pelagosa and/or Otranto paleosills) located in the southern Adriatic foredeep (Fig. 2a; see Pellen et al., 2017; Amadori et al., 2018; Manzi et al., 2020). Conversely, Winterberg et al. (2020) suggested that the over-deepened valleys on the southern slope of the Alps are related to Pleistocene glacial erosion. Although Winterberg et al. (2020) do not address the paleoenvironment during the Messinian, this interpretation does not rule out the possibility that (at least part of) Stage 3 sedimentation occurred in a PPAF con­ nected to the Mediterranean water mass and that no catastrophic reflooding occurred at the Miocene/Pliocene boundary. The conclusion of a non-catastrophic refilling was also drawn by Pellen et al. (2017) on the basis of the onshore Adriatic record (see subsection 3.7). 3.9. Greece Several MSC localities are reported from the Greek Ionian Islands (Corfu, Cephalonia and Zakynthos) and from Crete (Fig. 2b). On the NW coast of Corfu (Aghios Stefanos section), the PLG unit is missing and only a 32 m-thick cyclically-arranged terrigenous succes­ sion is present comprising three m-thick conglomerate beds alternating with fine-grained deposits rich in unspecified species of brackish water ostracods (Pierre et al., 2006). In the southern part of Zakynthos, an evaporitic succession composed of eight gypsum cycles (Kalamaki section) occurs above ma­ rine marly deposits (Karakitsios et al., 2017b). These gypsum beds were initially ascribed to the UG unit (Pierre et al., 2006) and later to the PLG (Karakitsios et al., 2017b). The gypsum unit is overlain by approxi­ mately ~13 m of siltstones and marls with scattered, cm-thick beds of sandstones, conglomerates and carbonates with nodular texture (Pierre et al., 2006; Karakitsios et al., 2017b). Although no ostracods are re­ ported from this interval, due to its stratigraphic position the post- evaporitic unit is correlated to the Lago-Mare phase (Karakitsios et al., 2017b). Except for the rare presence of marine nannofossils (Ceratolithus acutus together with Reticulofenestra zancleana) just below the Miocene/ Pliocene boundary, only reworked marine fauna has been reported from the post-evaporitic package (Karakitsios et al., 2017b). This dominantly terrigenous succession is unconformably overlain by the Zanclean Trubi Formation (Fig. 6e; Karakitsios et al., 2017b). The Upper Gypsum successions are commonly incomplete in many of the Caltanissetta Basin sections (Pasquasia-Capodarso, Casteltermini, Alimena, Nicosia, Siculiana-Marina; Decima and Sprovieri, 1973; Rou­ chy and Caruso, 2006; Manzi et al., 2009; Fig. 5i). In the most complete section, Eraclea Minoa (Fig. 3a), the Upper Gypsum Unit consists of 6 (Van der Laan et al., 2006) to 7 (Manzi et al., 2009) primary gypsum beds with a repetitive internal organization of facies (see Schreiber, 1997 and Manzi et al., 2009 for facies description) interbedded with marls and lenticular terrigenous sandstone bodies, gypsarenites and gypsrudites (Fig. 5h). Two of the terrigenous sandstone bodies are highlighted by Manzi et al. (2009) in the thick (~60 m), Cyprideis agrigentina-rich (Grossi et al., 2015), marly interval dividing gypsum VI and VII for its alleged astronomical significance (Fig. 3a; see subsection 2.2). 3.8. Sicily The MSC record is widely exposed on Sicily, mainly in the Calta­ nissetta Basin and in scattered locations on the Hyblean Plateau (i.e. Ragusa-Siracusa area) and the Messina area (Fig. 2b; Butler et al., 1995; Manzi et al., 2009; Sciuto et al., 2018). Like the Northern Apennines, it shows a complex distribution and variable stratigraphy that mirrors the structuring of Sicily into basins with different characters, geometries and depocenters which subsided at different times and rates (Butler et al., 1995; Catalano et al., 2013). This structural setting permitted the simultaneous deposition of shallow and intermediate-water sediments (Roveri et al., 2008b). Mostly found in the Caltanissetta Basin, these intermediate-water successions have for decades been considered the onshore counterpart of the offshore evaporitic trilogy seen in seismic data from the Western Mediterranean Basin (Decima and Wezel, 1973). More recently, Raad et al. (2021) attempted a similar onshore-offshore correlation but with the intermediate Central Mallorca Depression. The currently endorsed stratigraphic model (Fig. 4a), refined over the years by Decima and Wezel (1971, 1973), Decima et al. (1988), Butler et al. (1995), García-Veigas et al. (1995), Rouchy and Caruso (2006), Roveri et al. (2008b) and Manzi et al. (2009), envisages two ‘evaporitic cycles’. The ‘First cycle’, overlying both alluvial and deep-water sedi­ ments (Tripoli Fm., Licata Fm. and Terravecchia Fm.; see Maniscalco et al., 2019 and references therein), comprises the disputed Calcare di Base (Manzi et al., 2011, 2016b vs Caruso et al., 2015), PLG or Gessi di Cattolica Fm. (Decima and Wezel, 1973; Lugli et al., 2010) and the Halite Unit (Lugli et al., 1999). The ‘Second cycle’ comprises the Upper Gypsum (UG) or Gessi di Pasquasia Fm., which is only present in depocenters of the Caltanissetta Basin (see Manzi et al., 2009 for a detailed overview), sporadically overlain by the siliciclastic Arenazzolo Fm. (Decima and Wezel, 1973; Cita and Colombo, 1979). The whole succession is sealed by the Pliocene marine Trubi Fm. (Fig. 4a). The two evaporite cycles are separated by an erosional surface (MES) associated with an angular discordance broadly linked to the main Mediterranean drawdown event (e.g. Butler et al., 1995; Roveri et al., 2008b). Clauzon et al. (1996), however, placed the MES at the Arenazzolo Fm.-Trubi Fm. transition, implying that the entire evaporitic deposition in the Calta­ nissetta Basin pre-dated the offshore one, but they do not provide evi­ dence of erosion at that level. 3.7. Apennine system (2021) erroneously refer to Cita and Colombo (1979), where no erosion is mentioned at the M/P boundary in Eraclea Minoa. reported from the horizons containing these Paratethyan taxa (Bertini, 2006; Carnevale et al., 2006a; Popescu et al., 2007; Pellen et al., 2017). While the autochthony of ostracods, when considered, is unquestioned, the allochthonous vs autochthonous character of the other mentioned fossils is disputed and still unclear (see Chapter 5). 3.8. Sicily In more recent publications from the same research group, the MES is shifted towards the base of the Arenazzolo Fm. (e.g. Bache et al., 2012), again without evidence of major erosion, and different ages are assigned (see Fig. 4b and Grothe et al., 2018 for details). The bathymetric jump between the <100 m of water depth during the late Messinian and the >200 m at the base of the Trubi Fm. is often regarded as a key onshore evidence of the sudden and catastrophic Mediterranean-Atlantic re-connection at the Miocene/Pliocene bound­ ary (e.g. Caruso et al., 2020). However, the real depth of the base of the Trubi is all but obvious. In fact, variable estimates have been proposed based on the observed benthic foraminifera and/or psychrospheric os­ tracods at Capo Rossello and Eraclea Minoa: 200-500 m (Decima and Wezel, 1973), 600-800 m (Sgarrella et al., 1997, 1999; Barra et al., 1998), 1400-2400 m (Cita and Colombo, 1979). 3.7. Apennine system progradational and retrograda­ tional, respectively; Roveri et al., 1998, 2001, 2005, 2008a; Manzi et al., 2005, 2007, 2020). Allounit p-ev1 only accumulated in deep-water settings (e.g. Cusercoli, Sapigno, Maccarone and Trave sections; Roveri et al., 1998; Iaccarino et al., 2008; Cosentino et al., 2013) during the subaerial exposure of the basin margins (e.g. Vena del Gesso Basin, Monticino quarry, Pellen et al., 2017). It starts with resedimented clastic evaporites (i.e. Sapigno Fm.) followed by a coarsening- and shallowing- upward succession (i.e. di Tetto or San Donato Fm.) of mudstones with intercalated turbiditic sandstones (Fig. 5f) and a volcaniclastic marker bed dated initially by 40Ar-39Ar at ~5.5 Ma (Odin et al., 1997) and later by 238U-206Pb at 5.5320±0.0046/0.0074 Ma (Cosentino et al., 2013; Fig. 3a). Allounit p-ev2 (i.e. Colombacci Fm.) occurs in the deeper depocenters in 4/5 sedimentary cycles consisting of three > 5 m-thick coarse-grained bodies (conglomerates and sandstones) and two black- The Miocene/Pliocene boundary is variably expressed through the Apennine system: unconformable above the ostracod-bearing clays and highlighted by erosional surfaces draped by conglomeratic accumula­ tions (e.g. Stingeti section in Molise; Cosentino et al., 2018), conform­ able above 0.5-1 m-thick black mudstones similar to how it is observed in Piedmont and of equally unknown paleoenvironmental significance (e.g. Romagna area and Maccarone section; Roveri et al., 1998; Gennari et al., 2008) or conformable above the ostracod-rich mudstones (e.g. Maccarone and Fonte dei Pulcini sections; Cosentino et al., 2005, 2012, 2013; Sampalmieri et al., 2010). All p-ev1 deposits studied are almost devoid of in-situ biota, except for fish otoliths and three fish skeletons found in the upper substage 3.1 part of Cava Serredi (Carnevale et al., 2006b). The p-ev2/Colombacci deposits, instead, host typical Paratethyan assemblages of brackish- water mollusks, ostracods, dinocysts and fish (Bassetti et al., 2003; Bertini, 2006; Popescu et al., 2007; Grossi et al., 2008; Iaccarino et al., 2008; Cosentino et al., 2012, 2018; Schwarzhans et al., 2020). A diverse array of marine fossils (benthic and planktic foraminifera, calcareous nannofossils, dinocysts and fish otoliths and skeletons) has also been 11 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. section; Bonaduce and Sgarrella, 1999; Rouchy and Caruso, 2006; Londeix et al., 2007; Grossi et al., 2015; Fig. 3a). Calcareous nannofossils have been found along with the above organisms in a more northerly location by Maniscalco et al. (2019) and considered reworked. 3.7. Apennine system Above the last gypsum, the ~6-7 m-thick Arenazzolo Fm. is found, represented by reddish arkosic cross-laminated and poorly consolidated sand (Bonaduce and Sgarrella, 1999; Roveri et al., 2008b) and interpreted as the expression of a shallow-water delta, albeit without a sedimento­ logical investigation (e.g. Decima and Wezel, 1973; Cita and Colombo, 1979). The whole Stage 3 sequence is conformably overlain by the Zanclean marine Trubi Fm. in the basin center (e.g. at Eraclea Minoa and Capo Rossello; Fig. 6d; Brolsma, 1975; Cita and Colombo, 1979; Van Couvering et al., 2000; Rouchy and Caruso, 2006; Manzi et al., 2009; Fig. 6d) and unconformably in the shallower, marginal areas (Manzi et al., 2009; Roveri et al., 2019b). Only Decima and Wezel (1973) and Raad et al. (2021) report the Miocene/Pliocene transition in the key, intermediate water-representative section of Eraclea Minoa as erosive. However, they do not provide evidence (e.g. photographic documenta­ tion) for the presence of an erosional unconformity and, moreover, Raad et al. (2021) erroneously refer to Cita and Colombo (1979), where no erosion is mentioned at the M/P boundary in Eraclea Minoa. section; Bonaduce and Sgarrella, 1999; Rouchy and Caruso, 2006; Londeix et al., 2007; Grossi et al., 2015; Fig. 3a). Calcareous nannofossils have been found along with the above organisms in a more northerly location by Maniscalco et al. (2019) and considered reworked. Above the last gypsum, the ~6-7 m-thick Arenazzolo Fm. is found, represented by reddish arkosic cross-laminated and poorly consolidated sand (Bonaduce and Sgarrella, 1999; Roveri et al., 2008b) and interpreted as the expression of a shallow-water delta, albeit without a sedimento­ logical investigation (e.g. Decima and Wezel, 1973; Cita and Colombo, 1979). The whole Stage 3 sequence is conformably overlain by the Zanclean marine Trubi Fm. in the basin center (e.g. at Eraclea Minoa and Capo Rossello; Fig. 6d; Brolsma, 1975; Cita and Colombo, 1979; Van Couvering et al., 2000; Rouchy and Caruso, 2006; Manzi et al., 2009; Fig. 6d) and unconformably in the shallower, marginal areas (Manzi et al., 2009; Roveri et al., 2019b). Only Decima and Wezel (1973) and Raad et al. (2021) report the Miocene/Pliocene transition in the key, intermediate water-representative section of Eraclea Minoa as erosive. However, they do not provide evidence (e.g. photographic documenta­ tion) for the presence of an erosional unconformity and, moreover, Raad et al. 3.10. Cyprus MSC Stage 3 finds expression in a >1 km thick continental unit unconformable, through an erosional surface, above the pre-evaporitic, Stage 1 anhydrite-shale alternations (Radeff et al., 2016) and resedi­ mented gypsum-bearing Stage 2 deposits (Cosentino et al., 2010; Cipollari et al., 2013). This unit mainly consists of fluvial coarse- and fine-grained deposits representing channel fill and overbank deposits. Sporadically, some fine-grained intercalations are found containing a mixed brackish (ostracod) and marine (foraminifera and calcareous nannofossils) fauna. The ostracod fauna has affinity with the Para­ tethyan fauna but, unlike to many other Mediterranean onshore local­ ities, is poorly diversified, with monospecific assemblages of Cyprideis agrigentina (Avadan section and T-191 borehole; Cipollari et al., 2013) or with Cyprideis agrigentina accompanied by rare to abundant specimens of Loxoconcha muelleri, Euxinocyhere (Maeotocythere) praebaquana, and Loxoconcha sp. (Adana section; Faranda et al., 2013). Ostracods are often associated with Ammonia beccarii and rare Elphidium and Cri­ broelphidium, which are the only foraminifera considered as autoch­ thonous. Conversely, the entire nannoflora is interpreted as physically reworked (Cipollari et al., 2013; Faranda et al., 2013). The Handere Fm. is followed by early Zanclean marine sediments (Avadan Fm.) deposited, according to the paleoecology of the benthic foraminifera species recognized, at bathymetries ranging from 200 to 500 m (Cipollari et al., 2013). The lithological nature of the Miocene/ Pliocene boundary in the Adana Basin is not clear, but it occurs either above the continental or subaqueous, ostracod-bearing facies. A similar stratigraphic sequence is present in the subsurface. Here, however, chaotic gypsum-bearing deposits are not found and two halite bodies ~20 and ~170 m-thick are present, separated and followed by fluvial gravels, sands and silts (Cipollari et al., 2013). The sedimentary sequence overlying the last gypsum bed and assigned by Manzi et al. (2016a) to the Lago-Mare phase lacks a clear and rhythmic sedimentary cyclicity. In the Pissouri Basin this interval (up to 25-30 m-thick) mostly consists of conglomerates, sandstones, limestones, paleosols (which appear as dm to m-thick dark marly hori­ zons, in one case with pulmonated gastropods) and subordinated clay- marly horizons (Rouchy et al., 2001). By contrast, in the Polemi sec­ tions the clay-marly facies dominates this interval (Rouchy et al., 2001). 3.10. Cyprus The tectonically active, during the Miocene, thrust-top basin of Adana in southern Turkey (Radeff et al., 2017) retains the most com­ plete and better exposed easternmost successions of the MSC (Fig. 2b), whose deposits were attributed to the Handere Fm. (Cosentino et al., 2010; Radeff et al., 2016). i MSC deposits on Cyprus outcrop in the Pissouri, Psematismenos, Mesaoria and Polemi basins on the southerly fringe of the Troodos massif (Fig. 2b; Rouchy et al., 2001; Manzi et al., 2016a). According to Rouchy et al. (2001) and Orszag-Sperber et al. (2009), sediments belonging to all MSC stages of Roveri et al. (2014a) are preserved in the Cypriot basins. By contrast, Robertson et al. (1995) and Manzi et al. (2016a) considered that PLG evaporites on Cyprus are only present as fragments reworked within a chaotic unit (the Lower Gypsum and In­ termediate breccia units of Orszag-Sperber et al., 2009) and that the only in situ evaporites belong to the overlying Upper Gypsum Unit, which encompasses the whole of Stage 3 (Figs. 3a, 4a). A continuous, Eraclea Minoa-like section is not known in Cyprus (Manzi et al., 2016a). The best exposure of the lower 60 m of this unit is found in the Polemi Basin (Manzi et al., 2016a). It comprises up to six gypsum beds (the lower three of which are mainly selenitic, while the upper three are predom­ inantly laminated; Fig. 3a). Gypsum beds range in thickness from 1 to 6 m and are separated by laminated marls (Fig. 5j) occasionally inter­ bedded with conglomerates and sandstones (e.g. between the 5th and 6th gypsum layers; Rouchy, 1982; Rouchy et al., 2001; Manzi et al., 2016a). The sixth gypsum bed is reported by Rouchy et al. (2001) to be hollowed in the upper part with the cavities filled with overlying sediments. The similarity of the cyclicity and facies association of this Cyprus succession with the substage 3.1 interval of the Sicilian UG led Manzi et al. (2016a) to propose a bed-to-bed correlation and to recognize the substage 3.1/ 3.2 boundary at the top of the last gypsum bed (Fig. 3a). According to Orszag-Sperber et al. (2000) and Rouchy et al. (2001), this chro­ nostratigraphic boundary coincides with a Mediterranean-scale sea- level drop, a conclusion that arises from the interpretation of the cavities in the uppermost gypsum as the product of karstic dissolution following a prolonged period of subaerial exposure. 3.9. Greece A mixed (physically reworked) marine (foraminifera and dino­ cysts) and (in-situ) brackish biota (ostracods and dinocysts) of Para­ tethyan origin characterizes the marly interbeds from at least gypsum III upwards (following the investigations carried on the Eraclea Minoa MSC deposits on Crete (e.g. Meulenkamp et al., 1979; Delrieu et al., 1993; Cosentino et al., 2007; Roveri et al., 2008a; Zachariasse et al., 2008, 2011) were deposited in Miocene extensional, fault-bound basins driven by tectonic subsidence that ceased in the late Pliocene and Pleistocene (Van Hinsbergen and Meulenkamp, 2006). Because of the strong tectonic and eustatic sea-level-related fragmentation of the stratigraphic record, reconstructing the late Miocene stratigraphy of Crete has not been straightforward (Zachariasse et al., 2008, 2011). Several primary and clastic gypsum facies are recognized, but their 12 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. the euryhaline Aphanius crassicaudus are described from some of the substage 3.1 and 3.2 fine-grained facies and within the terrigenous laminae of some balatino gypsum (Orszag-Sperber et al., 2000; Rouchy et al., 2001; Orszag-Sperber, 2006; Manzi et al., 2016a). The upward change in diversity of the ostracod fauna seen elsewhere (e.g. Malaga, Nijar, Vera, Apennines and Eraclea Minoa) is not reported in Cyprus but this may be because no detailed study of ostracod assemblages in Stage 3 sediments has been published. The Miocene/Pliocene boundary, near Polemi village is described by Manzi et al. (2016a) as a sharp contact above a dark, organic-rich layer (Fig. 6f). It appears to be similar to the boundary reported from Piedmont (Fig. 6c; Trenkwalder et al., 2008; Dela Pierre et al., 2016) and Northern Apennines (Gennari et al., 2008; Grossi et al., 2008), if not for the presence, in Cyprus, of (possibly) in- situ Cyprideis agrigentina (Manzi et al., 2016a). A layer with the same field appearance, thickness (~ 1 m) and stratigraphic position is re­ ported in Pissouri by Rouchy et al. (2001), which they interpreted as a paleosol based on mottling, oxidized roots, carbonate concretions and plant fragments. correlation with the MSC stratigraphy is disputed (see Cosentino et al., 2007; Roveri et al., 2008a, 2014a; Zachariasse et al., 2008). Coarse- grained, mammal-bearing terrigenous facies irregularly alternating with marls are in places found unconformably overlying the gypsum and separated from the Pliocene facies by an erosion surface (see Meu­ lenkamp et al., 1979; Delrieu et al., 1993; Cosentino et al., 2007). 3.10. Cyprus In situ fresh-brackish water species of articulated mollusks (Limno­ cardiidae, Melanopsis), Paratethyan (Loxocorniculina djafarovi, Euxyno­ cythere praebaquana) and Mediterranean (Cyprideis agrigentina) ostracods and foraminifera (Ammonia beccarii), Characeae, abundant fragments of the marine euryhaline fish Clupeidae and a fish skeleton of 3.9. Greece In two localities on the Messar`a Plain, Cosentino et al. (2007) described a highly diversified ostracod fauna with Paratethyan affinity in some marly intervals. Messinian evaporites and/or Lago-Mare deposits are also reported from the North Aegean region onshore in the Strymon Basin (Snel et al., 2006; Suc et al., 2015; Karakitsios et al., 2017a) and Dardanelles region (Melinte-Dobrinescu et al., 2009) and offshore (Prinos-Nestos Basin; Karakitsios et al., 2017a), but recent integrated studies suggested that the sections studied by the above listed authors are older than the MSC (see Krijgsman et al., 2020a, 2020b). In particular, Krijgsman et al. (2020a) proposed that for most, if not all, of the MSC the North Aegean was a brackish water, mostly Paratethyan-fed basin restricted by the Cyclades sill to the south (Fig. 2a) and forming a passageway for Para­ tethyan overspill waters towards the Mediterranean. 3.12. Summary of the onshore Stage 3 record Most of the onshore Stage 3 records formed in shallow marginal Mediterranean basins, which underwent substantial uplift from the Messinian till nowadays and are assumed to have had their depocenter at ~200 to 50 m below the Atlantic level during the late Messinian (Roveri et al., 2014a, 2019a; Radeff et al., 2016, 2017). The Calta­ nissetta Basin (Sicily), some basins along the Apennines and (possibly) Cyprus represent, instead, possible onshore representative of interme­ diate basins. The nature and duration of these records is quite variable, 13 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Although the exact topography and hypsometry of the Messinian Mediterranean is difficult to reconstruct, this present-day geography is generally assumed to have been in place, with five main differences: (1) the Albor´an Basin was split into a Western (WAB) and Eastern Albor´an (EAB) by a volcanic arc (Booth-Rea et al., 2018); (2) the Tyrrhenian Basin was only partly opened (Lymer et al., 2018); (3) the precise depth and width of the ancient Sicily Sill are difficult to estimate, but may have been much deeper than today (~300 m; Meijer and Krijgsman, 2005Jolivet et al., 2006). Paleodepth estimations for the Messinian configuration range from 380 m (Just et al., 2011) to 430 m (Garcia- Castellanos et al., 2009); (4) one or two sills were present at the southern termination of the Adriatic foredeep (Pellen et al., 2017; Amadori et al., 2018; Manzi et al., 2020); (5) the North Aegean was partially isolated from the Mediterranean by the Cyclades Sill and with high Paratethys affinity (Krijgsman et al., 2020a). Following the schematic classification of the Messinian sub-basins by Roveri et al. (2014a), all these subbasins are regarded as either intermediate (i.e. relatively deep-water, 200–1000 m) or deep (water depth > 1000 m). and there are only six sections that may record an entire Stage 3 sequence (i.e. Sorbas, Nijar, Northern Apennines, Eraclea Minoa and Cyprus; Fig. 3a). Reasons for the fragmentary nature of the Stage 3 sedimentary record include different durations of subaerial exposure following the Stage 2 drawdown, local tectonics and associated syn- depositional erosion and deposition. One of the consequences of this is that any sedimentary cyclicity that resulted from orbital fluctuations is typically either less well developed or poorly preserved, making the chronology of Stage 3 rather uncertain or controversial in places. Despite this variability, several fairly consistent characteristics are widely expressed. 3.12. Summary of the onshore Stage 3 record These are: 1) Stage 3 sedimentation follows a period of intensive tectonic and/or eustatic-driven erosion of the margins, as demonstrated by the frequent presence of erosional unconformities and/or chaotic Stage 2 deposits (RLG unit); 2) Stage 3 lithologies are mostly terrigenous (conglomerates, sand­ stones and mudstones) and deposited in a variety of continental (fan delta, fluvial channels and alluvial plains) and shallow water envi­ ronments (endorheic lakes or water bodies connected with the Mediterranean water mass is the riddle). Carbonate intercalations are sometimes present (e.g. Sorbas Basin and Colombacci limestones in the Apennines). Stage 3 gypsum is only found in deeper-water intermediate basins of Caltanissetta in Sicily and Cyprus. ii Compared with the onshore domain, the offshore basins hold a far greater percentage of the total volume of MSC sediments (Ryan, 1973; Haq et al., 2020). The architecture, geometry and main lithologies of the MSC and younger deposits are well known thanks to the high density of seismic data and the fact that evaporites (halite particularly) are easily identified on seismic profiles due to their unusual seismic properties, especially compared to those of terrigenous and carbonate sediments (see Lofi et al., 2011a, 2011b; Lofi, 2018; Haq et al., 2020). However, the detailed lithological, sedimentological, paleontological and geochem­ ical nature and their chronostratigraphy are still poorly constrained offshore because these cannot be univocally defined on the basis of seismic data alone (Roveri et al., 2019b) and direct information about these deep MSC successions is limited to a small number of cores (16) drilled during the DSDP (Ryan et al., 1973; Hsü et al., 1978b) and ODP (Kastens et al., 1987; Comas et al., 1996; Emeis et al., 1996) drilling campaigns that penetrated exclusively the uppermost tens of meters of the deep MSC suite in very scattered localities (Fig. 2b). Only recently, access to industrial boreholes crossing the base of the halite in the deep Levant Basin has been granted, providing a rare glimpse of the deep MSC deposits in the easternmost part of the Mediterranean (Gvirtzman et al., 2017; Manzi et al., 2018; Meilijson et al., 2018, 2019). 3) A diversified fossil assemblage with Paratethyan affiliation (ostra­ cods, dinocysts, mollusks) is commonly found in the shallow-water sediments of Lago-Mare successions. Only in the intermediate Cal­ tanissetta Basin (Sicily) do these diversified Paratethyan forms (only ostracods) occur earlier, in the sediments from substage 3.1. 3.12. Summary of the onshore Stage 3 record Where these have been studied in detail, these assemblages typically show an increase in diversity with time (possibly every wet phase of the precession cycles). Some of these sediments also contain marine fossils and there is controversy over whether these are in situ and contemporaneous or reworked. 4) In outcrop, the Miocene/Pliocene boundary has four main sedi­ mentary expressions: erosive and followed by a conglomeratic lag (e. g. Malaga, Vera, Mallorca; Fig. 6a); conformable above continental facies (e.g. Nijar Basin; Fig. 6b); conformable above ostracod-rich mudstones (e.g. Eraclea Minoa; Fig. 6d); sharp contact above a black layer of still largely unknown paleoenvironmental significance (Piedmont, Apennines and Cyprus; Fig. 6c, f). 4) In outcrop, the Miocene/Pliocene boundary has four main sedi­ mentary expressions: erosive and followed by a conglomeratic lag (e. g. Malaga, Vera, Mallorca; Fig. 6a); conformable above continental facies (e.g. Nijar Basin; Fig. 6b); conformable above ostracod-rich mudstones (e.g. Eraclea Minoa; Fig. 6d); sharp contact above a black layer of still largely unknown paleoenvironmental significance (Piedmont, Apennines and Cyprus; Fig. 6c, f). The MSC is commonly described as tripartite (‘Messinian trilogy’ after Montadert et al., 1978) in the Western Mediterranean (Lower- Mobile-Upper units: LU-MU-UU, respectively). However, in the Ionian Basin is described as bipartite (MU-UU) by Camerlenghi et al. (2019) while according to Lofi et al. (2011a), Gvirtzman et al. (2013, 2017), Lofi (2018) and Camerlenghi et al. (2019), the Levant Basin consists of the MU and the UU is only present locally and possibly represented by evaporite-free terrigenous accumulations (Kartveit et al., 2019; Madof et al., 2019). The lack of many age constraints within the offshore MSC successions hampers unambiguous correlation with onshore sequences (Fig. 1a; Roveri et al., 2014a). Nevertheless, different authors have proposed onshore-offshore correlation of specific events (e.g. onset, Ochoa et al., 2015; and termination of the MSC, Biscaye et al., 1972, Iaccarino et al., 1999) and stratigraphic schemes (Decima and Wezel, 1971; Raad et al., 2021) based on and biostratigraphic evidence (Cosentino et al., 2006), 87Sr/86Sr isotope ratios (Roveri et al., 2014b; Gvirtzman et al., 2017; Manzi et al., 2018) and astronomical tuning of the deep seismic record (Ochoa et al., 2015, 2018; Manzi et al., 2018; Meilijson et al., 2018, 2019). 3.12. Summary of the onshore Stage 3 record Here we focus on the seismic and For a better understanding of how Stage 3 developed across the Mediterranean these marginal records now need to be considered alongside the evidence from intermediate to deep offshore settings. We note that alternative chronostratigraphic frameworks have been proposed for several onshore (Malaga, Sorbas, Mallorca, Apennines, Sicily) and offshore (Sites 134B, 976B, 978A) locations (see Fig. 4b for references), but we have omitted them as they are shown to rely on incorrect (bio)stratigraphic arguments (see Roveri et al., 2008c, Grothe et al., 2018 and subsection 5.5). 4. Offshore domain The offshore Mediterranean is a complex array of variable-depth and morphologically complex subbasins framed by morphological highs or sills. Traditionally it is divided into two main domains (Fig. 2a), the Western and Eastern Mediterranean, with the intervening divide (or Sicily sill) situated in the Sicily channel at present with a maximum depth of 316 m. The Albor´an Basin, the depressions on the Balearic Promontory, the Corsica, Valencia, Algero-Balearic, Liguro (or Sardo)- Provençal and Tyrrhenian basins belong to the "Western Mediterranean" (Fig. 2a). The Adriatic foredeep, the Ionian, Sirte, Aegean and Levant basins belong in the "Eastern Mediterranean" (Fig. 2a). Smaller-sized depressions, again surrounded by sills of variable depth, are identified and labelled within each of these subbasins. 14 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Fig. 7. Seismic profiles from intermediate- deep Western Mediterranean basins con­ taining MSC markers/units. (a) Seismic reflection line CAB01-104 from the WAB (modified from Booth-Rea et al., 2018). The line shows the variable geometry of the inferred M/P boundary, erosive in proximity of mud diapirs, (para)conformable in tectonically undisturbed sectors. Chaotic reflectors are occasionally imaged below the inferred M/P boundary. (b) Seismic profile SF12-09 imaging the lower slope of the south Algero-Balearic margin and part of the Algero-Balearic abyssal plain (modified from Mocnik et al., 2014). Here a high reflecting and horizontally stratified UU covers a thin layer of MU evidenced by salt diapirism. Note the concordant deformation of the UU and MU. (c) Line MS-39 from the abyssal plain of the Liguro-Provençal Basin showing the Messinian trilogy and non-erosive bot­ tom and top surfaces (BS and TS; Dal Cin et al., 2016). Halokinesis of MU gives rise to domes that deform the UU and PQ units. (d) Interpreted seismic profile from the lower- middle slope of the west Sardinian margin (modified from Dal Cin et al., 2016). Thin MU and UU are present on the lower slope, while on the middle slope (and upper slope here not shown) they converge in the margin erosion surface MES. (e) Line drawing of seismic line imaging from the Catalan margin (or Ebro Basin) to the abyss of the Liguro-Provençal basin (modified from Maillard et al., 2011b). Note the pinch out of the MU in the Valencia Basin and of the UU in the Ebro Basin, which is MSC free. 4.1. Western Albor´an Basin and westernmost East Albor´an Basin The Albor´an Basin has received particular attention because of its proximity to the Gibraltar Corridor (Estrada et al., 2011; Popescu et al., 2015 and references therein). Evaporites only occur on the eastern side of the EAB (which is treated in subsection 4.2; Fig. 2a). To the west of the volcanic archipelago (Booth-Rea et al., 2018, i.e. the WAB) and imme­ diately to the east on the western side of the EAB only terrigenous sediments occur in the MSC interval (Booth-Rea et al., 2018; de la Pe˜na et al., 2020). Sediments at the Miocene/Pliocene boundary appear in the seismic reflection data as parallel reflectors with increasing reflectivity (Comas et al., 1996, 1999; Booth-Rea et al., 2018). Locally, just below the M discontinuity, some of the reflectors suggest a chaotic seismic facies (Fig. 7a; Booth-Rea et al., 2018; Bulian et al., 2021). Miocene sediments with a maximum thickness of 100 m have been recovered from two out of nine holes drilled in the region (ODP 976B, 978A; Comas et al., 1996, 1999). These sediments mostly consist of claystones, silt­ stones and sandstones with Chondrites and Zoophycos ichnofacies at site 976B and include a conglomerate close to the Miocene/Pliocene boundary at Site 978A. The lack of age-diagnostic fossils hampers their correlation with the Geologic Time Scale (GTS). However, the presence, high in the Miocene section, of an oligotypic association of ostracods (Candona sp., Loxoconcha muelleri, and Cyprideis sp.) with different stages of growth (Site 978A; Iaccarino and Bossio, 1999) and Para­ tethyan dinocysts (including Galeacysta etrusca; see subsection 5.2; Popescu et al., 2015) indicates a latest Messinian (substage 3.2) age and brackish paleodepositional conditions. Foraminifera and nannofossils are also present, but all species recognized are of no help in narrowing down the paleoenvironmental interpretation because they are consid­ ered either definitely or likely to be reworked (Iaccarino and Bossio, 1999). By contrast, Popescu et al. (2015) interpreted some species of calcareous nannofossils (Reticulofenestra pseudoumbilicus, Discoaster quinqueramus, Ceratolithus acutus, Triquetrorhabdulus rugosus, Amaur­ olithus primus) and marine dinocysts as autochthonous. Two W-E-aligned erosional channels straddling the Strait of Gibraltar and stretching for 390 km from the easternmost Gulf of C´adiz (Atlantic Ocean) into the Albor´an Basin are clearly observed in seismic profiles (Garcia-Castellanos et al., 2009; Estrada et al., 2011). There is disagreement, however, concerning the timing and nature of their for­ mation. 4.2. Eastern Albor´an, Algero-Balearic and Liguro-Provençal basins 4.2. Eastern Albor´an, Algero-Balearic and Liguro-Provençal basins From the eastern margin of the EAB as far east as the Tyrrhenian coast of Italy, the Messinian (evaporites-bearing) trilogy LU-MU-UU is found and sealed by the PQ. The MU and UU are interpreted to fill the deepest depocenters (Algero-Balearic, Valencia and Liguro-Provençal basins; with minor interruptions due to seamounts) and the lower slope domain, where they comprise ~500 to ~800 m of UU and ~1000 m of MU/halite (Figs. 7b-d; Camerlenghi et al., 2009; Lofi et al., 2011a, 2011b; Geletti et al., 2014; Mocnik et al., 2014; Dal Cin et al., 2016; Lofi, 2018). Upslope, a thinner, possibly incomplete UU is locally described on the middle-upper continental slopes of Western Corsica (Guennoc et al., 2011) and Sardinia (Mocnik et al., 2014; Dal Cin et al., 2016) and the northern (Maillard et al., 2006) and southern (Maillard and Mauf­ fret, 2013; Mocnik et al., 2014; Dal Cin et al., 2016) flanks of the Balearic Promontory, even though the structural settings of these locations are mostly dominated by erosion (Fig. 7d). MSC evaporites are absent on the continental shelves bordering the deep Algero-Balearic and Liguro- Provençal basins, where the PQ directly overlies the MES which, in turn, cuts through the middle Miocene deposits (Gorini et al., 2005; Lofi et al., 2005). The only late Messinian sediments are present as Complex Units (Gulf of Lion, Bessis, 1986; Gorini et al., 2005; Lofi et al., 2005; Algerian Basin, Medaouri et al., 2014; Arab et al., 2016; Fig. 7e). CUs can have various origin (Lofi et al., 2011a, 2011b), but when identified at the outlet of drainage systems, they are commonly interpreted as Messinian clastics supplied by rivers (Lofi et al., 2005). In the Gulf of Lion, the MES is a high angle unconformity with substantial erosion along highly rugged relief thought to have been generated by fluvial incision (Lofi et al., 2005). In contrast, Roveri et al. (2014c) suggested that the drainage networks visible on the seismic could be of subaqueous origin. When not involved in MU-related deformation processes, the UU appears as a highly reflective series of flat reflectors alternating with less reflective, but concordant, reflectors (Figs. 7b-c; Lofi et al., 2011a, 2011b) aggrading in the basin center and onlapping the margins p y The nature of the Miocene/Pliocene boundary is also uncertain. 4. Offshore domain (f) Interpreted seismic profile Simbad 15 crossing the depocenter of the CMD showing all the MSC units and erosional surfaces (modified from Raad et al., 2021). (g) Interpreted seismic profile MYS40 illus­ trating the MU-UU-PQ units in the East- Sardinia Basin and Cornaglia Terrace, sepa­ rated by the MSC deposits-free Quirra Sea­ mounts (modified from Lymer et al., 2018). Fig. 7. Seismic profiles from intermediate- deep Western Mediterranean basins con­ taining MSC markers/units. (a) Seismic reflection line CAB01-104 from the WAB (modified from Booth-Rea et al., 2018). The line shows the variable geometry of the inferred M/P boundary, erosive in proximity of mud diapirs, (para)conformable in tectonically undisturbed sectors. Chaotic reflectors are occasionally imaged below the inferred M/P boundary. (b) Seismic profile SF12-09 imaging the lower slope of the south Algero-Balearic margin and part of the Algero-Balearic abyssal plain (modified from Mocnik et al., 2014). Here a high reflecting and horizontally stratified UU covers a thin layer of MU evidenced by salt diapirism. Note the concordant deformation of the UU and MU. (c) Line MS-39 from the abyssal plain of the Liguro-Provençal Basin showing the Messinian trilogy and non-erosive bot­ tom and top surfaces (BS and TS; Dal Cin et al., 2016). Halokinesis of MU gives rise to domes that deform the UU and PQ units. (d) Interpreted seismic profile from the lower- middle slope of the west Sardinian margin (modified from Dal Cin et al., 2016). Thin MU and UU are present on the lower slope, while on the middle slope (and upper slope here not shown) they converge in the margin erosion surface MES. (e) Line drawing of seismic line imaging from the Catalan margin (or Ebro Basin) to the abyss of the Liguro-Provençal basin (modified from Maillard et al., 2011b). Note the pinch out of the MU in the Valencia Basin and of the UU in the Ebro Basin, which is MSC free. (f) Interpreted seismic profile Simbad 15 crossing the depocenter of the CMD showing all the MSC units and erosional surfaces (modified from Raad et al., 2021). (g) Interpreted seismic profile MYS40 illus­ trating the MU-UU-PQ units in the East- Sardinia Basin and Cornaglia Terrace, sepa­ rated by the MSC deposits-free Quirra Sea­ mounts (modified from Lymer et al., 2018). 15 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. (Fig. 7a). 1 Lithostratigraphic and biostratigraphic information from DSDP and ODP cores are primarily extracted from the Scientific Shipboard Party documents, accessible from https://www.marum.de/en/Research/Cores-at-BCR.html. These documents are referenced in the text as follows: Ryan et al. (1973): DSDP 120-134; Hsü et al. (1978b): DSDP 371-378; Kastens et al. (1987): ODP 650- 656; Comas et al. (1996): ODP 974-979; Emeis et al. (1996): ODP 963-973. 4. Offshore domain In more undisturbed sectors these authors argue that the boundary is a paraconformity with no evidence of erosion (Fig. 7a). The lack of Messinian erosion in the shallow regions of the WAB margins has prompted the hypothesis that this area did not desiccate during the MSC (Booth-Rea et al., 2018; de la Pe˜na et al., 2020). This contradicts much of the interpretation made of the DSDP and ODP cores of this interval in the Albor´an Sea. The succession recovered by drilling from beneath the Pliocene comprises gravels that contain a mixed Miocene fauna. These sediments and their faunal content are thought to have been reworked from older sediments exposed as Albor´an substrate with no evidence of an extensive wet Lago Mare interval immediately before the Zanclean (Comas et al., 1996). geological (core-derived)1 properties of the Upper Unit (and laterally grading/interfingering sediments when present), stratigraphically below the Plio-Quaternary deposits (PQ) suggesting that it belongs to (at least part of) Stage 3. 4.1. Western Albor´an Basin and westernmost East Albor´an Basin 4.1. Western Albor´an Basin and westernmost East Albor´an Basin These incisions are classically considered to occur at the very top of the MSC suite (when present) and to be the consequence of the Zanclean megaflood (Garcia-Castellanos et al., 2009, 2020; Estrada et al., 2011 among others). More recently, Krijgsman et al. (2018) highlighted that an accurate age determination for these channel in­ cisions is lacking and that they might have been formed earlier during the MSC as a result of the Mediterranean-Atlantic gateway currents. Interpretation of E-W seismic profiles across the Albor´an Basin com­ bined with mammal records in Africa and Iberia led Booth-Rea et al. (2018) to suggest the existence of an emergent volcanic archipelago that temporarily connected southeastern Iberia with northern Africa, sepa­ rating an open marine, Atlantic-influenced West Albor´an Basin realm from a restricted, hydrologically complex Mediterranean realm to the east. 4.3. Valencia Basin (Fig. 7b; Camerlenghi et al., 2009; Lofi et al., 2011a, 2011b; Geletti et al., 2014; Mocnik et al., 2014; Dal Cin et al., 2016). The aggrading nature, shelf-ward thinning and the onlap terminations of the UU are interpreted as evidence of sedimentation in a lake with fluctuating base level (e.g. Lofi et al., 2005; Lofi et al., 2011a). In the abyssal plains (Figs. 2a, 7c), nine to ten cycles have been interpreted on high resolution seismic profiles as corresponding to gypsum-marl alternations (Geletti et al., 2014; Mocnik et al., 2014). At Sites 124 and 372, ~40-50 m of the UU have been drilled at the feet of the east Menorca continental rise and the northern Menorca slope, where 3-4 gypsum-marl cycles are recog­ nized (Fig. 2b; Ryan et al., 1973; Hsü et al., 1978a). Primary gypsum facies are widely overprinted by post-depositional diagenetic processes, but the still recognizable laminated and clastic primary textures indicate precipitation at the water-air interface and emplacement by gravity flows, respectively (Lugli et al., 2015). The marl interbeds are made from stiff to firm dolomitic mud containing substantial quantities of detrital material intercalated with current-bedded sandstones and, at Site 124, with diatomites (Ryan et al., 1973). Cyprideis sp. specimens are reported from some mudstone interbeds at Site 372, while dwarf planktonic foraminifera are present just below the Miocene/Pliocene boundary at Site 124 (Ryan et al., 1973). The Valencia Basin (VB; Fig. 2a) is an aborted rift formed during the late Oligocene-early Miocene opening of the back-arc Liguro-Provençal Basin (e.g. Jolivet et al., 2006). It is located between the Spanish Ebro Margin to the west and the Balearic Promontory to the east, while it grades into the Liguro-Provençal Basin to the E/NE (Fig. 7e; Maillard and Mauffret, 2006; Maillard et al., 2006). Numerous exploratory boreholes exist on the western Ebro margin. These boreholes, tied to seismic data, confirm that MSC-related sedi­ ments on the northwestern shelf are missing (Fig. 7e; Urgeles et al., 2011; Pellen et al., 2019). The only MSC feature present is a prominent erosional surface (the MES) incising Serravallian-early Messinian sedi­ ments (Urgeles et al., 2011). By contrast, on the southwestern and southern part of the margin, well data show the presence of evaporitic sediments (e.g. Delta L and Golfo de Valencia D1 boreholes; Del Olmo, 2011; Del Olmo and Martín, 2016; Lozano, 2016). 4.3. Valencia Basin 7e; see subsection 4.2), although it thins from ~1000 m to < 500 m. The UU is characterized by aggrading and onlapping geometries towards the slopes, where it also thins out until it disappears along the middle-upper slope (Fig. 7e; Maillard et al., 2006; Cameselle and Urgeles, 2017). Maillard et al. (2006), Urgeles et al. (2011), Cameselle et al. (2014) and Cameselle and Urgeles (2017) all stated that the UU formed during an important Mediterranean-level lowstand (~1000 m). Several Complex Units (CU), with different origin, have been observed and described as belonging to the MSC (Cameselle and Urgeles, 2017). Halite is present at the bottom of Hole 134 drilled within the UU (Ryan et al., 1973; Sage et al., 2005; Lugli et al., 2015). High-resolution seismic profiles from both the Algero-Balearic and Ligurian-Provençal basins confirm the presence of a halite layer high in the UU sequence (Geletti et al., 2014; Mocnik et al., 2014). This layer is up to 50 m thick (Dal Cin et al., 2016) and is correlated with an erosional surface (called IES: Intermediate Erosional Surface by Lofi et al., 2011a, 2011b) asso­ ciated with an angular unconformity which is better developed on the lower slope (Fig. 7d). Geletti et al. (2014) and Mocnik et al. (2014) interpreted this layer as autochtonous and indicative of at least one important sea level drop during UU deposition. However, this intra UU halite layer is always described from areas strongly affected by salt diapirism (just like in the Ionian Abyssal Plain; see subsection 4.6.1) and is never found in adjacent, undisturbed areas (see Camerlenghi et al., 2009; Geletti et al., 2014; Mocnik et al., 2014; Dal Cin et al., 2016), two features that may suggest it has an allochthonous origin. DSDP Site 122, drilled at the mouth of a valley incision, recovered a few meters of sand-gravels made of well-rounded basalt, marine lime­ stones, nodules of crystalline gypsum and mollusk fragments in a clay- silty matrix rich in deep-water benthonic foraminifera and early Plio­ cene nannofossils, all interpreted as erosional products of the VB seabed (Ryan et al., 1973). The uppermost Messinian in two industrial wells (Ibiza Marino and Cabriel boreholes; see Lozano, 2016) is represented by intercalations of clastic gypsum/anhydrite and marls (unit M8-P1 of Del Olmo and Martín, 2016). 4.3. Valencia Basin Del Olmo and Martín (2016) described these evaporites as primary selenites and ascribed them to MSC Stage 1 (their unit M7). Lozano (2016) described the same evaporitic deposits in the same boreholes as ‘white anhydrites’, leaving open the question as to whether the anhydrite is primary (sabhka’s) or secondary at the expense of a primary gypsum facies. On the eastern margin of the VB boreholes and seismic studies suggest there are no MSC units with only a prominent MES cutting pre-MSC sediments (Driussi et al., 2015; Raad et al., 2021). All authors conclude that the shelves of VB were exposed to subaerial erosion during and following the main drawdown. The Miocene/Pliocene boundary coincides with the top of the UU where present (labelled TES when erosional and TS when conformable; Lofi et al., 2011a, 2011b). In the abyssal plain-lower slope domain it appears to be undulating, although this geometry is related to the deformation of the underlying salt (Figs. 7b-c), and it actually corre­ sponds to a sharp surface lacking signs of erosion (Lofi et al., 2011a, 2011b; Geletti et al., 2014; Mocnik et al., 2014). By contrast, the UU-PQ boundary commonly appears strongly erosional in the middle-upper slope and shelf domain, where it coincides with the MES (Fig. 7d; Lofi et al., 2005; Maillard et al., 2006; Geletti et al., 2014; Mocnik et al., 2014). Among the six DSDP-ODP Sites drilled in this region (Fig. 2b), only Hole 975B recovered the Miocene/Pliocene boundary (Iaccarino and Bossio, 1999; Marsaglia and Tribble, 1999). Here the Messinian is a few centimeters thick and consists of banded micritic silty clays with minor calcareous siltstones to sandstones typified by a diverse faunal assemblage consisting of dwarf planktonic foraminifera, Ammonia tepida tests and brackish Paratethyan ostracods (Loxocorniculina djafarovi, Euxinocythere praebaquana, Amnicythere idonea, Leptocythere limbate, Candona sp., and Cyprideis sp.; Iaccarino and Bossio, 1999). MSC deposits are also absent along the slopes and, where present, consist of coarse- and fine-grained terrigenous facies filling valleys largely related to fluvial incision (Fig. 7e; Stampfli and H¨ocker, 1989). A different situation features in the depocenter. Despite its present- day depth of > 2000 m, no MU is observed in the depocenter, as the salt pinches-out at the edge of the deeper Provençal Basin (Fig. 7e). Only the seismic properties of UU suggest that it is roughly continuous from the Provençal Basin into the VB (Fig. 4.2. Eastern Albor´an, Algero-Balearic and Liguro-Provençal basins According to some authors, the “M” discontinuity is a high-amplitude reflector with evidence of erosion attributed to subaerial processes (Estrada et al., 2011; Urgeles et al., 2011) and locally (e.g. close to Site 121; Ryan et al., 1973) associated with an angular unconformity that abruptly truncates the upper Miocene deposits and morphological highs (Comas et al., 1999; Estrada et al., 2011; Garcia-Castellanos et al., 2020). Although the M-reflector was drilled at Sites 976B, 977A and 978A, a lithological contact was only recovered at Site 976B coinciding with a major erosional surface between the early Messinian and the base of the Pliocene (Bulian et al., 2021). Only at Site 978A (and possibly 977A) was a few meters of what may be the contact interval recovered (Comas et al., 1996). This comprises a 25 m-thick cemented succession containing pebbles of volcanic and sedimentary rocks likely to derive from the Albor´an substrate (46R, 620.9-630.67 mbsf, between the Pliocene-bearing core 45R and the Messinian-bearing core 47R; Comas et al., 1996). In contrast, Booth-Rea et al. (2018) concluded that the M- reflector is an unconformity only close to the mud diapirs and owes its erosive shape and angular discordance to the activity of these structures reflector is an unconformity only close to the mud diapirs and owes its erosive shape and angular discordance to the activity of these structures 1 Lithostratigraphic and biostratigraphic information from DSDP and ODP cores are primarily extracted from the Scientific Shipboard Party documents, accessible from https://www.marum.de/en/Research/Cores-at-BCR.html. These documents are referenced in the text as follows: Ryan et al. (1973): DSDP 120-134; Hsü et al. (1978b): DSDP 371-378; Kastens et al. (1987): ODP 650- 656; Comas et al. (1996): ODP 974-979; Emeis et al. (1996): ODP 963-973. When not involved in MU-related deformation processes, the UU appears as a highly reflective series of flat reflectors alternating with less reflective, but concordant, reflectors (Figs. 7b-c; Lofi et al., 2011a, 2011b) aggrading in the basin center and onlapping the margins 16 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. 4.4. Balearic Promontory 4.4. Balearic Promontory eastward migration of the Apennine subduction system from middle Miocene to Pliocene times (Gaullier et al., 2014; Lymer et al., 2018; Loreto et al., 2020 and references therein). Three main domains are traditionally identified (Lymer et al., 2018 and references therein): 1) the East Sardinia Basin, with present-day water depths between 200- 2000 m consisting of a system of seamounts and depressions that do not contain MSC sediments (Lymer et al., 2018); 2) the Cornaglia Terrace (2000-3000 m deep), a wide, flat area with dispersed structural highs; 3) the Tyrrhenian Basin s.s., with water depths varying from 3000-3600 m. Whether the Tyrrhenian Basin acquired the present-day topography and hypsometry before the MSC (Lymer et al., 2018) or at least part of it (e.g. Eastern Sardinia margin, where Site 654 is located, and Northern Tyrrhenian) was much shallower (possibly comparable to the Caltanissetta Basin; Roveri et al., 2014b) and underwent extension and subsidence during the Messinian-Pliocene (e.g. Kastens and Mascle, 1990; Loreto et al., 2020) is still unresolved. i Sticking out from the surrounding deep-water locations, the Balearic Promontory (BP; Fig. 2a) is a continental high that has undergone tec­ tonic extension since the late Serravallian (Roca and Guimera, 1992; Sabat et al., 2011). During the Messinian, it comprised in topographic lows/subbasins at different water depths and separated by structural highs/sills (Maillard et al., 2014; Driussi et al., 2015; Roveri et al., 2019b; Raad et al., 2021). The area has been the subject of multiple studies (Maillard et al., 2014; Driussi et al., 2015; Ochoa et al., 2015; Roveri et al., 2019b; Raad et al., 2021) and several controversies arose after the publication of Roveri et al. (2019b). i p The first controversy concerns the Messinian paleodepth of the BP’s subbasins. According to Roveri et al. (2019b) the subbasins were shallow during the Messinian and acquired today’s paleodepths following a strong post-MSC subsidence; Maillard and Mauffret (2011), Maillard et al. (2014) and Raad et al. (2021), instead, consider the tectonic movements in the BP to have been minor since the late Miocene (Messinian) and the region to have been already structured as it is today during the MSC. Well-to-seismic ties in the shallower basins closer to the Spanish coast (i.e. 4.4. Balearic Promontory Bajo Segura, San Pedro and Elche basins) comprise up to 14 Stage 1 primary gypsum-marl cycles similar to the onshore PLG unit (Lugli et al., 2010) truncated at the top by the MES (Soria et al., 2008a, 2008b; Ochoa et al., 2015). At first, Ochoa et al. (2015) concluded that all sediments overlying the MES are Pliocene in age. A later re-appraisal of the same downhole logging data and cuttings led Ochoa et al. (2018) to attribute the first ~13 m-thick micritic and evaporite-free sediments to the late Messinian (stage 2 or 3 of the MSC according to the authors). The MSC stratigraphy of the shallowest offshore basins of the BP closely resembles that described from cores and outcrops onshore Mallorca (see subsection 3.4; Roveri et al., 2019b). l The MSC units in seismic profiles from the Tyrrhenian Basin (Fig. 7g) are very similar to the ones described in the Algero-Balearic and Liguro- Provençal basins (Fig. 7b-c; Gaullier et al., 2014; Lymer et al., 2018). ODP Sites 652, 653 and 654 confirmed the seismic-inferred lithological nature of UU as consisting, of gypsum-mudstone alternations (8 are counted at Site 654; Kastens et al., 1987; Borsetti et al., 1990; Roveri et al., 2014b). Intercalations of ripple–cross-laminated, fine-grained, azoic sandstones occur within the mudstone intervals in places (Cita et al., 1990; Iaccarino and Bossio, 1999). In some mudstone samples, the ostracod Cyprideis sp. (Site 654) and Candona sp. (DSDP Hole 974B) and the foraminifera Ammonia beccarii and Ammonia tepida have been found, indicating a shallow-water (< 50 m) brackish environment (see sub­ sections 5.1 and 5.4; Cita et al., 1990; Iaccarino and Bossio, 1999). 87Sr/86Sr isotope ratios of UU gypsum and planktic foraminifera of the overlying Pliocene (Unit 1 at Site 654) show values much lower (from 0.708627 to 0.708745) and roughly equivalent (from 0.708935 to 0.709112) to coeval ocean water (~0.709020-30; McArthur et al., 2012), respectively (Müller et al., 1990; Müller and Mueller, 1991). Similar 87Sr/86Sr values were obtained from the gypsum cored at Site 652 (0.708626-0.708773; Müller and Mueller, 1991). High resolution seismic reflection data in the Central Mallorca Depression (CMD) highlighted up to 500 m of MSC deposits made of a Bedded Unit (BU) and a thin salt layer (Maillard et al., 2014; Driussi et al., 2015). This BU has never been drilled and, therefore, lacks lith­ ological and chronostratigraphic constraints. 4.4. Balearic Promontory Two contrasting chro­ nostratigraphic and lithological interpretations are proposed: Roveri et al. (2019b) ascribed these sediments to Stage 2 and 3 and suggested that only reworked evaporites and halite are present. By contrast, following the seismostratigraphic description of Maillard et al. (2014), Ochoa et al. (2015) and Raad et al. (2021) inferred the presence of Stage 1 gypsum also in the CMD. The Miocene/Pliocene boundary at DSDP Site 132 is placed above a cross-bedded sand rich in quartz, mica, pyrite, rounded fragments of gypsum and specimens of Ammonia beccarii and Elphidium macellus (Ryan et al., 1973). In the adjacent ODP Site 653 a similar sandstone is found slightly below the biostratigraphically-defined Messinian/Plio­ cene boundary and ~70 cm of grey mudstones with foraminifera and nannofossils of undisclosed provenance are squeezed in between (Cita et al., 1990). These mudstones also contain rare dwarf planktic fora­ minifera (Globorotalia acostaensis, Orbulina universa, and Globigerina bulbosa; Cita et al., 1990). gyp Raad et al. (2021) made a step forward by disclosing a possible tri­ partition of the BU unit (Fig. 7f). In their seismostratigraphic framework, Raad et al. (2021) noticed that the uppermost evaporite-bearing unit (called BU3), ~120 m-thick in the CMD, has geometric, stratigraphic and facies analogies with the astronomically-tuned UG unit of the Cal­ tanissetta Basin (Fig. 3a) that endorse its attribution to Stage 3. Similar to the UU in the deepest basins (see subsection 4.2), BU3 consists of up to 9 low- and medium-amplitude reflectors that are interpreted as alter­ nating terrigenous and gypsum beds (Maillard et al., 2014; Raad et al., 2021). Reflectors are parallel and continuous in the more distal areas, while they appear more chaotic in the more proximal sectors (Raad et al., 2021). The base of BU3 coincides with the erosional top of the salt, interpreted as created by salt exposure, dissolution and locally salt gliding towards the depocenter (Fig. 7f; Raad et al., 2021). By contrast, the top of BU3, which corresponds to the Miocene/Pliocene boundary, is largely flat without signs of erosion (Fig. 7f; Maillard et al., 2014; Raad et al., 2021). An irregular geometry is sometimes visible, but is likely to be related to deformation of the underlying salt (Fig. 7f; Raad et al., 2021). 4.6. Ionian Basin The deepest basin in the Mediterranean today is the Ionian Basin, with its lowest point at 5,267 meters. The so-called Ionian Abyssal Plain (IAP) is bounded on all sides by pre-MSC structural highs (Fig. 2a; Camerlenghi et al., 2019): the Malta Escarpment to the west; the Medina Escarpment to the south separating it from the Gulf of Sirt (Fig. 8a); the Gargano-Pelagosa and/or Otranto sill to the north dividing it from the Adriatic Foreland and, finally, an unnamed sill to the east, separating the IAP from the Levant Basin. Evidence from recent high-resolution seismic studies across the region have been used to support Stage 3 desiccation models (e.g. Hsü et al., 1978a, 1978b; Bowman, 2012; Micallef et al., 2018, 2019; Camerlenghi et al., 2019; Spatola et al., 2020). 4.4. Balearic Promontory Overall, the uppermost Messinian sediments of the Tyrrhenian Basin are interpreted as having been deposited in lakes with periodic episodes of increased salinity and dilution under the strong influence of high energy rivers and, perhaps ocassionally, of the Atlantic (Cita et al., 1990; Müller et al., 1990; Müller and Mueller, 1991). 4.3. Valencia Basin These are interpreted as turbidites sourced from the shelf and/or slope during a lowstand phase of the Mediterra­ nean base level (Del Olmo, 2011; Del Olmo and Martín, 2016; Cameselle and Urgeles, 2017). il Site 134 shows evidence of a “desiccation crack" cutting through a sandy silt layer interbedded with unaffected laminated halite (Hsü et al., 1973c). Unfortunately, the core photograph of this crack has been published in two different orientations (Hsü et al., 1973a, 1973b), leading both Hardie and Lowenstein (2004) and Lugli et al. (2015) to question the evidence for subaerial desiccation. Because of these am­ biguities, we suggest to dismiss this example from the book of evidence. In seismic profiles, the UU/PQ transition (M-reflector or TES) is locally both sharp and smooth (in more distal settings) and erosional (in more proximal settings; Fig. 7e). Maillard and Mauffret (2006) indicate that the smooth parts have been caused by increasing fresh water influx during the Lago-Mare phase, leading to dissolution of the evaporites, and the rough erosional segments are of subaerial origin. For Cameselle and Urgeles (2017), the top of the UU is a smooth and conformable downlap surface, representing the rapid inundation of the basin with only local minor erosional features. 17 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. F. Andreetto et al. 4.6.2. Malta Escarpment At the foot of the Malta Escarpment, Micallef et al. (2018, 2019) and Spatola et al. (2020) amalgamated the MU and UU into one seismic unit, Unit 3, which is separated from the Plio-Quaternary marine sediments (Unit 1) by Unit 2, a chaotic transparent seismic package (Fig. 8c). Unit 2 has a maximum thickness of 760-860 m, a volume of 1430–1620 km3 and a wedge-shaped geometry that thins eastwards, disappearing before reaching the IAP (Micallef et al., 2018). Micallef et al. (2018) and Spa­ tola et al. (2020) proposed a lithological/sedimentological interpreta­ tion of this chaotic body, suggesting it is composed of well-sorted sediments of the Pelagian Platform to the west of the Malta Escarpment, with coarser material at the mouth grading into more distal finer sedi­ ments. This chaotic body has recently been attributed to the Zanclean megaflood during its passage from the western to the eastern Mediter­ ranean via a gateway located in south-eastern Sicily (Micallef et al., 2018, 2019; Spatola et al., 2020). Given the rapidity of the reflooding (≤ 2 years, Garcia-Castellanos et al., 2009, 2020), this interpretation im­ plies rapid mass deposition. Other interpretations of this Unit 2 include being the result of extensive marine mass movement (Polonia et al., 2011), being folded UU (Butler et al., 2015) or being a complex unit built during lower sea level phases (Lofi et al., 2011a, 2011b). The UU and the overlying Zanclean (subunit PQc of Camerlenghi et al., 2019) reflectors are conformably folded throughout most of the abyssal plain, locally showing chaotic internal structure, irregular folding mimicking V-shaped incisions and truncations (Fig. 8b; Camer­ lenghi et al., 2019). These features are interpreted by Camerlenghi et al. (2019) as fluvial valleys carved in subaerially exposed evaporites by the Eso-Sahabi fluvial system, the closest fluvial drainage network to the area (see Micallef et al., 2018) that drained Libya in the late Miocene (Griffin, 2002) and has been traced across the Gulf of Sirt offshore (Sabato Ceraldi et al., 2010; Bowman, 2012). Several arguments coun­ teract this interpretation: 1) the coherent, deformation, mostly of post- Messinian age, of both the UU and the lower Zanclean; 2) the absence of fluvial facies above the bottom of the “valleys”, which instead correspond to a mudstone interval that underwent post-depositional dolomitization (Unit II; see below; Fig. 8b); 3) the unlikelihood that the Eso-Sahabi fluvial system managed to bypass the Medina Ridge divide (Fig. 4.5. Tyrrhenian Basin The Tyrrhenian Basin to the east of Sardinia is a back-arc basin that opened by continental rifting and oceanic spreading related to the 18 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. ( (caption on next page) 19 19 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Fig. 8. Seismic profiles from intermediate-deep Eastern Mediterranean basins containing MSC markers/units (see Fig. 7 for abbreviations). (a) High-resolution seismic line MS27 imaging the PQ and the uppermost MSC’s UU and MU in the Ionian Abyssal Plain and Gulf of Sirt (modified from Camerlenghi et al., 2019). Note how the MSC units are thinner, more difficult to distinguish and more deformed in the Sirt Abyssal Plain than in the IAP. PQ, UU and MU all onlap the structural highs of the Medina Ridge and VHS-2 sill. (b) High-resolution imaging of the lower part of the Plio-Quaternary (PQc unit) and upper part of the Messinian (UU) in the IAP (Camerlenghi et al., 2019). The MSC-PQ boundary is a highly irregular surface, describing apparent V-shaped incisions (symbol V) of controversial origin (see subsection 4.6.1 for insights). Note the coherent deformation of PQc with the underlying MSC sequence and the absence of fluvial facies within the incisions (Unit II is made of lower Pliocene dolomitic marls recovered in Site 374 drilled nearby the seismic line; see text). (c) Multichannel seismic reflection profile MEM-07-203 running approximately parallel to the Malta Escarpment and showing the relationship between Unit 2 of Micallef et al. (2018) with the overlying and underlying Zanclean and Messinian sediments, respectively (modified from Spatola et al., 2020). (d) Uninterpreted (left) and interpreted (right) seismic profiles showing the cyclic and channelized nature of the uppermost Messinian observed in the offshore Sirt Basin (modified from Bowman, 2012). (e) Interpreted 2D regional WNW–ESE seismic profile crossing the continental shelf and offshore Levant Basin and the Herodotus Abyssal Plain (Jagger et al., 2020). Note the lateral continuity of the Messinian MU. (f) Seismic profile from the Levant Basin showing the 6 sub-units distinguished inside the MU as well as its lower (N-reflector) and upper (M-reflector) boundaries (modified from Gvirtzman et al., 2013). (g) High-resolution seismic reflection image with wireline logs from Aphrodite-2 well illustrating that M-reflector previously considered as top evaporitic sequence and M/P boundary here consists of a ~100-m-thick unit (i.e. 4.5. Tyrrhenian Basin Unit 7 of Gvirtzman et al., 2017) in which different layers are distinguished (modified from Gvirtzman et al., 2017). (h) Interpreted and uninterpreted seismic profiles imaging the Mavqi’im and Afiq formations described in the canyons on the Levant continental margin (modified from Ben Moshe et al., 2020). bouncing of the drill bit at the (e.g. Sites 122, 124, 125, 132, 133 and 134; Ryan et al., 1973). Dolomitization was (Hsü et al., 1973a, 1973b; Ryan et al., 1973) and still is (e.g. Ryan, 2009) largely considered a "diagnostic feature of tidal (sabkha) sediments" (Friedman, 1973, pp. 705). Its occurrence at locations with present-day water depth exceeding 2000 m was therefore considered strong evidence that the Mediterranean floor was subaerially exposed prior to the Zanclean marine replenish­ ment (e.g. Ryan et al., 1973). It is, however, now widely accepted that dolomite precipitation is not exclusive of sabkha environments, but rather is a common process also in bottom sediments under a relatively deep water column (see Dela Pierre et al., 2012, 2014 and references therein). In the specific case of the offshore Mediterranean’s M/P boundary on the Ionian Abyssal Plain, already in the ‘70s dolomitization was thought to have occurred after burial (Hsü et al., 1978b), a conclusion recently reinforced by McKenzie et al. (2017). 4.6.2. Malta Escarpment 8a). Alternatively, the apparent incisions at the M/P boundary in the IAP may represent post-sedimentary folds resulting from post-Messinian tectonic and/or salt movements-driven deforma­ tion (e.g. Mocnik et al., 2018). At Site 374 the Miocene/Pliocene boundary has been recovered (Unit II), but its primary nature (likely a mudstone) is obscured by diagenesis (cementation by dolomite; Hsü et al., 1978b). A lithified dolostone at the (seismic) predicted depth of the M-Horizon is a characteristic of several sites. Sometimes this interval has been recovered (e.g. Sites 125 and 374; Ryan et al., 1973; Hsü et al., 1978b; Comas et al., 1996); at others the hard lithology is inferred by the torqueing of the drill string (resistance to turning) accompanied by 4.6.1. Ionian Abyssal Plain 4.6.1. Ionian Abyssal Plain The typical “trilogy” of seismic units representing the MSC deposi­ tion in the Western Mediterranean is recognized also in the IAP by Gallais et al. (2018) and Mocnik et al. (2018). However, Camerlenghi et al. (2019) states the MSC sequence in the IAP is ~1,300 m-thick and composed of only two units (Fig. 8a). The lowermost 150-700 m-thick Mobile Unit (MU) is seismically transparent without discernible bedding and with diapiric structures, all features diagnostic of halite. By contrast, the 350-1,000 m-thick Upper Unit (UU) alternates highly reflective with acoustically transparent reflectors (Figs. 8a-b), similar to those described of the UU sequences of the Western Mediterranean (Figs. 7b- c). These are therefore assumed to represent gypsum-mudstone cycles (Camerlenghi et al., 2019). The uppermost 80 m of UU has been recovered from DSDP Site 374 (Hsü et al., 1978b), confirming the presence of gypsum (both primary cumulate and clastic; Lugli et al., 2015) alternating with mudstones (Unit III; Hsü et al., 1978b). These mudstones are largely barren of in situ fossils. However, the presence of some foraminifera and siliceous microfossils led Cita et al. (1978) and Hsü et al. (1978a) to suggest that sporadic marine incursions, possibly from the Indian Ocean, took place during Stage 3. Similar to Site 372, the basal part of Hole 374 intercepted one thin (~3.5 m) halite layer within the UU (Hsü et al., 1978b). 4.6.3. Gulf of Sirt The Gulf of Sirt (or Sirt embayment; Figs. 2a, 8a), the offshore extension of the Sirt Basin onshore Libya (Griffin, 2002 and references therein), is cross-cut by high-density seismic and well datasets as a result of oil potential of the region (Fiduk, 2009). However, only few studies have tackled the MSC (e.g. Hallett, 2002; Fiduk, 2009; Bowman, 2012). The Gulf of Sirt (or Sirt embayment; Figs. 2a, 8a), the offshore extension of the Sirt Basin onshore Libya (Griffin, 2002 and references therein), is cross-cut by high-density seismic and well datasets as a result of oil potential of the region (Fiduk, 2009). However, only few studies have tackled the MSC (e.g. Hallett, 2002; Fiduk, 2009; Bowman, 2012). In the Sirt embayment the MSC unit(s) is unevenly distributed in sub- basins controlled by a pre-existing topography, there is little distinction between the MU and UU, the overall thickness of the MSC unit(s) is lower and the degree of deformation is higher than in the adjacent IAP (Fig. 8a; Camerlenghi et al., 2019). The presence of halite in the Sirt In the Sirt embayment the MSC unit(s) is unevenly distributed in sub- basins controlled by a pre-existing topography, there is little distinction between the MU and UU, the overall thickness of the MSC unit(s) is lower and the degree of deformation is higher than in the adjacent IAP (Fig. 8a; Camerlenghi et al., 2019). The presence of halite in the Sirt 20 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Meilijson et al. (2019)’s lithological interpretation of industrial bore­ holes slightly farther to the NE (Fig. 2b) give Unit 7 a significantly smaller thickness (5 m; Fig. 3b). Independent studies offshore Lebanon and Syria (Kartveit et al., 2019; Madof et al., 2019) describe a unit (Nahr Menashe complex) interpreted as a thicker (up to 300 m; Madof et al., 2019), but lateral equivalent of Gvirtzman et al. (2017)’s Unit 7. Based on its channelized morphology identified upslope near the Lebanese coast, Kartveit et al. (2019) and Madof et al. (2019) interpreted the Nahr Menashe Unit and the IMTS underneath as fluvial in origin, deposited/ formed on a subaerially exposed floor of the Levant Basin. Six (Madof et al., 2019) to eight (Madof and Connell, 2018) lobes were identified and are proposed to have stacked with precessional frequency. 4.6.3. Gulf of Sirt The Nahr Menashe sequence has been correlated by the same authors with the Abu Madi Fm. located within the Messinian canyons offshore Egypt (Abdel Aal et al., 2000; Loncke et al., 2006; Abdel-Fattah, 2014), the Handere Formation offshore Turkey (Radeff et al., 2017) and with the Eosahabi deposits offshore Libya (Bowman, 2012). This interpretation implies a low base-level during the final stage of the MSC. embayment is debated, but most authors think it is absent (see Fiduk, 2009; Sabato Ceraldi et al., 2010; Lofi, 2018; Jagger et al., 2020; Fig. 2b). Bowman (2012) distinguishes seven seismic units within the MSC-related sequence (Fig. 8d). On the basis of high-resolution 3D and 2D data, each seismic unit has been interpreted consisting of clastics filling erosional channels cutting up to 100 m deep and wide (Fig. 8d) and evaporites (gypsum and anhydrite) alternating with precessional cyclicity (Bowman, 2012). The presence of anhydrite in the topmost part of the sequence is confirmed by the B1 NC 35A well (Hallett, 2002). Sabato Ceraldi et al. (2010) and Bowman (2012) envisaged a three-step evolution of each unit: 1) evaporitic deposition during precession maxima in a dried out Sirt embayment; 2) erosion of the valleys during the arid-wet transition fed by the Eso-Sahabi paleofluvial system; 3) filling of the valleys with the fluvial sediments during the wet phase. Based on this chronostratigraphic interpretation, the evaporite cycles should be time equivalent to most of Stage 3, with the upper four seismic units reflecting the Lago-Mare phase (Fig. 1a). gi g Manzi et al. (2018) and Meilijson et al. (2018, 2019) attempted as­ tronomical dating of the abyssal MSC succession of the Levant Basin by integrating biostratigraphy on the pre-MU succession, reflector counting within the MU (only Meilijson et al., 2019) and well-log data (Fig. 3b). They achieved two contrasting results that gave rise to an outstanding controversy (Figs. 3b, 4a). Manzi et al. (2018) proposed that Stage 1 in the deep Levant is represented by a foraminifera-barren, evaporite-free shales interval labelled FBI (foraminifer barren interval) observed in the deep Aprodite-2 well and in the more proximal Myra-1 well. In this interpretation Unit 7 comprises the whole of Stage 3 (with the IMTS corresponding to the Stage 2/3 transition) and all halite deposition took place in ~50 kyr estimated during Stage 2 of the MSC (Fig. 1a; Roveri et al., 2014a). 4.7.1. Abyssal plain y Old seismic data in the Levant Basin show an up to 2 km-thick, high velocity, acoustically transparent sequence bounded by the N-reflector at the base and the M-reflector at the top (Figs. 8e-f; Ryan, 1978; Net­ zeband et al., 2006). This sequence thickens and extends for tens of ki­ lometers towards WNW and thins eastward along the continental margin (Fig. 8e), where the N and M-reflectors converge forming the condensed MSC section of the Mavqiim and Afiq formations (described in subsec­ tion 4.7.2; Gvirtzman et al., 2017; Manzi et al., 2018). High resolution 2D and 3D industrial seismic and well data from the southern Levant Basin revealed that this transparent sequence is largely made of pure halite with internal stratification picked out by diatomite, clay- and clastic-rich layers that allowed the division of the sequence into six sub- units, basinward-tilted and truncated at the top by the flat TES (Fig. 8f; Gvirtzman et al., 2013, 2015, 2017; Feng et al., 2016, 2017; Manzi et al., 2018; Meilijson et al., 2018, 2019). Clastic beds ~3 to 20 m-thick are abundant in the highly reflective and chaotic Unit 5 (i.e. Interbedded evaporites of Meilijson et al., 2019; MC2 unit of Feng et al., 2016; Figs. 3b, 4a), where they are interbedded with evaporites (probably halite with minor occurrences of anhydrite) varying in thickness from ~6 to 30 m (Manzi et al., 2018; Meilijson et al., 2019). Paleontological analyses of cuttings probably belonging to one of the clastic beds revealed the presence of a few mollusk fragments, ostracods, echinoid spines and a relatively rich assemblage of benthic and planktic forami­ nifera which Meilijson et al. (2019) concluded to be reworked. Based on seismic and well-log data, clastic intercalations (probably of clays, silts and sands) within a halite-dominated sequence are thought to persist in the overlying Unit 6, although they diminish in thickness and frequency (Gvirtzman et al., 2013; Feng et al., 2016; Meilijson et al., 2019). The expression of the end of the MSC is highly controversial. Until recently, the M-reflector of Ryan (1978) (later renamed as the Top Erosion Sur­ face, TES; Lofi et al., 2011a, 2011b) bounding Unit 6 at the top was considered to be the Miocene/Pliocene boundary (Fig. 8f; Ryan, 1978; Gvirtzman et al., 2013; Feng et al., 2016). Instead, Gvirtzman et al. 4.7. Levant Basin 4.7. Levant Basin 4.6.3. Gulf of Sirt By contrast, the FBI is not present in the Dolphin well targeted by Meilijson et al. (2019), which is located in an intermediate position between the Aprodite-2 and Myra-1 wells studied by Manzi et al., 2018; Fig. 2b). Instead, in the Dolphin well a relatively open- marine, foraminifera-rich sequence extends below the (conformable) base of the MU, placed in correspondence to a ~2 to 5.5 m-thick anhydrite/shale (Unit 0; Manzi et al., 2018 and Meilijson et al., 2018, respectively). Astronomical tuning of the ~33 cycles counted in the MU in the Dolphin well, which are assumed to be precessional, results in the Main Halite body (i.e. Unit 0-4 of Gvirtzman et al., 2013 and Manzi et al., 2018) spanning MSC Stage 1 and 2, the Interbedded Evaporites/ Unit 5 covering substage 3.1 and the Argillaceous Evaporites/Unit 6-7 to encompass the Lago-Mare phase (Figs. 3b, 4a). In this interpretation from Meilijson et al. (2019), halite deposition in the Levant Basin started in Stage 1 and persisted throughout the entire MSC, including Stage 3, during which more allochthonous material was delivered to the basin (Fig. 3b). Madof and Connell (2018) and Madof et al. (2019) also attempted an astronomical tuning of the Nahr Menashe Unit, concluding that it spans throughout substage 3.2 and part of substage 3.1. Feng et al. (2016) claim, however, that the impressive thickness of clastics found in the Levantine MU is more indicative of distinct short-term events (shorter than the precession cycle) associated with transport of extraordinary power and magnitude. 4.7.1. Abyssal plain Mavqi’im Fm (Fig. 8h). Ben Moshe et al. (2020) distinguished a lower sub-unit composed of clastic gypsum and with fore-stepping and down- stepping internal geometry typical of progradational wedges, and an upper sub-unit containing anhydrite fragments and marls with Lago- Mare fauna (e.g. Cyprideis torosa; Rosenfeld, 1977) and with seismic characteristics typical of a transgressive systems tract. Incisions are re­ ported throughout the Afiq Fm. at different depths, while erosional surfaces bound both sub-units (Ben Moshe et al., 2020). In particular, the surface capping the upper subunit and correlated to the M horizon or TES basinward (see subsection 4.7.1) shows dendritic drainage patterns of gullies and channels (Ben Moshe et al., 2020). - Abundant Cyprideis pannonica specimens were also recovered from DSDP Sites 375 and 376 drilled on the crest of the Florence Rise, west of Cyprus (Fig. 2b; Hsü et al., 1978b). - Abundant, well-preserved Ammonia tests and Cyprideis specimens are also known from Site 129A (Fig. 2b), occurring with dwarf plank­ tonic foraminifera (Ryan et al., 1973). g Ben Moshe et al. (2020) identify the variation of base level specif­ ically during Stage 3 by analyzing the morphology of truncation surfaces bounding the Afiq Formation on the continental margin of the Levant Basin. This suggests high amplitude fluctuations of base-level in the order of one hundred meters, with development of subaerial erosion surfaces and the deposition of clastics and incision by fluvial drainage systems that occurred during the lowstand phases, while aggradational units (of unknown lithological nature) accumulated during the high­ stand phases. According to their analysis, base level dropped down to a maximum 535 m during Afiq deposition (i.e. below the maximum 430 m estimated paleodepth of the Sicily Sill; Garcia-Castellanos et al., 2009), implying hydrological disconnection between the Eastern and Western basins at various times during Stage 3. A regression to 615-885 m is proposed to have occurred at the top of the Afiq Fm., pre-dating the abrupt refilling at the base of the Zanclean (e.g. Micallef et al., 2018, 2019; Garcia-Castellanos et al., 2020; Spatola et al., 2020). All the evidence listed above suggest that a base-level fall leading to subaerial exposure occurred at some point(s) during Stage 3 in the Eastern Mediterranean (Ryan, 2009). 4.7.2. Levant continental margin Evaporitic and non-evaporitic deposits are buried beneath PQ de­ posits (Yafo Formation) along the Levant continental margin, where they are mostly preserved within canyons carved underwater in pre- Messinian time (Druckman et al., 1995; Lugli et al., 2013). Within the Afiq canyon, Druckman et al. (1995) distinguished three formations in the Messinian sequence: the evaporitic Mavqi’im Formation, 115 m- thick and mostly composed of anhydrite in places interbedded with micritic limestones; the Be’eri Formation, cmprising gypsum; the Afiq Formation, varying in thickness from 30 to 90 m and consisting of conglomerates, sandstones and marls interpreted as representing fluvial and lacustrine-marsh environments (Druckman et al., 1995). The Afiq Fm. is only present in the deepest portions of the canyon where it overlies the Mavqi’im Fm. By contrast, the Be’eri gypsum is only found along the canyon shoulders covered by the Pliocene, at elevations > 600 m with respect to the Mavqi’im Fm. Based on Sr values, Druckman et al. (1995) attributed the Mavqi’im Fm. to MSC Stage 1, the Be’eri Fm. to substage 3.1 and the Afiq Fm. to the Lago-Mare phase. These authors also suggested that gypsum precipitation occurred under subaqueous conditions, with the water level ~600 m (i.e. the difference in altitude between the Mavqi’im and Be’eri fms.) higher during the deposition of the Be’eri Fm. Two base-level falls of approximately the same magnitude are thought to have occurred between the evaporitic phases and after Mavqi’im deposition. A lowstand phase was therefore in force during Afiq deposition (Druckman et al., 1995). Knowledge of the Stage 3 sequence offshore is mainly based on the integration of seismic interpretations and analysis of material recovered from fragmentary and unevenly distributed DSDP/ODP/industrial cores. ▪ MSC sediments are absent on the eroded continental shelves bordering the deep basins, except in the Messinian thalwegs and at their mouth. Here the PQ lies directly above the MES which, in turn, cuts through the pre-MSC deposits (Fig. 7e). A similar stratigraphic arrangement is found along the middle- upper slopes (Fig. 7d), although the presence of a thin, possibly incomplete UU in morphological depressions is sometimes postulated. Seamounts also lack MSC Stage 3 sedi­ ments and are strongly incised by the MES (Fig. 7g). ▪ The thick UU is widespread and roughly present everywhere in the abyssal plains from west of the Albor´an volcanic arc to the eastern edge of the Ionian Basin (Fig. 2b). 4.7.1. Abyssal plain (2017) showed that in higher resolution seismic data the M-reflector/ TES is a bundle of reflectors forming a distinct layer (Unit 7) overlying a truncation surface (i.e. Unit 6/7 boundary) that they re-labelled intra- Messinian truncation surface (IMTS; Fig. 8g) and ascribed to subaqueous dissolution rather than subaerial incision (e.g. Bertoni and Cartwright, 2007; Lofi et al., 2011a, 2011b; Kartveit et al., 2019; Madof et al., 2019). This conclusion was recently corroborated by the independent study of Kirkham et al. (2020). Analysis of gamma-ray and resistivity logs in three deep basin wells (Aphrodite-2 Myra-1 Sara-1; Fig 2b) and cor­ Late Messinian sediments have also been recovered at several DSDP (129, 375, 376; Ryan et al., 1973; Hsü et al., 1978b) and ODP Sites (965, 967, 968; Emeis et al., 1996), but the assignment of the retrieved sedi­ ments to seismostratigraphic units is problematic. Nevertheless, they provide several key nuggets of precious information about the Stage 3 paleoenvironment: - Sites 965 and 966, located roughly on the crest of the Eratosthenes Seamount, just south of Cyprus (Fig. 2b), recovered soil structures above the evaporites indicating exposure above sea level (Robertson, 1998a, 1998b; Maillard et al., 2011a; Reiche et al., 2016). i This conclusion was recently corroborated by the independent study of Kirkham et al. (2020). Analysis of gamma-ray and resistivity logs in three deep basin wells (Aphrodite-2, Myra-1, Sara-1; Fig. 2b) and cor­ relation with the Or-South-1 well (located between the deep basin and the shelf) showed that Unit 7 maintains a constant thickness of ~100 m- thick and consists of clastic-rich anhydrite of undisclosed provenance. i This conclusion was recently corroborated by the independent study of Kirkham et al. (2020). Analysis of gamma-ray and resistivity logs in three deep basin wells (Aphrodite-2, Myra-1, Sara-1; Fig. 2b) and cor­ relation with the Or-South-1 well (located between the deep basin and the shelf) showed that Unit 7 maintains a constant thickness of ~100 m- thick and consists of clastic-rich anhydrite of undisclosed provenance. - ODP Sites 967 and 968, located at the base of the northern and southern slope of Eratosthenes Seamount (Fig. 2b), respectively, 21 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. revealed the presence, within the MSC interval, of calcareous turbi­ dites with Cyprus-derived clasts and clays containing Ammonia tep­ ida, Cyprideis pannonica and pulmonate gastropods (Blanc-Valleron et al., 1998; Robertson, 1998a,b; Reiche et al., 2016). 4.7.1. Abyssal plain However, it must be kept in mind that both the Florence Rise and Eratosthenes Seamount are likely to have been much more elevated during the Messinian than they are today because of Pliocene-Quaternary subsidence related to the Cyprus sub­ duction zone (Robertson, 1998a, 1998b; Maillard et al., 2011a; Reiche et al., 2016). Sites 375 and 376 display several discrete layers of primary and clastic gypsum interbedded in the Cyprideis-rich mudstones (McCulloch and De Deckker, 1989; Lugli et al., 2015). This succession shares several similarities with sites drilled in the Western Mediterranean (e.g. ODP 654A) and Ionian Basin (DSDP 374), where they have been correlated with the seismic Upper Unit (see subsections 4.5 and 4.6). This may suggest that a Western Mediterranean-like gypsum-bearing UU was also locally deposited in the easternmost abyss of the Mediterranean (see Günes¸ et al., 2018). 4.7.2. Levant continental margin In the abyssal plains its seismic facies appears homogeneous, comprising parallel and relatively continuous high amplitude reflections (Figs. 7b- c). The UU pinches out towards the foot of continental slopes and seamounts (Figs. 7b, d-g), where it can be irregularly bedded or relatively well bedded (Lofi et al., 2011a, 2011b). The uppermost part of the Bedded Units (defined in depressions physically disconnected from the abyssal plains and, therefore, from the UU; e.g. CMD and Corsica Basin; Maillard et al., 2014; Thinon et al., 2016; Raad et al., 2021) shows seismic features comparable to those of the UU. l i However, combining stratigraphic, sedimentological and geochem­ ical (Sr isotopes) considerations, Lugli et al. (2013) revealed the clastic nature of both the Mavqi’im and Be’eri evaporites and suggested the persistent drowning of the canyon(s), filled with turbidites (Lugli et al., 2013). Due to the presence of clastic evaporites, Gvirtzman et al. (2017) suggested that the Mavqiim Formation is a condensed section encom­ passing MSC Stage 2 and early Stage 3, while the evaporite-free Afiq Formation represents the Lago-Mare phase. i ▪ Drill Sites revealed that the reflections of relatively high amplitude in seismic profiles correspond to gypsum and mudstone alternations with sporadic intercalations of massive to cross-bedded sandstones. Some mudstone interbeds contain low-diversity assemblages of benthic organisms (ostracods, foraminifera and diatoms) indicative of shallow to neritic en­ vironments. Except for dwarfed forms of planktonic forami­ nifera and the monospecific nannofossil assemblages described Ben Moshe et al. (2020) ascribed (at least part of) the Afiq Fm. to the whole of Stage 3 as a wedge-shape clastic complex lying on top of the Mavqi’im Fm. and with the basal surface corresponding to the correla­ tive conformity of the MES developed landward, at the expense of the 22 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Fig. 9. Photomicrographs of the key micro- and macrofossils featuring Stage 3 sedi­ ments. (a) Scanning electron microscope (SEM) photographs of the more common Paratethyan ostracod species in substage 3.1 and 3.2 sediments (compiled from Stoica et al., 2016, Cosentino et al., 2018 and Sciuto et al., 2018). (b) Photomicrographs of the Paratethyan dinoflagellate cyst Galea­ cysta etrusca under the optical microscope (left) and SEM (right) (modified from Do Couto et al., 2014 and Grothe et al., 2018). Scale=20 μm. 4.7.2. Levant continental margin (c) SEM microphotographs of the euryhaline, shallow-water benthic fora­ minifera Ammonia beccarii (1-spiral side, 2- umbilical side) and Ammonia tepida (3-spi­ ral side; Carnevale et al., 2019) and of the dwarf fauna of planktonic foraminifera from the Bajo Segura Basin (4; Corbí and Soria, 2016). (d) Photographs in polarized light (crossed nicols) of some specimens of Cera­ tolithus acutus (1-3) described in the Lago- Mare unit of Malaga (1-Do Couto et al., 2014), the Zorreras Mb. of Sorbas (2-Clau­ zon et al., 2015) and the Colombacci Fm. of the Northern Apennines (3-Popescu et al., 2017) and of destroyed (4) and intact (5) ascidian spicules of Micrascidiscus sp. (Golovina et al., 2019). Note that C. acutus specimens closely resemble ascidian spicules of Micrascidiscus sp., which may lead to misinterpretation of the C. acutus (see Golovina et al., 2019), and that pictures 1 and 2 are identical, despite they are attrib­ uted to samples taken from two different localities. (e) Articulated skeletons of marine fish from substage 3.1 mudstone horizons in Cyprus (1-Aphanius crassicaudus; Manzi et al., 2016a) and substage 3.2 deposits in Cava Serredi (2-Mugil cf. cephalus; Carnevale et al., 2018). Fig. 9. Photomicrographs and macrofossils featurin ments. (a) Scanning elec (SEM) photographs of th Paratethyan ostracod speci and 3.2 sediments (comp et al., 2016, Cosentino Sciuto et al., 2018). (b) Pho the Paratethyan dinoflage cysta etrusca under the o (left) and SEM (right) (m Couto et al., 2014 and Gro Scale=20 μm. (c) SEM mic the euryhaline, shallow-w minifera Ammonia beccarii umbilical side) and Ammo ral side; Carnevale et al., dwarf fauna of planktonic the Bajo Segura Basin (4; 2016). (d) Photographs i (crossed nicols) of some sp tolithus acutus (1-3) descr Mare unit of Malaga (1- 2014), the Zorreras Mb. o zon et al., 2015) and the C the Northern Apennines ( 2017) and of destroyed ( ascidian spicules of M (Golovina et al., 2019). N specimens closely resemble of Micrascidiscus sp., wh misinterpretation of the Golovina et al., 2019), an and 2 are identical, despit uted to samples taken fr localities. (e) Articulated sk fish from substage 3.1 mud Cyprus (1-Aphanius cra et al., 2016a) and substag Cava Serredi (2-Mugil cf. ce et al., 2018). 23 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. by Castradori (1998), the rest of planktonic foraminifera and nannofossils are largely regarded as reworked. impoverished. Monospecific assemblages of Cyprideis sp. 5. The paleontological perspective Paleontological data have been used to define and identify Stage 3 sediments, but have also been a source of profound contention over the interpretation of its paleoenvironmental and paleohydrological nature. Biotic groups impacted by the evolution of the gateways linking the Mediterranean with the Atlantic, Indian Ocean and Paratethys include marine species (e.g. foraminifera, calcareous nannofossils, fish) and brackish water-species (ostracods, fish, mollusks and dinocysts endemic or with affinity to species of the Paratethys region) that were unable to migrate when these corridors were closed, and terrestrial species (e.g. mammals) that, conversely, got across the gateway during periods of exposure (see Colombero et al., 2017; Booth-Rea et al., 2018; Mas et al., 2018b). Analysis of these faunal datasets provides key insights into likely gateway dimensions and the timing of their opening, restriction and closure (e.g. Palcu et al., 2017). Furthermore, they are a key constraint on the water sources likely to have been affecting the Medi­ terranean during MSC Stage 3. ; , ; , ; , ) Some authors consider the time when the Paratethyan ostracods arrived in the Mediterranean to be well constrained (e.g. Roveri et al., 2008a; Grossi et al., 2011; Cosentino et al., 2018) by the scarce occur­ rence of the first Paratethyan immigrant Loxoconcha muelleri 20 cm below the ash layer in the Colla di Votta section, which has a 238U-206Pb age of 5.5320±0.0074 Ma (Cosentino et al., 2013), and in the chaotic deposits of the Adana Basin, ascribed to Stage 2 (Faranda et al., 2013). Instead, the first appearance of Loxocorniculina djafarovi has been considered to coincide with the biofacies 1-2 shift and to have occurred Mediterranean-wide synchronously at 5.40 Ma (Roveri et al., 2008a; Grossi et al., 2011; Cosentino et al., 2013). Roveri et al. (2008a) also showed Biofacies 2 diversity as increasing linearly through the Lago- Mare phase, reaching its maximum diversity just beneath the Miocene/Pliocene boundary and before disappearing in the Pliocene. Following the claimed synchronicity of the FO of both Loxoconcha muelleri and Loxocorniculina djafarovi, Roveri et al. (2008a) and Grossi et al. (2011) recognized one biozone in each biofacies: the Loxoconcha muelleri Biozone, spanning from 5.59 to 5.40 Ma, and the Loxocorniculina djafarovi Biozone, whose boundaries correspond respectively to the first (5.40 Ma) and last occurrence (5.33 Ma) of L. djafarovi in the Mediter­ ranean. This biozonation, erected by Grossi et al. 4.7.2. Levant continental margin (Sites 372, 129A, 376, 654A, 967, 968; Ryan et al., 1973; Cita et al., 1990) or oli­ gospecific assemblages dominated by Cyprideis and rare specimens of Candona sp. (Hole 974B, Iaccarino and Bossio, 1999) and L. muelleri (Hole 978, Iaccarino and Bossio, 1999) are the more widely reported. Interestingly, these assemblages are always associated with Ammonia sp. tests and, in some cases, with other species of shallow-water, euryhaline benthic foraminifera (see subsection 5.4). Only in Hole 975, close to the M/P boundary is a more heterotypic ostracod assemblage found (Euxi­ nocythere praebaquana, Amnicythere idonea, Leptocythere limbate, Lox­ ocorniculina djafarovi, Candona sp., and Cyprideis sp.; Iaccarino and Bossio, 1999) and lacking of euryhaline benthic foraminifera. The likely cause of the widespread barrenness of ostracods in most of the offshore samples is perhaps because environmental conditions in the deep basins (depth and/or salinity) where not suitable to permit population by this benthic fauna (see below for the ecological requirements; e.g. Hsü et al., 1978b in reference to Site 374). Finally, one must bear in mind that studying these organisms require much more material (some hundreds of grams) than the quantity of core sediments usually processed (i.e. ~10 cm3; Iaccarino and Bossio, 1999). ▪ The deep Levant Basin contains a ~1.8-2.0 km-thick MU (Figs. 8e-f), consisting of 6 to 7 seismic units depending on the resolution of the seismic employed. In high resolution seismic data, the lateral equivalent of part of the UU is identified as a ~100-m-thick, clastic-rich, anhydrite layer (Unit 7 of Gvirtz­ man 1207) offshore Israel, thickening to 300 m offshore Lebanon (Nahr Menashe complex, Madof et al., 2019). The Levant Basin still has major controversies concerning the timing of halite deposition (~50 kyr vs ~550 kyr; Manzi et al., 2018 vs Meilijson et al., 2019), the origin of the clastic accu­ mulations overlying the halite (fluvial vs subaqueous) and the presence or absence of gypsum-mudstone cycles. l ▪ The deep Levant Basin contains a ~1.8-2.0 km-thick MU (Figs. 8e-f), consisting of 6 to 7 seismic units depending on the resolution of the seismic employed. In high resolution seismic data, the lateral equivalent of part of the UU is identified as a ▪ Apart from the halite flow-related deformation, the Miocene/ Pliocene boundary (i.e. UU/PQ transition) is conformable in intermediate (e.g. 5. The paleontological perspective (2011), is often used for dating incomplete successions (e.g. Vera Basin; Stoica et al., 2016; Caruso et al., 2020). However, the first appearance of a diversified ostracod assemblage (including Loxocorniculina djafarovi) occurred in already cycle 3 of the Sicilian Upper Gypsum at Eraclea Minoa (Fig. 3a; Grossi et al., 2015), which has an astronomical age of 5.45 Ma (Van der Laan et al., 2006) or 5.47 Ma (Manzi et al., 2009). Furthermore, the sudden appearance of Biofacies 2 and its linear, upward increase in di­ versity have not been recognized in localities like Nijar and Malaga, where biofacies 1 and 2 are found stacked in more than one lithological (possibly precession-controlled) cycle in the Lago-Mare succession (Bassetti et al., 2006; Guerra-Merch´an et al., 2010). These findings argue that the appearance of Paratethyan ostracods in the Mediterranean may 4.7.2. Levant continental margin Balearic Promontory) and deep (WAB, EAB, Algero-Balearic, Liguro-Provençal, Tyrrhenian, Ionian and Levant) depocenters, while it shows signs of erosion on the shelf domain and along the upper-middle continental slopes and seamounts. Clear arguments of floor exposure at the M/P boundary are absent in all drill sites but 978A. The paleoecology (salinity and depth ranges) of Stage 3 ostracods has been based on both observations of few species that still live in the Caspian and Black seas today and have affinities with the Stage 3 species and on the interpretation of sedimentological, geochemical and miner­ alogical data of the surrounding sediments (see Gliozzi and Grossi, 2008 and Grossi et al., 2008 for insights). Biofacies 1 is thought to represent very shallow water environments (i.e. <15 m) with salinity fluctuating between mesohaline and hypersaline when the euryhaline Cyprideis is dominant. Instead, more stable oligo-mesohaline water is inferred when the other species are more abundant in Biofacies 1. The variegated Biofacies 2, on the other hand, is thought to represent somewhat deeper environments (up to 100 m) and less salty conditions (oligo-low meso­ haline; Gliozzi and Grossi, 2008; Grossi et al., 2008; Caruso et al., 2020). 5.1. Ostracods Ostracods are by far the most prolific faunal group during Stage 3. Brackish species are known from both land sections and deep-sea cores across the whole Mediterranean (see Fig. 2b for sites and references; Fig. 9a). Two characteristic biofacies are commonly distinguished: Biofacies 1 (Bonaduce and Sgarrella, 1999) or Cyprideis assemblage (Iaccarino and Bossio, 1999) consists of an monospecific population of Cyprideis species or of an oligotypic population dominated by Cyprideis species alongside rare specimens of Tyrrhenocythere ruggierii, Loxoconcha kochi, Loxoconcha muelleri and Caspiocypris alta); Biofacies 2 (Bonaduce and Sgarrella, 1999) or Loxocorniculina djafarovi assemblage (Iaccarino and Bossio, 1999) has a higher species diversity characterized by the abundant occurrence of truly Paratethyan species belonging to the genera Amnicythere, Loxoconcha, Loxocauda, Cytheromorpha, Cyprinotus and Tyrrenhocythere (see species names in Fig. 9a). The number of spe­ cies reported in the onshore sections is variable, ranging from a dozen (e. g. Caruso et al., 2020) to more than sixty (e.g. Gliozzi et al., 2007; Grossi et al., 2008). This variability is not explained, but it may result from the application of different taxonomic concepts that resulted in the recog­ nition of more or fewer species (Stoica et al., 2016) or from local envi­ ronmental conditions that differed from basin to basin and resulted in different patterns of colonization. Compared to the onshore domain, the ostracod fauna offshore is 24 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. not have been synchronous, therefore casting serious doubts upon the biostratigraphic relevance of the Mediterranean ostracods. Eastern Paratethys water (respectively) in the Mediterranean and (relatively) high water level conditions (e.g. Pellen et al., 2017). g Except for Cyprideis specimens, where species attribution is debated (see discussion in Stoica et al., 2016), the affinity of all other ostracod species observed in Mediterranean Stage 3 sediments (Fig. 9a) with those of the Eastern Paratethys basins (i.e. Dacian, Euxinic and Caspian) has been demonstrated in several publications (e.g. Ruggieri, 1967; Gliozzi et al., 2007; Stoica et al., 2016; Sciuto et al., 2018). Only Bassetti et al. (2003, 2006) have questioned the Paratethyan affinity by sug­ gesting that species from the Northern Apennines and Nijar Basin have ambiguous affinities with Paratethyan fauna as described in the mainly Russian literature from the ’60-’70s. However, these differences be­ tween the late Messinian Mediterranean and Paratethyan ostracods resulted from misidentifications and/or a different use of species nomenclature (Stoica et al., 2016). 5.1. Ostracods Recently acquired knowledge of the Pontian assemblages of the Dacian, Euxinic and Caspian basins now permit to trace the provenance of Mediterranean Stage 3 ostracod spe­ cies from the entire Black Sea region (Stoica et al., 2016) and, for a few species, from the Dacian (Stoica et al., 2013; Lazarev et al., 2020), Caspian (Van Baak et al., 2016) and North Aegean (see references in Krijgsman et al., 2020a) basins. Palynological studies on the late Messinian Mediterranean dinocysts record are rather scarce, confined to a limited number of outcrops (Malaga Basin, Do Couto et al., 2014; Northern Apennines, Bertini, 2006; Popescu et al., 2007; Iaccarino et al., 2008; Cosentino et al., 2012; Pellen et al., 2017; Caltanissetta Basin, Londeix et al., 2007) and deep wells (976B, 977A, 978A and 134B, Popescu et al., 2015). These studies describe substage 3.1 as being barren of dinocysts. By contrast, substage 3.2 dinocyst assemblages are diverse particularly a few meters/tens of meters below the Miocene/Pliocene boundary and show recurrent ver­ tical variation in abundance between brackish, Paratethyan-type taxa and marine stenohaline and euryhaline species. Taxa with Paratethyan affinities are largely considered to be autochthonous by all aforemen­ tioned authors. The extent to which reworking may have affected the marine assemblages is more controversial and debated between none (in Malaga and in the Apennines; Popescu et al., 2007; Do Couto et al., 2014; Pellen et al., 2017), partial (in the uppermost part of the Sicilian Upper Gypsum; Londeix et al., 2007) and total (in the Apennines; e.g. Bertini, 2006; Iaccarino et al., 2008; Cosentino et al., 2012). Given the extent of the implications (i.e. re-establishment of a Mediterranean- Atlantic flow or connection earlier than the Zanclean; e.g. Pellen et al., 2017), this is an issue that will require further clarification. l The means by which the ostracods travelled from the Paratethys to and across the Mediterranean during Stage 3 is as crucial for recon­ structing the Stage 3 paleoenvironment as it is poorly addressed in onshore studies or overlooked in seismic and computational studies. Two migratory mechanisms have been suggested: i A key dinocyst influencing our understanding of the late Miocene Lago-Mare phase is Galeacysta etrusca (Fig. 9b; see Bertini and Corradini, 1998; Popescu et al., 2009 and Grothe et al., 2018 for more insights). 5.1. Ostracods This species was originally described from sediments in the Mediterra­ nean (Corradini and Biffi, 1988), but has since been discovered in much older deposits in Paratethys (Magyar et al., 1999a, 1999b). The earliest recorded occurrence of Galeacysta etrusca is in sediments from the Pannonian Basin dated at ~8 Ma (Magyar et al., 1999a, 1999b). It subsequently dispersed throughout Paratethys at ~6 Ma and was pre­ sent in the Black Sea throughout the MSC interval (Grothe et al., 2014, 2018). Despite a Mediterranean-Eastern Paratethys connection that is thought to have been established at ~6.1 Ma (Krijgsman et al., 2010; Van Baak et al., 2016; Grothe et al., 2020), G. etrusca is not found in the Mediterranean during MSC Stages 1, 2 and 3.1 (5.97-5.42 Ma; Bertini, 2006, Londeix et al., 2007, Manzi et al., 2007, Iaccarino et al., 2008, Gennari et al., 2013) and is only reported in the uppermost part of the Lago-Mare phase, very close to the transition to the Pliocene (e.g. Ber­ tini, 2006; Londeix et al., 2007; Popescu et al., 2007; Iaccarino et al., 2008; Cosentino et al., 2012; Pellen et al., 2017). This implies that Galeacysta etrusca may have migrated from Paratethys into the Medi­ terranean after 5.42 Ma or that environmental conditions in the Medi­ terranean and in its marginal basins were only suitable for this species (and more generally the whole dinocysts Paratethyan contingent) to proliferate in the uppermost Messinian. Several authors report multiple occurrences of Galeacysta etrusca within the Zanclean (e.g. Clauzon et al., 2005; Londeix et al., 2007; Popescu et al., 2007, 2015; Do Couto et al., 2014; Clauzon et al., 2015), but these interpretations are based on the use of an alternative stratigraphic model for the MSC sections (Fig. 4b; see Grothe et al., 2018 for details). 1) the aerial dispersion of ostracods through the migration of aquatic birds (Benson, 1978; Caruso et al., 2020); this hypothesis was proposed because, in a Mediterranean concluded to have been desiccated, it was the only possible migration mechanism. 5.1. Ostracods 2) direct aqueous migration by the ostracods themselves (which are planktonic in the larval stage) through the establishment of similar paleoenvironmental conditions; by this mechanism, the dispersion of Paratethyan ostracod fauna from right across the Mediterranean re­ quires E-W intraconnection and a Mediterranean water-level high enough to reach the marginal basins (Gliozzi et al., 2007; Stoica et al., 2016; Sciuto et al., 2018; Sciuto and Baldanza, 2020). 2016; Sciuto et al., 2018; Sciuto and Baldanza, 2020). Finally, Carnevale et al. (2006a, 2006b, 2008, 2018) recognized the Paratethyan affinity of the Mediterranean Stage 3 species but, in view of their occurrence with in-situ species of marine fish, they suggested that Stage 3 ostracods descended from a Paratethyan stock that migrated into the Mediterranean well before the MSC and survived the extreme salinity conditions of Stage 1 and 2 in marginal, fresher water refugia. In this scenario the brackish water ostracod assemblages found in Stage 3 have no paleoecological significance for Stage 3 paleoenvironment (Carnevale et al., 2006a, 2006b, 2008, 2018). However, there are two, unflagged problems with this hypothesis: 1) the Mediterranean-Central Paratethys connection through the Trans-Tethyan gateway in Slovenia already closed in the early Tortonian (Kov´aˇc et al., 2007; Sant et al., 2017; Palcu et al., 2017); 2) No Paratethyan ostracod species have been found in the Mediterranean before the MSC (see Gliozzi et al., 2007). 5.5. Calcareous nannofossils and the C. acutus conundrum The mixing of foraminifera species with different ecological and salinity requirements and the widespread agreement that the brackish Paratethyan fauna are autochthonous (see subsection 5.1) has always complicated the interpretation of the origin of the foraminiferal as­ semblages. Among the benthic species, Ammonia taxa and the other benthic euryhaline taxa are generally considered autochthonous because they are typically well-preserved and their ecological and salinity requirements could be compatible with those of the Paratethyan ostracods. The habitat of these benthic foraminifera today in environments both influenced by and disconnected from the open ocean indicates that the Stage 3 sediments in which they occur were deposited in a shallow- water environment subject to salinity fluctuations (Caruso et al., 2020 and references therein), but they do not provide insights into the water provenance. By contrast, the poor preservation, older age and low di­ versity of the bathyal taxa strongly suggest that these species are reworked (Bassetti et al., 2006; Iaccarino et al., 2008; Caruso et al., 2020). Their mode of life is also incompatible with the shallower water elements of the faunal assemblage. The planktic species which went extinct before the MSC are also undoubtedly reworked (Iaccarino et al., 2008; Caruso et al., 2020). It is more challenging to discriminate be­ tween in situ and reworked specimens of the long range Neogene taxa. Most of them are considered to be reworked because of their scarcity, their occurrence with in-situ brackish organisms and their poor preser­ vation (e.g. Iaccarino et al., 2008; Caruso et al., 2020). A more complex controversy surrounds the long-range dwarf specimens (Fig. 9c) occur­ ring in onshore substage 3.1 (di Tetto Fm. in the Trave section; Iaccarino et al., 2008) and Lago-Mare sediments (Upper Mb. of the Nijar Feos Fm., Fortuin and Krijgsman, 2003; Aguirre and S´anchez-Almazo, 2004; By contrast, some authors (i.e. Popescu et al., 2007, 2015; Do Couto et al., 2014; Clauzon et al., 2015; Pellen et al., 2017) described the nannofossil assemblage the Lago-Mare LM Unit in Malaga, the Zorreras Member in Sorbas, the uppermost di Tetto/Colombacci Fm. in some Apenninic localities and offshore in the Albor´an Basin as having good preservation and showing no erratic fluctuations, all characteristics that led to their interpretation as autochthonous and to the conclusion that these sediments were deposited in a Mediterranean already replenished of Atlantic water (Fig. 4b). 5.5. Calcareous nannofossils and the C. acutus conundrum Calcareous nannofossils are the fossil remains of coccolithophores, single-celled marine algae which dwell in the eutrophic and photic zone of the ocean (e.g. Ziveri et al., 2004). The potential recognition of ma­ rine calcareous nannofossils in marginal Stage 3 deposits would there­ fore have implications for the Mediterranean base-level and the hydrological riddle of MSC Stage 3. However, like foraminifera and dinocysts, the in situ versus reworking issue also impacts the nannoflora. Planktic foraminifera are represented both by species whose last occurrence pre-dates the MSC (e.g. Praeorbulina spp., Paragloborotalia partimlabiata, P. siakensis, Neogloquadrina atlantica praeatlantica, Globi­ gerinoides subquadratus, Globorotalia saheliana, Globorotalia conomiozea, Acarinina sp., Hedbergella sp.) and by taxa with extended biostratigraphic ranges (e.g. Sphaeroidinellopsis seminulina, Turborotalita quinqueloba, Globorotalia miotumida, Globoturborotalita decoraperta, Neogloboquadrina acostaensis, Neogloboquadrina spp., Orbulina universa, Globigerinoides tri­ lobus, Globigerinoides obliquus, Globorotalia scitula, Globigerina bulloides, G. Mediterranea and G. humerosa; see Fig. 2b for references). l MSC Stage 3 is crossed by three important nannofossil bio-events astronomically calibrated in the ocean record: the top of Discoaster quinqueramus at 5.537 Ma, the base of Ceratolithus acutus at 5.36 Ma and the top of Triquetrorhabdulus rugosus at 5.231 Ma (Backman et al., 2012; Agnini et al., 2017). Most of the (few) studies that addressed the nan­ noflora component of Stage 3 deposits did not report taxa belonging to the biozones defined by these bio-events, but only taxa of Cenozoic and Cretaceous age, clearly physically reworked (e.g. Sites 132, 134, 653, 654A, 967A, 969B, Ryan et al., 1973; Hsü et al., 1978b; Müller et al., 1990; Castradori, 1998; Piedmont Basin, Trenkwalder et al., 2008; Violanti et al., 2009; Trave, Fonte dei Pulcini and Stingeti sections and Mondragone well in the Apennines, Cosentino et al., 2006, 2012, 2018; Iaccarino et al., 2008). An exception is the nannoflora observed in the uppermost Messinian sediments at Sites 978A, 975B and 967A (Levant Basin; Fig. 2b). Here, among the plethora of reworked and long-ranging Neogene taxa, Castradori (1998) reported the anomalous abundance of Sphenolithus spp (mostly Sphenolithus gr abies/moriformis). Although the assemblage points to the absence of a primary marine signature, the unlikely possibility that reworking and/or sorting lies behind the observed peak of Sphenolithus spp. led Castradori (1998) to conclude that at least one incursion of marine water occurred during the (up­ permost) Lago-Mare. 5.3. Diatoms This fauna is variably inter­ preted as: Bassetti et al., 2006; Sorbas Basin, Roveri et al., 2019a; Bajo Segura Basin, Corbí and Soria, 2016; Colombacci Fm. in Northern Apennines localities, Casati et al., 1976; Colalongo et al., 1976; Rio and Negri, 1988; Popescu et al., 2007; Cyprus, Rouchy et al., 2001) and in some offshore localities (e.g. Sites 124, 125, 129A, 132, 134, 372, 376, 653, 974B, 975, 978; Cita, 1973; Cita et al., 1978; Kastens et al., 1987; Cita et al., 1990; Iaccarino and Bossio, 1999). This fauna is variably inter­ preted as: Bassetti et al., 2006; Sorbas Basin, Roveri et al., 2019a; Bajo Segura Basin, Corbí and Soria, 2016; Colombacci Fm. in Northern Apennines localities, Casati et al., 1976; Colalongo et al., 1976; Rio and Negri, 1988; Popescu et al., 2007; Cyprus, Rouchy et al., 2001) and in some offshore localities (e.g. Sites 124, 125, 129A, 132, 134, 372, 376, 653, 974B, 975, 978; Cita, 1973; Cita et al., 1978; Kastens et al., 1987; Cita et al., 1990; Iaccarino and Bossio, 1999). This fauna is variably inter­ preted as: 5.4. Foraminifera A reasonably diverse benthic and planktic foraminiferal assemblage containing no age-diagnostic taxa have been found co-occurring with the brackish Paratethyan fauna in both the onshore and offshore record throughout the Mediterranean (Fig. 2b for localities and references). 1) reworked and size-sorted during transport, therefore lacking any paleoenvironmental significance (e.g. Kastens et al., 1987; Iaccarino and Bossio, 1999; Fortuin and Krijgsman, 2003; Bassetti et al., 2006); g g The benthic foraminifera assemblage is dominated by euryhaline representatives of the genus Ammonia, which today dwell in marginal marine (lagoons, estuaries, fjords and deltas) and lacustrine environ­ ments at depths < 50 m and tolerate salinities of up to 50‰ (Milker and Schmiedl, 2012; Consorti et al., 2020). Ammonia tepida and Ammonia beccari (Fig. 9c) are by far the most abundant species in both onshore (see Fig. 2b for localities and references) and offshore (e.g. Site 968A, Blanc-Valleron et al., 1998; Sites 375, 376, 965-968, Orszag-Sperber, 2006) localities, where they co-occur with ostracods belonging to Bio­ facies 1. Other commonly occurring benthic euryhaline taxa are Elphi­ dium sp., Cribroelphidium excavatum, Haynesina sp., Nonion sp., Quinqueloculina sp., Discorbis sp. and Trichohyalus sp., Brizalina dentel­ lata, Bulimina echinate and Bolivina spp. (Ryan et al., 1973; Hsü et al., 1978a, 1978b; Rouchy et al., 2001, 2003, 2007; Iaccarino et al., 2008; Caruso et al., 2020). These species are frequently mixed with poorly preserved and older in age bathyal species (e.g. Caruso et al., 2020). 2) in situ and indicating normal marine conditions (Aguirre and S´anchez-Almazo, 2004; Braga et al., 2006) or temporary Atlantic in­ cursions (Rouchy et al., 2001); 3) in situ and indicative of high-stress environments (Keller and Abramovich, 2009), such as restricted and/or diluted marine environ­ ments (Corbí and Soria, 2016; Corbí et al., 2016, 2020). However, the paleoecological significance of dwarfism in foraminifer tests is not well understood and, given its potential implications for the Lago-Mare environment, it needs to be explored in greater detail. 5.3. Diatoms Dinoflagellate cysts (dinocysts) are the fossil remains of unicellular protists that live in the upper water column of many water bodies (e.g. Zonneveld et al., 2013; Mudie et al., 2017). They can be used as pale­ oenvironmental indicators and for biostratigraphy, providing the ages of speciation and extinction events, as well as supplying evidence of age diagnostic dispersals of characteristic taxa/assemblages. Influxes of these microorganisms into a basin may occur as the result of intercon­ nection with another basin and dinocysts can therefore be useful in­ dicators of the open gateways between adjacent basins and the resultant changes in conditions (e.g. Grothe et al., 2018). In the case of the MSC, presence of in situ marine and/or Paratethys dinocyst assemblages in a marginal basin are likely to indicate the presence of Atlantic and/or Among the fresh-brackish organisms found in Stage 3 sediments are also species of diatoms. To date (and to our knowledge), there are no onshore studies that have ever looked for these organisms. By contrast, two samples from DSDP Site 124 in the Algero-Balearic Basin (Fig. 2b) revealed the presence of littoral planktonic forms accompanied by brackish water, and even freshwater, euryhaline, benthonic, and epiphytic species in considerable numbers (Haj´os, 1973). Diatoms of undisclosed paleoecological significance are also reported from the ~60 cm-thick mudstone bed between an anhydrite and halite bed found in the last core of Site 134 (Ryan et al., 1973). According to Haj´os (1973) and Ryan (2009), the diatoms found in these drill cores attest to an 25 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. extremely low salinity and a base level in the Balearic and Valencia basins below wave action. Further study of these indicative species and a wider distribution is required to apply this interpretation more generally. Bassetti et al., 2006; Sorbas Basin, Roveri et al., 2019a; Bajo Segura Basin, Corbí and Soria, 2016; Colombacci Fm. in Northern Apennines localities, Casati et al., 1976; Colalongo et al., 1976; Rio and Negri, 1988; Popescu et al., 2007; Cyprus, Rouchy et al., 2001) and in some offshore localities (e.g. Sites 124, 125, 129A, 132, 134, 372, 376, 653, 974B, 975, 978; Cita, 1973; Cita et al., 1978; Kastens et al., 1987; Cita et al., 1990; Iaccarino and Bossio, 1999). 5.5. Calcareous nannofossils and the C. acutus conundrum Since the good preservation of the otoliths suggests they did not suffer physical reworking (Carnevale et al., 2006a, 2006b, 2008, 2018; Grunert et al., 2016), predators foraging in open marine settings and migrating to marginal environments are proposed as a way out of the enigma (Carnevale et al., 2008, 2018; Grunert et al., 2016; Colombero et al., 2017). However, Carnevale et al. (2006a) also rule out that so well preserved otoliths may have suffered post-mortem transport and action of the digestive acids in the stomach of predators. Rare findings of Myctophidae from Ciabot Cagna (3 species), Cava Serredi (1 species), Capanne di Bronzo (1 species) and Podere Torricella (6 species) (Carnevale et al., 2018) are all from sections where the host sediments have not been studied in sufficient detail to be clear about the in situ or reworked nature of the fossil assemblage. This lack of sedimentological uncertainty also extends to the stratigraphic position of many samples, because a stratigraphic log is provided for only a few sections (i.e. Ca’ Ciuccio, Cava Serredi and Moncucco; Carnevale et al., 2006a, 2006b). What this stratigraphic information suggests is that euryhaline fish taxa are widespread throughout substage 3.2, whereas strictly Myctophidae, which are an oceanic, marine stenohaline species, only occur very close to the base of the Pliocene, plausibly corresponding to the uppermost lithological cycle in substage 3.2 (~5.35-5.33 Ma; Carnevale et al., 2018). i Such findings (especially that of C. acutus) are in sharp disagreement with most of the existing literature and have resulted in an important debate amongst the MSC community (e.g. Popescu et al., 2007, 2008 vs Roveri et al., 2008c and Stoica et al., 2016 vs Popescu et al., 2017), not only for their paleoenvironmental implications (i.e. presence of Atlantic water in the Mediterranean), but also for the chronostratigraphic re­ percussions (Fig. 4b). The chronostratigraphic value of C. acutus lies in its short temporal distribution straddling the M/P boundary (astro­ chronologically calibrated at 5.332 Ma; Van Couvering et al., 2000; Lourens et al., 2004). However, the corresponding biozone is established in oceanic areas (Zone CNPL1: 5.36-5.05 Ma; Backman et al., 2012; Agnini et al., 2017) and is considered not applicable to the Mediterra­ nean region during the MSC due to the harsh physicochemical condi­ tions that are unsuitable for marine biota (Di Stefano and Sturiale, 2010). 5.5. Calcareous nannofossils and the C. acutus conundrum In addition, these authors reported the low abundance, but continuous presence of the biostratigraphic markers for the Zanclean Triquetrorhabdulus rugosus and Ceratolithus acutus (Fig. 9d) below the formally defined Miocene/Pliocene boundary (Van Couvering et al., 2000) in several onshore and offshore Mediterranean (as wells as Paratethyan) localities (see Popescu et al., 2017 for details and a 26 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. complete list of finding locations). substage 3.2 in a few marginal sections on the Italian peninsula (Ciabot Cagna in the Piedmont Basin; Cava Serredi and Podere Torricella in Tuscany; Capanne di Bronzo, La Vicenne and Ca’ Ciuccio in thrust-top basins of the Northern and Central Apennines). The Lago-Mare fish re­ mains mainly comprise otoliths of both euryhaline and stenohaline taxa indicative of marine, brackish, and freshwater habitats (Carnevale et al., 2018). Three articulated skeletons of the euryhaline marine taxa Mugil cf. cephalus (Fig. 9e), the marine Indo-Pacific species Spratelloides gracilis and of Gobius sp. have been identified at Cava Serredi in a horizon < 1 m below the Miocene/Pliocene boundary (Carnevale et al., 2006b). The dominant stenohaline families in these assemblages are Gobiidae, a family of demersal fish occupying shallow-water marine, brackish and freshwater environments, and Myctophidae, which are marine meso­ pelagic fish that live below 200 m depth during the day, but feed at night in surface waters. A recent review of the Tortonian-Zanclean Gobiidae of the Mediterranean (Schwarzhans et al., 2020) showed that the otoliths of this family, described by Carnevale et al. (2006a, 2008, 2018) and Grunert et al. (2016) as belonging to marine Atlantic species, instead belong to brackish and freshwater species of Paratethyan affinity inhabiting sheltered prodelta environments. In fact, no normal marine demersal taxa were recognized in these assemblages by Schwarzhans et al. (2020). As for the Myctophidae, the vast majority of the taxa belonging to this family were recovered in Moncucco and Verduno from alluvial plain silty mudstones along with terrestrial mammals (Dela Pierre et al., 2011; Colombero et al., 2017 and references therein), pointing to a physically reworked origin. When 87Sr/86Sr isotope ratios are measured (Carnevale et al., 2008; Grunert et al., 2016), the resulting Sr-based age of the otoliths is > 7 Ma, therefore further arguing against their in-situ origin. 5.6. Fish Fossil fish remains provide information about salinity and depth and have been used to contradict the brackish nature of the Lago-Mare de­ posits by Carnevale et al. (2006a, 2006b, 2008, 2018) and Grunert et al. (2016). Euryhaline fish species inhabit marine to brackish environments and dominate settings with strong salinity variations while stenohaline fish have specific salinity requirements (marine, brackish, or freshwater) and cannot survive under different conditions. Demersal fish (i.e. those living in or immediately above the sea floor) have specific depth re­ quirements, whereas pelagic fish occupy the water column within a wide range of depth variable from species to species. Fossil fish remains are found either as articulated or disarticulated skeletal parts, including teeth and otoliths, which are identified to the species level. Articulated fish skeletons typically indicate autochthonous deposition because of the difficulty in reworking and transporting intact skeletons. Otoliths and fish teeth are much more likely to be transported. i Otoliths and rare articulated skeletons (Fig. 9e) of marine and Par­ atethyan species have been reported from Stage 3 deposits, but commonly huge volumes of sediment are required to find even quite small numbers of these fossils (e.g. 20 tons from Moncucco, 6 tons from Cava Serredi, 700 kg from Capanne di Bronzo; Schwarzhans et al., 2020), much more than what is expected for normal marine deposits (i. e. < 30 kg; Agiadi et al., 2017; Karakitsios et al., 2017b). 5.5. Calcareous nannofossils and the C. acutus conundrum The interpretation of these nannofossil assemblages in the westernmost areas of the Mediterranean has been countered with several observations: (1) the observation of these age-diagnostic taxa is often not replicated by other studies (e.g. Roveri et al., 2008a; Van Baak et al., 2015; Krijgsman et al., 2020b); (2) Ceratolithus acutus is very rare also in fully marine open-ocean sediments (e.g. Di Stefano and Sturiale, 2010); (3) despite being rare in the late Messinian Mediterranean, this species has never been documented together with other long-range taxa, generally predominant in the assemblage, in Stage 3 deposits (see dis­ cussion in Krijgsman et al., 2020b). Recently, Golovina et al. (2019) showed that the morphology and size of C. acutus overlaps with the shape and dimensions of destroyed ascidian spicules (i.e. calcareous elements produced by benthic tunicates; Fig. 9d), providing an expla­ nation for erroneous identification of C. acutus in the Black Sea Basin (Golovina et al., 2019) and perhaps in the western Mediterranean Lago- Mare sediments as well. 5.7. Summary of the Stage 3 paleontological record In the inset is shown the Mediterranean Sr record for the entire MSC as well as the time-equivalent Eastern Paratethys record (modified after Andreetto et al., 2021). (b) Plot of δ34SSO4 and δ18OSO4 in Stage 3 gypsum and anhydrite beds from onshore and offshore localities (see Supplementary material 1 and subsection 6.2 for references). No measures are available from the marginal basins, where gypsum did not deposit during Stage 3. The dark blue and black rectangles represent the sulfate isotopic composition of the Global Messinian ocean and Stage 1 (PLG) evaporites, respectively. The light blue area represents the sulfate isotopic composition of mixtures of Messinian marine waters with non-marine sources. The red area represents the isotopic composition of the residual sulfate ion in a basin where marine Messinian sulfate is consumed by microbial sulfate reduction to produce H2S. The arrow represents the isotope trajectory of dissolved sulfate resulting from the mixing of residual 34S- enriched sulfate produced by MSR and 34S-depleted sulfate produced by H2S oxidation. All the published δ34SSO4 and δ18OSO4 values are provided corrected with the fractionation factors δ34S=+1.65‰ and δ18O=+3.5‰ to smooth the isotopic fractionation effects experienced by dissolved sulfate and to reason on values repro­ ducing the isotopic composition at the time of gypsum precipitation. (c) δD isotopes of C29 and C31n-alkanes and C37 and C38 long chain alkenones recorded in the Stage 3 gypsums and marls of the Eraclea Minoa section (modified from Vasiliev et al., 2017). Blue lines indicate the values recorded in the present day lacustrine settings for the n-alkanes (Sachse et al., 2006) and in the alkenones from the Mediterranean in the recent times (Van der Meer et al., 2007). Error bars indicate standard errors of the mean. Messinian Mediterranean brackish species with the same species that were simultaneously dwelling in the Eastern Paratethyan basins (Dacian, Euxinic and Caspian) and in the North Aegean. Since these organisms were not present in the Mediterranean at any time before the MSC, they are considered, with a broad consensus, as in situ. This conclusion is further corroborated by the mixing of adult and juvenile forms in the ostracod assemblages and by the good preservation of the specimens, which do not show typical evidence of physical reworking like abrasion, dissolution, or fragmentation. Still problematic is the time of their arrival in the Mediterranean and their likelihood as biostrati­ graphic tool. 6.1. Strontium isotope ratios (87Sr/86Sr) The available strontium isotope data for Stage 3 (Fig. 10a; Supple­ mentary material 1) derive from measurements on both Ca-bearing fossils (ostracod valves, mollusk shells, fish otoliths; Fig. 9a) and min­ erals (calcite and gypsum), where Sr2+ dissolved in an aqueous solution substitutes Ca atoms due to their similar ionic radius (e.g. Hajj et al., 2017). Here we screen the available dataset and discuss only results that (1) reflect the original primary isotopic signal, i.e. the isotopic signal of the fluid at time of shell calcification or mineral precipitation, and (2) for which timing of mineral precipitation can be constrained. This screening excludes bulk carbonate samples (e.g. Colombacci limestones; Bassetti et al., 2004), which contain carbonate compounds of various and/or unknown provenance, measurements from mollusk shells and otoliths (e.g. Carnevale et al., 2008; Grunert et al., 2016; Roveri et al., 2019a), because they are made of mineral phases easily altered during diagenesis (e.g. aragonite; Marcano et al., 2015), and data coming from reworked material (e.g. all reworked gypsum or transported forami­ nifera). 87Sr/86Sr isotope ratios have also been measured by Müller and Mueller (1991) and Roveri et al. (2014b) on the halite beds recovered at Sites 134, 374 and 376 (Ryan et al., 1973; Hsü et al., 1978b). Although they provide interesting interpretative aspects, we do not consider these Sr measurements because the position of Sr in the crystal lattice of halite is unknown and the removal of all contaminants, that is not a straight­ forward procedure (see Meilijson et al., 2019), is not clear it was ach­ ieved by Müller and Mueller (1991) and Roveri et al. (2014b). As a matter of fact, there is no consistency between data generated from roughly the same interval in Core 134 by Müller and Mueller (1991) (0.708968) and Roveri et al. (2014b) (0.708800-0.708896). Added to this is the uncertainty over the provenance of halite in Sites 134 and 374 (see subsections 4.2 and 4.6.1), which violates both criteria mentioned above. Marine assemblages are composed by foraminifera, nannofossils, dinocysts and calcareous nannofossils. Their reworked or in situ nature is in many cases contested but critical for paleoenvironmental interpre­ tation. The picture that emerges from our review is that an open marine signature is questionable in the foraminifera, nannofossils, dinocyst and fish records, as well as in other biotic groups (e.g. 5.7. Summary of the Stage 3 paleontological record The aquatic fossil record of MSC Stage 3 indicates that substage 3.1 in onshore sections is mostly barren, while diverse assemblages char­ acterize substage 3.2 deposits. By contrast, the deep record as a whole contains relatively few, low diversity assemblages. This might be as a consequence either of the limited sample locations recovered from the offshore areas (see Fig. 2b) or because the environmental conditions in the intermediate-deep basins were less favorable for sustaining the life forms typical of the onshore domain. Nevertheless, the assemblages that are found in both marginal and deep locations comprise mixed brackish and marine species. Substage 3.1 sediments contain articulated skeletons (Fig. 9e) of the marine fish species Lampanyctus licatae and Maurolicus muelleri, and the shallow water, euryhaline species Aphanius crassicaudus in the Lower Feos Member in the Nijar Basin (de la Chapelle and Gaudant, 1987) and the marls of the first UG cycle in the Polemi Basin (Manzi et al., 2016a; Fig. 3a). Cava Serredi (Tuscany), Verduno and Moncucco (Piedmont) are the only other localities in which fish remains (only otoliths) in (claimed) substage 3.1 sediments are known (Carnevale et al., 2006a, 2008, 2018; Grunert et al., 2016). Brackish species are mostly represented by ostracods and dinocysts (and mollusks here not addressed because poorly studied; see Esu, 2007 and Guerra-Merch´an et al., 2010). Prominent is the affinity of these late The more diverse and abundant ichtyofaunal record occurs in 27 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. (caption (caption on next page) 28 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Fig. 10. Isotopic record of MSC Stage 3 for the Mediterranean Basin. (a) Compilation of MSC Stage 3 87Sr/86Sr isotope data sourced from ostracod valves and gypsum crystals (see Supplementary material 1 and subsection 6.1 for references). Data are plotted with the global 87Sr/86Sr seawater curve (McArthur et al., 2012). Error bars indicate analytical error, which is so small in some cases that no error bars are visible at this scale. To not complicate the figure, horizontal error bars have not been added for the sections/cores unprovided of a chronostratigraphic framework and for which age uncertainties are present (i.e. all but Nijar and Vera basins, Eraclea Minoa and onshore Cyprus; see Fig. 3). Note that none of the 87Sr/86Sr isotope ratios but one from Nijar plot on the ocean curve. 6.1. Strontium isotope ratios (87Sr/86Sr) corals, echinoids and mammals) here not tackled (and for which we refer the reader to Dominici et al., 2018 and Carnevale et al., 2019). All marine represen­ tatives of the above mentioned categories were reintroduced into the Mediterranean only at the beginning of the Pliocene and at the expense of the Paratethys species that, instead, disappeared. Collectively, these observations lead us to conclude that the marine model as conceived by Carnevale et al. (2006a, 2006b, 2008, 2018) and Grunert et al. (2016) has no foundation and therefore will not be further discussed. 5.7. Summary of the Stage 3 paleontological record From our review it seems more likely that truly Para­ tethyan species of ostracods entered the Mediterranean already during substage 3.1, when they colonized intermediate-deep settings, while they entered the marginal basins at different times during substage 3.2. As for dinocysts, characteristic is their occurrence only in the uppermost Messinian. However, it must be noted that samples from the substage 3.1 interval are rarely processed for dinocysts, especially in age model- equipped sections (Fig. 3a). The route followed by the Paratethyan im­ migrants is equally contested and important for paleoenvironmental and paleohydrological interpretations. In view of a desiccated Mediterra­ nean, their migration can only have taken place passively by means of aquatic migratory birds. Conversely, the homogeneity of the ostracod assemblages throughout the Mediterranean marginal basins is more indicative of the presence of a water body fed by Eastern Paratethys and connecting all Mediterranean subbasins, therefore implying relatively high water-level conditions (at least at times when ostracod-bearing sediments deposited; see Andreetto et al., 2021). interpreting geochemical signals that respond to the presence or absence of an exchange with a chemically-unique water body. Four main geochemical proxies have been applied so far to MSC Stage 3 sedi­ mentary and paleontological records. These includes both radiogenic (Sr isotope ratios) and stable isotopes (sulfate and oxygen) measured on fossils and minerals and hydrogen isotopes on molecular biomarkers. This section summarizes the dataset available for geochemical proxies (Fig. 10; Supplementary material 1) and its interpretation(s) for MSC Stage 3. 6. The geochemical perspective The one published Sr isotope value for ostracods found within one of the marl interbeds at Eraclea Minoa also has a lower value outside the typical Sicily gypsum range (Grossi et al., 2015). This suggests that a different hydrological regime may also have characterised precession minima stages of the precessional cycle. The Sr isotope dataset for the Lago-Mare phase includes the lowest values measured on MSC sediments (~0.7085 from between gypsum VI and VII at Eraclea Minoa; Fig. 3a; Grossi et al., 2015) and the widest range of ratios spanning from 0.7085 to 0.7091, which is above coeval oceanic values (Fig. 10a). Again, the conspicuously high Sr isotope values in substage 3.2 come from two areas, the marginal basins of southern Spain (Andreetto et al., 2021 and references therein; Figs. 5a-c) and the intermediate Polemi Basin on Cyprus (McCulloch and De Deckker, 1989). The lower values are drawn from right across the intermediate-deep Mediterranean (Algero-Balearic, Sicily, Levant; Fig. 2a) and are therefore more likely to represent a Mediterranean-wide Sr isotope signal. The sulfate isotopic values reported by Longinelli (1979) and Pierre (1982) from the Upper Gypsum of Eraclea Minoa (Caltanissetta Basin, Sicily) are considerably more scattered than those from a recent study by García-Veigas et al., 2018; Fig. 10b). Such discrepancies are probably a consequence of different sample selection: García-Veigas et al. (2018) analyzed only pristine whitish selenite and balatino samples, while Longinelli (1979) and Pierre (1982) analyzed all types of gypsum- bearing samples such as “gypsiferous marl” and gypsum laminae inter­ calated in carbonate or diatomaceous intervals. These less pristine samples probably contain high quantities of 34S-depleted solid sulfides or diagenetic gypsum formed by oxidation of sulfides (see Liu et al., 2017 for more details on this process) and are therefore unlikely to be representative of the primary gypsum facies. Once these data are excluded, the Eraclea Minoa sulfate values (δ18OSO4 from 12.4 to 14.6‰ and δ34SSO4 from 21.0 to 22.3‰) suggest a marine origin of the sulfate and stable redox conditions during gypsum deposition (Fig 9.b; García- Veigas et al., 2018). Interestingly, the Eraclea Minoa sulfate values are in compliance with the isotopic values (δ18OSO4=15.2 to 16.8‰; δ34SSO4= 20.4 to 21.9‰) measured by Pierre (1982) in the Polemi Basin (Cyprus). g New Sr isotope data from Eastern Paratethys (i.e. Dacian and Caspian basins; Fig. 2b) are now available for the interval corresponding to MSC Stage 3 (inset Fig. 6. The geochemical perspective By contrast, the data display δ18OSO4 isotopic values that deviate substantially from marine δ18OSO4 values towards higher values (Fig. 10). This is consistent with the influence of sulfate produced by reoxidation of reduced sulfur compounds generated by microbial sulfate reduction (MSR; Kaplan and Rittenberg, 1964; Brunner and Bernasconi, 2005; Sim et al., 2011; Leavitt et al., 2013). The microbial use of SO4 2-leads to an equilibration of δ18OSO4 with ambient water oxygen, whereas the δ34SSO4 returns towards its initial value as a higher fraction of sulfide produced by MSR is re-oxidated. This mechanism has been suggested for Sites in the Algero-Balearic, Tyrrhenian and Ionian basins and offshore Cyprus (Pierre, 1974; Pierre and Fontes, 1978; Pierre and Rouchy, 1990). Although some authors have suggested that partial equilibration of sulfate oxygen toward δ18OH2O values of the basin enriched in heavy oxygen isotopes by evaporation have led to an in­ crease in δ18OSO4 values without significant changes in δ34SSO4 (Fontes et al., 1973; Pierre, 1974; Ricchiuto and McKenzie, 1978), this hy­ pothesis seems highly unlikely as the abiotic equilibration between sulfate and water oxygen take about 20 Myr at normal marine pH (Lloyd, 1968; Longinelli and Craig, 1967; Turchyn et al., 2006). More­ over, the microbial sulfate reduction process is supported by the pres­ ence of pyrite at Sites 132, 654A and 968 (Pierre, 1982; Pierre and Rouchy, 1990; Blanc-Valleron et al., 1998) and the existence of fila­ ments of possible microbial origin at Site 654A (Pierre and Rouchy, 1990). Most of the data characterizing substage 3.1 (Fig. 10a) are from the Eraclea Minoa gypsum (Fig. 5h). These data define a narrow range of Sr isotope ratios between 0.708747 and 0.708793 (García-Veigas et al., 2018). Similar values were reported from both Eraclea Minoa and the nearby Siculiana Marina section (0.708710-0.708760; Keogh and But­ ler, 1999; Fig. 5i). The dominance of Sicily samples gives the appearance of a consistent Sr isotope signal for gypsum beds. However, data points from elsewhere (Cyprus, Manzi et al., 2016a; DSDPs 122, 371 and 372 in the Algero-Balearic Basin, ODPs 652, 653 and 654 in the Tyrrhenian Basin, DSDP 374 in the Ionian Basin; Müller et al., 1990; Müller and Mueller, 1991; Roveri et al., 2014b) display a wider range (from ~0.7087 to 0.708847; Fig. 10a) that may indicate a different hydro­ logical regime for each basin (e.g. Müller et al., 1990; Müller and Mueller, 1991; Ryan, 2009). 6. The geochemical perspective The general trend of the Mediterranean 87Sr/86Sr isotope ratio dur­ ing the MSC deviates from the ocean curve towards the less radiogenic values of the major peri-Mediterranean rivers and Paratethys and returns abruptly to oceanic values at the Miocene/Pliocene boundary (Fig. 10a inset). This trend is regarded to reflect the progressive re­ striction of Mediterranean-Atlantic exchange and the relative increase in Variations in the water sources draining into the Mediterranean are expected to be reflected also in (geo)chemical properties of the paleo­ depositional environments. Important information about the nature of the connectivity framework of the Mediterranean can be gained by 29 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. the proportion of non-marine source waters (Topper et al., 2011; Roveri et al., 2014a). At first glance it seems that each MSC Stage was char­ acterized by a well-defined range of Sr ratios (Fig. 10a inset), an observation that led Roveri et al. (2014b) to attribute a chronostrati­ graphic value to MSC 87Sr/86Sr ratios. A closer look, however, shows that MSC substages are anything but homogeneous with respect to 87Sr/86Sr ratios. At least in the marginal basins, local lithological dif­ ferences in the catchments (each lithology carries a unique 87Sr/86Sr fingerprint; see subsection 8.1.1) explain the different Sr isotopic com­ positions from basin to basin (see Schildgen et al., 2014; Modestou et al., 2017; Andreetto et al., 2021), therefore arguing against the use of 87Sr/86Sr ratios for chronostratigraphic purposes in the MSC record. δ34S (+1.65‰) at earth surface temperatures (Thode and Monster, 1965; Lloyd, 1968; Warren, 2016), δ18O and δ34S isotopic values measured in gypsum should be corrected with the above mentioned fractionation factors in order to reconstruct the sulfate isotopic composition of the basin waters at the time of gypsum formation. gyp The deep Mediterranean samples exhibit a wide range of δ34SSO4, but the majority of samples display δ34SSO4 values between 18 and 22‰, strongly indicative of a marine origin of the sulfate forming the gypsum (Fig. 10b; Fontes et al., 1973; Pierre, 1974, 1982; Pierre and Fontes, 1978; Pierre and Rouchy, 1990; Blanc-Valleron et al., 1998). The δ34SSO4 values lower than marine sulfate in the dataset are generally considered to represent a greater influence of continental sulfate input to the basin (Fig. 10b; Pierre, 1974; Pierre and Fontes, 1978; Pierre and Rouchy, 1990). 6. The geochemical perspective 10a). The 87Sr/86Sr ratios of the Dacian Basin (0.708865-0.708982; Vasiliev et al., 2010; Grothe, 2016) are slightly lower than coeval ocean water (0.709020), but much higher than coeval Mediterranean values. However, the Dacian Basin is regarded as highly restricted from the Mediterranean throughout the MSC (Vasiliev et al., 2010). By contrast, the Caspian has very low values (0.708402 to 0.708473, Grothe et al., 2020) which are thought to reflect both the very low Sr isotope ratio of the Volga river (0.708020; Vasiliev et al., 2010 and references therein) and some input from the Mediterranean (Grothe et al., 2020). et al., 2020). 6.5. Summary of the Stage 3 geochemical dataset The variety of paleoenvironmental and connectivity proxies applied to MSC Stage 3 record provide valuable insights into the hydrological conditions during Stage 3. The more outstanding results from all dis­ cussed proxies are that: Both δDC29n-alkane and δDalkenones results (Fig. 10c) suggested that conditions in Sicily were significantly dryer than today, with highly enriched values of δDC29n-alkanes (up to −125‰). The δDalkenones varied between values suggesting evaporative conditions (−125‰) and values typical for present-day δDalkenones in the Mediterranean (−203‰) (Vasiliev et al., 2017). 1) Paleodepositional subaqueous environments where gypsum was precipitating and ostracods and biomarker-producers were thriving were strongly dominated by non-oceanic inputs; No time-equivalent biomarker data from the open ocean settings are currently available. Instead, Vasiliev et al. (2017) compared their Mediterranean data with data from the Black Sea (DSDP 42B Hole 380 and Taman peninsula; Vasiliev et al., 2013, 2015). The Upper Gypsum δDn-alkanes were more enriched when compared to their time equivalent deposits of the DSDP 42B 380 borehole of the Black Sea (−180‰). This probably reflects the more intracontinental position of the Black Sea which commonly translates into more depleted values for δDprecipitation used by the vegetation, resulting in more depleted δDC29n -alkanes. However, there is a 30 to 40‰ enrichment relative to present in the δDn- alkanes (i.e. δDprecipitation) in both Mediterranean and Paratethys domains, indicating concurrent changes in both areas during the latest phase of the MSC. 2) an indisputable marine signal is absent and only regained above the M/P boundary. Sulfate and oxygen isotopes are currently difficult to use for water provenance reconstruction because the non-marine sources (local and major rivers and Eastern Paratethys) that are likely to be of influence lack distinctive isotopic signatures and, especially for oxygen, respond to a combination of controls (e.g. temperature, rainfall, evaporation) with local variability. δ34SSO4 are claimed by several authors to be an evidence of the presence of an Atlantic inflow (δ34SSO4=22‰; Turchyn and Schrag, 2004) in a Mediterranean strongly affected by non-marine waters (Manzi et al., 2009, 2016a; García-Veigas et al., 2018 among others). However, the same values can be obtained by means of the recycling of PLG deposits (~23‰; Lu et al., 2001; Lugli et al., 2010; García-Veigas et al., 2018). Both the Mediterranean and Paratethyan samples contain δDalkenones with low values (~-200‰) (Fig. 10c) leading Vasiliev et al. 6.5. Summary of the Stage 3 geochemical dataset (2017) to suggest that either the surface water from the Upper Gypsum was derived from the Black Sea, or that the Mediterranean and Paratethys were exchanging surface water during gypsum precipitation. Similarity between the relative contribution of the C37, C38 and C39 alkenones at Eraclea Minoa and one of the Black Sea samples may suggest common alkenone producers for the two areas, again supporting the idea of a Mediterranean-Paratethys connection during Stage 3 (Vasiliev et al., 2017). i g , Similarities between the δDalkenones of the Upper Gypsum at Eraclea Minoa and coeval Black Sea sediments and δDn-alkanes similar to present- day marine settings, suggest that Eastern Paratethys and the Atlantic were simultaneously contributing to the Mediterranean hydrological budget. 87Sr/86Sr isotope ratios are a useful water-mass tracer because each water body carries a unique Sr isotope fingerprint (see subsection 8.1.1). Our plotting of Stage 3 87Sr/86Sr isotope values (Fig. 10a) high­ lights the large geographical variability of the values and the sharp di­ vision between Sr isotope ratios measured in marginal basins versus those in intermediate-deep water locations. This is only noticeable in substage 3.2, since no (or not enough) material suitable for Sr analysis is present in substage 3.1 deposits from the marginal basins. Some authors see this variability as an indication of isolated subbasins with unique hydrological conditions driven by their catchment rivers (e.g. Müller et al., 1990; Müller and Mueller, 1991; Ryan, 2009). If some degree of connection was present, it involved only neighbouring basins (e.g. Tyrrhenian subbasins; Müller et al., 1990; Müller and Mueller, 1991). A recent comparison of the Sr isotope record of the Spanish marginal ba­ sins of Sorbas, Nijar and Vera with the Sr isotope ratios likely to have typified the local riverine sources demonstrated that a local sources- mixed signal expected from an endorheic lake in that location is ab­ sent. In this instance mixing of intrabasinal water sources with a non- marine Mediterranean water mass is used to explain the measured values (Andreetto et al., 2021). If this explanation is more widely applicable, then it may result in a re-interpretation of the spread of Sr isotope data from the latest Messinian interval. 6.4. Oxygen isotopes Oxygen stable isotope data (δ18O) are available from bulk samples (Rouchy et al., 2001, 2003, 2007; Pierre et al., 2006; Cosentino et al., 2012), gypsum (Pierre and Fontes, 1978; Ricchiuto and McKenzie, 1978; Lugli et al., 2007), mollusk shells (Carnevale et al., 2008; Grunert et al., 2016) and ostracod valves (Cosentino et al., 2012; Grossi et al., 2015). Oxygen stable isotope data (δ18O) are available from bulk samples (Rouchy et al., 2001, 2003, 2007; Pierre et al., 2006; Cosentino et al., 2012), gypsum (Pierre and Fontes, 1978; Ricchiuto and McKenzie, 1978; Lugli et al., 2007), mollusk shells (Carnevale et al., 2008; Grunert et al., 2016) and ostracod valves (Cosentino et al., 2012; Grossi et al., 2015). For all the sub-basins for which there is latest Messinian data (e.g. Sites 974 and 975; Eraclea Minoa section, Sicily; Aghios Stefanos sec­ tion, Corfu; Kalamaki section, Zakynthos; Pissouri Basin, Cyprus; Rou­ chy et al., 2001, Pierre et al., 2006), each has its own range of oxygen isotopic compositions and its own degree of variability. Values from above the Miocene/Pliocene boundary regain seawater values of 0.3 to 1 ‰ (e.g. Pierre et al., 2006). For all the sub-basins for which there is latest Messinian data (e.g. Sites 974 and 975; Eraclea Minoa section, Sicily; Aghios Stefanos sec­ tion, Corfu; Kalamaki section, Zakynthos; Pissouri Basin, Cyprus; Rou­ chy et al., 2001, Pierre et al., 2006), each has its own range of oxygen isotopic compositions and its own degree of variability. Values from above the Miocene/Pliocene boundary regain seawater values of 0.3 to 1 ‰ (e.g. Pierre et al., 2006). To conclude, geochemical proxies have great potential to test the different scenarios, but data are currently too numerically and geographically limited to be robust. In marginal marine settings and lakes, the controls over δ18O are poorly constrained as oxygen does not respond simply to the freshwater flux, but to a combination of variables such as temperature, rainfall and evaporation (e.g. Placzek et al., 2011). Freshwater input may contribute to the signal, resulting in δ18O more negative than seawater (0.3‰ to 0.8‰ SMOW; Dettman et al., 2004), but under prevailing evaporating conditions it is likely that the δ18O will be primarily influenced by 6.5. Summary of the Stage 3 geochemical dataset A final speculative insight from this biomarker dataset is that the relative contribution of alkenones found in the Upper Gypsum of Eraclea Minoa is strikingly similar to present-day open marine samples, even though Emiliania huxleyi, the principal ocean alkenone producer today, did not exist in the late Miocene. Vasiliev et al. (2017) suggested that this could imply the existence of a connection to the open ocean during Upper Gypsum deposition in Sicily (i.e. throughout Stage 3; Fig. 3a). 6.3. Hydrogen isotopes on molecular biomarkers Sulfur isotopic investigations have been carried out only on sulfate minerals (gypsum and more rarely anhydrite) of the MSC Stage 3 de­ posits with samples drawn from both onshore intermediate sequences (Caltanissetta Basin and Cypriot basins) and deep basinal records (Sites 122, 124, 125A, 132, 134, 372, 374, 375, 376, 652, 653, 654, 968, 969, 970; Fig. 10b; Fontes et al., 1973; Pierre, 1974, 1982; Pierre and Fontes, 1978; Ricchiuto and McKenzie, 1978; Pierre and Rouchy, 1990; Blanc- Valleron et al., 1998). Because the incorporation of dissolved sulfate into gypsum produces a nearly constant fractionation of δ18O (+3.5‰) and From the point of view of the application of organic geochemistry proxies, the Miocene Mediterranean Basin received little attention so far, with biomarker-based proxies that have been mostly applied to (a limited number of) pre-MSC sequences (Tzanova et al., 2015; Herbert et al., 2016; Mayser et al., 2017; Natalicchio et al., 2017, 2019; Vasiliev et al., 2019) and pre-Stage 3 sedimentary records (Lower Evaporites on Sicily, Andersen et al., 2001; Vena del Gesso Basin, Sinninghe Damst´e 30 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. evaporation, leading to δ18O more positive than seawater (e.g. Dettman et al., 2004), making any data very difficult to interpret. Furthermore, the lack of a unique δ18O signature for each water source makes oxygen isotopes a difficult tracer proxy to use. et al., 1995 and Vasiliev et al., 2017; Levant Basin, Meilijson et al., 2019). To date, only one study analyzed Stage 3 samples (Vasiliev et al., 2017). This study used compound specific hydrogen isotope (δD) ana­ lyses, measured on both terrestrial (long chain C29 and C31n-alkanes; Sachse et al., 2006) and aquatic (alkenones; Englebrecht and Sachs, 2005) biomarkers from the gypsum beds of the Upper Gypsum at Eraclea Minoa to reconstruct the hydrological cycle during gypsum precipitation. 7. Paleoenvironmental scenarios for freshening the salt giant: desiccated versus full Mediterranean The riddle of the Mediterranean environmental and hydrological conditions during Stage 3 is a highly debated topic and it is key to un­ derstanding the means by which open marine conditions were restored 31 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. interpreted as subaerial in origin; (3) the clastic fans at the outlet of the valleys onlapped by Stage 3 deposits and interpreted as fluvial accu­ mulations (e.g. Lofi et al., 2005; Maillard et al., 2006; Pellen et al., 2019). A number of studies have tried to quantify the magnitude of the sea-level fall by compensating for the isostatic vertical motion since the Messinian to obtain the original depth of the erosional features and Messinian deposits. However, this depends on the assumptions about when the drawdown occurred relative to the halite precipitation: before (e.g. Cartwright and Jackson, 2008; Bache et al., 2009, 2012), during (e. g. Ryan, 2008, 2009) or after (e.g. Ryan, 1978; Bertoni and Cartwright, 2007; Lofi et al., 2011a, 2011b). How shallow the Mediterranean became during Stage 2 is also a matter of disagreement. Estimates in the Western Mediterranean vary from a maximum drawdown of 2500 m (Ryan, 1976) to 1000-1500 m (Bache et al., 2012) in the Gulf of Lion, 800-1200 m in the Balearic promontory (Mas et al., 2018b) and 400 m in the Ebro delta region (Frey-Martinez et al., 2004). A later backstripping analysis of this delta yielded a drawdown of ~1300 m (Urgeles et al., 2011). East of the Sicily sill, backstripping studies estimated base-level drops of 1800-2000 m in the Ionian basin (Micallef et al., 2018, 2019; Camerlenghi et al., 2019; Spatola et al., 2020), 800-900 m in the Adri­ atic foredeep and Po plain (Ghielmi et al., 2013; Amadori et al., 2018), 800-1300 m (Ben-Gal et al., 2005), 600 (Druckman et al., 1995) and 800 m (Cartwright and Jackson, 2008) in the Levant Basin. i at the base of the Zanclean and on the potential impact that the Atlantic- Mediterranean re-connection had on the Atlantic and global climate (Flecker et al., 2015; Capella et al., 2019). In this chapter, the paleo­ environmental scenarios, in terms of base-level position (desiccated or full Mediterranean) and hydrological configuration (connections to the Atlantic and/or Paratethys), proposed for the Mediterranean during Stage 3 are described, as well as the different timings of the reflooding (instantaneous, gradual, step-like increments). 7.1. Stage 2 (5.59-5.55 Ma): formation of the Mediterranean salt giant Numerical modelling based on hydrological budget calculations shows that in order to reach salinity levels compatible with halite saturation and to accumulate the substantial thicknesses of halite observed in the seismic profiles (Ryan, 1973; Haq et al., 2020), the Atlantic-Mediterranean gateway needs to have permitted inflow from the Atlantic, but may have completely blocked outflow (Blanc, 2002; Krijgsman and Meijer, 2008). Numerical models also showed that without Atlantic inflow into the Mediterranean Sea its base level is forced to drop on time scales in the order of a few thousand years by virtue of the basin’s negative hydrological budget, where more water is lost to the atmosphere by evaporation than is received from rainfall and river runoff (e.g. Meijer and Krijgsman, 2005; Krijgsman and Meijer, 2008; Simon et al., 2017). The idea of a drawdown is supported by several arguments: (1) the widespread presence, from the margins to the slopes, of the Messinian Erosional Surface cutting through Stage 1 and pre-MSC deposits and canyon incisions following today’s drainage net­ works (e.g. Chumakov, 1973; Clauzon, 1982; Lofi et al., 2005, 2011a, 2011b; Loget et al., 2006; Maillard et al., 2006, 2020; Estrada et al., 2011; Just et al., 2011; Urgeles et al., 2011; Amadori et al., 2018; Lymer et al., 2018; Cazzini et al., 2020; Figs. 5e, 7e); (2) their morphology None of these quantifications could unequivocally constrain the timing of the drawdown within the MSC sequence, but numerical modeling studies show that, if the blocking of the outflow was controlled by a tectonic uplift counteracted by inflow erosion across the Strait of Gibraltar, then the expected drawdown of the Mediterranean Sea should be moderate (< 400 m; and possibly harmonic) due to an equilibrium between incision and uplift before the complete blocking of inflow and larger (up to complete desiccation) only after tectonic uplift overcame incision rates (Garcia-Castellanos and Villase˜nor, 2011). The same model suggests that the initiation of halite precipitation might overlap in time with the late primary gypsum deposition, right before the full disconnection from the Atlantic Ocean. The interpretation of the deep evaporites and their associated seismic markers (erosional surfaces and deep engravings along the shelf-slope Fig. 11. (a), (b) Schematic W-E profiles across the Mediterranean Basin showing the contrasting paleoenvironmental, paleohydrological and paleoconnectivity interpretations proposed for Stage 3. 7.1. Stage 2 (5.59-5.55 Ma): formation of the Mediterranean salt giant When a water flow is present (green arrow) from and/or to an extra-Mediterranean water mass (i.e., A: Atlantic Ocean; I: Indian Ocean; P: Eastern Paratethys), the direction of the arrow gives the direction of flow. For simplicity, water added by the major and local rivers is not shown, but it adds to the hydrological budget at any time in each scenario. Note the main difference between the isolated (a) and density-stratified (b) scenario lies in the connectivity framework (Atlantic connection closed and negligible influence from the Paratethys in the isolated scenario; influence from both Atlantic and Paratethys in the density-stratified scenario), which affects the position of the base level of the Mediterranean water mass and its hydrochemistry (see extensive discussion in sub­ section 7.2). Abbreviations: Sp.: SE Spain; V-B: Valencia Basin; Tyr: Tyrrhenian Basin; Calt: Caltanissetta Basin; IAP: Ionian Abyssal Plain; GS: Gulf of Sirt; Cyp: Cyprus; Ada: Adana Basin. See Fig. 2 for the geographic position of each basin. (c) Schematic plot showing the evolution of the Mediterranean base-level during Stage 3 according to both the isolated (red line) and half-full (black line) scenarios. The critical sills for controlling intra- and extra-Mediterranean connectivity are also shown. Fig. 11. (a), (b) Schematic W-E profiles across the Mediterranean Basin showing the contrasting paleoenvironmental, paleohydrological and paleoconnectivity interpretations proposed for Stage 3. When a water flow is present (green arrow) from and/or to an extra-Mediterranean water mass (i.e., A: Atlantic Ocean; I: Indian Ocean; P: Eastern Paratethys), the direction of the arrow gives the direction of flow. For simplicity, water added by the major and local rivers is not shown, but it adds to the hydrological budget at any time in each scenario. Note the main difference between the isolated (a) and density-stratified (b) scenario lies in the connectivity framework (Atlantic connection closed and negligible influence from the Paratethys in the isolated scenario; influence from both Atlantic and Paratethys in the density-stratified scenario), which affects the position of the base level of the Mediterranean water mass and its hydrochemistry (see extensive discussion in sub­ section 7.2). Abbreviations: Sp.: SE Spain; V-B: Valencia Basin; Tyr: Tyrrhenian Basin; Calt: Caltanissetta Basin; IAP: Ionian Abyssal Plain; GS: Gulf of Sirt; Cyp: Cyprus; Ada: Adana Basin. See Fig. 2 for the geographic position of each basin. 7. Paleoenvironmental scenarios for freshening the salt giant: desiccated versus full Mediterranean The low-salinity Stage 3 followed the hypersaline Stage 2 and the transition between the two likely influences the plausibility of the various paleoenvironmental scenarios proposed for the terminal stage. We therefore first summarize the current understanding of the configuration of the Mediterranean during Stage 2 and the enduring controversies (see Roveri et al., 2014a for a more extensive review). 7.1. Stage 2 (5.59-5.55 Ma): formation of the Mediterranean salt giant 7.2.1. An isolated Mediterranean dotted by sabkhas and lakes i The first and long-lasting paleoenvironmental interpretation of the evaporite-bearing UG/UU units and (possibly) time-equivalent evapo­ rite-free units (e.g. LM Unit in Malaga, Sorbas and Zorreras Mb. in Sorbas, Feos Fm. in Nijar, Cassano Spinola Conglomerates in Piedmont, San Donato/Colombacci fms. in the Apennines, Handere Fm. in Turkey) envisaged their sedimentation in a Mediterranean mostly isolated from the Paratethys (which may have added water only to some basins in the Eastern Mediterranean) and totally isolated from the Atlantic where, in each subbasin, continental settings (e.g. alluvial plains, river channels, alluvial fans, playa lakes, sabkhas) alternated/interfingered with shallow, endorheic lakes (Figs. 11a, c; e.g. Ruggieri, 1962, 1967; Decima and Sprovieri, 1973; Decima and Wezel, 1973; Friedman, 1973; Hsü et al., 1973a, 1973b, 1973c, Hsü et al., 1978a, 1978b; Ryan et al., 1973; Selli, 1973; Sturani, 1973; Sissingh, 1976; Benson, 1978; Bossio et al., 1978; Cita et al., 1978, 1990; Ricchiuto and McKenzie, 1978; Ryan, 1978, 2008, 2009; Cita and Colombo, 1979; Orszag-Sperber and Rou­ chy, 1979; Ghibaudo et al., 1985; Müller et al., 1990; Benson and Rakic- El Bied, 1991; Benson et al., 1991; Müller and Mueller, 1991; Orszag- Sperber et al., 2000; Rouchy et al., 2001, 2003, 2007; Blanc, 2002; Lofi et al., 2005, Lofi et al., 2011b; Bassetti et al., 2006; Rouchy and Caruso, 2006; Bertoni and Cartwright, 2007; Cameselle and Urgeles, 2017; Amadori et al., 2018; Camerlenghi et al., 2019; Kartveit et al., 2019; Madof et al., 2019; Ben Moshe et al., 2020; Caruso et al., 2020; Cazzini et al., 2020; Raad et al., 2021). The full disconnection is also supported by observations that support an abrupt Zanclean reflooding (e.g. Blanc, 2002; Micallef et al., 2018, 2019; Garcia-Castellanos et al., 2020; Spa­ tola et al., 2020), since a rapid outburst flood requires a large sea level difference prior to the flood that can only be developed in a scenario of a full Mediterranean-Atlantic disconnection (Garcia-Castellanos et al., 2009; Garcia-Castellanos and Villase˜nor, 2011). Although rarely explicitly stated, all these studies must assume that: The main problems with the isolated scenario lasting throughout Stage 3 are: (1) it does not provide an explanation neither for the ho­ mogeneity of Paratethyan ostracod assemblages in the marginal basins (e.g. 7.1. Stage 2 (5.59-5.55 Ma): formation of the Mediterranean salt giant Haj´os, 1973; Gliozzi and Grossi, 2008) that allowed alternated conditions suitable for gypsum to precipitate and Paratethyan biota and euryhaline benthic foraminifera to thrive were related to changes in the local freshwater budget; 3) The marine isotopic signals in UU/UG gypsum (Fig. 10) are entirely the reflection of the lithologies that are leached by continental wa­ ters in surficial and/or underground drainage areas (e.g. Ryan, 2009; Raad et al., 2021); 4) Stage 3 gypsum precipitated in extremely shallow-water (playa lakes) to completely dried environments (sabkhas) and the excessive sulfate necessary is completely derived from “clastic reworking, dissolution, re-precipitation and diagenesis of materials belonging to the PLG and halite of the previous MSC Stage 2” (Ryan, 2009). Observations supporting a Mediterranean isolated throughout Stage 3 and only at the mercy of local freshwater inputs (Fig. 11a) are: (1) the lack of evidence for in situ marine fauna and flora in UU (e.g. Ryan et al., 1973; Hsü et al., 1978a; Cita et al., 1990; Ryan, 2009; Lofi et al., 2011a); (2) the shallow-water mode of life and highly likely in-situ nature of ostracods and euryhaline, shallow-water benthic foraminifera observed in DSDP/ODP wells from intermediate and deep basins (e.g. Cita et al., 1978; Iaccarino and Bossio, 1999; Figs. 9a-c); (3) the bathymetric contrast (up to several hundred meters) between the late Messinian paleoenvironments and the marine Zanclean on top (e.g. Cita and Colombo, 1979; Bonaduce and Sgarrella, 1999; Caruso et al., 2020); (4) the presence of paleosols in Cyprus (Orszag-Sperber et al., 2000; Rouchy et al., 2001) and on the crest of the Eratosthenes seamount (Robertson, 1998a, 1998b); (5) the erosional features preserved both offshore on the continental shelves and lower-middle slope domain and interpreted in most seismic stratigraphic studies as the result of subaerial exposure (e. g. Lofi et al., 2005; Lofi et al., 2011b; Lymer et al., 2018; Ben Moshe et al., 2020); (6) the pinching out of the UU/BU units towards evaporite- free pre-Messinian structural highs (Figs. 7b-g; Figs. 8a, e; Ryan, 2009; Lymer et al., 2018; Camerlenghi et al., 2019; Raad et al., 2021); (7) the more abundant terrigenous clasts and reworked calcareous fossils in Stage 3 samples compared to the overlying, deep-water Pliocene (Ryan et al., 1973; Hsü et al., 1978b; Ryan, 2009); (8) the erosional nature of the M-reflector/TES/IMTS in the Levant Basin (Figs. 8e-g), by some linked to subaerial exposure of the Levant seafloor (e.g. 7.2. Stage 3 (5.55-5.33 Ma): resumption of (upper) gypsum precipitation and Paratethys fauna invasion 7.2. Stage 3 (5.55-5.33 Ma): resumption of (upper) gypsum precipitation and Paratethys fauna invasion 7.1. Stage 2 (5.59-5.55 Ma): formation of the Mediterranean salt giant (c) Schematic plot showing the evolution of the Mediterranean base-level during Stage 3 according to both the isolated (red line) and half-full (black line) scenarios. The critical sills for controlling intra- and extra-Mediterranean connectivity are also shown. 32 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. 1) all Paratethyan biota (and possibly other organisms of undisclosed provenance like diatoms) migrated passively via aquatic migratory birds across the entire Mediterranean (Fig. 11a; Benson, 1978; Ben­ son and Rakic-El Bied, 1991; Caruso et al., 2020); systems) is not straightforward. Recently, it was suggested that the deep evaporitic facies and the seismic morphological features could have been produced without a significant drop of the Mediterranean base- level, therefore promoting the persistence of a relatively deep-water Mediterranean basin even during halite deposition (Lugli et al., 2013, 2015; Roveri et al., 2014b). For example, Roveri et al. (2014c) proposed that downslope flows of dense, hypersaline waters sourced from evap­ oration in shallower water areas could have generated both the observed shelf-slope erosion and have created a deep brine, supersaturated in the ions necessary for precipitating halite. These subaqueous hyperpycnal flows are consistent with the observed clastic evaporites that filled the Levant margin canyons (Lugli et al., 2013) and, more generally, with the widespread presence of Complex Units at the outlet of the MES drainage systems (see Lofi et al., 2005, 2011a, 2011b; Lofi, 2018). These sedi­ ments are dominated by reworked PLG that would have been exposed by a sea-level fall as little as 200 m (Lugli et al., 2010). However, the hy­ persaline environment that is presumed to be established by these hyperpycnal flows during the deposition of the RLG is in contrast with the occurrence of the Paratethyan ostracod L. muelleri within the clastic evaporites (RLG) in several marginal sections (e.g. Adana Basin, Faranda et al., 2013; Radeff et al., 2016, 2017). 2) chemical and physical conditions (brackish water and water depth not exceeding 100 m; e.g. Haj´os, 1973; Gliozzi and Grossi, 2008) that allowed alternated conditions suitable for gypsum to precipitate and Paratethyan biota and euryhaline benthic foraminifera to thrive were related to changes in the local freshwater budget; 2) chemical and physical conditions (brackish water and water depth not exceeding 100 m; e.g. 7.1. Stage 2 (5.59-5.55 Ma): formation of the Mediterranean salt giant Bertoni and Cartwright, 2007; Lofi et al., 2011a, 2011b; Maillard et al., 2011a) before the emplacement of deposits interpreted as fluvial from seismic observations (Bowman, 2012; Radeff et al., 2017; Leila et al., 2018; Kartveit et al., 2019; Madof et al., 2019). Furthermore, (9) isolated hydrological circuits with unique chemical composition are regarded by Camerlenghi et al. (2019) as the most plausible explanation for the W-E change in the MSC sedimentary expression in the deep basins, repre­ sented by the trilogy LU-MU-UU in the Algero-Balearic and Liguro- Provençal basins, missing the LU in the Tyrrhenian and (possibly) Ionian basins, by terrigenous deposits with hiatuses in the WAB and Adriatic foredeep and by halite, anhydrite and clastics in the Levant Basin (Interbedded and Argillaceous evaporites of Meilijson et al., 2019; Fig 3b) Whatever the state of Mediterranean base-level during Stage 2, the more commonly used chronostratigraphic model for the MSC (Fig. 1a; Roveri et al., 2014a) states that massive halite precipitation ceased at 5.55 Ma and was superseded by an environment that, with precession periodicity (Fig. 3a), cycled between gypsum precipitation and condi­ tions that saw fresh-brackish organisms thriving. The question is whether these conditions cycled homogeneously in several isolated lakes or in basins largely connected to the same Atlantic and Eastern Paratethys-influenced water mass (Fig. 11). 7.2.2. The half-full, density-stratified Mediterranean scenarios 7.2.2. The half-full, density-stratified Mediterranean scenarios 7.2.2. The half-full, density-stratified Mediterranean scenarios An alternative concept to the isolated scenario envisages the Medi­ terranean connected with the Atlantic and/or the Eastern Paratethys and relatively full of water connecting the different subbasins (Fig. 11b). To our knowledge, this scenario was first developed by McCulloch and De Deckker (1989) on the basis of the similar 87Sr/86Sr ratios from marginal (Spain and Cyprus) and deep (Levantine and Algero-Balearic) basins. This intuition was a significant departure from the far more in vogue desiccated scenario (see conclusion of Hsü et al., 1973b), and for this was long overlooked. Sr isotope ratios lower than contemporary ocean water led McCulloch and De Deckker (1989) to conclude that a brackish water mass created by the mixing of water from the peri-Mediterranean rivers (e.g. Nile, Rhˆone and African rivers that no longer flow today, etc.; see Griffin, 2002 and Gladstone et al., 2007) with water of the Eastern Paratethys filled the Mediterranean, resembling the Caspian Sea today. This conclusion is consistent with the impoverished (or absent) marine fauna and flora of Stage 3 sediments and the enhanced assemblage of fresh-brackish water biota (see subsection 5.7; Figs. 9a-c), but is prob­ lematic as a viable origin for Stage 3 gypsum to precipitate at depth. Furthermore, climate models for the late Miocene fail to fill the Medi­ terranean Basin with fluvial and Paratethys waters alone (Gladstone et al., 2007; Marzocchi et al., 2016, 2019; Simon et al., 2017). A marine contribution is therefore required to fill the Mediterranean (Marzocchi et al., 2016). In the event, the contribution is most likely to have derived from the Atlantic via the Gibraltar Corridor (Flecker et al., 2015; Booth- Rea et al., 2018; Krijgsman et al., 2018) either through a karst system (Krijgsman et al., 2018) or an emerged volcanic archipelago in the Albor´an Basin (Booth-Rea et al., 2018). In fact, although an Indian Ocean contribution was proposed (Cita et al., 1978; Hsü et al., 1978a) and the possibility discussed (Ryan, 2009; Vai, 2016), palinspastic re­ constructions concluded that the Neo-Tethys Mediterranean-Indian Ocean connection via southern Turkey and Iran already closed before the Tortonian (R¨ogl, 1998; Popov et al., 2004; Gargani et al., 2008; Bialik et al., 2019; Gülyüz et al., 2020), while a seaway via the Red Sea and Gulf of Aden, although not completely ruled out (e.g. 7.2.2. The half-full, density-stratified Mediterranean scenarios Schütz, 1994; Bosworth et al., 2005; Gargani et al., 2008; Ryan, 2009), is highly contested (e.g. Meulenkamp and Sissingh, 2003; Segev et al., 2017). A Mediterranean step-wise refilled and at times filled with water up to the marginal belt agrees with: (1) Paratethyan biota being present only in intermediate-deeper settings during substage 3.1, but more widespread including marginal settings during substage 3.2; (2) the W-E homogeneity of Paratethyan ostracod assemblages around the Medi­ terranean marginal belt (Gliozzi et al., 2007; Stoica et al., 2016; Sciuto et al., 2018; Sciuto and Baldanza, 2020; Fig. 9a); (3) the presence, in marginal basins, of Paratethyan fish (Bannikov et al., 2018; Schwarz­ hans et al., 2020), dinocysts (e.g. Pellen et al., 2017; Fig. 9b) and bio­ markers (Vasiliev et al., 2017; Fig. 10c); (4) the occurrence of a monospecific assemblage of abundant Sphenolithus spp. just below the M/P boundary at ODP Sites 978, 975 and 967 (Castradori, 1998); (5) the requirement of water from the Mediterranean to explain the Sr isotope ratios of ostracods that inhabited marginal subaqueous environments (Andreetto et al., 2021); (6) the Atlantic-like sulfate values (although variably diluted and affected by microbial processes; Fig. 10b) of the UU/UG gypsum beds (García-Veigas et al., 2018); (7) the presence of long chain alkenones in the Sicilian UG beds similar to those observed in present-day marine settings (Fig. 10c; Vasiliev et al., 2017). i In light of this, Roveri et al. (2014c), Gvirtzman et al. (2017), Vasi­ liev et al. (2017), García-Veigas et al. (2018) and Grothe et al. (2020) suggested that the Mediterranean was likely density-stratified during this interval as a result of the simultaneous influx of isotopically- different marine and non-marine (major Mediterranean rivers and Eastern Paratethys) water sources (Fig. 11b). This connectivity frame­ work resulted in a brackish layer carrying low-salinity (mostly Para­ tethyan) biota (Gliozzi et al., 2007; Stoica et al., 2016; Grothe et al., In light of this, Roveri et al. (2014c), Gvirtzman et al. (2017), Vasi­ liev et al. (2017), García-Veigas et al. (2018) and Grothe et al. (2020) suggested that the Mediterranean was likely density-stratified during this interval as a result of the simultaneous influx of isotopically- different marine and non-marine (major Mediterranean rivers and Eastern Paratethys) water sources (Fig. 11b). 7.2.1. An isolated Mediterranean dotted by sabkhas and lakes i Gliozzi et al., 2007; Stoica et al., 2016), an aspect difficult to explain when fauna migration takes place passively via either birds or wind, nor for the biomarkers (Vasiliev et al., 2017), which cannot be transported effectively by aquatic birds; (2) it does not explain the mismatch between 87Sr/86Sr isotope ratios measured on marginal os­ tracods and Sr values expected from endorheic lakes fed with local 33 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. (Figs. 5e, 6a, e) and continental/lacustrine facies (Figs. 5a-b, d-g) widespread around the margins and shelves and suggestive of a Medi­ terranean base-level somewhat lower than the Atlantic level suggesting a one-way inflow from both the Atlantic and the Eastern Paratethys after Stage 2 (e.g. Marzocchi et al., 2016; Figs. 11b, c), a connectivity configuration that effectively translates in a half-full Mediterranean (e.g. Krijgsman and Meijer, 2008). Refilling as a result of persistent Atlantic inflow, in part perhaps because of the latest Messinian deglaciation (see subsection 2.2; Van der Laan et al., 2006; Hilgen et al., 2007), would have resulted in the establishment of two-way exchange first with the Paratethys at some point during the Lago-Mare phase and later, i.e. slightly before or at the Messinian/Zanclean boundary, with the Atlantic Ocean (Fig. 11c; Marzocchi et al., 2016). The moment the Mediterra­ nean base-level reached the sill with the adjacent water body (Para­ tethys and Atlantic) and a two-way exchange was initiated, the density contrast will have prompted an enhanced inflow into the Mediterranean (Marzocchi et al., 2016). The overall transgressive trend leading to the Zanclean marine replenishment was accompanied by base-level fluctu­ ations in the order of 400±100 m every precessional cycle (Fig. 11c; Fortuin and Krijgsman, 2003; Ben Moshe et al., 2020; Andreetto et al., 2021). These fluctuations are ascribed to switch in the Mediterranean freshwater budget driven by the African summer monsoon and Atlantic winter storms (e.g. Marzocchi et al., 2015, 2019; Simon et al., 2017). Since higher freshwater discharge rates occur at precession minima times and their Stage 3 sedimentary expression is considered to be the mudstone intervals (Fig. 3a; Manzi et al., 2009), mudstone interbeds (both onshore and offshore; e.g. Figs. 5h-j) represent the highstand ep­ isodes (e.g. Manzi et al., 2009; Roveri et al., 2008a; Omodeo-Sal´e et al., 2012; Fig. 3), while continental facies onshore (e.g. conglomerates in the Apennines; Fig. 7.2.1. An isolated Mediterranean dotted by sabkhas and lakes i 5g) and offshore (clastic beds in the Levant Basin) and gypsum beds (Algero-Balearic, Liguro-Provencal, CMD, Tyrrhenian, Caltanissetta, Ionian, Sirte and Polemi-Pissouri basins; Figs. 5h-j) represent the lowstand (e.g. Roveri et al., 2008a; Manzi et al., 2009; Meilijson et al., 2019; Fig. 3). If Atlantic was the major source of sulfate for Stage 3 gypsum (e.g. García-Veigas et al., 2018) and an intervening, relatively shallow (Sicily) sill was establishing Western and Eastern Mediterranean division during the MSC (e.g. Garcia-Castellanos et al., 2009, 2020; Micallef et al., 2018), the presence of Stage 3 gypsum to the east of the Sicily sill (Fig. 2b) implies that the Mediterranean base level never dropped below the (maximum estimated) paleodepth of the sill (i. e. ~430 m; Garcia-Castellanos et al., 2009) during Stage 3 and Western and Eastern Mediterranean remained connected also during the arid (lowstand) phases of the precession cycles. ii freshwaters (e.g. Andreetto et al., 2021); 3) it misses to substantiate, with geochemical arguments, the precipitation of gypsum in lakes, a process that is everything but straightforward (see Warren, 2016 for insights); 4) except for the salt-bearing basins, the source(s) of solutes which makes freshwater-fed endorheic lakes brackish and causes similar physico-chemical conditions to exist in each lake is also difficult to explain in the context of a Mediterranean only at the mercy of local rivers. 7.2.2. The half-full, density-stratified Mediterranean scenarios This connectivity frame­ work resulted in a brackish layer carrying low-salinity (mostly Para­ tethyan) biota (Gliozzi et al., 2007; Stoica et al., 2016; Grothe et al., 2018, 2020; Figs. 9a-b) to lay on top of a more saline layer formed by Atlantic-derived seawater from which UU/UG gypsum (Figs. 5h-j, 7b-g, 8a-d), that facies analyses demonstrated to result from subaqueous deposition (Hardie and Lowenstein, 2004; Lugli et al., 2015), precipi­ tated at intermediate and greater depths (e.g. García-Veigas et al., 2018). A dense, anoxic deep-water mass, possibly inherited from Stage 2, is envisaged at the bottom of the Mediterranean by Marzocchi et al. (2016) and García-Veigas et al. (2018), albeit without conclusive ar­ guments, and by Gvirtzman et al. (2017) following the observation that the tilted halite body of the Levant Basin was simultaneously eroded landward and preserved basinward (Fig. 8f). Major problems also exist with the half-full stratified scenario: (1) it does not provide a proper mechanism for gypsum precipitation at several hundreds, or thousands, meters water depth; (2) it fails to explain how unquestionable shallow-water (< 50 m) benthic organisms This scenario accounts for the erosive/non-depositional features 34 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Pliocene sediments (e.g. Caruso et al., 2020; Fig. 6d). Pliocene sediments (e.g. Caruso et al., 2020; Fig. 6d). such as Ammonia tepida and Cyprideis sp. could survive at hundreds of meters of depth and beyond; (3) it does not provide an explanation for the high abundance of coarse-grained detritus at intermediate and deep- water locations, especially when compared to deep-water Pliocene samples, as well as for the broad absence of MSC deposits in the shelf domain; (4) a persistent Atlantic inflow without outflow seems to be a configuration that cannot be maintained stable for ~200 kyr. Indeed, models coupling the inflow of marine waters with the erosion of the gateway channel concluded that, if the Mediterranean level was lowered by at least several hundred meters below present sea level, any small overtopping of water from the Atlantic would inevitably trigger a fast refill of the basin that, if responsible for the erosion trough the Albor´an Basin, should have involved an unprecedented water discharge and be completed within a few years or less (Garcia-Castellanos et al., 2020 and references therein). The scenario arising from Meilijson et al., 2019, Figs. 3b, 4a) is also problematic for a high base-level Mediterranean. 7.3. Demise of the MSC (5.33 Ma): the Zanclean marine replenishment The conspicuous and abrupt transition to normal marine sediments in the Mediterranean is globally and historically important because it is the origin of the stratigraphic position of the Miocene/Pliocene boundary (Van Couvering et al., 2000). From an ocean perspective, it is not an ideal stratigraphic location being difficult to locate from biozone data even in the adjacent Atlantic (Hodell et al., 2001; Krijgsman et al., 2004; Van den Berg et al., 2015). However, from a Mediterranean perspective it provides a clear and unambiguous end to the MSC and the restoration of normal marine conditions. All evidence show that the onset of the Zanclean marine replenishment followed a period of relative lowstand that exposed all the Mediterranean margins (see subsection 3.12; Figs. 6a-b, f) and kept intermediate and deep basins underwater (see subsection 4.8). Yet again, the key dispute concerns the exact depth of the Mediterranean base level preceding the Miocene/Pliocene transition. 8. Methods and proxies to better reconstruct base level and connectivity changes Chronological uncertainty and spatial variability limit the use of both sedimentological and paleontological information to achieve a comprehensive and coherent basin-wide interpretation of the conditions and drivers of Stage 3 environments and water levels. Alternative methods are therefore required to clarify connectivity relationships and constrain base-level conditions. This section explores the principles and potential of geochemical, backstripping and numerical modelling tech­ niques that could be used to further test existing hypotheses and enhance understanding of the complex environmental conditions experienced by the Mediterranean during the latest Messinian. Building on the isolated Mediterranean scenario, base level imme­ diately before the early Zanclean was more than thousand kilometers below eustatic sea level (Fig. 10e; e.g. Hsü et al., 1973a; Blanc, 2002; Loget et al., 2006; Garcia-Castellanos et al., 2009; P´erez-Asensio et al., 2012; García-Alix et al., 2016; Amadori et al., 2018; Micallef et al., 2018, 2019; Camerlenghi et al., 2019; Kartveit et al., 2019; Madof et al., 2019; Ben Moshe et al., 2020; Caruso et al., 2020; Cazzini et al., 2020; Mas and Forn´os, 2020; Spatola et al., 2020). Hydrodynamic erosional models allowed a reinterpretation of the erosional features across the strait of Gibraltar (Campillo et al., 1992; Blanc, 2002) suggesting that a sudden breach of the Mediterranean-Atlantic divide at Gibraltar resulted in a vast cascade of Atlantic water that refilled the entire Mediterranean in less than 2 years (i.e. rates of ten meters per day) spilling first into the Western Mediterranean (see the extensive review in Garcia-Castellanos et al., 2020) and then, after reaching the level of the Sicily sill, pouring into the Eastern Mediterranean (Micallef et al., 2018, 2019; Ben Moshe et al., 2020; Spatola et al., 2020). This concept of catastrophic refilling has led to terms such as “Zanclean flood” or “deluge”. Evidence sup­ porting the catastrophic flood mechanism mostly comes from the seismic reflection dataset and includes: 1) the presence of >250 m deep and 390-km-long incisions on both sides of the Gibraltar Strait (Garcia- Castellanos et al., 2020); 2) the detection of (allegedly) Pliocene-aged chaotic sedimentary bodies stretching for kilometers in the Albor´an Basin (Garcia-Castellanos et al., 2020 and references therein) and Ionian Basin at the foot of the Malta Escarpment (Micallef et al., 2018, 2019; Spatola et al., 2020; Fig. 8c). A further argument is the bathymetric jump of several hundred meters between the late Messinian and the early 7.2.2. The half-full, density-stratified Mediterranean scenarios In order to simultaneously reach precipitation of gypsum and halite in different basins sharing the same water, the water has to be of high salinity and stratified. Simon and Meijer (2017) demonstrated that this can be achieved with slow overturning circulation, but it is currently unclear how realistic this process is. Instantaneous sea level rise is not the only possible refilling model. Bache et al. (2012) suggested the reflooding occurred in two steps at ~5.60 Ma, accompanied by a moderate (≤500 m) rise, followed by a rapid rise of 600-900 m at around 5.46 Ma tracking the deposition of the deep basin evaporites and resulting from the collapse of the Gibraltar divide. There is also the reconnection model that follows from a Stage 3 Mediterranean that is already relatively full and with the base level possibly oscillating of 400 ± 100 m with precessional frequency (Fig. 10h; Fortuin and Krijgsman, 2003; Ben Moshe et al., 2020; Andreetto et al., 2021). In this case, only a sea level rise of a few hundred meters is required to restore the Mediterranean to the Atlantic level (Fig. 10h), which was hypothesized to have occurred in the last pre­ cessional cycle of the Messinian (Marzocchi et al., 2016; Fig. 3a). In detail, the re-establishment of a fully marine faunal diversity and oceanic geochemistry (e.g. 87Sr/86Sr ratios and δ18O) occurred more gradually over one or more precessional cycles in the earliest Zanclean (e.g. Iaccarino et al., 1999; Pierre et al., 1998, 2006; Cipollari et al., 2013; Roveri et al., 2019a; Bulian et al., 2021). This suggests that stressed ecological conditions at first only suitable for opportunistic organisms to survive (e.g. Bulian et al., 2021) developed (or persisted) in the Mediterranean as marine replenishment occurred (e.g. Rouchy et al., 2003). One possible mechanism for achieving this may be the physico- chemical turnover in the water column triggered by the re-established two-way exchange with the Atlantic which, for reasons that are largely unknown, took time (at least oneprecession cycle; Pierre et al., 2006) to displace surficial Paratethyan water and restore normal marine conditions (Marzocchi et al., 2016). 8.1. Geochemical proxies In this scenario, differences in the 87Sr/86Sr ratios between basins are likely the reflection of the different 87Sr/86Sr ratio of the local input in each basin (Andreetto et al., 2021). The application of numerical models assists to identify and quantify the different water sources feeding the basin(s) in question and (e.g. Placzek et al., 2011; Topper et al., 2011, 2014; Doebbert et al., 2014; Rossi et al., 2015b; Modestou et al., 2017; Grothe et al., 2020; Andreetto et al., 2021). Hydrogen isotopes. Organic geochemistry biomarker-based tools can be used as independent proxies for reconstructing sea surface tempera­ tures, relative changes in the basin hydrology and, indirectly, salinity. Basin water properties are reflected in a variety of life forms. Different types of organisms produce specific organic compounds that serve as molecular biomarkers. These large biomolecules record the changes in the hydrogen isotopic composition of the water used by different groups of biomarker producers (i.e. different organisms). The principle behind the method is to measure δD on biomarkers produced in Mediterranean Sea waters (e.g. alkenones, produced by a few species of haptophyte coccolithophores algae) during the MSC and compare the results with the δD signals retrieved from biomarkers produced in the open ocean ideally at the same time intervals. The influence of precipitation on the changes in hydrological budget can be monitored by measuring the δD of long chain n-alkanes (Sachse et al., 2006), biomarkers predominantly produced by higher terrestrial plants that rely on precipitation for plant growth, therefore reflecting the changes in the δD of the precipitation. The extreme base level drop(s) suggested for the Mediterranean during the MSC would, in principle, indicate a negative precipitation (P) + runoff (R) – evaporation (E) ratio. Such a negative water budget (E>P+R) results in waters increasingly enriched in δD whereas, a pos­ itive water balance (E<P+R) results instead in a negative shift of δD values. The analysis of compound specific δD of alkenones, long and short chain n-alkanes can be used to constrain E/(P+E) relationships. Sulfate isotopes. When sulfate-bearing minerals precipitate in a basin they uptake dissolved S and O with δ34SSO4 and δ18OSO4 isotopic composition that, once corrected for the fractionation effects during liquid-solid transition (see subsection 6.2), can be assimilated to that of the mother brine. 8.1. Geochemical proxies Radiogenic strontium isotopes. Radiogenic strontium isotope ratio (87Sr/86Sr) is a widely applied geochemical tool in provenance studies, including the reconstruction of the hydrological circuit and connectivity of basins with little or null oceanic entries. Its potential to detect the provenance of the hydrological fluxes derives from the unique 87Sr/86Sr ratio that typifies each water source and from the negligible effects of isotopic fractionation during the liquid-solid transition (see Hajj et al., 2017). Mineral phases precipitating in endorheic lakes uptake Sr with 87Sr/86Sr ratio that reflects the mixing of all feeding surficial and un­ derground streams and whose 87Sr/86Sr fingerprint hinges on the composition and age of watershed bedrock (see Peucker-Ehrenbrink and Fiske, 2019; Andreetto et al., 2021 and references therein). When river water mixes with seawater such as in the oceans, semi-enclosed basins or estuaries, mineral phases uptake Sr with oceanic 87Sr/86Sr ratios because the high oceanic Sr concentration (~7.8 mg/l today; Veizer, 1989) masks the impact of the ~100 times lower concentrated conti­ nental Sr-sources (~0.0780 mg/l; Palmer and Edmond, 1992). This is valid as long as a certain ratio of continental-marine water mixing is fulfilled, beyond which 87Sr/86Sr ratios deviate towards the 87Sr/86Sr ratios of the non-marine source(s) (Ingram and Sloan, 1992). For the 35 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. Mediterranean to attain non-marine 87Sr/86Sr ratios (like during the MSC), Topper et al. (2014) calculated a mixing of at least 1:4 (Atlantic: freshwater) to be required. cycling and/or freshwater riverine inputs (e.g. Utrilla et al., 1992; Lu et al., 2001; Turchyn et al., 2009) (Fig. 10b). Untangling the relative importance of these processes is key to understanding the Mediterra­ nean sulfur isotope record and gleaning paleoenvironmental insights into Stage 3. q If Mediterranean subbasins hosted endorheic lakes (Figs. 10c, e), the 87Sr/86Sr isotope ratios measured on ostracod valves or gypsum crystals of each lake are expected to generate a scattered distribution by virtue of the different geology in the hinterland of each basin. By contrast, some degree of connection between different basins and the Mediterranean water mass (Figs. 10d, f) is expected to result in more homogeneous 87Sr/86Sr ratios because, although isotopically-different, local rivers mix with a water mass that has the same 87Sr/86Sr value and (much higher) Sr concentration for each basin (Andreetto et al., 2021). 8.1. Geochemical proxies The higher concentrated source of sulfate is seawater (with present-day δ34SSO4=21.15±0.15‰ and δ18OSO4=8.67±0.21‰, Johnston et al., 2014; with Messinian values of ~22±0.2‰ for the δ34SSO4 and ~9±2‰ for the δ18OSO4; Turchyn and Schrag, 2004; Mar­ kovic et al., 2016; Masterson et al., 2016). Significantly higher inputs from the ~1000 times less concentrated riverine freshwater (both sur­ ficial and underground) with respect to the ocean water (more than 1:5 according to Lu et al., 2001) can modify the marine δ34SSO4 and δ18OSO4 isotopic composition of the mother brine (Utrilla et al., 1992; Lu et al., 2001) and have it deviated from that of the ocean (Lu et al., 2001). This deviation is normally towards lower values, because river-derived dis­ solved sulfate is generally depleted in heavy isotopes 34S and 18O compared to oceanic sulfate because these isotopes mainly come from the oxidation of 34S-depleted pyrite (FeS2) on the continents and to a lesser extent from the dissolution of older sulfate-bearing minerals (Claypool et al., 1980; Turchyn and Schrag, 2004; Burke et al., 2018). However, when marine sulfate is preferentially leached in the catch­ ment, 34S of the freshwater-dissolved sulfate and [SO4 2-] likely increase, therefore reducing the continental-marine mixing ratio necessary to deviate the resulting sulfate isotopic signature away from marine values. Unfortunately, the sulfate isotopic composition is not provided for a number of major Mediterranean rivers (Burke et al., 2018) nor for the Eastern Paratethys and it is hardly assessed with the catchment-forming lithologies (Liu et al., 2017; Burke et al., 2018), making sulfate isotopes still an unsuitable tracer of non-marine water provenance in Mediter­ ranean subbasins. 8.2. Backstripping analyses Backstripping uses paleobathymetry, sea level and sediment thick­ ness to quantify the tectonic and isostatic components of subsidence. If tectonic subsidence or uplift history are known relative to the current position and depth of paleoshoreline markers, an inverse approach al­ lows base level to be estimated. A number of approaches have been applied to the MSC, using erosional surfaces (e.g. Amadori et al., 2018), terraces (Micallef et al., 2018) or fluvial network characteristics (Urgeles et al., 2011) as paleoshoreline indicators. The relief on erosional features has also been used to estimate minimum base-level variation (Frey- Martinez et al., 2004). Apart from the quantitative constraints on base level that back­ stripping provides, consideration of the regional implications of isostatic subsidence and the gravitational impact of redistributing water masses (such as in the cascading model of Roveri et al., 2014c; Fig. 10b) and evaporite precipitation is important in gateway regions like Gibraltar, which due to their shallow and restricted nature are exceptionally sen­ sitive to vertical motions. Here, both flexural effects and gravitational effects on local sea level on the Atlantic side of the strait has the po­ tential to influence Mediterranean-Atlantic connectivity driving paleo­ environmental changes in the basin itself (Coulson et al., 2019). Deviation of δ34SSO4 and δ18OSO4 from the marine average can also be the result of isotopic fractionation during microbial sulfate reduction (MSR; Fritz et al., 1989; Berner, 1999). MSR produces 34S-depleted hydrogen sulfide (~0 to 70‰ lighter than initial sulfate; Brunner and Bernasconi, 2005; Sim et al., 2011; Leavitt et al., 2013) and induces the enrichment in 34S and 18O of the residual sulfate pool (Kaplan and Rit­ tenberg, 1964; Thode and Monster, 1965; Turchyn et al., 2006; Wort­ mann et al., 2007). Therefore, if isotopically light H2S produced by MSR leaves the system as a sulfide mineral (most likely pyrite), the resulting dissolved sulfate would have δ34SSO4 and δ18OSO4 isotopic signatures higher than the oceanic one (Brunner et al., 2005). However, if the MSR- produced H2S is re-oxidized back to sulfate through abiotic or microbial sulfide oxidation, isotopically light sulfate will be brought back to the 34S-enriched sulfate pool, producing little or no enrichment in 34S observed in the resulting sulfate (Gomes and Johnston, 2017 and ref­ erences therein; Pellerin et al., 2019). Slight deviations from marine δ18OSO4 and δ34SSO4 values of sulfate reflect both biological sulfur 8.3. Modelling Numerical models can be used complementary to lab- and field- based studies, or to find answers to open questions by testing the physical plausibility of hypotheses and their compatibility with the available sedimentological/stratigraphic/paleontological/geochemical data, which have to constrain model results and not adjust to it. For example, whether gypsum beds in marginal/intermediate basins can precipitate at the same time as the halite in deep basins is an intriguing question that circulates in the MSC literature (e.g. Van Couvering et al., 1976), but whether this is physically and geochemically possible is yet to be answered. In a first model analysis, Simon and Meijer (2017) found that the required stratification can indeed be achieved for specific 36 Earth-Science Reviews 216 (2021) 103577 F. Andreetto et al. conditions including a slow overturning circulation. A different approach is needed to determine whether such slow circulation is to be expected or if other scenarios should be considered. A thermo-haline stratification that enables coeval precipitation of two evaporites for a considerable time span might also influence the degree of heterogeneity of other parameters, such as strontium concentration. Previous models showed the influence of the Atlantic Ocean and major rivers (Topper et al., 2014) and of evaporation (Flecker et al., 2002) on the Sr value of a basin with restricted oceanic inflow and assuming a homogeneous dis­ tribution of strontium throughout the water column (Flecker et al., 2002; Topper et al., 2011, 2014; Modestou et al., 2017), but it is un­ certain if this holds true in conditions of water stratification. New in­ sights into this behavior would have consequences for the way the strontium dataset (Fig. 10a) must be interpreted. Another loose end where the model approach can provide insight relates to the question whether a high water level could have been reached without an inflow from the Atlantic. Climate simulations conducted by Gladstone et al. (2007), Simon et al. (2017) and Marzocchi et al. (2019) indicate that this is not possible with today’s bathymetry. A quantitative analysis exploring the Mediterranean water level reached in different situations (i.e. with or without an Atlantic or Paratethys in and outflow) and with information on the Mediterranean hypsometry that may be provided by isostatic restoration of the seafloor topography (flexural backstripping) could help understanding how the Messinian Salinity Crisis ended. contains in situ biota assemblages of Paratethyan provenance implying brackish water conditions. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.earscirev.2021.103577. 8.3. Modelling More problematic is the differentiation of in situ and reworked marine microfauna and flora and the paleoecological significance of dwarfism in marine calcareous microfossils/algae. These have important repercussions for the Mediterranean connectivity and base-level reconstruction. The geochemical dataset for Stage 3, particularly strontium isotopes and hydrogen isotopes on biomarkers, is both demonstrably valuable in providing key constraints on connectivity and environmental condi­ tions, and frustratingly inadequate in terms of data distribution. It has great potential as a constraint on quantitative sensitivity analysis of the different hydrochemistry scenarios that follow from the endmember Stage 3 hypotheses, but substantially more data is required. An approach which combines geological, geochemical, geophysical and paleontological data with numerical modelling (e.g. climate simu­ lations, backstripping analyses and paleoceanographic models) will provide a more accurate reconstruction of Mediterranean paleogeog­ raphy and the processes that occurred during the final phase of the Messinian Salinity Crisis. Declaration of Competing Interest The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consul­ tancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. 9. Certainties, open problems and future directions Our understanding of the nature of MSC Stage 3 has evolved considerably over the last fifty years. However, there are still such disparate models for the paleoenvironmental conditions and basin connectivity that led to Stage 3 deposition and that express the chal­ lenges that the study of this interval presents: it is a relatively short interval and its sedimentary expression varies spatially. It is no surprise that the main point of contention lies in reconciling the observations from seismic profiles and well data, which are largely interpreted as favoring the desiccated scenario, with the sedimentological, paleonto­ logical and geochemical data from the marginal basins record, largely discontinuous and unaddressed from seismic-based and computational- based studies and mainly supporting the full-Mediterranean hypothesis. Data availability The compilation of strontium, sulfate and hydrogen isotope data plotted in figure 9, as well as some of the available, and here not (graphically) shown, oxygen isotope values is accessible in separate excel spreadsheets (Supplementary material 1). Acknowledgments To a large extent this mismatch is the result of the lack of intersection of the two datasets. Some Stage 3 onshore localities are meticulously studied from the stratigraphic, sedimentological, paleontological and geochemical point of view, showing remarkably consistent and ho­ mogenous trends and patterns (as previously highlighted by Roveri et al., 2008a). However, changes at precessional and subprecessional scale are difficult to trace from one exposure to another and are well below the level of seismic resolution, making onshore-offshore correla­ tion at this scale impossible. Even correlation between onshore sections is problematic since most of the stratigraphic sections are incomplete, with erosion surfaces at the bottom and/or top (i.e. the Miocene/Plio­ cene boundary), and this lack of stratigraphic continuity frustrates at­ tempts to constrain ages by cyclostratigraphy. A future focus on strengthening the available chronostratigraphic framework (Fig. 3) and making it inclusive of the fragmented outcrops is required to better understand the paleoenvironmental and paleohydrological changes suffered by the Mediterranean marginal belt through time. The suc­ cessful drilling of the three IODP proposals currently in the scheduling pool (see Camerlenghi and Aloisi, 2020), all of which propose to recover Stage 3 sediments, will also provide transformative information enabling far better offshore-onshore correlation and interpretation of currently enigmatic seismic data. In the meantime, re-evaluation of existing DSDP and ODP material, particularly through the application of more novel geochemical techniques and, where possible, access to ma­ terial collected during industrial drilling would be helpful for under­ standing the deep Mediterranean Basin during this interval. We thank the entire SALTGIANT community for many profitable workshops that inspired the development of this manuscript. This research was supported by the project SALTGIANT-Understanding the Mediterranean Salt Giant, a European project which has received funding from the European Union’s Horizon 2020 research and inno­ vation program, under the Marie Sklodowska-Curie [grant agreement No 765256]. We greatly thank the two reviewers Domenico Cosentino and William Ryan and the editor Alessandra Negri for the fruitful comments provided that led to a substantial improvement of the manuscript. 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Carcass characteristics and meat quality of male pigs submitted to surgical or immunological castration
Anais da Academia Brasileira de Ciências
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Carcass characteristics and meat quality of male pigs submitted to surgical or immunological castration HENRIQUE C.M. MUNIZ, EDUARDA S. DE LIMA, LUCIANE I. SCHNEIDER, DANIELA R. KLEIN, LEONARDO T. DA ROCHA, JOSÉ L. NÖRNBERG, ARLEI R.B. DE QUADROS & VLADIMIR DE OLIVEIRA Abstract: The objective of this study was to evaluate carcass characteristics and meat quality of surgically castrated and immunocastrated male pigs. Data were collected from 24 surgically castrated pigs and 24 immunocastrated male entire originating from the commercial line (Agroceres x Topigs), receiving isonutritive diets and were housed in the same experimental shed, slaughtered at 177 days old, with a slaughter weight of 127.8 (± 6.5) and 135.1 (± 7.8) kg, respectively. Carcass and meat quality characteristics such as carcass yield, lean meat yield, fat content, backfat thickness, muscle depth, pH, meat coloring and, drip, thawing and cooking loss, were evaluated. Immunocastrated pigs have heavier carcass, higher meat yield and lower fat thickness when compared to surgically castrated animals. Regarding meat quality, the immunocastrated presented a lighter meat, less reddness and less water holding capacity. In conclusion, this study confi rmed that the carcass of male pigs, submitted immunocastration, has lower yield, however, more meat and less fat content, when compared to carcass of male pigs surgically castrated. Meat quality showed peculiar characteristics for immunocastrated pigs, and can be used more for sausage production, since it retains less water and have smaller redness color compared to surgically castrated pigs. Key words: Castration methods, immunocastration, swine, surgically castrated, water holding capacity. An Acad Bras Cienc (2021) 93(4): e20200130 DOI 10.1590/0001-3765202120200130 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal An Acad Bras Cienc (2021) 93(4): e20200130 DOI 10.1590/0001-3765202120200130 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal An Acad Bras Cienc (2021) 93(4) Animals and feeding Data were collected from 24 surgically castrated pigs, on the third day of life, and 24 immunocastrated male entire originating pigs from the commercial line (Agroceres x Topigs), receiving isonutritive diets and were housed in 24 pens separated according to sex and weight, in the same experimental shed, slaughtered at 177 days old, with a slaughter weight (SW) of 127.8 (± 6.5) and 135.1 (± 7.8) kg, respectively. The experimental design was in completely Castration’s management The piglets, which underwent surgical castration, were sedated with administration of 4 mg.kg-1 of tramadol and 1 mg.kg-1 of midazolam, intramuscularly. After 10 minutes, the skin of the scrotal sac was cleaned and local anesthesia in each spermatic cord, using 0.5 ml of 2% lidocaine without epinephrine. The surgical procedure was performed 5 minutes after the application of the anesthetic, with the cleaning and disinfection of the skin of the scrotum with an iodine- based antiseptic solution, with a longitudinal cut and removal of the testicles. After surgical castration, no piglet had any complications, and it was not necessary to use anti-inflammatory or anti-biotic However, it is known that immunocastration generally results in ranging between studies, for variables such as meat yield and carcass fat content, color of meat samples and water holding capacity (Batorek et al. 2012, Aluwé et al. 2013). Thus, the objective of this study was to evaluate carcass characteristics and meat quality of surgically castrated and immunocastrated male pigs. MATERIALS AND METHODS The male pigs destined of the immunocastration, were vaccinated by a technical team (Vivax®, Zoetis, Brazil). The vaccination protocol consisted of the subcutaneous application of 2 ml (200 μh GnRH conjugated to protein per milliliter) of the vaccine, the first and second doses were performed eight and four weeks before slaughter, respectively. According to Dunshea et al. (2013), the first dose is of a sensitizing nature, where the animal will have contact with the anti-GnRH antigen, and the second dose will cause it to produce anti-GnRH antibodies. The experiment was carried out at the Setor de Suínos linked to the Zootechnics Department of the Federal University of Santa Maria, Rio Grande do Sul state, Brazil. The procedures adopted in the study was approved by the Ethics Committee on the Use of Animals (protocol number 2874110618), from the same institution. INTRODUCTION anesthesia and or analgesia, it has gained space and popularity in the pig production industry (Martins et al. 2013). It is a procedure, where induces the formation of specific antibodies that bind to and neutralize GnRH, reducing activity in the hypothalamic-pituitary-gonadal axis (Thun et al. 2006). It usually consists of applying two doses of the vaccine, the fi rst dose prepares the immune system, while the second dose stimulates the animal to produce specifi c antibodies (Dunshea et al. 2001). Castration is a widespread procedure in pig farming recommended preventing pig meat from having an unpleasant taste and odor to the human palate, which may cause a reduction in your consumption (Pauly et al. 2010). Normally, castration is performed in the early days of life and without the use of analgesics and or anesthetics. However, with the growing concern in relation to animal welfare, new alternatives have been increasingly used. The immunocastration vaccine contributes to improving animal welfare during its productive life and performance in the fattening The immunocastration is an alternative technique to surgical castration, without An Acad Bras Cienc (2021) 93(4) CARCASS AND MEAT QUALITY IN MALE PIGS HENRIQUE C.M. MUNIZ et al. period (Weiler et al. 2013, Muniz et al. 2019). Characteristics that indicate meat quality as shear force and color are similar between castrated and immunocastrated pigs (Gispert et al. 2010, Batorek et al. 2012). The carcass yield in immunocastrated pigs can be up to 3.0% lower than that observed in castrated pigs (Dunshea et al. 2013). However, immunocastration increases the amount of meat compared to surgically castrated pigs (Caldara et al. 2012). randomised, having two treatments, castrated surgically and immunologically. The animals received a diet plan consisting of four diets balanced according to (NRC 2012; Table I). An Acad Bras Cienc (2021) 93(4)  e20200130  3 | 9 Slaughter and data collection The animals were fasted for eight hours before the transport. At the slaughterhouse, pigs were weighed individually, and housed in collective pens with free access to water. The time between transport and slaughter was approximately twelve hours. Using standard handling An Acad Bras Cienc (2021) 93(4)  e20200130  2 | 9 CARCASS AND MEAT QUALITY IN MALE PIGS HENRIQUE C.M. MUNIZ et al. Table I. Composition and calculated nutritional values of the diets fed to pigs in the growing and finishing phases. Ingredients Diets, % G1 G2 F1 F2 Corn 64.48 68.13 69.74 75.14 Soyabean meal (46% CP) 30.41 27.03 25.46 21.83 Soya oil 1.94 1.79 1.73 0.40 L-Lysine 0.32 0.31 0.29 0.27 DL-Methionine 0.14 0.11 0.13 0.07 L-Threonine 0.11 0.09 0.10 0.07 Limestone 0.74 0.77 0.78 0.76 Dicalcium phosphate 1.33 1.20 1.19 0.92 Salt 0.43 0.48 0.48 0.45 Mineral premix1 0.05 0.05 0.05 0.05 Vitamin premix2 0.05 0.05 0.05 0.05 Composition nutritional and energy G1 G2 F1 F2 Crude Protein, % 18.85 17.61 16.77 15.81 Metabolizable energy (ME), MJ.kg-1 13.82 13.82 13.82 13.60 SID lysine (Lys), % 1.00 0.92 0.88 0.79 Lys: ME, (g.MJ-1) 0.73 0.66 0.64 0.57 Calcium, % 0.70 0.67 0.64 0.59 Standardized phosphorus, % 0.35 0.33 0.32 0.28 Sodium, % 0.17 0.19 0.19 0.18 G1– 24 days (30-50kg); G2– 28 days (50-80kg); F1– 25 days (80-105kg); F2– 26 days (105-130kg); 1Composition per kg of product: calcium: 98.800mg; cobalt: 185mg; copper: 15.750mg; iron: 26.250mg; iodine: 1.470mg; manganese: 41.850mg; zinc: 77.999mg; selenium: 105mg; 2 Composition per kg of product: folic acid: 116.55mg; pantothenic acid: 2.333mg; biotin: 5.28mg; niacin: 5.600mg; pyridoxine: 175mg; riboflavin: 933.3mg; thiamine: 175mg; Vit. A: 1.225.000 U.I.; Vit. D3: 315.000 U.I.; Vit. E: 1.400mg; Vit. K3: 700mg; Vit. B12: 6.825mg. tion and calculated nutritional values of the diets fed to pigs in the growing and finishing phases. G1– 24 days (30-50kg); G2– 28 days (50-80kg); F1– 25 days (80-105kg); F2– 26 days (105-130kg); 1Composition per kg of product: calcium: 98.800mg; cobalt: 185mg; copper: 15.750mg; iron: 26.250mg; iodine: 1.470mg; manganese: 41.850mg; zinc: 77.999mg; selenium: 105mg; 2 Composition per kg of product: folic acid: 116.55mg; pantothenic acid: 2.333mg; biotin: 5.28mg; niacin: 5.600mg; pyridoxine: 175mg; riboflavin: 933.3mg; thiamine: 175mg; Vit. A: 1.225.000 U.I.; Vit. D3: 315.000 U.I.; Vit. E: 1.400mg; Vit. K3: 700mg; Vit. B12: 6.825mg. Slaughter and data collection G1– 24 days (30-50kg); G2– 28 days (50-80kg); F1– 25 days (80-105kg); F2– 26 days (105-130kg); 1Composition per kg of product: calcium: 98.800mg; cobalt: 185mg; copper: 15.750mg; iron: 26.250mg; iodine: 1.470mg; manganese: 41.850mg; zinc: 77.999mg; selenium: 105mg; 2 Composition per kg of product: folic acid: 116.55mg; pantothenic acid: 2.333mg; biotin: 5.28mg; niacin: 5.600mg; pyridoxine: 175mg; riboflavin: 933.3mg; thiamine: 175mg; Vit. A: 1.225.000 U.I.; Vit. D3: 315.000 U.I.; Vit. E: 1.400mg; Vit. K3: 700mg; Vit. B12: 6.825mg. pre-slaughter to minimize stress, the pigs were stunned with electrical system, followed immediately by bleeding, scalding, mechanical depilation and evisceration. The carcass were weighed and divided lengthwise. meter, this device compensates the temperature simultaneously due to an integrated sensor that allows accurate pH measurement. The pH meter calibration was carried out by immersing his sensor in solutions with pH 4 and 7, according to the device manual. The gradient and displacement values were greater than 50 mV / pH and less than 60 mV, respectively, demonstrating that the electrode is functioning properly. Among the readings performed, the After were collected hot carcass weight (HCW) and cold carcass (CCW) (without head and feet); pH 45 minutes post-slaughter (pH-45) and pH 24 hours post-slaughter (pH-24), measured in the ham using the Testo-205® portable pH CARCASS AND MEAT QUALITY IN MALE PIGS HENRIQUE C.M. MUNIZ et al. pH meter was packed in its cap, filled with KCl- based gel, to prevent disbalances. within normality were used to obtain the mean of each sample. In the region of the third and fourth ribs in the caudal-cranial direction, a cross section was performed, where the muscle depth (MD) of Longissimus lumborum and backfat thickness were measured, the entire area was drawn on butter paper for the calculation of loin eye area (LEA) and loin fat area (LFA). For the analysis of color, drip loss, thawing loss, cooking loss and shear force, samples of Longissimus lumborum muscle were collected from the left half- carcass. The carcass yield (CY) and carcass lean meat (CLM), were estimated through equations described by Irgang et al. (1998). The color intensities (L, a* and b*) were evaluated with 2.5 cm thick samples. Three measurements were taken at different sample points, using the Minolta Sensing Spectrophotometer CR-400, with 10 grade of standard observer, D65 illuminant and 11-mm aperture, calibrated with a background white. Statistical analyses All statistical analyzes were performed in Minitab® 2018 statistical software. The qualitative and quantitative data of carcasses of swine, were submitted to analysis of variance by the General Linear Model procedure. Were analyzed, with sexual categories as fixed factor, and carcass weight as co-variable for the following variables carcass yield, carcass lean meat, loin fat area, backfat thickness, loin eye area and muscle depth. The differences between means were compared by the Fisher test and considered significant if P <0.05. Post-slaughter analysis The water-holding capacity was measured according to the methodology of Honikel (1987), comprising the sum of the variables, drip loss, thawing loss and cooking loss. Drip loss was determined using a 150 g meat sample approximately, suspended and wrapped with a plastic bag, without contact, in a cold chamber, being weighed at the beginning and after forty- eight hours. Samples of proportion similar to those used for drip loss (150 g), were submitted to freezing after twenty-four hours of the slaughter, for thawing loss determination (frozen samples weight – thawed samples weight). The samples of 150 g in natura were then cooked in an oven, until the internal temperature reached 70 ºC, and weighed, for cooking loss quantification (in natura samples weight - cooked samples weight). An Acad Bras Cienc (2021) 93(4)  e20200130  4 | 9 RESULTS The carcass traits differed (P <0.05) among the sexual categories. The swine immunocastrated (IM) showed superiority, for hot carcass weight (HCW), cold carcass weight (CCW), carcass lean meat (CLM), loin eye area (LEA) and muscle depth (MD). While swine male surgically castrated (SC), were higher for carcass yield (CY), loin fat area (LFA), backfat thickness (Table II). The pH45, pH24 and shear force did not differ (P >0.05) between the castration groups. However, for meat coloration, only the variable b* (yellowness) did not differ statistically; L (luminosity) and a* (redness) were significantly different (P <0.05), indicating higher values for IM and SC, respectively (Table II). For the The Shear force was determined, with a Warner-Bratzler Shear machine, in six cores 12.5 x 25.0 mm diameter and width, respectively, were removed parallel to the longitudinal orientation of the muscle fibers, per meat sample. A boxplot was used to identify and exclude outliers, results An Acad Bras Cienc (2021) 93(4)  e20200130  4 | 9 CARCASS AND MEAT QUALITY IN MALE PIGS HENRIQUE C.M. MUNIZ et al. Table II. Carcass characteristics, meat quality and water holding capacity determined in the Longissimus lumborum muscle from swine male surgically castrated and immunocastrated. SC IM SEM p-value Carcass Characteristics (n) 24 24 Slaughter weight, Kg 127.80 135.10 1.475 0.001 Hot carcass weight, Kg 93.11 96.45 1.095 0.037 Cold carcass weight, Kg 88.51 92.01 1.065 0.025 Carcass yield, % sw* 72.88 71.38 0.240 0.001 Carcass lean meat, % sw* 57.88 59.93 0.413 0.001 Loin fat area, cm² sw* 24.13 20.23 0.792 0.001 Backfat thickness, mm sw* 16.18 12.89 0.651 0.001 Loin eye area, cm² sw* 56.52 60.63 1.190 0.019 Muscle depth, mm sw* 71.83 74.84 0.748 0.007 Meat Quality (n) 24 24 pH-45 6.49 6.54 0.053 0.493 pH-24 6.02 5.95 0.044 0.270 L* 52.86 55.65 0.619 0.004 a* 7.75 6.72 0.352 0.049 b* 12.94 12.64 0.309 0.496 Shear Force (N) 33.94 36.07 2.460 0.547 Water holding capacity (n) 24 24 Drip Loss (%) 2.21 2.92 0.203 0.019 Thawing loss (%) 5.06 7.28 0.544 0.008 Cooking loss (%) 23.22 25.91 0.903 0.044 SC: Male surgically castrated, IM: Male submitted to immunocastration; SEM: Standard error mean; Backfat thickness: was measured in the region of the third and fourth ribs in the caudal-cranial direction; L*: Luminosity, a*: redness, b*: yellowness; pH- 45: forty-five minutes after slaughter; pH-24: twenty-four hours after slaughter. RESULTS SW*: Variables in which the slaughter weight was utilized with co-variable for analysis of variance. Table II. Carcass characteristics, meat quality and water holding capacity determined in the Longissimus lumborum muscle from swine male surgically castrated and immunocastrated. SC: Male surgically castrated, IM: Male submitted to immunocastration; SEM: Standard error mean; Backfat thickness: was measured in the region of the third and fourth ribs in the caudal-cranial direction; L*: Luminosity, a*: redness, b*: yellowness; pH- 45: forty-five minutes after slaughter; pH-24: twenty-four hours after slaughter. SW*: Variables in which the slaughter weight was utilized with co-variable for analysis of variance. Sellier 2011, Trefan et al. 2013). According to Pauly et al. (2009), the IM have higher intestine, kidney and liver weights. In addition, the total weight of the reproductive organs of the IM is greater, due to the presence of the testicles, and attached glands of the reproductive system, which have not yet reduced to the same proportion as in SC (Skrlep et al. 2010, Batorek et al. 2012). This accounts for the differences between sexual categories, for slaughter weight and hot carcass water-holding capacity variables, IM in relation to SC, obtained higher values (P < 0.05) for drip, thawing and cooking loss (Table II). DISCUSSION Quantitative and qualitative characteristics of pig carcasses are highly influenced by body weight, castration method and genetics (Kouba & An Acad Bras Cienc (2021) 93(4)  e20200130  5 | 9 CARCASS AND MEAT QUALITY IN MALE PIGS HENRIQUE C.M. MUNIZ et al. weight, resulting in as well the higher carcass yield for SC. explained by the slaughter weight. According to Tikk et al. (2008) the meat of heavier carcasses swines, tend to have a lower intensity of red, being confirmed by this study, where IM had higher slaughter weight and lower intensity of a* variable. According to our results, IM were superior to SC for CLM, MD and LEA measurements, these variables have a positive correlation (Rehfeldt & Kuhn 2006). The likely explanation for this would be, male pigs submitted to immunocastration have greater anabolic potential, a result of the presence of gonadal hormones during most of the fattening period. Causing higher protein deposition in the carcass, resulting in value higher CLM after slaughter in relation to SC, that because they do not have testicles, they perform greater lipid deposition. (Pauly et al. 2009, Boler et al. 2012, Muniz et al. 2019). Batorek et al. (2012) demonstrated through meta-analytic study that entire male pigs have high values for shear force. The compensatory growth that occurs after the second dose of the vaccine in swines immunocastrated, produces a higher in vivo protein turnover, increasing post- mortem proteolysis which makes the meat from immunocastrated swine softer (Therkildsen et al. 2004, Lametsch et al. 2006). Generating similarity in the values found, for the variable shear force between IM and SC, as demonstrated in our study. The amount of fat in pig carcasses is influenced by the castration method, due to changes in the animal’s lipid metabolism (Kouba & Sellier 2011). Both forms of castration, surgical or vaccine-induced, reduce steroid hormones levels in the blood, resulting in increased lipid deposition (Schreurs et al. 2008, Fàbrega et al. 2010). However, in the IM increases the rate of lipid deposition after the second dose of the vaccine, 28 days before slaughter, (Lealiifano et al. 2011). Already SC, have this lipid metabolism from the third day of life, this may be the explanation for the presence the highest backfat thickness and loin fat area, resulting in a fatter carcass, for this sexual category. Unlike Boler et al. An Acad Bras Cienc (2021) 93(4)  e20200130  6 | 9 CARCASS AND MEAT QUALITY IN MALE PIGS BOLER DD, KILLEFER J, MEEUWSE DM, KING VL, MCKEITH FK & DILGER AC. 2012. Effects of slaughter time post- second injection on carcass cutting yields and bacon characteristics of immunologically castrated male pigs. J Anim Sci 90: 334-344. losses of water by cooking in samples whose pH was less than 6.0. These higher losses found may be switched on to the acceleration of the denaturation process of myofibrillar proteins during cooking, due to pH and temperature, justifying for the values obtained for cooking loss in our study. BOLER DD, PULS CL, CLARK DL, ELLIS M, SCHROEDER AL, MATZAT PD, KILLEFER J, MCKEITH FK & DILGER AC. 2014. Effects of immunological castration (Improvest) on changes in dressing percentage and carcass characteristics of finishing pigs. J Anim Sci 91: 359-368. CONCLUSION CALDARA FR, SANTOS VMO, SANTIAGO JC, ALMEIDA PAZ ICL, GARCIA RG, VARGAS JUNIOR FM, SANTOS LS & NÄÄS IA. 2012. Propriedades físicas e sensoriais da carne suína PSE. Rev Bras Saúde Prod Ani 13(3): 815-824. CALDARA FR, SANTOS VMO, SANTIAGO JC, ALMEIDA PAZ ICL, GARCIA RG, VARGAS JUNIOR FM, SANTOS LS & NÄÄS IA. 2012. Propriedades físicas e sensoriais da carne suína PSE. Rev Bras Saúde Prod Ani 13(3): 815-824. In conclusion, this study confirmed that the carcass of male pigs, submitted immunocastration, has a lower yield. However, immunocastrated pigs presented higher amounts of meat and less fat content when compared to carcass of male pigs surgically castrated. The meat of immunocastrated pigs retains less water and has smaller redness color compared to surgically castrated pigs, and therefore can be recommended for sausage production. DUNSHEA FR ET AL. 2013. The effect of immunization against GnRF on nutrient requirements of male pigs: A review. Animal 7(11): 1769-1778. DUNSHEA FR ET AL. 2013. The effect of immunization against GnRF on nutrient requirements of male pigs: A review. Animal 7(11): 1769-1778. DUNSHEA FR ET AL. 2001. Vaccination of boars with a GnRH vaccine (Improvac) eliminates boar taint and increases growth performance. J Anim Sci 79: 2524-2535. DUNSHEA FR ET AL. 2001. Vaccination of boars with a GnRH vaccine (Improvac) eliminates boar taint and increases growth performance. J Anim Sci 79: 2524-2535. GISPERT M, OLIVER MA, VELARDE A, SUAREZ P, PÉREZ J & FONT-I- FURNOLS M. 2010. Carcass and meat quality characteristics of immunocastrated male, surgically castrated male, entire male and female pigs. Meat Sci 85: 664-670. HONIKEL. 1987. How to measure the water-holding capacity of meat? Recommendation of standardized methods. In: Tarrant PV et al. (Eds), Evaluation and control of meat quality in pigs, Dordrecht, The Netherlands: Martinus Nijhof, p. 129-142. HONIKEL. 1987. How to measure the water-holding capacity of meat? Recommendation of standardized methods. In: Tarrant PV et al. (Eds), Evaluation and control of meat quality in pigs, Dordrecht, The Netherlands: Martinus Nijhof, p. 129-142. Acknowledgments This study was carried out with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001 and the financial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process 425055/2016-7). HUFF-LONERGAN E & LONERGAN SM. 2005. Mechanisms of water-holding capacity of meat: The role of postmortem biochemical and structural changes. Meat Sci 71: 194-204. IRGANG R, GUIDONI AL, BERLITZ D & CORSO C. 1998. Medidas de espessura de toucinho e de profundidade de músculo para estimar rendimento de carne magra em carcaças de suínos. Rev Bras Zoot 27(5): 928-935. IRGANG R, GUIDONI AL, BERLITZ D & CORSO C. 1998. Medidas de espessura de toucinho e de profundidade de músculo para estimar rendimento de carne magra em carcaças de suínos. Rev Bras Zoot 27(5): 928-935. DISCUSSION (2014), who state that immunocastration does not affect water-holding in the carcass. Our study found differences, in the variables that quantify water holding capacity between the sexual categories. The value found for CLM in our study, may be the explanation for the lower water holding capacity, obtained by IM. According to Schiavon & Emmans (2000), protein deposition retains more as, twice as much water as lipid deposition. With this, the IM has more water to be lost in industrial processes, such as cooling (drip loss), freezing followed by thawing (thawing loss) and cooking (cooking loss), validating the values found by our study. Color is a subjective indication of meat quality for the consumer, although the color indicators described in the literature are inconsistent (Aluwé et al. 2013). The IM obtained lower pH-24 and higher drop in pH post-mortem, however, nothing statistically significant. The pH drop causes, electrostatic repulsion between myofilaments, increasing the distance between myofibrils, and with that the refraction of light (Swatland 2004), thus increasing the value for variable L, in samples this sexual category. For variable a*, the difference obtained can be The drop in pH post-mortem results in myofibrillar distancing, interfering with the loss of inter and intracellular water (Swatland 2004, Huff-Lonergan & Lonergan 2005). In a study by Bertram et al. 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Meat Science 86: 498-504. MUNIZ HCM, LIMA ES, SCHNEIDER LI, KLEIN DR, ROCHA LT, NÖRNBERG JL, QUADROS ARB & OLIVEIRA V. 2021. Carcass characteristics and meat quality of male pigs submitted to surgical or immunological castration. An Acad Bras Cienc 93: e20200130. DOI 10.1590/0001-3765202120200130. Manuscript received on January 31, 2020; accepted for publication on June 26, 2020 Manuscript received on January 31, 2020; accepted for publication on June 26, 2020 REHFELDT C & KUHN G. 2006. Consequences of birth weight for postnatal growth performance and carcass quality in pigs as related to myogenesis. J Anim Sci 84: 113-123. HENRIQUE C.M. MUNIZ et al. HENRIQUE C.M. MUNIZ et al. 1Universidade Federal de Santa Maria, Departamento de Zootecnia, Cidade Universitária, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil 2Universidade Federal de Santa Maria, Departamento de Tecnologia e Ciência dos Alimentos, Cidade Universitária, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil 1Universidade Federal de Santa Maria, Departamento de Zootecnia, Cidade Universitária, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil 2Universidade Federal de Santa Maria, Departamento de Tecnologia e Ciência dos Alimentos, Cidade Universitária, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil 1Universidade Federal de Santa Maria, Departamento de Zootecnia, Cidade Universitária, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil 2Universidade Federal de Santa Maria, Departamento de Tecnologia e Ciência dos Alimentos, Cidade Universitária, Av. Roraima, 1000, 97105-900 Santa Maria, RS, Brazil An Acad Bras Cienc (2021) 93(4)  e20200130  9 | 9 ) 93(4)  e20200130  8 | 9  Manuscript received on January 31, 2020; accepted for publication on June 26, 2020 HENRIQUE C.M. MUNIZ1 https://orcid.org/0000-0002-4512-4424 EDUARDA S. DE LIMA2 https://orcid.org/0000-0001-5959-1501 LUCIANE I. SCHNEIDER1 https://orcid.org/0000-0002-8542-6937 DANIELA R. KLEIN1 https://orcid.org/0000-0002-0429-8268 LEONARDO T. DA ROCHA1 https://orcid.org/0000-0003-2362-7589 JOSÉ L. NÖRNBERG2 https://orcid.org/0000-0002-8366-4480 ARLEI R.B. DE QUADROS1 https://orcid.org/0000-0001-6090-3410 VLADIMIR DE OLIVEIRA1 https://orcid.org/0000-0002-9292-8943 HENRIQUE C.M. MUNIZ1 https://orcid.org/0000-0002-4512-4424 EDUARDA S. DE LIMA2 https://orcid.org/0000-0001-5959-1501 LUCIANE I. SCHNEIDER1 https://orcid.org/0000-0002-8542-6937 DANIELA R. KLEIN1 https://orcid.org/0000-0002-0429-8268 LEONARDO T. DA ROCHA1 https://orcid.org/0000-0003-2362-7589 JOSÉ L. NÖRNBERG2 https://orcid.org/0000-0002-8366-4480 ARLEI R.B. DE QUADROS1 https://orcid.org/0000-0001-6090-3410 VLADIMIR DE OLIVEIRA1 https://orcid.org/0000-0002-9292-8943 HENRIQUE C.M. MUNIZ1 https://orcid.org/0000-0002-4512-4424 EDUARDA S. DE LIMA2 https://orcid.org/0000-0001-5959-1501 LUCIANE I. SCHNEIDER1 https://orcid.org/0000-0002-8542-6937 DANIELA R. KLEIN1 https://orcid.org/0000-0002-0429-8268 LEONARDO T. DA ROCHA1 https://orcid.org/0000-0003-2362-7589 JOSÉ L. NÖRNBERG2 https://orcid.org/0000-0002-8366-4480 ARLEI R.B. DE QUADROS1 https://orcid.org/0000-0001-6090-3410 VLADIMIR DE OLIVEIRA1 https://orcid.org/0000-0002-9292-8943 HENRIQUE C.M. MUNIZ1 https://orcid.org/0000-0002-4512-4424 SCHIAVON S & EMMANS GC. 2000. A model to predict water intake of a pig growing in a known environment on a known diet. Br J Nutr 84: 873-883. SCHREURS NM, GARCIA F, JURIE C, AGABRIEL J, MICOL D, BAUCHART D, LISTRAT A & PICARD B. 2008. Meta-analysis of the effect of animal maturity on muscle characteristics in different muscles, breeds, and sexes of cattle. J Anim Sci 86: 2872-2887. SKRLEP M, SEGULA B, ZAJEC M, KASTELIC M, KOSOROK S, FAZARINC G & CANDEK-POTOKAR M. 2010. Effect of immunocastration (Improvac ®) in fattening pigs I: growth performance, reproductive organs and malodorous com pounds. Slov Vet Res 47(2): 57-64. SWATLAND HJ. 2004. Progress in understanding the paleness of meat with a low pH. S Afr J Anim Sci 34 (Supplement 2): 1-7. THERKILDSEN M, VESTERGAARD M, BUSK H, JENSEN MT, RIIS B, KARLSSON AH, KRISTENSEN L, ERTBJERG P & OKSBJERG N. 2004. Compensatory growth in slaughter pigs – in vitro muscle protein turnover at slaughter, circulating An Acad Bras Cienc (2021) 93(4)  e20200130  8 | 9 CARCASS AND MEAT QUALITY IN MALE PIGS Author contributions Henrique da Costa Mendes Muniz, contributed to performed the experiment, analyzed the data, writing of the manuscript and edited, for the periodic; Vladimir de Oliveira acted as a master’s advisor for Henrique da Costa Mendes Muniz, participating in the acquisition the financial resources of this project research, analysis the data and revised the manuscript for intellectual content; Eduarda S. de Lima, Luciane I. Schneider, Daniela R. Klein and Leonardo T. da Rocha assisted in performing the experiment and revised the manuscript; José L. Nörnberg and Arlei R.B. de Quadros revised the manuscript for intellectual content. All the authors have read and approved the final manuscript.
https://openalex.org/W2918036813
https://jneurodevdisorders.biomedcentral.com/track/pdf/10.1186/s11689-016-9137-x
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Journal of Neurodevelopmental Disorders reviewer acknowledgement 2015
Journal of neurodevelopmental disorders
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REVIEWER ACKNOWLEDGEMENT Open Access Journal of Neurodevelopmental Disorders reviewer acknowledgement 2015 Joseph Piven © 2016 Piven. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Piven Journal of Neurodevelopmental Disorders (2016) 8:3 DOI 10.1186/s11689-016-9137-x Piven Journal of Neurodevelopmental Disorders (2016) 8:3 DOI 10.1186/s11689-016-9137-x Contributing reviewers We thank all of the reviewers who have contributed to the journal in volume 7 (2015). High quality and timely reviews are critical to the overall quality of the journal. We are committed to providing a unique and important outlet for scholarship regarding neurodevelopmental disorders and are indebted to the outstanding reviewers who have contributed their time over the last year in helping us to reach this goal. John Constantino United States of America Cheryl Corcoran United States of America Jacqueline Crawley United States of America Julie Daniels United States of America Petrus de Vries South Africa William DeBassio United States of America Adele Dimian United States of America Anastasia Dimitropoulos United States of America Ilan Dinstein Israel Dennis Drayna United States of America Inge-Marie Eigsti United States of America Jed Elison United States of America Susan Faja United States of America Reinhold Feldmann Germany Susanna Fryer United States of America Oscar Gershanik Argentina Simon Gregory United States of America Amanda Hampton Wray United States of America Pamela Heaton United Kingdom Melissa Hines United Kingdom Lisa M Jacola United States of America Anna Jansen Belgium Chris Jarrold United Kingdom Loydie Jerome-Majewska Canada John Constantino United States of America Cheryl Corcoran United States of America Jacqueline Crawley United States of America Julie Daniels United States of America Petrus de Vries South Africa William DeBassio United States of America Adele Dimian United States of America Anastasia Dimitropoulos United States of America Ilan Dinstein Israel Dennis Drayna United States of America Inge-Marie Eigsti United States of America Jed Elison United States of America Susan Faja United States of America Reinhold Feldmann Germany Susanna Fryer United States of America Oscar Gershanik Argentina Simon Gregory United States of America Amanda Hampton Wray United States of America Pamela Heaton United Kingdom Melissa Hines United Kingdom Lisa M Jacola United States of America Anna Jansen Belgium Chris Jarrold United Kingdom Loydie Jerome-Majewska Canada Ted Abel Susan Faja United States of America Reinhold Feldmann Germany Susanna Fryer United States of America Oscar Gershanik Argentina Simon Gregory United States of America Amanda Hampton Wray United States of America Pamela Heaton United Kingdom Melissa Hines United Kingdom Lisa M Jacola United States of America Anna Jansen Belgium Chris Jarrold United Kingdom Loydie Jerome-Majewska Canada Ted Abel United States of America Ted Abel United States of America Kevin Antshel United States of America Simon Baron-Cohen United Kingdom Carrie Bearden United States of America Nan Bernstein Ratner United States of America Thomas Bourgeron France Randal Carpenter United States of America Tony Charman United Kingdom Katarzyna Chawarska United States of America Rina Cianfaglione United Kingdom Claire Coles United States of America John Colombo United States of America Correspondence: jneurodevdisorders@b Carolina Institute for Developmental Dis at Chapel Hill, 100 Renee Lynne Court, Ted Abel United States of America Kevin Antshel United States of America Simon Baron-Cohen United Kingdom Carrie Bearden United States of America Nan Bernstein Ratner United States of America Thomas Bourgeron France Randal Carpenter United States of America Tony Charman United Kingdom Katarzyna Chawarska United States of America Rina Cianfaglione United Kingdom Claire Coles United States of America John Colombo United States of America Correspondence: jneurodevdisorders@b Carolina Institute for Developmental Di at Chapel Hill, 100 Renee Lynne Court, Correspondence: jneurodevdisorders@biomedcentral.com Carolina Institute for Developmental Disabilities, University of North Carolina at Chapel Hill, 100 Renee Lynne Court, Carrboro, NC 27510, USA © 2016 Piven. 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https://openalex.org/W4361833670
https://figshare.com/articles/journal_contribution/Supplementary_Figure_4_from_Prediction_and_Genetic_Demonstration_of_a_Role_for_Activator_E2Fs_in_Myc-Induced_Tumors/22390641/1/files/39836172.pdf
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Supplementary Figure 3 from Prediction and Genetic Demonstration of a Role for Activator E2Fs in Myc-Induced Tumors
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Combined Datasets Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 High E2F2 Low E2F2 Percent of Tumor Samples * * * A GSE 7390 Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 Low E2F2 High E2F2 Percent of Tumor Samples * * * GSE 2990 75 100 High E2F2 Low E2F2 r B C Combined Datasets Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 High E2F2 Low E2F2 Percent of Tumor Samples * * * A GSE 7390 Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 Low E2F2 High E2F2 Percent of Tumor Samples * * * GSE 2990 75 100 High E2F2 Low E2F2 r B C A Combined Datasets Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 High E2F2 Low E2F2 Percent of Tumor Samples * * * A Combined Datasets Lum B Lum A Basal ErbB2 Normal 0 25 50 Percent of Samp * * GSE 7390 Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 Low E2F2 High E2F2 Percent of Tumor Samples * * * GSE 2990 Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 High E2F2 Low E2F2 Percent of Tumor Samples * * * B C GSE 7390 Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 Low E2F2 High E2F2 Percent of Tumor Samples * * * B B Percent of Tumor S C GSE 2990 Lum B Lum A Basal ErbB2 Normal 0 25 50 75 100 High E2F2 Low E2F2 Percent of Tumor Samples * * * C Percent of Tumor S
https://openalex.org/W2771688822
https://research.bond.edu.au/files/28863075/Risk_of_bias_reporting_in_the_recent_animal_focal_cerebral_ischaemia_literature.pdf
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Aligning Pathology Assessment in a Learner-Cantered Undergraduate Medical Curriculum
Biomedical journal of scientific & technical research
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General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Methods: We developed and used text analytic approaches to automatically ascertain re- porting of measures to reduce risk of bias from full-text articles describing animal experi- ments inducing middle cerebral artery occlusion (MCAO) or modelling lacunar stroke. Results: Compared with previous assessments, there were improvements in the reporting of measures taken to reduce risks of bias in the MCAO literature but not in the lacunar stroke literature. Accuracy of automated annotation of risk of bias in the MCAO literature was 86% (randomization), 94% (blinding) and 100% (sample size calculation); and in the lacunar stroke literature accuracy was 67% (randomization), 91% (blinding) and 96% (sample size calculation). Methods: We developed and used text analytic approaches to automatically ascertain re- porting of measures to reduce risk of bias from full-text articles describing animal experi- ments inducing middle cerebral artery occlusion (MCAO) or modelling lacunar stroke. Results: Compared with previous assessments, there were improvements in the reporting of measures taken to reduce risks of bias in the MCAO literature but not in the lacunar stroke literature. Accuracy of automated annotation of risk of bias in the MCAO literature was 86% (randomization), 94% (blinding) and 100% (sample size calculation); and in the lacunar stroke literature accuracy was 67% (randomization), 91% (blinding) and 96% (sample size calculation). Discussion: There remains substantial opportunity for improvement in the reporting of an- imal research modelling stroke, particularly in the lacunar stroke literature. Further, auto- mated tools perform sufficiently well to identify whether studies report blinded assessment of outcome, but improvements are required in the tools to ascertain whether randomization and a sample size calculation were reported. Bond University Research Repository Aligning Pathology Assessment in a Learner-Centered Undergraduate Medical Curriculum Doshi, Neelam; Tepper, Carmel; Wright, Robert Gordon Published in: Biomedical Journal of Scientific and Technical Research DOI: 10.26717/BJSTR.2017.01.000435 Licence: Unspecified Link to output in Bond University research repository. Recommended citation(APA): Doshi, N., Tepper, C., & Wright, R. G. (2017). Aligning Pathology Assessment in a Learner-Centered Undergraduate Medical Curriculum. Biomedical Journal of Scientific and Technical Research, 1(5). https://doi.org/10.26717/BJSTR.2017.01.000435 Bond University Research Repository Aligning Pathology Assessment in a Learner-Centered Undergraduate Medical Curriculum Doshi, Neelam; Tepper, Carmel; Wright, Robert Gordon Published in: Biomedical Journal of Scientific and Technical Research DOI: 10.26717/BJSTR.2017.01.000435 Licence: Unspecified Link to output in Bond University research repository. Recommended citation(APA): Doshi, N., Tepper, C., & Wright, R. G. (2017). Aligning Pathology Assessment in a Learner-Centered Undergraduate Medical Curriculum. Biomedical Journal of Scientific and Technical Research, 1(5). https://doi.org/10.26717/BJSTR.2017.01.000435 Bond University Research Repository Doshi, Neelam; Tepper, Carmel; Wright, Robert Gordon Published in: Biomedical Journal of Scientific and Technical Research Link to output in Bond University research repository. Recommended citation(APA): Doshi, N., Tepper, C., & Wright, R. G. (2017). Aligning Pathology Assessment in a Learner-Centered Undergraduate Medical Curriculum. Biomedical Journal of Scientific and Technical Research, 1(5). https://doi.org/10.26717/BJSTR.2017.01.000435 Recommended citation(APA): Doshi, N., Tepper, C., & Wright, R. G. (2017). Aligning Pathology Assessment in a Learner-Centered Undergraduate Medical Curriculum. Biomedical Journal of Scientific and Technical Research, 1(5). https://doi.org/10.26717/BJSTR.2017.01.000435 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. For more information, or if you believe that this document breaches copyright, please contact the Bond University rese coordinator. Download date: 24 Oct 2024 Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 Review Article Risk of bias reporting in the recent animal focal cerebral ischaemia literature Zsanett Bahor1, Jing Liao1, Malcolm R. Macleod1, Alexandra Bannach-Brown1, Sarah K. McCann1, Kimberley E. Wever2, James Thomas3, Thomas Ottavi4, David W. Howells4, Andrew Rice5, Sophia Ananiadou6 and Emily Sena1 1Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor’s Building, Little France Crescent, Edinburgh, U.K.; 2SYRCLE, Nijmegen Institute for Health Sciences, Radboud University Medical Center, Nijmegen, Netherlands; 3Evidence for Policy and Practice Information and Coordinating (EPPI)-Centre, Social Science Research Unit, UCL Institute of Education, University of London, London, U.K.; 4School of Medicine, University of Tasmania, Hobart, TAS, Australia; 5Pain Research, Department of Surgery and Cancer, Imperial College, London, U.K.; 6School of Computer Science and National Centre for Text Mining, University of Manchester, 131 Princess Street, Manchester, U.K. Correspondence: Malcolm Macleod (malcolm macleod@ed ac uk) Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 Received: 20 April 2017 Revised: 19 August 2017 Accepted: 07 September 2017 Version of Record published: 13 October 2017 Background: Findings from in vivo research may be less reliable where studies do not re- port measures to reduce risks of bias. The experimental stroke community has been at the forefront of implementing changes to improve reporting, but it is not known whether these efforts are associated with continuous improvements. Our aims here were firstly to validate an automated tool to assess risks of bias in published works, and secondly to assess the reporting of measures taken to reduce the risk of bias within recent literature for two exper- imental models of stroke. Risk of bias reporting in the recent animal focal cerebral ischaemia literature Zsanett Bahor1, Jing Liao1, Malcolm R. Macleod1, Alexandra Bannach-Brown1, Sarah K. McCann1, Kimberley E. Wever2, James Thomas3, Thomas Ottavi4, David W. Howells4, Andrew Rice5, Sophia Ananiadou6 and Emily Sena1 1Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor’s Building, Little France Crescent, Edinburgh, U.K.; 2SYRCLE, Nijmegen Institute for Health Sciences, Radboud University Medical Center, Nijmegen, Netherlands; 3Evidence for Policy and Practice Information and Coordinating (EPPI)-Centre, Social Science Research Unit, UCL Institute of Education, University of London, London, U.K.; 4School of Medicine, University of Tasmania, Hobart, TAS, Australia; 5Pain Research, Department of Surgery and Cancer, Imperial College, London, U.K.; 6School of Computer Science and National Centre for Text Mining, University of Manchester, 131 Princess Street, Manchester, U.K. Correspondence: Malcolm Macleod (malcolm.macleod@ed.ac.uk) c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). it is apparent that studies which do not report, for instance, randomization of animals to group or the blinded assess- ment of outcome, give inflated estimates of treatment effect [5]. Identifying the true prevalence of measures to reduce the risk of bias is not straightforward because it may be that scientists have taken account of these measures but not, for whatever reason, reported this in their manuscript. Recognition of this has led to the development of reporting standards for in vivo research including the ARRIVE guidelines [6] and the Landis checklist [7]. it is apparent that studies which do not report, for instance, randomization of animals to group or the blinded assess- ment of outcome, give inflated estimates of treatment effect [5]. Identifying the true prevalence of measures to reduce the risk of bias is not straightforward because it may be that scientists have taken account of these measures but not, for whatever reason, reported this in their manuscript. Recognition of this has led to the development of reporting standards for in vivo research including the ARRIVE guidelines [6] and the Landis checklist [7]. The in vivo stroke research community have been amongst the first to adopt best practice guidelines for the conduct and reporting of animal studies. Following the demonstration that the reporting of risks of bias in in vivo stroke research was low [8], Good Laboratory Practice guidelines for in vivo stroke research were published in 2009 [9] and were adopted as editorial policy by a number of Journals including ‘Stroke’. Subsequently, Minnerup et al. [10] have shown a substantial increase in the prevalence of reported randomization of group allocation, blinded conduct of the experiment, blinded assessment of outcome and sample size calculations in the years from 2010 to 2013. Recent research suggests that improvement may been limited to specific journals reporting predominantly stroke research [11]. In 2014, Pedder et al. [12] reported a systematic review of animal studies in lacunar stroke published between 1992 and 2011, where the reporting of measures to reduce risks of bias was in general better than had been observed in the middle cerebral artery occlusion (MCAO) literature in 2007 [8]. It is not known, however whether the quality of reporting in lacunar stroke has improved since that time Figures 1–3. Ascertainment of improvement in reporting might serve as an important outcome measure for efforts to improve performance at the level of a journal, funder, or institution, as part of a research improvement activity. However, one problem with this approach is that ascertainment of reporting of measures to reduce risks of bias in published work is a time-consuming process, requiring review of full-text by at least two independent assessors to minimize risk of human error and taking around 1 h for each manuscript. We have therefore set out to develop text mining tools to automatically ascertain the reporting of key measures to reduce risks of bias in published works describing animal research modelling of MCAO and lacunar stroke. Methods This development work was not guided by a study protocol. Criteria for successful performance of a text mining system were developed at a consensus meeting organized by the SLIM (Systematic Living Information Machine) consortium in April 2016 (prior to the commencement of this project), which recommended that a tool performing with sensitivity and specificity of at least 0.80 could make a valuable contribution. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Introduction There has been much recent interest in strategies to improve the usefulness of findings from biomedical research [1]. This has been occasioned by a growing realization that many ‘findings’ in the published literature cannot be replicated, either in single replication attempts [2,3] or in formal replication studies ([4], https://osf.io/e81xl/wiki/home/). There are a number of potential reasons for this, including low positive predictive values of the origi- nator study, publication bias, heterogeneity of treatment effects due to the variable presence of unknown modifying variables, overreliance on tests of statistical rather than biological significance and flexibility in study protocols and statistical analysis plans. Notwithstanding these possibilities, it may be that findings reported from laboratory research are overstated because individual studies are at risk of bias, leading to an overstatement of treatment effects. Across the animal modelling of a range of neurological conditions 2525 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons A ib i Li 4 0 (CC BY) Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 Identification of relevant manuscripts p For MCAO studies, we searched PUBMED on 4 October 2016 with the terms <middle cerebral artery occlusion> OR <MCAO>, limited to animals, with a date of record completion later than 4 October 2014. For lacunar stroke studies, we searched PUBMED on 11 January 2017 using the terms employed in Pedder et al., limited to a deposition date between 6 March 2012 (the date of the search which informed the Pedder review) and 11 January 2017 for the terms ((< Biological Models> OR <Animal Disease Models> OR <Animal Models> OR <Animals> OR <mammals> OR <primates> OR <mice> OR <rats>) AND (<cerebrovascular disorders> OR <basal ganglia cerebrovascu- lar disease> OR <brain ischemia> OR <cerebrovascular accident> OR <brain infarction> OR <brain hypoxia ischemia> OR <brain edema> OR <cerebrovascular accident> OR <vascular dementia> OR <intracranial arte- rial diseases> OR <intracranial vasospasm>) AND (<Stroke* [TW]> OR <cerebrovasc* [TW]> OR <cerebral vasc* [TW]> OR <cerebral* [TW]>) AND (<micro vessel [tw]> OR <small vessel [tw]> OR <perforat* vessel [tw]> OR <arteriole [tw]> OR <lacunar stroke [tw]> OR <lacunar infarct [tw]> OR <small stroke* [tw]> OR <small occlusion* [tw]> OR <small disease* [tw]> OR <micro stroke* [tw]> OR <micro occlusion* [tw]> OR <micro disease* [tw]> OR <lacun* infarct* [tw]> OR <lacun* lesion* [tw]> OR <lacun* stroke* [tw]> OR <small infarct* [tw]> OR <small lesion* [tw]> OR <small stroke* [tw]> OR <subcortical infarct* [tw]> OR <subcortical lesion* [tw]> OR <subcortical stroke* [tw]> OR <deep infarct* [tw]> OR <deep lesion* [tw]> OR <deep stroke* [tw]> OR <silent infarct* [tw]> OR <silent lesion* [tw]> OR <silent stroke* [tw])) NOT (<heart [tiab]> OR <bone [tiab]> OR <eye [tiab]> OR <lung [tiab]> OR <kidney [tiab]> OR <liver [tiab]> OR <renal [tiab]> OR <intestine* [tiab]> OR <spinal [tiab]> OR <pulmonary [tiab]> OR <hepatic [tiab]> OR <global [tiab]> OR <AD [tw]> OR <PD [tw]> OR <Alzheimer* [tw]> OR <Parkinson* [tw]> OR <epilepsy [tw]> OR <MS [tw]> OR <Multiple Sclerosis [tw]), limited to animals. Each of these search results was screened by a single investigator using the NC3Rs/CAMARADES SyRF facility (app.syrf.org.uk) for inclusion. We also extracted the journal in which the work was published. Full-text pdf articles were retrieved through the reference management software EndNote X8 using our institutional full-text subscriptions, and subsequently converted to text files using PDF Extractor SDK. 2526 Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 ure 1. Reporting of risks of bias in the recent MCAO literature. Identification of relevant manuscripts porting of (A) blinding, (B) randomization and (C) sample size calculation in the middle cerebral artery occlusion literature in month epochs to October 2016. Values represent mean and 95% Clopper–Pearson exact confidence intervals. gure 1 Reporting of risks of bias in the recent MCAO literature Figure 1. Reporting of risks of bias in the recent MCAO literature. Figure 1. Reporting of risks of bias in the recent MCAO literature. Reporting of (A) blinding, (B) randomization and (C) sample size calculation in the middle cerebral artery occlusion literature in 6-month epochs to October 2016. Values represent mean and 95% Clopper–Pearson exact confidence intervals. Figure 1. Reporting of risks of bias in the recent MCAO literature. Reporting of (A) blinding, (B) randomization and (C) sample size calculation in the middle cerebral artery occlusion literature in 6-month epochs to October 2016. Values represent mean and 95% Clopper–Pearson exact confidence intervals. g linding, (B) randomization and (C) sample size calculation in the middle cerebral artery occlusion literature in o October 2016. Values represent mean and 95% Clopper–Pearson exact confidence intervals. Development of ‘regular expressions’ Development of ‘regular expressions’ p g p In the context of a systematic review of studies describing animal models of psychosis, we collected phrases associated with the reporting of randomization, blinded assessment of outcome and sample size calculations and used these to develop target regular expressions (“RegEx”s). These were informally tested through application to a second dataset 2527 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 Reporting of risks of bias in the recent literature compared with previous estimates. ng of blinding, randomization and sample size calculation in the MCAO literature collated from systematic reviews and d in 2007 in Sena et al. [8] (column 1), from the journal Stroke from 2010 to 2013 reported by Minnerup et al. [10] (column m 2014 to 2016 reported here (column 3). (B) Reporting of blinding, randomization and sample size calculation in the al lacunar stroke literature from the systematic review of 2012 [12] (column 1), and from 2012 to 2017 reported here Figure 3. Reporting of risks of bias in the recent literature compared with previous estimates. (A) Reporting of blinding randomization and sample size calculation in the MCAO literature collated from systematic reviews Figure 3. Reporting of risks of bias in the recent literature compared with previous estimates. Figure 3. Reporting of risks of bias in the recent literature compared with previous estimates. (A) Reporting of blinding, randomization and sample size calculation in the MCAO literature collated from systematic reviews and summarized in 2007 in Sena et al. [8] (column 1), from the journal Stroke from 2010 to 2013 reported by Minnerup et al. [10] (column 2), and from 2014 to 2016 reported here (column 3). (B) Reporting of blinding, randomization and sample size calculation in the experimental lacunar stroke literature from the systematic review of 2012 [12] (column 1), and from 2012 to 2017 reported here (column 2). (A) Reporting of blinding, randomization and sample size calculation in the MCAO literature collated from systematic reviews and summarized in 2007 in Sena et al. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Development of ‘regular expressions’ [8] (column 1), from the journal Stroke from 2010 to 2013 reported by Minnerup et al. [10] (column 2), and from 2014 to 2016 reported here (column 3). (B) Reporting of blinding, randomization and sample size calculation in the experimental lacunar stroke literature from the systematic review of 2012 [12] (column 1), and from 2012 to 2017 reported here (column 2). of 1173 full-text publications describing in vivo research where risks of bias had been previously extracted [13], and no changes were made. We then applied these tools to the converted full-text of stroke publications using the Grepl function in R, resulting in a call of ‘true’ or ‘false’ for each of the measures to reduce risk of bias. We then conducted manual (‘gold standard’) ascertainment of reporting of measures to reduce risk of bias for each publication. To com- pare the text analytic with human ‘gold standard’, we calculated sensitivity (effectiveness of the tool in identifying positives), specificity (effectiveness of the tool in identifying negatives) and accuracy (number of publications cor- rectly labelled as a proportion of the total number of publications).The R code is available at GitHub (link provided on publication). We present the prevalence of reporting of randomization of animals to experimental groups, blinded assessment of outcome and sample size calculation, along with exact binomial confidence intervals (derived using cii in STATA) for each dataset and, for the MCAO literature, in quartiles of PUBMED accession date and by journal. Least squares linear regression was used to understand the correlation between time of publication and the reporting of measures taken to reduce the risk of bias. 2528 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 eporting of risks of bias by Journal. f (A) blinding, (B) randomization and (C) sample size calculation in the middle cerebral artery occlusion literature in to October 2016 by journal of publication for journals with five or more relevant manuscripts, ranked by performance. sent mean and 95% Clopper–Pearson exact confidence intervals. The grey shaded bar represents the 95% confidence e overall performance across 918 manuscripts. thor(s). Development of ‘regular expressions’ This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons 4 0 (CC BY) Figure 2. Reporting of risks of bias by Journal. Reporting of (A) blinding, (B) randomization and (C) sample size calculation in the middle cerebral artery the 2 years to October 2016 by journal of publication for journals with five or more relevant manuscripts, ra Values represent mean and 95% Clopper–Pearson exact confidence intervals. The grey shaded bar represent interval of the overall performance across 918 manuscripts. c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed Attribution License 4.0 (CC BY). Fi 2 R ti f i k f bi b J l Figure 2. Reporting of risks of bias by Journal. g p g y Reporting of (A) blinding, (B) randomization and (C) sample size calculation in the middle cerebral artery occlusion literature in the 2 years to October 2016 by journal of publication for journals with five or more relevant manuscripts, ranked by performance. Values represent mean and 95% Clopper–Pearson exact confidence intervals. The grey shaded bar represents the 95% confidence interval of the overall performance across 918 manuscripts. Reporting of (A) blinding, (B) randomization and (C) sample size calculation in the middle cerebral artery occlusion literature in the 2 years to October 2016 by journal of publication for journals with five or more relevant manuscripts, ranked by performance. Values represent mean and 95% Clopper–Pearson exact confidence intervals. The grey shaded bar represents the 95% confidence interval of the overall performance across 918 manuscripts. 2529 c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4 0 (CC BY) c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Results In 2259 studies reporting animal models of psychosis, the RegEx had performed above our pre-defined performance criteria (sensitivity of 0.80 and specificity of 0.91 for randomization, sensitivity of 0.83 and specificity of 0.95 for blinding, and sensitivity of 1.00 and specificity of 0.96 for sample size calculation). In a second dataset of in vivo studies from leading U.K. institutions [13], the tool was somewhat less efficient in calling papers as randomized (sensitivity of 1.00 and specificity of 0.62), but blinding and sample size calculation ascertainment were still above our successful performance criteria level of 0.80 (sensitivity of 0.88 and specificity of 0.98 for blinding, and sensitivity of 0.88 and specificity of 0.97 for sample size calculation). y For the MCAO search we identified 1311 publications, of which 1152 met our inclusion criteria. We were able to retrieve full-text pdfs for 918 of these. The RegEx called 670 of 918 MCAO studies (73%) as reporting randomization, 621 (68%) as reporting the blinded assessment of outcome and 21 (2%) as reporting a sample size calculation. Compared with manual assessment, sensi- tivity was 1.00 (randomization), 0.99 (blinding) and 0.26 (sample size calculation), with specificity of 0.67 (random- ization), 0.77 (blinded assessment of outcome) and 0.99 (sample size calculation). Accuracy was 87% (randomization), 90% (blinded assessment of outcome) and 95% (sample size calculation) (Table 1). p Reporting of randomization and of sample size calculations was highest in the last quartile and lowest in the first quartile, but using least squares linear regression we found no significant change over time. In journals represented in the dataset by five publications or more, reporting of randomization ranged from 33% to 100% (median 76%), of blinded outcome assessment from 17% to 100% (median 70%), and of sample size calculations from 0% to 17% (median 0%). Overall, reporting of these measures to reduce risk of bias showed substantial improvements from those observed in a selection of stroke systematic reviews reported in 2007. In the 50 publications carried in the journal Stroke, the performance in 2014–2016 was comparable with that reported from 2013 by Minnerup. For the lacunar search we identified 492 publications, of which 61 met our inclusion criteria and of which we were able to retrieve full-text for 46. Results This lower rate of inclusion (12% vs 87% for MCAO) reflects the reduced sensitivity of the search terms used, reflecting in turn the heterogeneity in the terms used to describe animal experiments modelling lacunar stroke. The RegEx called 31 of 46 lacunar studies (67%) as reporting randomization, 28 (61%) as reporting the blinded assessment of outcome and 2 (4%) as reporting a sample size calculation. Compared with manual ascertainment, sen- sitivity was 1.00 for each of randomization and blinded assessment of outcome, and 0.50 for sample size calculation, with specificity of 0.48 (randomization), 0.81 (blinded assessment of outcome) and 1.00 (sample size calculation). Accuracy was 67% (randomization), 91% (blinded assessment of outcome) and 96% (sample size calculation). True prevalence of randomization was 37%, of blinding 53% and of sample size calculations 9%. These are essentially un- changed from the Pedder review of the lacunar stroke literature up to 2012, showing no improvements in reporting of measures to reduce risk of bias over time. No journals were represented in the dataset by five publications or more. Development of ‘regular expressions’ Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 Table 1 Summary of performance of RegEx compared with gold standard of manual human ascertainment Prevalence (RegEx) Prevalence (gold standard) Sensitivity Specificity Accuracy MCAO (n=918) Randomization 73% 60% 1.00 0.67 87% Blinding 68% 59% 0.99 0.77 90% Sample size calculation 2% 6% 0.26 0.99 95% Lacunar (n=46) Randomization 67% 37% 1.00 0.48 67% Blinding 61% 53% 1.00 0.81 91% Sample size calculation 4% 9% 0.50 1.00 96% Accuracy is calculated as the number of publications correctly labelled as a proportion of the total number of publications. Table 1 Summary of performance of RegEx compared with gold standard of manual human ascertainment of performance of RegEx compared with gold standard of manual human ascertainment Table 1 Summary of performance of RegEx compared with gold standard of manual huma c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Discussion There have been improvements in reporting of measures to reduce risks of bias in the MCAO literature over the last 10 years, but we did not observe similar improvements in the in vivo lacunar stroke literature. For both MCAO and lacunar stroke the reporting of a sample size calculation remained low, and there remains substantial room for 2530 Clinical Science (2017) 131 2525–2532 https://doi.org/10.1042/CS20160722 improvement. Within the MCAO literature performance differed between journals, for instance, in journals con- tributing five or more publications the rate of reporting of randomization ranged from 33% to 100%, of blinding from 17% to 100%, and of sample size calculation from 0% to 17%. There were no substantial differences, in aggre- gate, in 239 manuscripts published in journals publishing fewer than five included publications compared with 679 manuscripts published in journals publishing five or more included publications (blinding 63% (less than 5) vs 69% (5 or more), randomization 72% vs 73%, sample size calculation 1% vs 3%). p While it has been argued that an experiment not guided by a sample size calculation is not in itself at risk of bias, we consider the presentation of a sample size calculation to give much greater confidence in the results presented. Firstly, in the presence of publication bias, underpowered experiments will lead to a literature that overstates observed effects [14]. Secondly, a sample size calculation gives greater confidence that the experiment is presented as originally planned, rather than group size being incrementally increased until an arbitrary level of statistical significance is achieved. Finally, the positive predictive value of an experiment is increased when a study has adequate statistical power [15], and without a sample size calculation this is less likely to be the case. y Our study has limitations. Firstly, screening and gold standard risk of bias annotations were performed by a single investigator, and this may have led to errors. Secondly, we were only able to perform data extraction for manuscripts where full-text pdf was available for automated retrieval or pdf could be converted to text, and it may be that the reporting of risks of bias is different in journals for which we do not enjoy an institutional subscription. Discussion Moreover, the automated tool did not take into account the existence of any supplementary materials that might have been associated with a publication and may have contained the reporting of methodology pertaining to the measures to reduce risk of bias. Such supplementary materials are not always returned during automatic text retrieval; but in any case reporting guidelines usually recommend that such items be covered in the main text. Finally, the performance of the RegEx for randomization and for sample size calculations did not perform as well for stroke studies as had been the case in other datasets. Problems related mainly to ‘overcalling’ of blinding and randomization (low specificity) and ‘undercalling’ of sample size calculations (low sensitivity). It may be that there are domain specific differences in language used, and these will be incorporated into further iterations of the RegEx used. Future work will include the integration with text mining tools which support screening (such as app.syrf.org.uk) and reduce the amount of annotations required by using topic analysis and active learning [16,17]; and crowdsourced annotations performed in the context of reviews using the SyRF platform will contribute to the pool of annotated data available for tool development. However, for the reporting of the blinded assessment of outcome our RegEx performed at or around the level which had previously been determined as the threshold above which it could be used for the automated ascertainment of the reporting of measures to reduce risk of bias in in vivo research. Such ascertainment, providing as it does almost real-time reporting of the performance of a journal (or an institution, a funder or a nation), might serve as an indicator of changes in the quality of published research, and be used to guide audit for improvement activity, for instance the application of Plan-Do-Study-Act cycles [18] to research activity. Funding g This work was funded by a joint grant from UK MRC and Wellcome Trust administered by the MRC as MR/N015665/1. Competing Interests The authors declare that there are no competing interests associated with the manuscript. Author Contribution Z.B., J.T., M.R.M., A.R., S.A. and E.S. conceived the experiment. Z.B., J.L., M.R.M. and E.S. designed the experiment. M.R.M. conducted the systematic searches, screened for inclusion. M.R.M., A.B.-B., S.K.M., K.E.W., D.W.H. and T.O. attributed risks of bias manually. Z.B. developed the RegEx tools, J.L. performed the pdf to text conversion and ran the R code. 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This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). c⃝2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).